Therapeutic compositions

[0001] The present application is a continuation of U.S. application Ser. No. 14/282,769, filed May 20, 2014, which is a continuation of U.S. application Ser. No. 13/626,196, filed Sep. 25, 2012, which is a continuation of U.S. application Ser. No. 12/721,413, filed Mar. 10, 2010, which is a continuation of U.S. application Ser. No. 10/548,611, filed Aug. 22, 2006, which is the National Stage of International Application No. PCT/US2004/007070, filed Mar. 8, 2004, which claims the benefit of Application No. 60/452,682, filed Mar. 7, 2003; Application No. 60/462,894, filed Apr. 14, 2003; and Application No. 60/465,665, filed Apr. 25, 2003; Application No. 60/463,772, filed Apr. 17, 2003; Application No. 60/465,802, filed Apr. 25, 2003; Application No. 60/493,986, filed Aug. 8, 2003; Application No. 60/494,597, filed Aug. 11, 2003; Application No. 60/506,341, filed Sep. 26, 2003; Application No. 60/518,453, filed Nov. 7, 2003; Application No. 60/454,265, filed Mar. 12, 2003; Application No. 60/454,962, filed Mar. 13, 2003; Application No. 60/455,050, filed Mar. 13, 2003; Application No. 60/469,612, filed May 9, 2003; Application No. 60/510,246, filed Oct. 9, 2003; Application No. 60/510,318, filed Oct. 10, 2003. The contents of the above applications are hereby incorporated by reference in their entirety.

[0002] The invention relates to RNAi and related methods, e.g., methods of making and using iRNA agents.

[0003] RNA interference or “RNAi” is a term initially coined by Fire and co-workers to describe the observation that double-stranded RNA (dsRNA) can block gene expression when it is introduced into worms (Fire et al. (1998) Nature 391, 806-811). Short dsRNA directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. RNAi may involve mRNA degradation.

[0004] A number of advances related to the application of RNAi to the treatment of subjects are disclosed herein. For example, the invention features iRNA agents targeted to specific genes; palindromic iRNA agents; iRNA agents having non canonical monomer pairings; iRNA agents having particular structures or architectures e.g., the Z—X—Y or asymmetrical iRNA agents described herein; drug delivery conjugates for the delivery of iRNA agents; amphipathic substances for the delivery of iRNA agents, as well as iRNA agents having chemical modifications for optimizing a property of the iRNA agent. The invention features each of these advances broadly as well as in combinations. For example, an iRNA agent targeted to a specific gene can also include one or more of a palindrome, non canonical, Z—X—Y, or asymmetric structure. Other nonlimiting examples of combinations include an asymmetric structure combined with a chemical modification, or formulations or methods or routes of delivery combined with, e.g., chemical modifications or architectures described herein. The iRNA agents of the invention can include any one of these advances, or pairwise or higher order combinations of the separate advances.

[0005] In one aspect, the invention features iRNA agents that can target more than one RNA region, and methods of using and making the iRNA agents.

[0006] In another aspect, an iRNA agent includes a first and second sequence that are sufficiently complementary to each other to hybridize. The first sequence can be complementary to a first target RNA region and the second sequence can be complementary to a second target RNA region.

[0007] In one embodiment, the first and second sequences of the iRNA agent are on different RNA strands, and the mismatch between the first and second sequences is less than 50%, 40%, 30%, 20%, 10%, 5%, or 1%.

[0008] In another embodiment, the first and second sequences of the iRNA agent are on the same RNA strand, and in a related embodiment more than 50%, 60%, 70%, 80%, 90%, 95%, or 1% of the iRNA agent is in bimolecular form.

[0009] In another embodiment, the first and second sequences of the iRNA agent are fully complementary to each other.

[0010] In one embodiment, the first target RNA region is encoded by a first gene and the second target RNA region is encoded by a second gene, and in another embodiment, the first and second target RNA regions are different regions of an RNA from a single gene. In another embodiment, the first and second sequences differ by at least 1 and no more than 6 nucleotides.

[0011] In certain embodiments, the first and second target RNA regions are on transcripts encoded by first and second sequence variants, e.g., first and second alleles, of a gene. The sequence variants can be mutations, or polymorphisms, for example.

[0012] In certain embodiments, the first target RNA region includes a nucleotide substitution, insertion, or deletion relative to the second target RNA region.

[0013] In other embodiments, the second target RNA region is a mutant or variant of the first target RNA region.

[0014] In certain embodiments, the first and second target RNA regions comprise viral, e.g., HCV, or human RNA regions. The first and second target RNA regions can also be on variant transcripts of an oncogene or include different mutations of a tumor suppressor gene transcript. In one embodiment, the oncogene, or tumor suppressor gene is expressed in the liver. In addition, the first and second target RNA regions correspond to hot-spots for genetic variation.

[0015] In another aspect, the invention features a mixture of varied iRNA agent molecules, including one iRNA agent that includes a first sequence and a second sequence sufficiently complementary to each other to hybridize, and where the first sequence is complementary to a first target RNA region and the second sequence is complementary to a second target RNA region. The mixture also includes at least one additional iRNA agent variety that includes a third sequence and a fourth sequence sufficiently complementary to each other to hybridize, and where the third sequence is complementary to a third target RNA region and the fourth sequence is complementary to a fourth target RNA region. In addition, the first or second sequence is sufficiently complementary to the third or fourth sequence to be capable of hybridizing to each other. In one embodiment, at least one, two, three or all four of the target RNA regions are expressed in the liver. Exemplary RNAs are transcribed from the apoB-100 gene, glucose-6-phosphatase gene, beta catenin gene, or an HCV gene.

[0016] In certain embodiments, the first and second sequences are on the same or different RNA strands, and the third and fourth sequences are on same or different RNA strands.

[0017] In one embodiment, the mixture further includes a third iRNA agent that is composed of the first or second sequence and the third or fourth sequence.

[0018] In one embodiment, the first sequence is identical to at least one of the second, third and fourth sequences, and in another embodiment, the first region differs by at least 1 but no more than 6 nucleotides from at least one of the second, third and fourth regions.

[0019] In certain embodiments, the first target RNA region comprises a nucleotide substitution, insertion, or deletion relative to the second, third or fourth target RNA region.

[0020] The target RNA regions can be variant sequences of a viral or human RNA, and in certain embodiments, at least two of the target RNA regions can be on variant transcripts of an oncogene or tumor suppressor gene. In one embodiment, the oncogene or tumor suppressor gene is expressed in the liver.

[0021] In certain embodiments, at least two of the target RNA regions correspond to hot-spots for genetic variation.

[0022] In one embodiment, the iRNA agents of the invention are formulated for pharmaceutical use. In one aspect, the invention provides a container (e.g., a vial, syringe, nebulizer, etc) to hold the iRNA agents described herein.

[0023] Another aspect of the invention features a method of making an iRNA agent. The method includes constructing an iRNA agent that has a first sequence complementary to a first target RNA region, and a second sequence complementary to a second target RNA region. The first and second target RNA regions have been identified as being sufficiently complementary to each other to be capable of hybridizing. In one embodiment, the first and second target RNA regions are on transcripts expressed in the liver.

[0024] In certain embodiments, the first and second target RNA regions can correspond to two different regions encoded by one gene, or to regions encoded by two different genes.

[0025] Another aspect of the invention features a method of making an iRNA agent composition. The method includes obtaining or providing information about a region of an RNA of a target gene (e.g., a viral or human gene, or an oncogene or tumor suppressor, e.g., p53), where the region has high variability or mutational frequency (e.g., in humans). In addition, information about a plurality of RNA targets within the region is obtained or provided, where each RNA target corresponds to a different variant or mutant of the gene (e.g., a region including the codon encoding p53 248Q and/or p53 249S). The iRNA agent is constructed such that a first sequence is complementary to a first of the plurality of variant RNA targets (e.g., encoding 249Q) and a second sequence is complementary to a second of the plurality of variant RNA targets (e.g., encoding 249S). The first and second sequences are sufficiently complementary to hybridize. In certain embodiments, the target gene can be a viral or human gene expressed in the liver.

[0026] In one embodiment, sequence analysis, e.g., to identify common mutants in the target gene, is used to identify a region of the target gene that has high variability or mutational frequency. For example, sequence analysis can be used to identify regions of apoB-100 or beta catenin that have high variability or mutational frequency. In another embodiment, the region of the target gene having high variability or mutational frequency is identified by obtaining or providing genotype information about the target gene from a population. In another embodiment, the genotype information can be from a population suffering from a liver disorder, such as hepatocellular carcinoma or hepatoblastoma.

[0027] Another aspect of the invention features a method of modulating expression, e.g., downregulating or silencing, a target gene, by providing an iRNA agent that has a first sequence and a second sequence sufficiently complementary to each other to hybridize. In addition, the first sequence is complementary to a first target RNA region and the second sequence is complementary to a second target RNA region.

[0028] In one embodiment, the iRNA agent is administered to a subject, e.g., a human.

[0029] In another embodiment, the first and second sequences are between 15 and 30 nucleotides in length.

[0030] In one embodiment, the method of modulating expression of the target gene further includes providing a second iRNA agent that has a third sequence complementary to a third target RNA region. The third sequence can be sufficiently complementary to the first or second sequence to be capable of hybridizing to either the first or second sequence.

[0031] Another aspect of the invention features a method of modulating expression, e.g., downregulating or silencing, a plurality of target RNAs, each of the plurality of target RNAs corresponding to a different target gene. The method includes providing an iRNA agent selected by identifying a first region in a first target RNA of the plurality and a second region in a second target RNA of the plurality, where the first and second regions are sufficiently complementary to each other to be capable of hybridizing.

[0032] In another aspect of the invention, an iRNA agent molecule includes a first sequence complementary to a first variant RNA target region and a second sequence complementary to a second variant RNA target region, and the first and second variant RNA target regions correspond to first and second variants or mutants of a target gene. In certain embodiments, the target gene is an apoB-100, beta catenin, or glucose-6 phosphatase gene.

[0033] In one embodiment, the target gene is a viral gene (e.g., an HCV gene), tumor suppressor or oncogene.

[0034] In another embodiment, the first and second variant target RNA regions include allelic variants of the target gene.

[0035] In another embodiment, the first and second variant RNA target regions comprise mutations (e.g., point mutations) or polymorphisms of the target gene.

[0036] In one embodiment, the first and second variant RNA target regions correspond to hot-spots for genetic variation.

[0037] Another aspect of the invention features a plurality (e.g., a panel or bank) of iRNA agents. Each of the iRNA agents of the plurality includes a first sequence complementary to a first variant target RNA region and a second sequence complementary to a second variant target RNA region, where the first and second variant target RNA regions correspond to first and second variants of a target gene. In certain embodiments, the variants are allelic variants of the target gene.

[0038] Another aspect of the invention provides a method of identifying an iRNA agent for treating a subject. The method includes providing or obtaining information, e.g., a genotype, about a target gene, providing or obtaining information about a plurality (e.g., panel or bank) of iRNA agents, comparing the information about the target gene to information about the plurality of iRNA agents, and selecting one or more of the plurality of iRNA agents for treating the subject. Each of the plurality of iRNA agents includes a first sequence complementary to a first variant target RNA region and a second sequence complementary to a second variant target RNA region, and the first and second variant target RNA regions correspond to first and second variants of the target gene. The target gene can be an endogenous gene of the subject or a viral gene. The information about the plurality of iRNA agents can be the sequence of the first or second sequence of one or more of the plurality.

[0039] In certain embodiments, at least one of the selected iRNA agents includes a sequence capable of hybridizing to an RNA region corresponding to the target gene, and at least one of the selected iRNA agents comprises a sequence capable of hybridizing to an RNA region corresponding to a variant or mutant of the target gene.

[0040] In one aspect, the invention relates to compositions and methods for silencing genes expressed in the liver, e.g., to treat disorders of or related to the liver. An iRNA agent composition of the invention can be one which has been modified to alter distribution in favor of the liver.

[0041] In another aspect, the invention relates to iRNA agents that can target more than one RNA region, and methods of using and making the iRNA agents. In one embodiment, the RNA is from a gene that is active in the liver, e.g., apoB-100, glucose-6-phosphatase, beta-catenin, or Hepatitis C virus (HCV).

[0042] In another aspect, an iRNA agent includes a first and second sequence that are sufficiently complementary to each other to hybridize. The first sequence can be complementary to a first target RNA region and the second sequence can be complementary to a second target RNA region. For example, the first sequence can be complementary to a first target apoB-100 RNA region and the second sequence can be complementary to a second target apoB-100 RNA region.

[0043] In one embodiment, the first target RNA region is encoded by a first gene, e.g., a gene expressed in the liver, and the second target RNA region is encoded by a second gene, e.g., a second gene expressed in the liver. In another embodiment, the first and second target RNA regions are different regions of an RNA from a single gene, e.g., a single gene that is at least expressed in the liver. In another embodiment, the first and second sequences differ by at least one and no more than six nucleotides.

[0044] In another embodiment, sequence analysis, e.g., to identify common mutants in the target gene, is used to identify a region of the target gene that has high variability or mutational frequency. For example, sequence analysis can be used to identify regions of aopB-100 or beta catenin that have high variability or mutational frequency. In another embodiment, the region of the target gene having high variability or mutational frequency is identified by obtaining or providing genotype information about the target gene from a population. In particular, the genotype information can be from a population suffering from a liver disorder, such as hepatocellular carcinoma or hepatoblastoma.

[0045] In another aspect, the invention features a method for reducing apoB-100 levels in a subject, e.g., a mammal, such as a human. The method includes administering to a subject an iRNA agent which targets apoB-100. The iRNA agent can be one described here, and can be a dsRNA that has a sequence that is substantially identical to a sequence of the apoB-100 gene. The iRNA can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length. In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length.

[0046] In a preferred embodiment, the subject is treated with an iRNA agent which targets one of the sequences listed in Tables 5 and 6. In a preferred embodiment it targets both sequences of a palindromic pair provided in Tables 5 and 6. The most preferred targets are listed in descending order of preferrability, in other words, the more preferred targets are listed earlier in Tables 5 and 6.

[0047] In a preferred embodiment the iRNA agent will include regions, or strands, which are complementary to a pair in Tables 5 and 6. In a preferred embodiment the iRNA agent will include regions complementary to the palindromic pairs of Tables 5 and 6 as a duplex region.

[0048] In a preferred embodiment the duplex region of the iRNA agent will target a sequence listed in Tables 5 and 6 but will not be perfectly complementary with the target sequence, e.g., it will not be complementary at at least 1 base pair. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, in total, or per strand, which do not hybridize with the target sequence

[0049] In a preferred embodiment the iRNA agent includes overhangs, e.g., 3′ or 5′ overhangs, preferably one or more 3′ overhangs. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

[0050] The iRNA agent that targets apoB-100 can be administered in an amount sufficient to reduce expression of apoB-100 mRNA. In one embodiment, the iRNA agent is administered in an amount sufficient to reduce expression of apoB-100 protein (e.g., by at least 2%, 4%, 6%, 10%, 15%, 20%). Preferably, the iRNA agent does not reduce expression of apoB-48 mRNA or protein. This can be effected, e.g., by selection of an iRNA agent which specifically targets the nucleotides subject to RNA editing in the apoB-100 transcript.

[0051] The iRNA agent that targets apoB-100 can be administered to a subject, wherein the subject is suffering from a disorder characterized by elevated or otherwise unwanted expression of apoB-100, elevated or otherwise unwanted levels of cholesterol, and/or disregulation of lipid metabolism. The iRNA agent can be administered to an individual at risk for the disorder to delay onset of the disorder or a symptom of the disorder. These disorders include HDL/LDL cholesterol imbalance; dyslipidemias, e.g., familial combined hyperlipidemia (FCHL), acquired hyperlipidemia; hypercholestorolemia; statin-resistant hypercholesterolemia; coronary artery disease (CAD) coronary heart disease (CHD) atherosclerosis. In one embodiment, the iRNA that targets apoB-100 is administered to a subject suffering from statin-resistant hypercholesterolemia.

[0052] The apoB-100 iRNA agent can be administered in an amount sufficient to reduce levels of serum LDL-C and/or HDL-C and/or total cholesterol in a subject. For example, the iRNA is administered in an amount sufficient to decrease total cholesterol by at least 0.5%, 1%, 2.5%, 5%, 10% in the subject. In one embodiment, the iRNA agent is administered in an amount sufficient to reduce the risk of myocardial infarction the subject.

[0053] In a preferred embodiment the iRNA agent is administered repeatedly. Administration of an iRNA agent can be carried out over a range of time periods. It can be administered daily, once every few days, weekly, or monthly. The timing of administration can vary from patient to patient, depending on such factors as the severity of a patient's symptoms. For example, an effective dose of an iRNA agent can be administered to a patient once a month for an indefinite period of time, or until the patient no longer requires therapy. In addition, sustained release compositions containing an iRNA agent can be used to maintain a relatively constant dosage in the patient's blood.

[0054] In one embodiment, the iRNA agent can be targeted to the liver, and apoB expression level are decreased in the liver following administration of the apoB iRNA agent. For example, the iRNA agent can be complexed with a moiety that targets the liver, e.g., an antibody or ligand that binds a receptor on the liver.

[0055] The iRNA agent, particularly an iRNA agent that targets apoB, beta-catenin or glucose-6-phosphatase RNA, can be targeted to the liver, for example by associating, e.g., conjugating the iRNA agent to a lipophilic moiety, e.g., a lipid, cholesterol, oleyl, retinyl, or cholesteryl residue (see Table 1). Other lipophilic moieties that can be associated, e.g., conjugated with the iRNA agent include cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, bomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. In one embodiment, the iRNA agent can be targeted to the liver by associating, e.g., conjugating, the iRNA agent to a low-density lipoprotein (LDL), e.g., a lactosylated LDL. In another embodiment, the iRNA agent can be targeted to the liver by associating, e.g., conjugating, the iRNA agent to a polymeric carrier complex with sugar residues.

[0056] In another embodiment, the iRNA agent can be targeted to the liver by associating, e.g., conjugating, the iRNA agent to a liposome complexed with sugar residues. A targeting agent that incorporates a sugar, e.g., galactose and/or analogues thereof, is particularly useful. These agents target, in particular, the parenchymal cells of the liver (see Table 1). In a preferred embodiment, the targeting moiety includes more than one galactose moiety, preferably two or three. Preferably, the targeting moiety includes 3 galactose moieties, e.g., spaced about 15 angstroms from each other. The targeting moiety can be lactose. A lactose is a glucose coupled to a galactose. Preferably, the targeting moiety includes three lactoses. The targeting moiety can also be N-Acetyl-Galactosamine, N-Ac-Glucosamine. A mannose, or mannose-6-phosphate targeting moiety can be used for macrophage targeting.

[0057] The targeting agent can be linked directly, e.g., covalently or non covalently, to the iRNA agent, or to another delivery or formulation modality, e.g., a liposome. E.g., the iRNA agents with or without a targeting moiety can be incorporated into a delivery modality, e.g., a liposome, with or without a targeting moiety.

[0058] It is particularly preferred to use an iRNA conjugated to a lipophilic molecule to conjugate to an iRNA agent that targets apoB, beta-catenin or glucose-6-phosphatase iRNA targeting agent.

[0059] In one embodiment, the iRNA agent has been modified, or is associated with a delivery agent, e.g., a delivery agent described herein, e.g., a liposome, which has been modified to alter distribution in favor of the liver. In one embodiment, the modification mediates association with a serum albumin (SA), e.g., a human serum albumin (HSA), or a fragment thereof.

[0060] The iRNA agent, particularly an iRNA agent that targets apoB, beta-catenin or glucose-6-phosphatase RNA, can be targeted to the liver, for example by associating, e.g., conjugating the iRNA agent to an SA molecule, e.g., an HSA molecule, or a fragment thereof. In one embodiment, the iRNA agent or composition thereof has an affinity for an SA, e.g., HSA, which is sufficiently high such that its levels in the liver are at least 10, 20, 30, 50, or 100% greater in the presence of SA, e.g., HSA, or is such that addition of exogenous SA will increase delivery to the liver. These criteria can be measured, e.g., by testing distribution in a mouse in the presence or absence of exogenous mouse or human SA.

[0061] The SA, e.g., HSA, targeting agent can be linked directly, e.g., covalently or noncovalently, to the iRNA agent, or to another delivery or formulation modality, e.g., a liposome. E.g., the iRNA agents with or without a targeting moiety can be incorporated into a delivery modality, e.g., a liposome, with or without a targeting moiety.

[0062] It is particularly preferred to use an iRNA conjugated to an SA, e.g., an HSA, molecule wherein the iRNA agent is an apoB, beta-catenin or glucose-6-phosphatase iRNA targeting agent.

[0063] In another aspect, the invention features, a method for reducing glucose-6-phosphatase levels in a subject, e.g., a mammal, such as a human. The method includes administering to a subject an iRNA agent which targets glucose-6-phosphatase. The iRNA agent can be a dsRNA that has a sequence that is substantially identical to a sequence of the glucose-6-phosphatase gene.

[0064] In a preferred embodiment, the subject is treated with an iRNA agent which targets one of the sequences listed in Table 7. In a preferred embodiment it targets both sequences of a palindromic pair provided in Table 7. The most preferred targets are listed in descending order of preferrability, in other words, the more preferred targets are listed earlier in Table 7.

[0065] In a preferred embodiment the iRNA agent will include regions, or strands, which are complementary to a pair in Table 7. In a preferred embodiment the iRNA agent will include regions complementary to the palindromic pairs of Table 7 as a duplex region.

[0066] In a preferred embodiment the duplex region of the iRNA agent will target a sequence listed in Table 7 but will not be perfectly complementary with the target sequence, e.g., it will not be complementary at at least 1 base pair. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, in total, or per strand, which do not hybridize with the target sequence

[0067] In a preferred embodiment the iRNA agent includes overhangs, e.g., 3′ or 5′ overhangs, preferably one or more 3′ overhangs. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

[0068] Table 7 refers to sequences from human glucose-6-phosphatase. Table 8 refers to sequences from rat glucose-6-phosphatase. The sequences from table 8 can be used, e.g., in experiments with rats or cultured rat cells.

[0069] In a preferred embodiment iRNA agent can have any architecture, e.g., architecture described herein. E.g., it can be incorporated into an iRNA agent having an overhang structure, overall length, hairpin vs. two-strand structure, as described herein. In addition, monomers other than naturally occurring ribonucleotides can be used in the selected iRNA agent.

[0070] The iRNA that targets glucose-6-phosphatase can be administered in an amount sufficient to reduce expression of glucose-6-phosphatase mRNA.

[0071] The iRNA that targets glucose-6-phosphatase can be administered to a subject to inhibit hepatic glucose production, for the treatment of glucose-metabolism-related disorders, such as diabetes, e.g., type-2-diabetes mellitus. The iRNA agent can be administered to an individual at risk for the disorder to delay onset of the disorder or a symptom of the disorder.

[0072] In other embodiments, iRNA agents having sequence similarity to the following genes can also be used to inhibit hepatic glucose production. These other genes include “forkhead homologue in rhabdomyosarcoma (FKHR); glucagon; glucagon receptor; glycogen phosphorylase; PPAR-Gamma Coactivator (PGC-1); Fructose-1,6-bisphosphatase; glucose-6-phosphate locator; glucokinase inhibitory regulatory protein; and phosphoenolpyruvate carboxykinase (PEPCK).

[0073] In one embodiment, the iRNA agent can be targeted to the liver, and RNA expression levels of the targeted genes are decreased in the liver following administration of the iRNA agent.

[0074] The iRNA agent can be one described herein, and can be a dsRNA that has a sequence that is substantially identical to a sequence of a target gene. The iRNA can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length. In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length

[0075] In another aspect, the invention features a method for reducing beta-catenin levels in a subject, e.g., a mammal, such as a human. The method includes administering to a subject an iRNA agent that targets beta-catenin. The iRNA agent can be one described herein, and can be a dsRNA that has a sequence that is substantially identical to a sequence of the beta-catenin gene. The iRNA can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length. In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length.

[0076] In a preferred embodiment, the subject is treated with an iRNA agent which targets one of the sequences listed in Table 9. In a preferred embodiment it targets both sequences of a palindromic pair provided in Table 9. The most preferred targets are listed in descending order of preferrability, in other words, the more preferred targets are listed earlier in Table 9.

[0077] In a preferred embodiment, the subject is treated with an iRNA agent which targets one of the sequences listed in Table 9. In a preferred embodiment it targets both sequences of a palindromic pair provided in Table 9. The most preferred targets are listed in descending order of preferrability, in other words, the more preferred targets are listed earlier in Table 9.

[0078] In a preferred embodiment the iRNA agent will include regions, or strands, which are complementary to a pair in Table 9. In a preferred embodiment the iRNA agent will include regions complementary to the palindromic pairs of Table 9 as a duplex region.

[0079] In a preferred embodiment the duplex region of the iRNA agent will target a sequence listed in Table 9 but will not be perfectly complementary with the target sequence, e.g., it will not be complementary at at least 1 base pair. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, in total, or per strand, which do not hybridize with the target sequence

[0080] In a preferred embodiment the iRNA agent includes overhangs, e.g., 3′ or 5′ overhangs, preferably one or more 3′ overhangs. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

[0081] The iRNA agent that targets beta-catenin can be administered in an amount sufficient to reduce expression of beta-catenin mRNA. In one embodiment, the iRNA agent is administered in an amount sufficient to reduce expression of beta-catenin protein (e.g., by at least 2%, 4%, 6%, 10%, 15%, 20%).

[0082] The iRNA agent that targets beta-catenin can be administered to a subject, wherein the subject is suffering from a disorder characterized by unwanted cellular proliferation in the liver or of liver tissue, e.g., metastatic tissue originating from the liver. Examples include, a benign or malignant disorder, e.g., a cancer, e.g., a hepatocellular carcinoma (HCC), hepatic metastasis, or hepatoblastoma.

[0083] The iRNA agent can be administered to an individual at risk for the disorder to delay onset of the disorder or a symptom of the disorder

[0084] In a preferred embodiment the iRNA agent is administered repeatedly. Administration of an iRNA agent can be carried out over a range of time periods. It can be administered daily, once every few days, weekly, or monthly. The timing of administration can vary from patient to patient, depending on such factors as the severity of a patient's symptoms. For example, an effective dose of an iRNA agent can be administered to a patient once a month for an indefinite period of time, or until the patient no longer requires therapy. In addition, sustained release compositions containing an iRNA agent can be used to maintain a relatively constant dosage in the patient's blood.

[0085] In one embodiment, the iRNA agent can be targeted to the liver, and beta-catenin expression level are decreased in the liver following administration of the beta-catenin iRNA agent. For example, the iRNA agent can be complexed with a moiety that targets the liver, e.g., an antibody or ligand that binds a receptor on the liver.

[0086] In another aspect, the invention provides methods to treat liver disorders, e.g., disorders characterized by unwanted cell proliferation, hematological disorders, disorders characterized by inflammation disorders, and metabolic or viral diseases or disorders of the liver. A proliferation disorder of the liver can be, for example, a benign or malignant disorder, e.g., a cancer, e.g., a hepatocellular carcinoma (HCC), hepatic metastasis, or hepatoblastoma. A hepatic hematology or inflammation disorder can be a disorder involving clotting factors, a complement-mediated inflammation or a fibrosis, for example. Metabolic diseases of the liver can include dyslipidemias, and irregularities in glucose regulation. Viral diseases of the liver can include hepatitis C or hepatitis B. In one embodiment, a liver disorder is treated by administering one or more iRNA agents that have a sequence that is substantially identical to a sequence in a gene involved in the liver disorder.

[0087] In one embodiment an iRNA agent to treat a liver disorder has a sequence which is substantially identical to a sequence of the beta-catenin or c-jun gene. In another embodiment, such as for the treatment of hepatitis C or hepatitis B, the iRNA agent can have a sequence that is substantially identical to a sequence of a gene of the hepatitis C virus or the hepatitis B virus, respectively. For example, the iRNA agent can target the 5′ core region of HCV. This region lies just downstream of the ribosomal toe-print straddling the initiator methionine. Alternatively, an iRNA agent of the invention can target any one of the nonstructural proteins of HCV: NS3, 4A, 4B, 5A, or 5B. For the treatment of hepatitis B, an iRNA agent can target the protein X (HBx) gene, for example.

[0088] In a preferred embodiment, the subject is treated with an iRNA agent which targets one of the sequences listed in Table 10. In a preferred embodiment it targets both sequences of a palindromic pair provided in Table 10. The most preferred targets are listed in descending order of preferrability, in other words, the more preferred targets are listed earlier in Table 10.

[0089] In a preferred embodiment the iRNA agent will include regions, or strands, which are complementary to a pair in Table 10. In a preferred embodiment the iRNA agent will include regions complementary to the palindromic pairs of Table 10 as a duplex region.

[0090] In a preferred embodiment the duplex region of the iRNA agent will target a sequence listed in Table 10, but will not be perfectly complementary with the target sequence, e.g., it will not be complementary at at least 1 base pair. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, in total, or per strand, which do not hybridize with the target sequence

[0091] In a preferred embodiment the iRNA agent includes overhangs, e.g., 3′ or 5′ overhangs, preferably one or more 3′ overhangs. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

[0092] In another aspect, an iRNA agent can be administered to modulate blood clotting, e.g., to reduce the tendency to form a blood clot. In a preferred embodiment the iRNA agent targets Factor V expression, preferably in the liver. One or more iRNA agents can be used to target a wild type allele, a mutant allele, e.g., the Leiden Factor V allele, or both. Such administration can be used to treat or prevent venous thrombosis, e.g., deep vein thrombosis or pulmonary embolism, or another disorder caused by elevated or otherwise unwanted expression of Factor V, in, e.g., the liver. In one embodiment the iRNA agent can treat a subject, e.g., a human who has Factor V Leiden or other genetic trait associated with an unwanted tendency to form blood clots.

[0093] In a preferred embodiment administration of an iRNA agent which targets Factor V is with the administration of a second treatment, e.g., a treatment which reduces the tendency of the blood to clot, e.g., the administration of heparin or of a low molecular weight heparin.

[0094] In one embodiment, the iRNA agent that targets Factor V can be used as a prophylaxis in patients, e.g., patients with Factor V Leiden, who are placed at risk for a thrombosis, e.g., those about to undergo surgery, in particular those about to undergo high-risk surgical procedures known to be associated with formation of venous thrombosis, those about to undergo a prolonged period of relative inactivity, e.g., on a motor vehicle, train or airplane flight, e.g., a flight or other trip lasting more than three or five hours. Such a treatment can be an adjunct to the therapeutic use of low molecular weight (LMW) heparin prophylaxis.

[0095] In another embodiment, the iRNA agent that targets Factor V can be administered to patients with Factor V Leiden to treat deep vein thrombosis (DVT) or pulmonary embolism (PE). Such a treatment can be an adjunct to (or can replace) therapeutic uses of heparin or coumadin. The treatment can be administered by inhalation or generally by pulmonary routes.

[0096] In a preferred embodiment, an iRNA agent administered to treat a liver disorder is targeted to the liver. For example, the iRNA agent can be complexed with a targeting moiety, e.g., an antibody or ligand that recognizes a liver-specific receptor.

[0097] The invention also includes preparations, including substantially pure or pharmaceutically acceptable preparations of iRNA agents which silence any of the genes discussed herein and in particular for any of apoB-100, glucose-6-phosphatase, beta-catenin, factor V, or any of the HVC genes discussed herein.

[0098] The methods and compositions of the invention, e.g., the methods and compositions to treat diseases and disorders of the liver described herein, can be used with any of the iRNA agents described. In addition, the methods and compositions of the invention can be used for the treatment of any disease or disorder described herein, and for the treatment of any subject, e.g., any animal, any mammal, such as any human.

[0099] In another aspect, the invention features, a method of selecting two sequences or strands for use in an iRNA agent. The method includes:

[0100] providing a first candidate sequence and a second candidate sequence;

[0101] determining the value of a parameter which is a function of the number of palindromic pairs between the first and second sequence, wherein a palindromic pair is a nucleotide on said first sequence which, when the sequences are aligned in anti-parallel orientation, will hybridize with a nucleotide on said second sequence;

[0102] comparing the number with a predetermined reference value, and if the number has a predetermined relationship with the reference, e.g., if it is the same or greater, selecting the sequences for use in an iRNA agent. In most cases each of the two sequences will be completely complementary with a target sequence (though as described elsewhere herein that may not always be the case, there may not be perfect complementarity with one or both of the target sequences) and will have sufficient complementarity with each other to form a duplex. The parameter can be derived e.g., by directly determining the number of palindromic pairs, e.g., by inspection or by the use of a computer program which compares or analyses sequence. The parameter can also be determined less directly, and include e.g., calculation of or measurement of the Tm or other value related to the free energy of association or dissociation of a duplex.

[0103] In a preferred embodiment the determination can be performed on a target sequence, e.g., a genomic sequence. In such embodiments the selected sequence is converted to its complement in the iRNA agent.

[0104] In a preferred embodiment the first and second sequences are selected from the sequence of a single target gene. In other embodiments the first sequence is selected from the sequence of a first target gene and the second sequence is selected from the target of a second target gene.

[0105] In a preferred embodiment the method includes comparing blocks of sequence, e.g., blocks which are between 15 and 25 nucleotides in length, and preferably 19, 20, or 21, and most preferably 19 nucleotides in length, to determine if they are suitable for use, e.g., if they possess sufficient palindromic pairs.

[0106] In a preferred embodiment the first and second sequences are divided into a plurality of regions, e.g., terminal regions and a middle region disposed between the terminal regions and where in the reference value, or the predetermined relationship to the reference value, is different for at least two regions. E.g., the first and second sequences, when aligned in anti-parallel orientation, are divided into terminal regions each of a selected number of base pairs, e.g., 2, 3, 4, 5, or 6, and a middle region, and the reference value for the terminal regions is higher than for the middle regions. In other words, a higher number or proportion of palindromic pairs is required in the terminal regions.

[0107] In a preferred embodiment the first and second sequences are gene sequences thus the complements of the sequences will be used in a iRNA agent.

[0108] In a preferred embodiment hybridize means a classical Watson-Crick pairing. In other embodiments hybridize can include non-Watson-Crick paring, e.g., parings seen in micro RNA precursors.

[0109] In a preferred embodiment the method includes the addition of nucleotides to form overhangs, e.g., 3′ or 5′ overhangs, preferably one or more 3′ overhangs. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

[0110] In a preferred embodiment the method is used to select all or part of a iRNA agent. The selected sequences can be incorporated into an iRNA agent having any architecture, e.g., an architecture described herein. E.g., it can be incorporated into an iRNA agent having an overhang structure, overall length, hairpin vs. two-strand structure, as described herein. In addition, monomers other than naturally occurring ribonucleotides can be used in the selected iRNA agent.

[0111] Preferred iRNA agents of this method will target genes expressed in the liver, e.g., one of the genes disclosed herein, e.g., apo B, Beta catenin, an HVC gene, or glucose 6 phosphatase.

[0112] In another aspect, the invention features, an iRNA agent, determined, made, or selected by a method described herein.

[0113] The methods and compositions of the invention, e.g., the methods and iRNA compositions to treat liver-based diseases described herein, can be used with any dosage and/or formulation described herein, as well as with any route of administration described herein.

[0114] The invention also provides for the use of an iRNA agent which includes monomers which can form other than a canonical Watson-Crick pairing with another monomer, e.g., a monomer on another strand.

[0115] The use of “other than canonical Watson-Crick pairing” between monomers of a duplex can be used to control, often to promote, melting of all or part of a duplex. The iRNA agent can include a monomer at a selected or constrained position that results in a first level of stability in the iRNA agent duplex (e.g., between the two separate molecules of a double stranded iRNA agent) and a second level of stability in a duplex between a sequence of an iRNA agent and another sequence molecule, e.g., a target or off-target sequence in a subject. In some cases the second duplex has a relatively greater level of stability, e.g., in a duplex between an anti-sense sequence of an iRNA agent and a target mRNA. In this case one or more of the monomers, the position of the monomers in the iRNA agent, and the target sequence (sometimes referred to herein as the selection or constraint parameters), are selected such that the iRNA agent duplex is has a comparatively lower free energy of association (which while not wishing to be bound by mechanism or theory, is believed to contribute to efficacy by promoting disassociation of the duplex iRNA agent in the context of the RISC) while the duplex formed between an anti-sense targeting sequence and its target sequence, has a relatively higher free energy of association (which while not wishing to be bound by mechanism or theory, is believed to contribute to efficacy by promoting association of the anti-sense sequence and the target RNA).

[0116] In other cases the second duplex has a relatively lower level of stability, e.g., in a duplex between a sense sequence of an iRNA agent and an off-target mRNA. In this case one or more of the monomers, the position of the monomers in the iRNA agent, and an off-target sequence, are selected such that the iRNA agent duplex is has a comparatively higher free energy of association while the duplex formed between a sense targeting sequence and its off-target sequence, has a relatively lower free energy of association (which while not wishing to be bound by mechanism or theory, is believed to reduce the level of off-target silencing by contribute to efficacy by promoting disassociation of the duplex formed by the sense strand and the off-target sequence).

[0117] Thus, inherent in the structure of the iRNA agent is the property of having a first stability for the intra-iRNA agent duplex and a second stability for a duplex formed between a sequence from the iRNA agent and another RNA, e.g., a target mRNA. As discussed above, this can be accomplished by judicious selection of one or more of the monomers at a selected or constrained position, the selection of the position in the duplex to place the selected or constrained position, and selection of the sequence of a target sequence (e.g., the particular region of a target gene which is to be targeted). The iRNA agent sequences which satisfy these requirements are sometimes referred herein as constrained sequences. Exercise of the constraint or selection parameters can be, e.g., by inspection, or by computer assisted methods. Exercise of the parameters can result in selection of a target sequence and of particular monomers to give a desired result in terms of the stability, or relative stability, of a duplex.

[0118] Thus, in one aspect, the invention features, an iRNA agent which includes: a first sequence which targets a first target region and a second sequence which targets a second target region. The first and second sequences have sufficient complementarity to each other to hybridize, e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. In a duplex region of the iRNA agent, at a selected or constrained position, the first target region has a first monomer, and the second target region has a second monomer. The first and second monomers occupy complementary or corresponding positions. One, and preferably both monomers are selected such that the stability of the pairing of the monomers contribute to a duplex between the first and second sequence will differ form the stability of the pairing between the first or second sequence with a target sequence.

[0119] Usually, the monomers will be selected (selection of the target sequence may be required as well) such that they form a pairing in the iRNA agent duplex which has a lower free energy of dissociation, and a lower Tm, than will be possessed by the paring of the monomer with its complementary monomer in a duplex between the iRNA agent sequence and a target RNA duplex.

[0120] The constraint placed upon the monomers can be applied at a selected site or at more than one selected site. By way of example, the constraint can be applied at more than 1, but less than 3, 4, 5, 6, or 7 sites in an iRNA agent duplex.

[0121] A constrained or selected site can be present at a number of positions in the iRNA agent duplex. E.g., a constrained or selected site can be present within 3, 4, 5, or 6 positions from either end, 3′ or 5′ of a duplexed sequence. A constrained or selected site can be present in the middle of the duplex region, e.g., it can be more than 3, 4, 5, or 6, positions from the end of a duplexed region.

[0122] The iRNA agent can be selected to target a broad spectrum of genes, including any of the genes described herein.

[0123] In a preferred embodiment the iRNA agent has an architecture (architecture refers to one or more of overall length, length of a duplex region, the presence, number, location, or length of overhangs, sing strand versus double strand form) described herein.

[0124] E.g., the iRNA agent can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length and there is a duplex region of about 19 pairs. In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length.

[0125] In some embodiment the duplex region of the iRNA agent will have, mismatches, in addition to the selected or constrained site or sites. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, which do not form canonical Watson-Crick pairs or which do not hybridize. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

[0126] The monomers can be selected such that: first and second monomers are naturally occurring ribonucleotides, or modified ribonucleotides having naturally occurring bases, and when occupying complementary sites either do not pair and have no substantial level of H-bonding, or form a non canonical Watson-Crick pairing and form a non-canonical pattern of H bonding, which usually have a lower free energy of dissociation than seen in a canonical Watson-Crick pairing, or otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing. When one (or both) of the iRNA agent sequences duplexes with a target, the first (or second) monomer forms a canonical Watson-Crick pairing with the base in the complementary position on the target, or forms a non canonical Watson-Crick pairing having a higher free energy of dissociation and a higher Tm than seen in the paring in the iRNA agent. The classical Watson-Crick parings are as follows: A-T, G-C, and A-U. Non-canonical Watson-Crick pairings are known in the art and can include, U-U, G-G, G-Atrans, G-Acis, and GU.

[0127] The monomer in one or both of the sequences is selected such that, it does not pair, or forms a pair with its corresponding monomer in the other sequence which minimizes stability (e.g., the H bonding formed between the monomer at the selected site in the one sequence and its monomer at the corresponding site in the other sequence are less stable than the H bonds formed by the monomer one (or both) of the sequences with the respective target sequence. The monomer in one or both strands is also chosen to promote stability in one or both of the duplexes made by a strand and its target sequence. E.g., one or more of the monomers and the target sequences are selected such that at the selected or constrained position, there is are no H bonds formed, or a non canonical pairing is formed in the iRNA agent duplex, or otherwise they otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing, but when one (or both) sequences form a duplex with the respective target, the pairing at the selected or constrained site is a canonical Watson-Crick pairing.

[0128] The inclusion of such a monomers will have one or more of the following effects: it will destabilize the iRNA agent duplex, it will destabilize interactions between the sense sequence and unintended target sequences, sometimes referred to as off-target sequences, and duplex interactions between the a sequence and the intended target will not be destabilized.

[0129] By way of example:

[0130] the monomer at the selected site in the first sequence includes an A (or a modified base which pairs with T), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., G. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.

[0131] the monomer at the selected site in the first sequence includes U (or a modified base which pairs with A), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., U or G. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.

[0132] The monomer at the selected site in the first sequence includes a G (or a modified base which pairs with C), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., G, Acis, Atrans, or U. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.

[0133] The monomer at the selected site in the first sequence includes a C (or a modified base which pairs with G), and the monomer in at the selected position in the second sequence is chosen a monomer which will not pair or which will form a non-canonical pairing. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.

[0134] In another embodiment a non-naturally occurring or modified monomer or monomers are chosen such that when a non-naturally occurring or modified monomer occupies a positions at the selected or constrained position in an iRNA agent they exhibit a first free energy of dissociation and when one (or both) of them pairs with a naturally occurring monomer, the pair exhibits a second free energy of dissociation, which is usually higher than that of the pairing of the first and second monomers. E.g., when the first and second monomers occupy complementary positions they either do not pair and have no substantial level of H-bonding, or form a weaker bond than one of them would form with a naturally occurring monomer, and reduce the stability of that duplex, but when the duplex dissociates at least one of the strands will form a duplex with a target in which the selected monomer will promote stability, e.g., the monomer will form a more stable pair with a naturally occurring monomer in the target sequence than the pairing it formed in the iRNA agent.

[0135] An example of such a pairing is 2-amino A and either of a 2-thio pyrimidine analog of U or T.

[0136] When placed in complementary positions of the iRNA agent these monomers will pair very poorly and will minimize stability. However, a duplex is formed between 2 amino A and the U of a naturally occurring target, or a duplex is between 2-thio U and the A of a naturally occurring target or 2-thio T and the A of a naturally occurring target will have a relatively higher free energy of dissociation and be more stable. This is shown in the FIG. 1.

[0137] The pair shown in FIG. 1 (the 2-amino A and the 2-s U and T) is exemplary. In another embodiment, the monomer at the selected position in the sense strand can be a universal pairing moiety. A universal pairing agent will form some level of H bonding with more than one and preferably all other naturally occurring monomers. An example of a universal pairing moiety is a monomer which includes 3-nitro pyrrole. (Examples of other candidate universal base analogs can be found in the art, e.g., in Loakes, 2001, NAR 29: 2437-2447, hereby incorporated by reference. Examples can also be found in the section on Universal Bases below.) In these cases the monomer at the corresponding position of the anti-sense strand can be chosen for its ability to form a duplex with the target and can include, e.g., A, U, G, or C.

[0138] In another aspect, the invention features, an iRNA agent which includes: a sense sequence, which preferably does not target a sequence in a subject, and an anti-sense sequence, which targets a target gene in a subject. The sense and anti-sense sequences have sufficient complementarity to each other to hybridize, e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. In a duplex region of the iRNA agent, at a selected or constrained position, the monomers are selected such that:

[0139] the monomer in the sense sequence is selected such that, it does not pair, or forms a pair with its corresponding monomer in the anti-sense strand which minimizes stability (e.g., the H bonding formed between the monomer at the selected site in the sense strand and its monomer at the corresponding site in the anti-sense strand are less stable than the H bonds formed by the monomer of the anti-sense sequence and its canonical Watson-Crick partner or, if the monomer in the anti-sense strand includes a modified base, the natural analog of the modified base and its canonical Watson-Crick partner);

[0140] the monomer is in the corresponding position in the anti-sense strand is selected such that it maximizes the stability of a duplex it forms with the target sequence, e.g., it forms a canonical Watson-Crick paring with the monomer in the corresponding position on the target stand;

[0141] optionally, the monomer in the sense sequence is selected such that, it does not pair, or forms a pair with its corresponding monomer in the anti-sense strand which minimizes stability with an off-target sequence.

[0142] The inclusion of such a monomers will have one or more of the following effects: it will destabilize the iRNA agent duplex, it will destabilize interactions between the sense sequence and unintended target sequences, sometimes referred to as off-target sequences, and duplex interactions between the anti-sense strand and the intended target will not be destabilized.

[0143] The constraint placed upon the monomers can be applied at a selected site or at more than one selected site. By way of example, the constraint can be applied at more than 1, but less than 3, 4, 5, 6, or 7 sites in an iRNA agent duplex.

[0144] A constrained or selected site can be present at a number of positions in the iRNA agent duplex. E.g., a constrained or selected site can be present within 3, 4, 5, or 6 positions from either end, 3′ or 5′ of a duplexed sequence. A constrained or selected site can be present in the middle of the duplex region, e.g., it can be more than 3, 4, 5, or 6, positions from the end of a duplexed region.

[0145] The iRNA agent can be selected to target a broad spectrum of genes, including any of the genes described herein.

[0146] In a preferred embodiment the iRNA agent has an architecture (architecture refers to one or more of overall length, length of a duplex region, the presence, number, location, or length of overhangs, sing strand versus double strand form) described herein.

[0147] E.g., the iRNA agent can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length and there is a duplex region of about 19 pairs. In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length.

[0148] In some embodiment the duplex region of the iRNA agent will have, mismatches, in addition to the selected or constrained site or sites. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, which do not form canonical Watson-Crick pairs or which do not hybridize. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

[0149] One or more selection or constraint parameters can be exercised such that: monomers at the selected site in the sense and anti-sense sequences are both naturally occurring ribonucleotides, or modified ribonucleotides having naturally occurring bases, and when occupying complementary sites in the iRNA agent duplex either do not pair and have no substantial level of H-bonding, or form a non-canonical Watson-Crick pairing and thus form a non-canonical pattern of H bonding, which generally have a lower free energy of dissociation than seen in a Watson-Crick pairing, or otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing. When one, usually the anti-sense sequence of the iRNA agent sequences forms a duplex with another sequence, generally a sequence in the subject, and generally a target sequence, the monomer forms a classic Watson-Crick pairing with the base in the complementary position on the target, or forms a non-canonical Watson-Crick pairing having a higher free energy of dissociation and a higher Tm than seen in the paring in the iRNA agent. Optionally, when the other sequence of the iRNA agent, usually the sense sequences forms a duplex with another sequence, generally a sequence in the subject, and generally an off-target sequence, the monomer fails to forms a canonical Watson-Crick pairing with the base in the complementary position on the off target sequence, e.g., it forms or forms a non-canonical Watson-Crick pairing having a lower free energy of dissociation and a lower Tm.

[0150] By way of example:

[0151] the monomer at the selected site in the anti-sense stand includes an A (or a modified base which pairs with T), the corresponding monomer in the target is a T, and the sense strand is chosen from a base which will not pair or which will form a noncanonical pair, e.g., G;

[0152] the monomer at the selected site in the anti-sense stand includes a U (or a modified base which pairs with A), the corresponding monomer in the target is an A, and the sense strand is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., U or G;

[0153] the monomer at the selected site in the anti-sense stand includes a C (or a modified base which pairs with G), the corresponding monomer in the target is a G, and the sense strand is chosen a monomer which will not pair or which will form a non-canonical pairing, e.g., G, A_cis, A_trans, or U; or

[0154] the monomer at the selected site in the anti-sense stand includes a G (or a modified base which pairs with C), the corresponding monomer in the target is a C, and the sense strand is chosen from a monomer which will not pair or which will form a non-canonical pairing.

[0155] In another embodiment a non-naturally occurring or modified monomer or monomers is chosen such that when it occupies complementary a position in an iRNA agent they exhibit a first free energy of dissociation and when one (or both) of them pairs with a naturally occurring monomer, the pair exhibits a second free energy of dissociation, which is usually higher than that of the pairing of the first and second monomers. E.g., when the first and second monomers occupy complementary positions they either do not pair and have no substantial level of H-bonding, or form a weaker bond than one of them would form with a naturally occurring monomer, and reduce the stability of that duplex, but when the duplex dissociates at least one of the strands will form a duplex with a target in which the selected monomer will promote stability, e.g., the monomer will form a more stable pair with a naturally occurring monomer in the target sequence than the pairing it formed in the iRNA agent.

[0156] An example of such a pairing is 2-amino A and either of a 2-thio pyrimidine analog of U or T. As is discussed above, when placed in complementary positions of the iRNA agent these monomers will pair very poorly and will minimize stability. However, a duplex is formed between 2 amino A and the U of a naturally occurring target, or a duplex is formed between 2-thio U and the A of a naturally occurring target or 2-thio T and the A of a naturally occurring target will have a relatively higher free energy of dissociation and be more stable.

[0157] The monomer at the selected position in the sense strand can be a universal pairing moiety. A universal pairing agent will form some level of H bonding with more than one and preferably all other naturally occurring monomers. An examples of a universal pairing moiety is a monomer which includes 3-nitro pyrrole. Examples of other candidate universal base analogs can be found in the art, e.g., in Loakes, 2001, NAR 29: 2437-2447, hereby incorporated by reference. In these cases the monomer at the corresponding position of the anti-sense strand can be chosen for its ability to form a duplex with the target and can include, e.g., A, U, G, or C.

[0158] In another aspect, the invention features, an iRNA agent which includes:

[0159] a sense sequence, which preferably does not target a sequence in a subject, and an anti-sense sequence, which targets a plurality of target sequences in a subject, wherein the targets differ in sequence at only 1 or a small number, e.g., no more than 5, 4, 3 or 2 positions. The sense and anti-sense sequences have sufficient complementarity to each other to hybridize, e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. In the sequence of the anti-sense strand of the iRNA agent is selected such that at one, some, or all of the positions which correspond to positions that differ in sequence between the target sequences, the anti-sense strand will include a monomer which will form H-bonds with at least two different target sequences. In a preferred example the anti-sense sequence will include a universal or promiscuous monomer, e.g., a monomer which includes 5-nitro pyrrole, 2-amino A, 2-thio U or 2-thio T, or other universal base referred to herein.

[0160] In a preferred embodiment the iRNA agent targets repeated sequences (which differ at only one or a small number of positions from each other) in a single gene, a plurality of genes, or a viral genome, e.g., the HCV genome.

[0161] An embodiment is illustrated in the FIGS. 2 and 3.

[0162] In another aspect, the invention features, determining, e.g., by measurement or calculation, the stability of a pairing between monomers at a selected or constrained position in the iRNA agent duplex, and preferably determining the stability for the corresponding pairing in a duplex between a sequence form the iRNA agent and another RNA, e.g., a target sequence. The determinations can be compared. An iRNA agent thus analyzed can be used in the development of a further modified iRNA agent or can be administered to a subject. This analysis can be performed successively to refine or design optimized iRNA agents.

[0163] In another aspect, the invention features, a kit which includes one or more of the following an iRNA described herein, a sterile container in which the iRNA agent is disclosed, and instructions for use.

[0164] In another aspect, the invention features, an iRNA agent containing a constrained sequence made by a method described herein. The iRNA agent can target one or more of the genes referred to herein.

[0165] iRNA agents having constrained or selected sites, e.g., as described herein, can be used in any way described herein. Accordingly, they iRNA agents having constrained or selected sites, e.g., as described herein, can be used to silence a target, e.g., in any of the methods described herein and to target any of the genes described herein or to treat any of the disorders described herein. iRNA agents having constrained or selected sites, e.g., as described herein, can be incorporated into any of the formulations or preparations, e.g., pharmaceutical or sterile preparations described herein. iRNA agents having constrained or selected sites, e.g., as described herein, can be administered by any of the routes of administration described herein.

[0166] The term “other than canonical Watson-Crick pairing” as used herein, refers to a pairing between a first monomer in a first sequence and a second monomer at the corresponding position in a second sequence of a duplex in which one or more of the following is true: (1) there is essentially no pairing between the two, e.g., there is no significant level of H bonding between the monomers or binding between the monomers does not contribute in any significant way to the stability of the duplex; (2) the monomers are a non-canonical paring of monomers having a naturally occurring bases, i.e., they are other than A-T, A-U, or G-C, and they form monomer-monomer H bonds, although generally the H bonding pattern formed is less strong than the bonds formed by a canonical pairing; or (3) at least one of the monomers includes a non-naturally occurring bases and the H bonds formed between the monomers is, preferably formed is less strong than the bonds formed by a canonical pairing, namely one or more of A-T, A-U, G-C.

[0167] The term “off-target” as used herein, refers to a sequence other than the sequence to be silenced.

[0168] Universal Bases: “wild-cards”; shape-based complementarity

[0169] Bi-stranded, multisite replication of a base pair between difluorotoluene and adenine: confirmation by ‘inverse’ sequencing. Liu, D.; Moran, S.; Kool, E. T. Chem. Biol., 1997, 4, 919-926)

[0170]

[0171] (Importance of terminal base pair hydrogen-bonding in 3′-end proofreading by the Klenow fragment of DNA polymerase I. Morales, J. C.; Kool, E. T. Biochemistry, 2000, 39, 2626-2632)

[0172] (Selective and stable DNA base pairing without hydrogen bonds. Matray, T, J.; Kool, E. T. J. Am. Chem. Soc., 1998, 120, 6191-6192)

[0173]

[0174] (Difluorotoluene, a nonpolar isostere for thymine, codes specifically and efficiently for adenine in DNA replication. Moran, S. Ren, R. X.-F.; Rumney I V, S.; Kool, E. T. J. Am. Chem. Soc., 1997, 119, 2056-2057)

[0175]

[0176] (Structure and base pairing properties of a replicable nonpolar isostere for deoxyadenosine. Guckian, K. M.; Morales, J. C.; Kool, E. T. J. Org. Chem., 1998, 63, 9652-9656)

[0177]

[0178] (Universal bases for hybridization, replication and chain termination. Berger, M.; Wu. Y.; Ogawa, A. K.; McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. Nucleic Acids Res., 2000, 28, 2911-2914)

[0179]

• [0180] (1. Efforts toward the expansion of the genetic alphabet: Information storage and replication with unnatural hydrophobic base pairs. Ogawa, A. K.; Wu, Y.; McMinn, D. L.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 3274-3287. 2. Rational design of an unnatural base pair with increased kinetic selectivity. Ogawa, A. K.; Wu. Y.; Berger, M.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 8803-8804)

[0181]

[0182] (Efforts toward expansion of the genetic alphabet: replication of DNA with three base pairs. Tae, E. L.; Wu, Y.; Xia, G.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2001, 123, 7439-7440)

[0183]

[0184] (1. Efforts toward expansion of the genetic alphabet: Optimization of interbase hydrophobic interactions. Wu, Y.; Ogawa, A. K.; Berger, M.; McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 7621-7632. 2. Efforts toward expansion of genetic alphabet: DNA polymerase recognition of a highly stable, self-pairing hydrophobic base. McMinn, D. L.; Ogawa. A. K.; Wu, Y.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 1999, 121, 11585-11586)

[0185] (A stable DNA duplex containing a non-hydrogen-bonding and non-shape complementary base couple: Interstrand stacking as the stability determining factor. Brotschi, C.; Haberli, A.; Leumann, C, J. Angew. Chem. Int. Ed., 2001, 40, 3012-3014)

[0186] (2,2′-Bipyridine Ligandoside: A novel building block for modifying DNA with intra-duplex metal complexes. Weizman, H.; Tor, Y. J. Am. Chem. Soc., 2001, 123, 3375-3376)

[0187]

[0188] (Minor groove hydration is critical to the stability of DNA duplexes. Lan, T.; McLaughlin, L. W. J. Am. Chem. Soc., 2000, 122, 6512-13)

[0189]

[0190] (Effect of the Universal base 3-nitropyrrole on the selectivity of neighboring natural bases. Oliver, J. S.; Parker, K. A.; Suggs, J. W. Organic Lett., 2001, 3, 1977-1980. 2. Effect of the 1-(2′-deoxy-β-D-ribofuranosyl)-3-nitropyrrol residue on the stability of DNA duplexes and triplexes. Amosova, O.; George J.; Fresco, J. R. Nucleic Acids Res., 1997, 25, 1930-1934. 3. Synthesis, structure and deoxyribonucleic acid sequencing with a universal nucleosides: 1-(2′-deoxy-β-D-ribofuranosyl)-3-nitropyrrole. Bergstrom, D. E.; Zhang, P.; Toma, P. H.; Andrews, P. C.; Nichols, R. J. Am. Chem. Soc., 1995, 117, 1201-1209)

[0191]

[0192] (Model studies directed toward a general triplex DNA recognition scheme: a novel DNA base that binds a CG base-pair in an organic solvent. Zimmerman, S. C.; Schmitt, P. J. Am. Chem. Soc., 1995, 117, 10769-10770)

[0193]

[0194] (A universal, photocleavable DNA base: nitropiperonyl 2′-deoxyriboside. J. Org. Chem., 2001, 66, 2067-2071)

[0195]

[0196] (Recognition of a single guanine bulge by 2-acylamino-1,8-naphthyridine. Nakatani, K.; Sando, S.; Saito, I. J. Am. Chem. Soc., 2000, 122, 2172-2177. b. Specific binding of 2-amino-1,8-naphthyridine into single guanine bulge as evidenced by photooxidation of GC doublet, Nakatani, K.; Sando, S.; Yoshida, K.; Saito, I. Bioorg. Med. Chem. Lett., 2001, 11, 335-337)

[0197]
Other universal bases can have the following formulas:

[0198]

[0199] wherein:

[0200] Q is N or CR⁴⁴;

[0201] Q′ is N or CR⁴⁵;

[0202] Q″ is N or CR⁴⁷;

[0203] Q′″ is N or CR⁴⁹;

[0204] Q^iv is N or CR⁵⁰;

[0205] R⁴⁴ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^b, or NR^bR^c, C₁-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, or when taken together with R⁴⁵ forms —OCH₂O—;

[0206] R⁴⁵ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^b, or NR^bR^c, C₁-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, or when taken together with R⁴⁴ or R⁴⁶ forms —OCH₂O—;

[0207] R⁴⁶ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^b, or NR^bR^c, C₁-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, or when taken together with R⁴⁵ or R⁴⁷ forms —OCH₂O—;

[0208] R⁴⁷ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^b, or NR^bR^c, C₁-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, or when taken together with R⁴⁶ or R⁴⁸ forms —OCH2O—;

[0209] R⁴⁸ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^b, or NR^bR^c, C₁-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, or when taken together with R⁴⁷ forms —OCH2O—;

[0210] R⁴⁹ R⁵⁰, R⁵¹, R⁵², R⁵³, R⁵⁴, R⁵⁷, R⁵⁸, R⁵⁹, R⁶⁰, R⁶¹, R⁶², R⁶³, R⁶⁴, R⁶⁵, R⁶⁶, R⁶⁷, R⁶⁸, R⁶⁹, R⁷⁰, R⁷¹, and R⁷² are each independently selected from hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^b, or NR^bR^c, C₁-C₆ alkyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, NC(O)R¹⁷, or NC(O)R^∘;

[0211] R⁵⁵ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^b, or NR^bR^c, C₁-C₆ alkyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, NC(O)R¹⁷, or NC(O)R^∘, or when taken together with R⁵⁶ forms a fused aromatic ring which may be optionally substituted;

[0212] R⁵⁶ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^b, or NR^bR^c, C₁-C₆ alkyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, NC(O)R¹⁷, or NC(O)R^∘, or when taken together with R⁵⁵ forms a fused aromatic ring which may be optionally substituted;

[0213] R¹⁷ is halo, NH₂, NHR^b, or NR^bR^c;

[0214] R^b is C₁-C₆ alkyl or a nitrogen protecting group;

[0215] R^c is C₁-C₆ alkyl; and

[0216] R^∘ is alkyl optionally substituted with halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^b, or NR^bR^c, C₁-C₆ alkyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, NC(O)R¹⁷, or NC(O)R^∘.

[0217] Examples of universal bases include:

[0218]

[0219] In one aspect, the invention features methods of producing iRNA agents, e.g., sRNA agents, e.g. an sRNA agent described herein, having the ability to mediate RNAi. These iRNA agents can be formulated for administration to a subject.

[0220] In another aspect, the invention features a method of administering an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, to a subject (e.g., a human subject). The method includes administering a unit dose of the iRNA agent, e.g., a sRNA agent, e.g., double stranded sRNA agent that (a) the double-stranded part is 19-25 nucleotides (nt) long, preferably 21-23 nt, (b) is complementary to a target RNA (e.g., an endogenous or pathogen target RNA), and, optionally, (c) includes at least one 3′ overhang 1-5 nucleotide long. In one embodiment, the unit dose is less than 1.4 mg per kg of bodyweight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight, and less than 200 nmole of RNA agent (e.g. about 4.4×10¹⁶ copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of RNA agent per kg of bodyweight.

[0221] The defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with the target RNA. The unit dose, for example, can be administered by injection (e.g., intravenous or intramuscular), an inhaled dose, or a topical application. Particularly preferred dosages are less than 2, 1, or 0.1 mg/kg of body weight.

[0222] In a preferred embodiment, the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered a single time.

[0223] In one embodiment, the effective dose is administered with other traditional therapeutic modalities. In one embodiment, the subject has a viral infection and the modality is an antiviral agent other than an iRNA agent, e.g., other than a double-stranded iRNA agent, or sRNA agent. In another embodiment, the subject has atherosclerosis and the effective dose of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, is administered in combination with, e.g., after surgical intervention, e.g., angioplasty.

[0224] In one embodiment, a subject is administered an initial dose and one or more maintenance doses of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). The maintenance dose or doses are generally lower than the initial dose, e.g., one-half less of the initial dose. A maintenance regimen can include treating the subject with a dose or doses ranging from 0.01 μg to 1.4 mg/kg of body weight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of bodyweight per day. The maintenance doses are preferably administered no more than once every 5, 10, or 30 days.

[0225] In one embodiment, the iRNA agent pharmaceutical composition includes a plurality of iRNA agent species. In another embodiment, the iRNA agent species has sequences that are non-overlapping and non-adjacent to another species with respect to a naturally occurring target sequence. In another embodiment, the plurality of iRNA agent species is specific for different naturally occurring target genes. In another embodiment, the iRNA agent is allele specific.

[0226] The inventors have discovered that iRNA agents described herein can be administered to mammals, particularly large mammals such as nonhuman primates or humans in a number of ways.

[0227] In one embodiment, the administration of the iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, composition is parenteral, e.g. intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser that delivers a metered dose. Selected modes of delivery are discussed in more detail below.

[0228] The invention provides methods, compositions, and kits, for rectal administration or delivery of iRNA agents described herein.

[0229] Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes a an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of a iRNA agent described herein, e.g., a iRNA agent having a double stranded region of less than 40, and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered rectally, e.g., introduced through the rectum into the lower or upper colon. This approach is particularly useful in the treatment of, inflammatory disorders, disorders characterized by unwanted cell proliferation, e.g., polyps, or colon cancer.

[0230] In some embodiments the medication is delivered to a site in the colon by introducing a dispensing device, e.g., a flexible, camera-guided device similar to that used for inspection of the colon or removal of polyps, which includes means for delivery of the medication.

[0231] In one embodiment, the rectal administration of the iRNA agent is by means of an enema. The iRNA agent of the enema can be dissolved in a saline or buffered solution.

[0232] In another embodiment, the rectal administration is by means of a suppository. The suppository can include other ingredients, e.g., an excipient, e.g., cocoa butter or hydropropylmethylcellulose.

[0233] The invention also provides methods, compositions, and kits for oral delivery of iRNA agents described herein.

[0234] Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of a iRNA described herein, e.g., a iRNA agent having a double stranded region of less than 40 and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered orally.

[0235] Oral administration can be in the form of tablets, capsules, gel capsules, lozenges, troches or liquid syrups. In a preferred embodiment the composition is applied topically to a surface of the oral cavity.

[0236] The invention also provides methods, compositions, and kits for buccal delivery of iRNA agents described herein.

[0237] Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of iRNA agent having a double stranded region of less than 40 and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered to the buccal cavity. The medication can be sprayed into the buccal cavity or applied directly, e.g., in a liquid, solid, or gel form to a surface in the buccal cavity. This administration is particularly desirable for the treatment of inflammations of the buccal cavity, e.g., the gums or tongue, e.g., in one embodiment, the buccal administration is by spraying into the cavity, e.g., without inhalation, from a dispenser, e.g., a metered dose spray dispenser that dispenses the pharmaceutical composition and a propellant.

[0238] The invention also provides methods, compositions, and kits for ocular delivery of iRNA agents described herein.

[0239] Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of a iRNA agent described herein, e.g., a sRNA agent having a double stranded region of less than 40 and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered to ocular tissue.

[0240] The medications can be applied to the surface of the eye or nearby tissue, e.g., the inside of the eyelid. It can be applied topically, e.g., by spraying, in drops, as an eyewash, or an ointment. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser that delivers a metered dose.

[0241] The medication can also be administered to the interior of the eye, and can be introduced by a needle or other delivery device which can introduce it to a selected area or structure.

[0242] Ocular treatment is particularly desirable for treating inflammation of the eye or nearby tissue.

[0243] The invention also provides methods, compositions, and kits for delivery of iRNA agents described herein to or through the skin.

[0244] Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of a iRNA agent described herein, e.g., a sRNA agent having a double stranded region of less than 40 and preferably less than 30 nucleotides and one or two 1-3 nucleotide single strand 3′ overhangs can be administered directly to the skin.

[0245] The medication can be applied topically or delivered in a layer of the skin, e.g., by the use of a microneedle or a battery of microneedles which penetrate into the skin, but preferably not into the underlying muscle tissue.

[0246] In one embodiment, the administration of the iRNA agent composition is topical. In another embodiment, topical administration delivers the composition to the dermis or epidermis of a subject. In other embodiments the topical administration is in the form of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids or powders. A composition for topical administration can be formulated as a liposome, micelle, emulsion, or other lipophilic molecular assembly.

[0247] In another embodiment, the transdermal administration is applied with at least one penetration enhancer. In other embodiments, the penetration can be enhanced with iontophoresis, phonophoresis, and sonophoresis. In another aspect, the invention provides methods, compositions, devices, and kits for pulmonary delivery of iRNA agents described herein.

[0248] Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of iRNA agent, e.g., a sRNA agent having a double stranded region of less than 40, preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered to the pulmonary system. Pulmonary administration can be achieved by inhalation or by the introduction of a delivery device into the pulmonary system, e.g., by introducing a delivery device which can dispense the medication.

[0249] The preferred method of pulmonary delivery is by inhalation. The medication can be provided in a dispenser which delivers the medication, e.g., wet or dry, in a form sufficiently small such that it can be inhaled. The device can deliver a metered dose of medication. The subject, or another person, can administer the medication.

[0250] Pulmonary delivery is effective not only for disorders which directly affect pulmonary tissue, but also for disorders which affect other tissue.

[0251] iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder, crystal, or aerosol for pulmonary delivery.

[0252] In another aspect, the invention provides methods, compositions, devices, and kits for nasal delivery of iRNA agents described herein. Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of iRNA agent, e.g., a sRNA agent having a double stranded region of less than 40 and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered nasally. Nasal administration can be achieved by introduction of a delivery device into the nose, e.g., by introducing a delivery device which can dispense the medication.

[0253] The preferred method of nasal delivery is by spray, aerosol, liquid, e.g., by drops, of by topical administration to a surface of the nasal cavity. The medication can be provided in a dispenser which delivery of the medication, e.g., wet or dry, in a form sufficiently small such that it can be inhaled. The device can deliver a metered dose of medication. The subject, or another person, can administer the medication.

[0254] Nasal delivery is effective not only for disorders which directly affect nasal tissue, but also for disorders which affect other tissue

[0255] iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder, crystal, or for nasal delivery.

[0256] In another embodiment, the iRNA agent is packaged in a viral natural capsid or in a chemically or enzymatically produced artificial capsid or structure derived therefrom.

[0257] In one aspect, of the invention, the dosage of a pharmaceutical composition including a iRNA agent is administered in order to alleviate the symptoms of a disease state, e.g., cancer or a cardiovascular disease.

[0258] In another aspect, gene expression in a subject is modulated by administering a pharmaceutical composition including a iRNA agent. In other embodiments, a subject is treated with the pharmaceutical composition by any of the methods mentioned above. In another embodiment, the subject has cancer.

[0259] An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) composition can be administered as a liposome. For example, the composition can be prepared by a method that includes: (1) contacting a iRNA agent with an amphipathic cationic lipid conjugate in the presence of a detergent; and (2) removing the detergent to form a iRNA agent and cationic lipid complex. In one embodiment, the detergent is cholate, deoxycholate, lauryl sarcosine, octanoyl sucrose, CHAPS (3-[(3-cholamidopropyl)-di-methylamine]-2-hydroxyl-1-propane), novel-θ-D-glucopyranoside, lauryl dimethylamine oxide, or octylglucoside. The iRNA agent can be an sRNA agent. The method can include preparing a composition that includes a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

[0260] In another aspect, a subject is treated by administering a defined amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent) composition that is in a powdered form. In one embodiment, the powder is a collection of microparticles. In one embodiment, the powder is a collection of crystalline particles. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

[0261] In one aspect, a subject is treated by administering a defined amount of a iRNA agent composition that is prepared by a method that includes spray-drying, i.e. atomizing a liquid solution, emulsion, or suspension, immediately exposing the droplets to a drying gas, and collecting the resulting porous powder particles. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

[0262] In one aspect, the iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), is provided in a powdered, crystallized or other finely divided form, with or without a carrier, e.g., a micro- or nano-particle suitable for inhalation or other pulmonary delivery. In one embodiment, this includes providing an aerosol preparation, e.g., an aerosolized spray-dried composition. The aerosol composition can be provided in and/or dispensed by a metered dose delivery device.

[0263] In another aspect, a subject is treated for a condition treatable by inhalation. In one embodiment, this method includes aerosolizing a spray-dried iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) composition and inhaling the aerosolized composition. The iRNA agent can be an sRNA. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

[0264] In another aspect, the invention features a method of treating a subject that includes: administering a composition including an effective/defined amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), wherein the composition is prepared by a method that includes spray-drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques

[0265] In another aspect, the invention features a method that includes: evaluating a parameter related to the abundance of a transcript in a cell of a subject; comparing the evaluated parameter to a reference value; and if the evaluated parameter has a preselected relationship to the reference value (e.g., it is greater), administering a iRNA agent (or a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes a iRNA agent or precursor thereof) to the subject. In one embodiment, the iRNA agent includes a sequence that is complementary to the evaluated transcript. For example, the parameter can be a direct measure of transcript levels, a measure of a protein level, a disease or disorder symptom or characterization (e.g., rate of cell proliferation and/or tumor mass, viral load)

[0266] In another aspect, the invention features a method that includes: administering a first amount of a composition that comprises an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) to a subject, wherein the iRNA agent includes a strand substantially complementary to a target nucleic acid; evaluating an activity associated with a protein encoded by the target nucleic acid; wherein the evaluation is used to determine if a second amount should be administered. In a preferred embodiment the method includes administering a second amount of the composition, wherein the timing of administration or dosage of the second amount is a function of the evaluating. The method can include other features described herein.

[0267] In another aspect, the invention features a method of administering a source of a double-stranded iRNA agent (ds iRNA agent) to a subject. The method includes administering or implanting a source of a ds iRNA agent, e.g., a sRNA agent, that (a) includes a double-stranded region that is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to a target RNA (e.g., an endogenous RNA or a pathogen RNA), and, optionally, (c) includes at least one 3′ overhang 1-5 nt long. In one embodiment, the source releases ds iRNA agent over time, e.g. the source is a controlled or a slow release source, e.g., a microparticle that gradually releases the ds iRNA agent. In another embodiment, the source is a pump, e.g., a pump that includes a sensor or a pump that can release one or more unit doses.

[0268] In one aspect, the invention features a pharmaceutical composition that includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) including a nucleotide sequence complementary to a target RNA, e.g., substantially and/or exactly complementary. The target RNA can be a transcript of an endogenous human gene. In one embodiment, the iRNA agent (a) is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to an endogenous target RNA, and, optionally, (c) includes at least one 3′ overhang 1-5 nt long. In one embodiment, the pharmaceutical composition can be an emulsion, microemulsion, cream, jelly, or liposome.

[0269] In one example the pharmaceutical composition includes an iRNA agent mixed with a topical delivery agent. The topical delivery agent can be a plurality of microscopic vesicles. The microscopic vesicles can be liposomes. In a preferred embodiment the liposomes are cationic liposomes.

[0270] In another aspect, the pharmaceutical composition includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) admixed with a topical penetration enhancer. In one embodiment, the topical penetration enhancer is a fatty acid. The fatty acid can be arachidonic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C_1-10 alkyl ester, monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.

[0271] In another embodiment, the topical penetration enhancer is a bile salt. The bile salt can be cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate, polyoxyethylene-9-lauryl ether or a pharmaceutically acceptable salt thereof.

[0272] In another embodiment, the penetration enhancer is a chelating agent. The chelating agent can be EDTA, citric acid, a salicyclate, a N-acyl derivative of collagen, laureth-9, an N-amino acyl derivative of a beta-diketone or a mixture thereof.

[0273] In another embodiment, the penetration enhancer is a surfactant, e.g., an ionic or nonionic surfactant. The surfactant can be sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether, a perfluorchemical emulsion or mixture thereof.

[0274] In another embodiment, the penetration enhancer can be selected from a group consisting of unsaturated cyclic ureas, 1-alkyl-alkones, 1-alkenylazacyclo-alakanones, steroidal anti-inflammatory agents and mixtures thereof. In yet another embodiment the penetration enhancer can be a glycol, a pyrrol, an azone, or a terpenes.

[0275] In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a form suitable for oral delivery. In one embodiment, oral delivery can be used to deliver an iRNA agent composition to a cell or a region of the gastro-intestinal tract, e.g., small intestine, colon (e.g., to treat a colon cancer), and so forth. The oral delivery form can be tablets, capsules or gel capsules. In one embodiment, the iRNA agent of the pharmaceutical composition modulates expression of a cellular adhesion protein, modulates a rate of cellular proliferation, or has biological activity against eukaryotic pathogens or retroviruses. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablets, capsules or gel capsules in a mammalian stomach. In a preferred embodiment the enteric material is a coating. The coating can be acetate phthalate, propylene glycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxy propyl methylcellulose phthalate or cellulose acetate phthalate.

[0276] In another embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer. The penetration enhancer can be a bile salt or a fatty acid. The bile salt can be ursodeoxycholic acid, chenodeoxycholic acid, and salts thereof. The fatty acid can be capric acid, lauric acid, and salts thereof.

[0277] In another embodiment, the oral dosage form of the pharmaceutical composition includes an excipient. In one example the excipient is polyethyleneglycol. In another example the excipient is precirol.

[0278] In another embodiment, the oral dosage form of the pharmaceutical composition includes a plasticizer. The plasticizer can be diethyl phthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethyl citrate.

[0279] In one aspect, the invention features a pharmaceutical composition including an iRNA agent and a delivery vehicle. In one embodiment, the iRNA agent is (a) is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to an endogenous target RNA, and, optionally, (c) includes at least one 3′ overhang 1-5 nucleotides long.

[0280] In one embodiment, the delivery vehicle can deliver an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) to a cell by a topical route of administration. The delivery vehicle can be microscopic vesicles. In one example the microscopic vesicles are liposomes. In a preferred embodiment the liposomes are cationic liposomes. In another example the microscopic vesicles are micelles.

[0281] In one aspect, the invention features a method for making a pharmaceutical composition, the method including: (1) contacting an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent) with a amphipathic cationic lipid conjugate in the presence of a detergent; and (2) removing the detergent to form a iRNA agent and cationic lipid complex.

[0282] In another aspect, the invention features a pharmaceutical composition produced by a method including: (1) contacting an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent) with a amphipathic cationic lipid conjugate in the presence of a detergent; and (2) removing the detergent to form a iRNA agent and cationic lipid complex. In one embodiment, the detergent is cholate, deoxycholate, lauryl sarcosine, octanoyl sucrose, CHAPS (3-[(3-cholamidopropyl)-di-methylamine]-2-hydroxyl-1-propane), novel-θ-D-glucopyranoside, lauryl dimethylamine oxide, or octylglucoside. In another embodiment, the amphipathic cationic lipid conjugate is biodegradable. In yet another embodiment the pharmaceutical composition includes a targeting ligand.

[0283] In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in an injectable dosage form. In one embodiment, the injectable dosage form of the pharmaceutical composition includes sterile aqueous solutions or dispersions and sterile powders. In a preferred embodiment the sterile solution can include a diluent such as water; saline solution; fixed oils, polyethylene glycols, glycerin, or propylene glycol.

[0284] In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in oral dosage form. In one embodiment, the oral dosage form is selected from the group consisting of tablets, capsules and gel capsules. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablets, capsules or gel capsules in a mammalian stomach. In a preferred embodiment the enteric material is a coating. The coating can be acetate phthalate, propylene glycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxy propyl methyl cellulose phthalate or cellulose acetate phthalate. In one embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer, e.g., a penetration enhancer described herein.

[0285] In another embodiment, the oral dosage form of the pharmaceutical composition includes an excipient. In one example the excipient is polyethyleneglycol. In another example the excipient is precirol.

[0286] In another embodiment, the oral dosage form of the pharmaceutical composition includes a plasticizer. The plasticizer can be diethyl phthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethyl citrate.

[0287] In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a rectal dosage form. In one embodiment, the rectal dosage form is an enema. In another embodiment, the rectal dosage form is a suppository.

[0288] In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a vaginal dosage form. In one embodiment, the vaginal dosage form is a suppository. In another embodiment, the vaginal dosage form is a foam, cream, or gel.

[0289] In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a pulmonary or nasal dosage form. In one embodiment, the iRNA agent is incorporated into a particle, e.g., a macroparticle, e.g., a microsphere. The particle can be produced by spray drying, lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination thereof. The microsphere can be formulated as a suspension, a powder, or an implantable solid.

[0290] In one aspect, the invention features a spray-dried iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) composition suitable for inhalation by a subject, including: (a) a therapeutically effective amount of a iRNA agent suitable for treating a condition in the subject by inhalation; (b) a pharmaceutically acceptable excipient selected from the group consisting of carbohydrates and amino acids; and (c) optionally, a dispersibility-enhancing amount of a physiologically-acceptable, water-soluble polypeptide.

[0291] In one embodiment, the excipient is a carbohydrate. The carbohydrate can be selected from the group consisting of monosaccharides, disaccharides, trisaccharides, and polysaccharides. In a preferred embodiment the carbohydrate is a monosaccharide selected from the group consisting of dextrose, galactose, mannitol, D-mannose, sorbitol, and sorbose. In another preferred embodiment the carbohydrate is a disaccharide selected from the group consisting of lactose, maltose, sucrose, and trehalose.

[0292] In another embodiment, the excipient is an amino acid. In one embodiment, the amino acid is a hydrophobic amino acid. In a preferred embodiment the hydrophobic amino acid is selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. In yet another embodiment the amino acid is a polar amino acid. In a preferred embodiment the amino acid is selected from the group consisting of arginine, histidine, lysine, cysteine, glycine, glutamine, serine, threonine, tyrosine, aspartic acid and glutamic acid.

[0293] In one embodiment, the dispersibility-enhancing polypeptide is selected from the group consisting of human serum albumin, α-lactalbumin, trypsinogen, and polyalanine.

[0294] In one embodiment, the spray-dried iRNA agent composition includes particles having a mass median diameter (MMD) of less than 10 microns. In another embodiment, the spray-dried iRNA agent composition includes particles having a mass median diameter of less than 5 microns. In yet another embodiment the spray-dried iRNA agent composition includes particles having a mass median aerodynamic diameter (MMAD) of less than 5 microns.

[0295] In certain other aspects, the invention provides kits that include a suitable container containing a pharmaceutical formulation of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). In certain embodiments the individual components of the pharmaceutical formulation may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical formulation separately in two or more containers, e.g., one container for an iRNA agent preparation, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition. The kit can also include a delivery device.

[0296] In another aspect, the invention features a device, e.g., an implantable device, wherein the device can dispense or administer a composition that includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), e.g., a iRNA agent that silences an endogenous transcript. In one embodiment, the device is coated with the composition. In another embodiment the iRNA agent is disposed within the device. In another embodiment, the device includes a mechanism to dispense a unit dose of the composition. In other embodiments the device releases the composition continuously, e.g., by diffusion. Exemplary devices include stents, catheters, pumps, artificial organs or organ components (e.g., artificial heart, a heart valve, etc.), and sutures.

[0297] As used herein, the term “crystalline” describes a solid having the structure or characteristics of a crystal, i.e., particles of three-dimensional structure in which the plane faces intersect at definite angles and in which there is a regular internal structure. The compositions of the invention may have different crystalline forms. Crystalline forms can be prepared by a variety of methods, including, for example, spray drying.

[0298] As used herein, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between a compound of the invention and a target RNA molecule. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed. The non-target sequences typically differ by at least 5 nucleotides.

[0299] In one embodiment, an iRNA agent is “sufficiently complementary” to a target RNA, e.g., a target mRNA, such that the iRNA agent silences production of protein encoded by the target mRNA. In another embodiment, the iRNA agent is “exactly complementary” to a target RNA, e.g., the target RNA and the iRNA agent anneal, preferably to form a hybrid made exclusively of Watson-Crick basepairs in the region of exact complementarity. A “sufficiently complementary” target RNA can include an internal region (e.g., of at least 10 nucleotides) that is exactly complementary to a target RNA. Moreover, in some embodiments, the iRNA agent specifically discriminates a single-nucleotide difference. In this case, the iRNA agent only mediates RNAi if exact complementary is found in the region (e.g., within 7 nucleotides of) the single-nucleotide difference.

[0300] As used herein, the term “oligonucleotide” refers to a nucleic acid molecule (RNA or DNA) preferably of length less than 100, 200, 300, or 400 nucleotides.

[0301] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The materials, methods, and examples are illustrative only and not intended to be limiting. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, useful methods and materials are described below. Other features and advantages of the invention will be apparent from the accompanying drawings and description, and from the claims. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference. In case of conflict, the present specification, including definitions, will control.

[0310] Double-stranded (dsRNA) directs the sequence-specific silencing of mRNA through a process known as RNA interference (RNAi). The process occurs in a wide variety of organisms, including mammals and other vertebrates.

[0311] It has been demonstrated that 21-23 nt fragments of dsRNA are sequence-specific mediators of RNA silencing, e.g., by causing RNA degradation. While not wishing to be bound by theory, it may be that a molecular signal, which may be merely the specific length of the fragments, present in these 21-23 nt fragments recruits cellular factors that mediate RNAi. Described herein are methods for preparing and administering these 21-23 nt fragments, and other iRNAs agents, and their use for specifically inactivating gene function. The use of iRNAs agents (or recombinantly produced or chemically synthesized oligonucleotides of the same or similar nature) enables the targeting of specific mRNAs for silencing in mammalian cells. In addition, longer dsRNA agent fragments can also be used, e.g., as described below.

[0312] Although, in mammalian cells, long dsRNAs can induce the interferon response which is frequently deleterious, sRNAs do not trigger the interferon response, at least not to an extent that is deleterious to the cell and host. In particular, the length of the iRNA agent strands in an sRNA agent can be less than 31, 30, 28, 25, or 23 nt, e.g., sufficiently short to avoid inducing a deleterious interferon response. Thus, the administration of a composition of sRNA agent (e.g., formulated as described herein) to a mammalian cell can be used to silence expression of a target gene while circumventing the interferon response. Further, use of a discrete species of iRNA agent can be used to selectively target one allele of a target gene, e.g., in a subject heterozygous for the allele.

[0313] Moreover, in one embodiment, a mammalian cell is treated with an iRNA agent that disrupts a component of the interferon response, e.g., double stranded RNA (dsRNA)-activated protein kinase PKR. Such a cell can be treated with a second iRNA agent that includes a sequence complementary to a target RNA and that has a length that might otherwise trigger the interferon response.

[0314] In a typical embodiment, the subject is a mammal such as a cow, horse, mouse, rat, dog, pig, goat, or a primate. The subject can be a dairy mammal (e.g., a cow, or goat) or other farmed animal (e.g., a chicken, turkey, sheep, pig, fish, shrimp). In a much preferred embodiment, the subject is a human, e.g., a normal individual or an individual that has, is diagnosed with, or is predicted to have a disease or disorder.

[0315] Further, because iRNA agent mediated silencing persists for several days after administering the iRNA agent composition, in many instances, it is possible to administer the composition with a frequency of less than once per day, or, for some instances, only once for the entire therapeutic regimen. For example, treatment of some cancer cells may be mediated by a single bolus administration, whereas a chronic viral infection may require regular administration, e.g., once per week or once per month.

[0316] A number of exemplary routes of delivery are described that can be used to administer an iRNA agent to a subject. In addition, the iRNA agent can be formulated according to an exemplary method described herein.

iRNA Agent Structure

[0317] Described herein are isolated iRNA agents, e.g., RNA molecules, (double-stranded; single-stranded) that mediate RNAi. The iRNA agents preferably mediate RNAi with respect to an endogenous gene of a subject or to a gene of a pathogen.

[0318] An “RNA agent” as used herein, is an unmodified RNA, modified RNA, or nucleoside surrogate, all of which are defined herein (see, e.g., the section below entitled RNA Agents). While numerous modified RNAs and nucleoside surrogates are described, preferred examples include those which have greater resistance to nuclease degradation than do unmodified RNAs. Preferred examples include those which have a 2′ sugar modification, a modification in a single strand overhang, preferably a 3′ single strand overhang, or, particularly if single stranded, a 5′ modification which includes one or more phosphate groups or one or more analogs of a phosphate group.

[0319] An “iRNA agent” as used herein, is an RNA agent which can, or which can be cleaved into an RNA agent which can, down regulate the expression of a target gene, preferably an endogenous or pathogen target RNA. While not wishing to be bound by theory, an iRNA agent may act by one or more of a number of mechanisms, including post-transcriptional cleavage of a target mRNA sometimes referred to in the art as RNAi, or pre-transcriptional or pre-translational mechanisms. An iRNA agent can include a single strand or can include more than one strands, e.g., it can be a double stranded iRNA agent. If the iRNA agent is a single strand it is particularly preferred that it include a 5′ modification which includes one or more phosphate groups or one or more analogs of a phosphate group.

[0320] The iRNA agent should include a region of sufficient homology to the target gene, and be of sufficient length in terms of nucleotides, such that the iRNA agent, or a fragment thereof, can mediate down regulation of the target gene. (For ease of exposition the term nucleotide or ribonucleotide is sometimes used herein in reference to one or more monomeric subunits of an RNA agent. It will be understood herein that the usage of the term “ribonucleotide” or “nucleotide”, herein can, in the case of a modified RNA or nucleotide surrogate, also refer to a modified nucleotide, or surrogate replacement moiety at one or more positions.) Thus, the iRNA agent is or includes a region which is at least partially, and in some embodiments fully, complementary to the target RNA. It is not necessary that there be perfect complementarity between the iRNA agent and the target, but the correspondence must be sufficient to enable the iRNA agent, or a cleavage product thereof, to direct sequence specific silencing, e.g., by RNAi cleavage of the target RNA, e.g., mRNA.

[0321] Complementarity, or degree of homology with the target strand, is most critical in the antisense strand. While perfect complementarity, particularly in the antisense strand, is often desired some embodiments can include, particularly in the antisense strand, one or more but preferably 6, 5, 4, 3, 2, or fewer mismatches (with respect to the target RNA). The mismatches, particularly in the antisense strand, are most tolerated in the terminal regions and if present are preferably in a terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides of the 5′ and/or 3′ terminus. The sense strand need only be sufficiently complementary with the antisense strand to maintain the over all double strand character of the molecule.

[0322] As discussed elsewhere herein, an iRNA agent will often be modified or include nucleoside surrogates in addition to the RRMS. Single stranded regions of an iRNA agent will often be modified or include nucleoside surrogates, e.g., the unpaired region or regions of a hairpin structure, e.g., a region which links two complementary regions, can have modifications or nucleoside surrogates. Modification to stabilize one or more 3′- or 5′-terminus of an iRNA agent, e.g., against exonucleases, or to favor the antisense sRNA agent to enter into RISC are also favored. Modifications can include C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol), special biotin or fluorescein reagents that come as phosphoramidites and that have another DMT-protected hydroxyl group, allowing multiple couplings during RNA synthesis.

[0323] iRNA agents include: molecules that are long enough to trigger the interferon response (which can be cleaved by Dicer (Bernstein et al. 2001. Nature, 409:363-366) and enter a RISC (RNAi-induced silencing complex)); and, molecules which are sufficiently short that they do not trigger the interferon response (which molecules can also be cleaved by Dicer and/or enter a RISC), e.g., molecules which are of a size which allows entry into a RISC, e.g., molecules which resemble Dicer-cleavage products. Molecules that are short enough that they do not trigger an interferon response are termed sRNA agents or shorter iRNA agents herein. “sRNA agent or shorter iRNA agent” as used herein, refers to an iRNA agent, e.g., a double stranded RNA agent or single strand agent, that is sufficiently short that it does not induce a deleterious interferon response in a human cell, e.g., it has a duplexed region of less than 60 but preferably less than 50, 40, or 30 nucleotide pairs. The sRNA agent, or a cleavage product thereof, can down regulate a target gene, e.g., by inducing RNAi with respect to a target RNA, preferably an endogenous or pathogen target RNA.

[0324] Each strand of an sRNA agent can be equal to or less than 30, 25, 24, 23, 22, 21, or 20 nucleotides in length. The strand is preferably at least 19 nucleotides in length. For example, each strand can be between 21 and 25 nucleotides in length. Preferred sRNA agents have a duplex region of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, and one or more overhangs, preferably one or two 3′ overhangs, of 2-3 nucleotides.

[0325] In addition to homology to target RNA and the ability to down regulate a target gene, an iRNA agent will preferably have one or more of the following properties:

[0326] (1) it will be of the Formula 1, 2, 3, or 4 set out in the RNA Agent section below;

[0327] (2) if single stranded it will have a 5′ modification which includes one or more phosphate groups or one or more analogs of a phosphate group;

[0328] (3) it will, despite modifications, even to a very large number, or all of the nucleosides, have an antisense strand that can present bases (or modified bases) in the proper three dimensional framework so as to be able to form correct base pairing and form a duplex structure with a homologous target RNA which is sufficient to allow down regulation of the target, e.g., by cleavage of the target RNA;

[0329] (4) it will, despite modifications, even to a very large number, or all of the nucleosides, still have “RNA-like” properties, i.e., it will possess the overall structural, chemical and physical properties of an RNA molecule, even though not exclusively, or even partly, of ribonucleotide-based content. For example, an iRNA agent can contain, e.g., a sense and/or an antisense strand in which all of the nucleotide sugars contain e.g., 2′ fluoro in place of 2′ hydroxyl. This deoxyribonucleotide-containing agent can still be expected to exhibit RNA-like properties. While not wishing to be bound by theory, the electronegative fluorine prefers an axial orientation when attached to the C2′ position of ribose. This spatial preference of fluorine can, in turn, force the sugars to adopt a C_3′-endo pucker. This is the same puckering mode as observed in RNA molecules and gives rise to the RNA-characteristic A-family-type helix. Further, since fluorine is a good hydrogen bond acceptor, it can participate in the same hydrogen bonding interactions with water molecules that are known to stabilize RNA structures. (Generally, it is preferred that a modified moiety at the 2′ sugar position will be able to enter into H-bonding which is more characteristic of the OH moiety of a ribonucleotide than the H moiety of a deoxyribonucleotide. A preferred iRNA agent will: exhibit a C_3′-endo pucker in all, or at least 50, 75, 80, 85, 90, or 95% of its sugars; exhibit a C_3′-endo pucker in a sufficient amount of its sugars that it can give rise to a the RNA-characteristic A-family-type helix; will have no more than 20, 10, 5, 4, 3, 2, or 1 sugar which is not a C₃′-endo pucker structure. These limitations are particularly preferably in the antisense strand;

[0330] (5) regardless of the nature of the modification, and even though the RNA agent can contain deoxynucleotides or modified deoxynucleotides, particularly in overhang or other single strand regions, it is preferred that DNA molecules, or any molecule in which more than 50, 60, or 70% of the nucleotides in the molecule, or more than 50, 60, or 70% of the nucleotides in a duplexed region are deoxyribonucleotides, or modified deoxyribonucleotides which are deoxy at the 2′ position, are excluded from the definition of RNA agent.

[0331] A “single strand iRNA agent” as used herein, is an iRNA agent which is made up of a single molecule. It may include a duplexed region, formed by intra-strand pairing, e.g., it may be, or include, a hairpin or pan-handle structure. Single strand iRNA agents are preferably antisense with regard to the target molecule. In preferred embodiments single strand iRNA agents are 5′ phosphorylated or include a phosphoryl analog at the 5′ prime terminus. 5′-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′); 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)—O-5′-). (These modifications can also be used with the antisense strand of a double stranded iRNA.)

[0332] A single strand iRNA agent should be sufficiently long that it can enter the RISC and participate in RISC mediated cleavage of a target mRNA. A single strand iRNA agent is at least 14, and more preferably at least 15, 20, 25, 29, 35, 40, or 50 nucleotides in length. It is preferably less than 200, 100, or 60 nucleotides in length.

[0333] Hairpin iRNA agents will have a duplex region equal to or at least 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region will preferably be equal to or less than 200, 100, or 50, in length. Preferred ranges for the duplex region are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length. The hairpin will preferably have a single strand overhang or terminal unpaired region, preferably the 3′, and preferably of the antisense side of the hairpin. Preferred overhangs are 2-3 nucleotides in length.

[0334] A “double stranded (ds) iRNA agent” as used herein, is an iRNA agent which includes more than one, and preferably two, strands in which interchain hybridization can form a region of duplex structure.

[0335] The antisense strand of a double stranded iRNA agent should be equal to or at least, 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides in length. It should be equal to or less than 200, 100, or 50, nucleotides in length. Preferred ranges are 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.

[0336] The sense strand of a double stranded iRNA agent should be equal to or at least 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides in length. It should be equal to or less than 200, 100, or 50, nucleotides in length. Preferred ranges are 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.

[0337] The double strand portion of a double stranded iRNA agent should be equal to or at least, 14, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 60 nucleotide pairs in length. It should be equal to or less than 200, 100, or 50, nucleotides pairs in length. Preferred ranges are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length.

[0338] In many embodiments, the ds iRNA agent is sufficiently large that it can be cleaved by an endogenous molecule, e.g., by Dicer, to produce smaller ds iRNA agents, e.g., sRNAs agents

[0339] It may be desirable to modify one or both of the antisense and sense strands of a double strand iRNA agent. In some cases they will have the same modification or the same class of modification but in other cases the sense and antisense strand will have different modifications, e.g., in some cases it is desirable to modify only the sense strand. It may be desirable to modify only the sense strand, e.g., to inactivate it, e.g., the sense strand can be modified in order to inactivate the sense strand and prevent formation of an active sRNA/protein or RISC. This can be accomplished by a modification which prevents 5′-phosphorylation of the sense strand, e.g., by modification with a 5′-O-methyl ribonucleotide (see Nykänen et al., (2001) ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309-321.) Other modifications which prevent phosphorylation can also be used, e.g., simply substituting the 5′-OH by H rather than O-Me. Alternatively, a large bulky group may be added to the 5′-phosphate turning it into a phosphodiester linkage, though this may be less desirable as phosphodiesterases can cleave such a linkage and release a functional sRNA 5′-end. Antisense strand modifications include 5′ phosphorylation as well as any of the other 5′ modifications discussed herein, particularly the 5′ modifications discussed above in the section on single stranded iRNA molecules.

[0340] It is preferred that the sense and antisense strands be chosen such that the ds iRNA agent includes a single strand or unpaired region at one or both ends of the molecule. Thus, a ds iRNA agent contains sense and antisense strands, preferable paired to contain an overhang, e.g., one or two 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3 nucleotides. Most embodiments will have a 3′ overhang. Preferred sRNA agents will have single-stranded overhangs, preferably 3′ overhangs, of 1 or preferably 2 or 3 nucleotides in length at each end. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. 5′ ends are preferably phosphorylated.

[0341] Preferred lengths for the duplexed region is between 15 and 30, most preferably 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., in the sRNA agent range discussed above. sRNA agents can resemble in length and structure the natural Dicer processed products from long dsRNAs. Embodiments in which the two strands of the sRNA agent are linked, e.g., covalently linked are also included. Hairpin, or other single strand structures which provide the required double stranded region, and preferably a 3′ overhang are also within the invention.

[0342] The isolated iRNA agents described herein, including ds iRNA agents and sRNA agents can mediate silencing of a target RNA, e.g., mRNA, e.g., a transcript of a gene that encodes a protein. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such a gene is also referred to as a target gene. In general, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNAs other than mRNA, e.g., tRNAs, and viral RNAs, can also be targeted.

[0343] As used herein, the phrase “mediates RNAi” refers to the ability to silence, in a sequence specific manner, a target RNA. While not wishing to be bound by theory, it is believed that silencing uses the RNAi machinery or process and a guide RNA, e.g., an sRNA agent of 21 to 23 nucleotides.

[0344] As used herein, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between a compound of the invention and a target RNA molecule. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed. The non-target sequences typically differ by at least 5 nucleotides.

[0345] In one embodiment, an iRNA agent is “sufficiently complementary” to a target RNA, e.g., a target mRNA, such that the iRNA agent silences production of protein encoded by the target mRNA. In another embodiment, the iRNA agent is “exactly complementary” (excluding the RRMS containing subunit(s)) to a target RNA, e.g., the target RNA and the iRNA agent anneal, preferably to form a hybrid made exclusively of Watson-Crick basepairs in the region of exact complementarity. A “sufficiently complementary” target RNA can include an internal region (e.g., of at least 10 nucleotides) that is exactly complementary to a target RNA. Moreover, in some embodiments, the iRNA agent specifically discriminates a single-nucleotide difference. In this case, the iRNA agent only mediates RNAi if exact complementary is found in the region (e.g., within 7 nucleotides of) the single-nucleotide difference.

[0346] As used herein, the term “oligonucleotide” refers to a nucleic acid molecule (RNA or DNA) preferably of length less than 100, 200, 300, or 400 nucleotides.

[0347] RNA agents discussed herein include otherwise unmodified RNA as well as RNA which have been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, preferably as occur naturally in the human body. The art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et al., (1994) Summary: the modified nucleosides of RNA, Nucleic Acids Res. 22: 2183-2196. Such rare or unusual RNAs, often termed modified RNAs (apparently because the are typically the result of a post transcriptionally modification) are within the term unmodified RNA, as used herein. Modified RNA as used herein refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occur in nature, preferably different from that which occurs in the human body. While they are referred to as modified “RNAs,” they will of course, because of the modification, include molecules which are not RNAs. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to the presented in the correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone. Examples of all of the above are discussed herein.

[0348] Much of the discussion below refers to single strand molecules. In many embodiments of the invention a double stranded iRNA agent, e.g., a partially double stranded iRNA agent, is required or preferred. Thus, it is understood that that double stranded structures (e.g. where two separate molecules are contacted to form the double stranded region or where the double stranded region is formed by intramolecular pairing (e.g., a hairpin structure)) made of the single stranded structures described below are within the invention. Preferred lengths are described elsewhere herein.

[0349] As nucleic acids are polymers of subunits or monomers, many of the modifications described below occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or the a non-linking O of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many, and in fact in most cases it will not. By way of example, a modification may only occur at a 3′ or 5′ terminal position, may only occur in a terminal regions, e.g. at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of an RNA or may only occur in a single strand region of an RNA. E.g., a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal regions, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5′ end or ends can be phosphorylated.

[0350] In some embodiments it is particularly preferred, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, or in both. E.g., it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3′ or 5′ overhang will be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2′ OH group of the ribose sugar, e.g., the use of deoxyribonucleotides, e.g., deoxythymidine, instead of ribonucleotides, and modifications in the phosphate group, e.g., phosphothioate modifications. Overhangs need not be homologous with the target sequence.

[0351] Modifications and nucleotide surrogates are discussed below.

[0352]

[0353] The scaffold presented above in Formula 1 represents a portion of a ribonucleic acid. The basic components are the ribose sugar, the base, the terminal phosphates, and phosphate internucleotide linkers. Where the bases are naturally occurring bases, e.g., adenine, uracil, guanine or cytosine, the sugars are the unmodified 2′ hydroxyl ribose sugar (as depicted) and W, X, Y, and Z are all O, Formula 1 represents a naturally occurring unmodified oligoribonucleotide.

[0354] Unmodified oligoribonucleotides may be less than optimal in some applications, e.g., unmodified oligoribonucleotides can be prone to degradation by e.g., cellular nucleases. Nucleases can hydrolyze nucleic acid phosphodiester bonds. However, chemical modifications to one or more of the above RNA components can confer improved properties, and, e.g., can render oligoribonucleotides more stable to nucleases. Umodified oligoribonucleotides may also be less than optimal in terms of offering tethering points for attaching ligands or other moieties to an iRNA agent.

[0355] Modified nucleic acids and nucleotide surrogates can include one or more of:

[0356] (i) alteration, e.g., replacement, of one or both of the non-linking (X and Y) phosphate oxygens and/or of one or more of the linking (W and Z) phosphate oxygens (When the phosphate is in the terminal position, one of the positions W or Z will not link the phosphate to an additional element in a naturally occurring ribonucleic acid. However, for simplicity of terminology, except where otherwise noted, the W position at the 5′ end of a nucleic acid and the terminal Z position at the 3′ end of a nucleic acid, are within the term “linking phosphate oxygens” as used herein.);

[0357] (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar, or wholesale replacement of the ribose sugar with a structure other than ribose, e.g., as described herein;

[0358] (iii) wholesale replacement of the phosphate moiety (bracket I) with “dephospho” linkers;

[0359] (iv) modification or replacement of a naturally occurring base;

[0360] (v) replacement or modification of the ribose-phosphate backbone (bracket II);

[0361] (vi) modification of the 3′ end or 5′ end of the RNA, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, e.g. a fluorescently labeled moiety, to either the 3′ or 5′ end of RNA.

[0362] The terms replacement, modification, alteration, and the like, as used in this context, do not imply any process limitation, e.g., modification does not mean that one must start with a reference or naturally occurring ribonucleic acid and modify it to produce a modified ribonucleic acid bur rather modified simply indicates a difference from a naturally occurring molecule.

[0363] It is understood that the actual electronic structure of some chemical entities cannot be adequately represented by only one canonical form (i.e. Lewis structure). While not wishing to be bound by theory, the actual structure can instead be some hybrid or weighted average of two or more canonical forms, known collectively as resonance forms or structures. Resonance structures are not discrete chemical entities and exist only on paper. They differ from one another only in the placement or “localization” of the bonding and nonbonding electrons for a particular chemical entity. It can be possible for one resonance structure to contribute to a greater extent to the hybrid than the others. Thus, the written and graphical descriptions of the embodiments of the present invention are made in terms of what the art recognizes as the predominant resonance form for a particular species. For example, any phosphoroamidate (replacement of a nonlinking oxygen with nitrogen) would be represented by X═O and Y═N in the above figure.

[0364] Specific modifications are discussed in more detail below.

[0365] The Phosphate Group

[0366] The phosphate group is a negatively charged species. The charge is distributed equally over the two non-linking oxygen atoms (i.e., X and Y in Formula 1 above). However, the phosphate group can be modified by replacing one of the oxygens with a different substituent. One result of this modification to RNA phosphate backbones can be increased resistance of the oligoribonucleotide to nucleolytic breakdown. Thus while not wishing to be bound by theory, it can be desirable in some embodiments to introduce alterations which result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.

[0367] Examples of modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. Unlike the situation where only one of X or Y is altered, the phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligoribonucleotides diastereomers. Diastereomer formation can result in a preparation in which the individual diastereomers exhibit varying resistance to nucleases. Further, the hybridization affinity of RNA containing chiral phosphate groups can be lower relative to the corresponding unmodified RNA species. Thus, while not wishing to be bound by theory, modifications to both X and Y which eliminate the chiral center, e.g. phosphorodithioate formation, may be desirable in that they cannot produce diastereomer mixtures. Thus, X can be any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl). Thus Y can be any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl). Replacement of X and/or Y with sulfur is preferred.

[0368] The phosphate linker can also be modified by replacement of a linking oxygen (i.e., W or Z in Formula 1) with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at a terminal oxygen (position W (3′) or position Z (5′). Replacement of W with carbon or Z with nitrogen is preferred.

[0369] Candidate agents can be evaluated for suitability as described below.

[0370] The Sugar Group

[0371] A modified RNA can include modification of all or some of the sugar groups of the ribonucleic acid. E.g., the 2′ hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents. While not being bound by theory, enhanced stability is expected since the hydroxyl can no longer be deprotonated to form a 2′ alkoxide ion. The 2′ alkoxide can catalyze degradation by intramolecular nucleophilic attack on the linker phosphorus atom. Again, while not wishing to be bound by theory, it can be desirable to some embodiments to introduce alterations in which alkoxide formation at the 2′ position is not possible.

[0372] Examples of “oxy”-2′ hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_nCH₂CH₂OR; “locked” nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by a methylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE (AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino) and aminoalkoxy, O(CH₂)_nAMINE, (e.g., AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino). It is noteworthy that oligonucleotides containing only the methoxyethyl group (MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nuclease stabilities comparable to those modified with the robust phosphorothioate modification.

[0373] “Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, which are of particular relevance to the overhang portions of partially ds RNA); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); NH(CH₂CH₂NH)_nCH₂CH₂-AMINE (AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino), —NHC(O)R (R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino functionality. Preferred substitutents are 2′-methoxyethyl, 2′-OCH3, 2′-O-allyl, 2′-C-allyl, and 2′-fluoro.

[0374] The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified RNA can include nucleotides containing e.g., arabinose, as the sugar.

[0375] Modified RNA's can also include “abasic” sugars, which lack a nucleobase at C-1′. These abasic sugars can also be further contain modifications at one or more of the constituent sugar atoms.

[0376] To maximize nuclease resistance, the 2′ modifications can be used in combination with one or more phosphate linker modifications (e.g., phosphorothioate). The so-called “chimeric” oligonucleotides are those that contain two or more different modifications.

[0377] The modification can also entail the wholesale replacement of a ribose structure with another entity at one or more sites in the iRNA agent. These modifications are described in section entitled Ribose Replacements for RRMSs.

[0378] Candidate modifications can be evaluated as described below.

[0379] Replacement of the Phosphate Group

[0380] The phosphate group can be replaced by non-phosphorus containing connectors (cf. Bracket I in Formula 1 above). While not wishing to be bound by theory, it is believed that since the charged phosphodiester group is the reaction center in nucleolytic degradation, its replacement with neutral structural mimics should impart enhanced nuclease stability. Again, while not wishing to be bound by theory, it can be desirable, in some embodiment, to introduce alterations in which the charged phosphate group is replaced by a neutral moiety.

[0381] Examples of moieties which can replace the phosphate group include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. Preferred replacements include the methylenecarbonylamino and methylenemethylimino groups.

[0382] Candidate modifications can be evaluated as described below.

[0383] Replacement of Ribophosphate Backbone

[0384] Oligonucleotide-mimicking scaffolds can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates (see Bracket II of Formula 1 above). While not wishing to be bound by theory, it is believed that the absence of a repetitively charged backbone diminishes binding to proteins that recognize polyanions (e.g. nucleases). Again, while not wishing to be bound by theory, it can be desirable in some embodiment, to introduce alterations in which the bases are tethered by a neutral surrogate backbone.

[0385] Examples include the mophilino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates. A preferred surrogate is a PNA surrogate.

[0386] Candidate modifications can be evaluated as described below.

[0387] Terminal Modifications

[0388] The 3′ and 5′ ends of an oligonucleotide can be modified. Such modifications can be at the 3′ end, 5′ end or both ends of the molecule. They can include modification or replacement of an entire terminal phosphate or of one or more of the atoms of the phosphate group. E.g., the 3′ and 5′ ends of an oligonucleotide can be conjugated to other functional molecular entities such as labeling moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon, boron or ester). The functional molecular entities can be attached to the sugar through a phosphate group and/or a spacer. The terminal atom of the spacer can connect to or replace the linking atom of the phosphate group or the C-3′ or C-5′ O, N, S or C group of the sugar. Alternatively, the spacer can connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNAs). These spacers or linkers can include e.g., —(CH₂)_n—, —(CH₂)_nN—, —(CH₂)_nO—, —(CH₂)_nS—, O(CH₂CH₂O)_nCH₂CH₂OH (e.g., n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents. When a spacer/phosphate-functional molecular entity-spacer/phosphate array is interposed between two strands of iRNA agents, this array can substitute for a hairpin RNA loop in a hairpin-type RNA agent. The 3′ end can be an —OH group. While not wishing to be bound by theory, it is believed that conjugation of certain moieties can improve transport, hybridization, and specificity properties. Again, while not wishing to be bound by theory, it may be desirable to introduce terminal alterations that improve nuclease resistance. Other examples of terminal modifications include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic carriers (e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, bomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles).

[0389] Terminal modifications can be added for a number of reasons, including as discussed elsewhere herein to modulate activity or to modulate resistance to degradation. Terminal modifications useful for modulating activity include modification of the 5′ end with phosphate or phosphate analogs. E.g., in preferred embodiments iRNA agents, especially antisense strands, are 5′ phosphorylated or include a phosphoryl analog at the 5′ prime terminus. 5′-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′); 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)—O-5′-).

[0390] Terminal Modifications Useful for Increasing Resistance to Degradation Include

[0391] Terminal modifications can also be useful for monitoring distribution, and in such cases the preferred groups to be added include fluorophores, e.g., fluorscein or an Alexa dye, e.g., Alexa 488. Terminal modifications can also be useful for enhancing uptake, useful modifications for this include cholesterol. Terminal modifications can also be useful for cross-linking an RNA agent to another moiety; modifications useful for this include mitomycin C.

[0392] Candidate modifications can be evaluated as described below.

[0393] The Bases

[0394] Adenine, guanine, cytosine and uracil are the most common bases found in RNA. These bases can be modified or replaced to provide RNA's having improved properties. E.g., nuclease resistant oligoribonucleotides can be prepared with these bases or with synthetic and natural nucleobases (e.g., inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine) and any one of the above modifications. Alternatively, substituted or modified analogs of any of the above bases, e.g., “unusual bases” and “universal bases,” can be employed. Examples include without limitation 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases. Further purines and pyrimidines include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613.

[0395] Generally, base changes are less preferred for promoting stability, but they can be useful for other reasons, e.g., some, e.g., 2,6-diaminopurine and 2 amino purine, are fluorescent. Modified bases can reduce target specificity. This should be taken into consideration in the design of iRNA agents.

[0396] Candidate modifications can be evaluated as described below.

[0397] Evaluation of Candidate RNA's

[0398] One can evaluate a candidate RNA agent, e.g., a modified RNA, for a selected property by exposing the agent or modified molecule and a control molecule to the appropriate conditions and evaluating for the presence of the selected property. For example, resistance to a degradent can be evaluated as follows. A candidate modified RNA (and preferably a control molecule, usually the unmodified form) can be exposed to degradative conditions, e.g., exposed to a milieu, which includes a degradative agent, e.g., a nuclease. E.g., one can use a biological sample, e.g., one that is similar to a milieu, which might be encountered, in therapeutic use, e.g., blood or a cellular fraction, e.g., a cell-free homogenate or disrupted cells. The candidate and control could then be evaluated for resistance to degradation by any of a number of approaches. For example, the candidate and control could be labeled, preferably prior to exposure, with, e.g., a radioactive or enzymatic label, or a fluorescent label, such as Cy3 or Cy5. Control and modified RNA's can be incubated with the degradative agent, and optionally a control, e.g., an inactivated, e.g., heat inactivated, degradative agent. A physical parameter, e.g., size, of the modified and control molecules are then determined. They can be determined by a physical method, e.g., by polyacrylamide gel electrophoresis or a sizing column, to assess whether the molecule has maintained its original length, or assessed functionally. Alternatively, Northern blot analysis can be used to assay the length of an unlabeled modified molecule.

[0399] A functional assay can also be used to evaluate the candidate agent. A functional assay can be applied initially or after an earlier non-functional assay, (e.g., assay for resistance to degradation) to determine if the modification alters the ability of the molecule to silence gene expression. For example, a cell, e.g., a mammalian cell, such as a mouse or human cell, can be co-transfected with a plasmid expressing a fluorescent protein, e.g., GFP, and a candidate RNA agent homologous to the transcript encoding the fluorescent protein (see, e.g., WO 00/44914). For example, a modified dsRNA homologous to the GFP mRNA can be assayed for the ability to inhibit GFP expression by monitoring for a decrease in cell fluorescence, as compared to a control cell, in which the transfection did not include the candidate dsRNA, e.g., controls with no agent added and/or controls with a non-modified RNA added. Efficacy of the candidate agent on gene expression can be assessed by comparing cell fluorescence in the presence of the modified and unmodified dsRNA agents.

[0400] In an alternative functional assay, a candidate dsRNA agent homologous to an endogenous mouse gene, preferably a maternally expressed gene, such as c-mos, can be injected into an immature mouse oocyte to assess the ability of the agent to inhibit gene expression in vivo (see, e.g., WO 01/36646). A phenotype of the oocyte, e.g., the ability to maintain arrest in metaphase II, can be monitored as an indicator that the agent is inhibiting expression. For example, cleavage of c-mos mRNA by a dsRNA agent would cause the oocyte to exit metaphase arrest and initiate parthenogenetic development (Colledge et al. Nature 370: 65-68, 1994; Hashimoto et al. Nature, 370:68-71, 1994). The effect of the modified agent on target RNA levels can be verified by Northern blot to assay for a decrease in the level of target mRNA, or by Western blot to assay for a decrease in the level of target protein, as compared to a negative control. Controls can include cells in which with no agent is added and/or cells in which a non-modified RNA is added.

[0401] The oligoribonucleotides and oligoribonucleosides used in accordance with this invention may be with solid phase synthesis, see for example “Oligonucleotide synthesis, a practical approach”, Ed. M. J. Gait, IRL Press, 1984; “Oligonucleotides and Analogues, A Practical Approach”, Ed. F. Eckstein, IRL Press, 1991 (especially Chapter 1, Modern machine-aided methods of oligodeoxyribonucleotide synthesis, Chapter 2, Oligoribonucleotide synthesis, Chapter 3, 2′-O-Methyloligoribonucleotide-s: synthesis and applications, Chapter 4, Phosphorothioate oligonucleotides, Chapter 5, Synthesis of oligonucleotide phosphorodithioates, Chapter 6, Synthesis of oligo-2′-deoxyribonucleoside methylphosphonates, and. Chapter 7, Oligodeoxynucleotides containing modified bases. Other particularly useful synthetic procedures, reagents, blocking groups and reaction conditions are described in Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48, 2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49, 6123-6194, or references referred to therein.

[0402] Modification described in WO 00/44895, WO01/75164, or WO002/44321 can be used herein.

[0403] The disclosure of all publications, patents, and published patent applications listed herein are hereby incorporated by reference.

[0404] Phosphate Group References

[0405] The preparation of phosphinate oligoribonucleotides is described in U.S. Pat. No. 5,508,270. The preparation of alkyl phosphonate oligoribonucleotides is described in U.S. Pat. No. 4,469,863. The preparation of phosphoramidite oligoribonucleotides is described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878. The preparation of phosphotriester oligoribonucleotides is described in U.S. Pat. No. 5,023,243. The preparation of borano phosphate oligoribonucleotide is described in U.S. Pat. Nos. 5,130,302 and 5,177,198. The preparation of 3′-Deoxy-3′-amino phosphoramidate oligoribonucleotides is described in U.S. Pat. No. 5,476,925. 3′-Deoxy-3′-methylenephosphonate oligoribonucleotides is described in An, H, et al. J. Org. Chem. 2001, 66, 2789-2801. Preparation of sulfur bridged nucleotides is described in Sproat et al. Nucleosides Nucleotides 1988, 7,651 and Crosstick et al. Tetrahedron Lett. 1989, 30, 4693.

[0406] Sugar Group References

[0407] Modifications to the 2′ modifications can be found in Verma, S. et al. Annu. Rev. Biochem. 1998, 67, 99-134 and all references therein. Specific modifications to the ribose can be found in the following references: 2′-fluoro (Kawasaki et. al., J. Med. Chem., 1993, 36, 831-841), 2′-MOE (Martin, P. Helv. Chim. Acta 1996, 79, 1930-1938), “LNA” (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310).

[0408] Replacement of the Phosphate Group References

[0409] Methylenemethylimino linked oligoribonucleosides, also identified herein as MMI linked oligoribonucleosides, methylenedimethylhydrazo linked oligoribonucleosides, also identified herein as MDH linked oligoribonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified herein as amide-3 linked oligoribonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified herein as amide-4 linked oligoribonucleosides as well as mixed backbone compounds having, as for instance, alternating MMI and PO or PS linkages can be prepared as is described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and in published PCT applications PCT/US92/04294 and PCT/US92/04305 (published as WO 92/20822 WO and 92/20823, respectively). Formacetal and thioformacetal linked oligoribonucleosides can be prepared as is described in U.S. Pat. Nos. 5,264,562 and 5,264,564. Ethylene oxide linked oligoribonucleosides can be prepared as is described in U.S. Pat. No. 5,223,618. Siloxane replacements are described in Cormier, J. F. et al. Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements are described in Tittensor, J. R. J. Chem. Soc. C1971, 1933. Carboxymethyl replacements are described in Edge, M. D. et al. J. Chem. Soc. Perkin Trans. 1 1972, 1991. Carbamate replacements are described in Stirchak, E. P. Nucleic Acids Res. 1989, 17, 6129.

[0410] Replacement of the Phosphate-Ribose Backbone References

[0411] Cyclobutyl sugar surrogate compounds can be prepared as is described in U.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate can be prepared as is described in U.S. Pat. No. 5,519,134. Morpholino sugar surrogates can be prepared as is described in U.S. Pat. Nos. 5,142,047 and 5,235,033, and other related patent disclosures. Peptide Nucleic Acids (PNAs) are known per se and can be prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. No. 5,539,083.

[0412] Terminal Modification References

[0413] Terminal modifications are described in Manoharan, M. et al. Antisense and Nucleic Acid Drug Development 12, 103-128 (2002) and references therein.

[0414] Bases References

[0415] N-2 substituted purine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,459,255. 3-Deaza purine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,457,191. 5,6-Substituted pyrimidine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,614,617. 5-Propynyl pyrimidine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,484,908. Additional references can be disclosed in the above section on base modifications.

[0416] Preferred iRNA Agents

[0417] Preferred RNA agents have the following structure (see Formula 2 below):

[0418]

[0419] Referring to Formula 2 above, R¹, R², and R³ are each, independently, H, (i.e. abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, 7-deazaguanine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases.

[0420] R⁴, R⁵, and R⁶ are each, independently, OR⁸, O(CH₂CH₂O)_mCH₂CH₂OR⁸; O(CH₂)_nR⁹; O(CH₂)_nOR⁹, H; halo; NH₂; NHR⁸; N(R⁸)₂; NH(CH₂CH₂NH)_mCH₂CH₂NHR⁹; NHC(O)R⁸; cyano; mercapto, SR⁸; alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl, alkynyl, each of which may be optionally substituted with halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido; or R⁴, R⁵, or R⁶ together combine with R⁷ to form an [—O—CH₂—] covalently bound bridge between the sugar 2′ and 4′ carbons.

[0421] A¹ is:

[0422]
H; OH; OCH₃; W¹; an abasic nucleotide; or absent;

[0423] (a preferred A1, especially with regard to anti-sense strands, is chosen from 5′-monophosphate ((HO)₂(O)P—O-5′), 5′-diphosphate ((HO)₂(O)P—O—P(HO)(O)—O-5′), 5′-triphosphate ((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-monothiophosphate (phosphorothioate; (HO)₂(S)P—O-5′), 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO)₂(O)P—S-5′); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH₂)(O)P—O-5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—O-5′-, (OH)₂(O)P-5′-CH₂—), 5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH₂—), ethoxymethyl, etc., e.g. RP(OH)(O)—O-5′-)).

[0424] A² is:

[0425]

[0426] A³ is:

[0427]
and

[0428] A⁴ is:

[0429]
H; 4; an inverted nucleotide; an abasic nucleotide; or absent.

[0430] W¹ is OH, (CH₂)_nR¹⁰, (CH₂)_nNHR¹⁰, (CH₂)_n OR¹⁰, (CH₂)_n SR¹⁰; O(CH₂)_nR¹⁰; O(CH₂)_nOR¹⁰, O(CH₂)_nNR¹⁰, O(CH₂)_nSR¹⁰; O(CH₂)_nSS(CH₂)_nOR¹⁰, O(CH₂)_nC(O)OR¹⁰, NH(CH₂)_nR¹⁰; NH(CH₂)_nNR¹⁰; NH(CH₂)_nOR¹⁰, NH(CH₂)_nSR¹⁰; S(CH₂)_nR¹⁰, S(CH₂)_nNR¹⁰, S(CH₂)_nOR¹⁰, S(CH₂)_nSR¹⁰O(CH₂CH₂O)_mCH₂CH₂OR¹⁰; O(CH₂CH₂O)_mCH₂CH₂NHR¹⁰, NH(CH₂CH₂NH)_mCH₂CH₂NHR¹⁰; Q-R¹⁰, O-Q-R¹⁰ N-Q-R¹⁰, S-Q-R¹⁰ or —O—. W⁴ is O, CH₂, NH, or S.

[0431] X¹, X², X³, and X⁴ are each, independently, O or S.

[0432] Y¹, Y², Y³, and Y⁴ are each, independently, OH, O⁻, OR⁸, S, Se, BH₃⁻, H, NHR⁹, N(R⁹)₂ alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted.

[0433] Z¹, Z², and Z³ are each independently O, CH₂, NH, or S. Z⁴ is OH, (CH₂)_nR¹⁰, (CH₂)_nNHR¹⁰, (CH₂)_n OR¹⁰, (CH₂)_n SR¹⁰; O(CH₂)_nR¹⁰; O(CH₂)_nOR¹⁰, O(CH₂)_nNR¹⁰, O(CH₂)_nR¹⁰, O(CH₂)_nSS(CH₂)_nOR¹⁰, O(CH₂)_nC(O)OR¹⁰; NH(CH₂)_nR¹⁰; NH(CH₂)_nNR¹⁰; NH(CH₂)_nOR¹⁰, NH(CH₂)_nSR¹⁰; S(CH₂)_nR¹⁰, S(CH₂)_nNR¹⁰, S(CH₂)_nOR¹⁰, S(CH₂)_nSR¹⁰ O(CH₂CH₂O)_mCH₂CH₂OR¹⁰, O(CH₂CH₂O)_mCH₂CH₂NHR¹⁰, NH(CH₂CH₂NH)_mCH₂CH₂NHR¹⁰; Q-R¹⁰, O-Q-R¹⁰ N-Q-R¹⁰, S-Q-R¹⁰.

[0434] x is 5-100, chosen to comply with a length for an RNA agent described herein.

[0435] R⁷ is H; or is together combined with R⁴, R⁵, or R⁶ to form an [—O—CH₂—] covalently bound bridge between the sugar 2′ and 4′ carbons.

[0436] R⁸ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, amino acid, or sugar; R⁹ is NH₂, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid; and R¹⁰ is H; fluorophore (pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes); sulfur, silicon, boron or ester protecting group; intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipohilic carriers (cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, bomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino; alkyl, cycloalkyl, aryl, aralkyl, heteroaryl; radiolabelled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles); or an RNA agent. m is 0-1,000,000, and n is 0-20. Q is a spacer selected from the group consisting of abasic sugar, amide, carboxy, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, biotin or fluorescein reagents.

[0437] Preferred RNA agents in which the entire phosphate group has been replaced have the following structure (see Formula 3 below):

[0438]

[0439] Referring to Formula 3, A¹⁰-A⁴⁰ is L-G-L; A¹⁰ and/or A⁴⁰ may be absent, in which L is a linker, wherein one or both L may be present or absent and is selected from the group consisting of CH₂(CH₂)_g; N(CH₂)_g; O(CH₂)_g; S(CH₂)_g. G is a functional group selected from the group consisting of siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.

[0440] R¹⁰, R²⁰, and R³⁰ are each, independently, H, (i.e. abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, 7-deazaguanine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3 carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases.

[0441] R⁴⁰, R⁵⁰, and R⁶⁰ are each, independently, OR⁸, O(CH₂CH₂O)_mCH₂CH₂OR⁸; O(CH₂)_nR⁹; O(CH₂)_nOR⁹, H; halo; NH₂; NHR⁸; N(R⁸)₂; NH(CH₂CH₂NH)_mCH₂CH₂R⁹; NHC(O)R⁸; cyano; mercapto, SR⁷; alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl, alkynyl, each of which may be optionally substituted with halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido groups; or R⁴⁰, R⁵⁰, or R⁶⁰ together combine with R⁷⁰ to form an [—O—CH₂—] covalently bound bridge between the sugar 2′ and 4′ carbons.

[0442] x is 5-100 or chosen to comply with a length for an RNA agent described herein.

[0443] R⁷⁰ is H; or is together combined with R⁴⁰, R⁵⁰, or R⁶⁰ to form an [—O—CH₂—] covalently bound bridge between the sugar 2′ and 4′ carbons.

[0444] R⁸ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, amino acid, or sugar; and R⁹ is NH₂, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid. m is 0-1,000,000, n is 0-20, and g is 0-2.

[0445] Preferred nucleoside surrogates have the following structure (see Formula 4 below):
SLR¹⁰⁰(M-SLR²⁰⁰)_x-M-SLR³⁰⁰   FORMULA 4

[0446] S is a nucleoside surrogate selected from the group consisting of mophilino, cyclobutyl, pyrrolidine and peptide nucleic acid. L is a linker and is selected from the group consisting of CH₂(CH₂)_g; N(CH₂)_g; O(CH₂)_g; S(CH₂)_g; —C(O)(CH₂)_n— or may be absent. M is an amide bond; sulfonamide; sulfinate; phosphate group; modified phosphate group as described herein; or may be absent.

[0447] R¹⁰⁰, R²⁰⁰, and R³⁰⁰ are each, independently, H (i.e., abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, 7-deazaguanine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil substituted 1,2,4,-triazoles, 2-pyridinones, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3 carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases.

[0448] x is 5-100, or chosen to comply with a length for an RNA agent described herein; and g is 0-2.

[0449] Nuclease Resistant Monomers

[0450] In one aspect, the invention features a nuclease resistant monomer, or a an iRNA agent which incorporates a nuclease resistant monomer (NMR), such as those described herein and those described in copending, co-owned U.S. Provisional Application Ser. No. 60/469,612, filed on May 9, 2003, which is hereby incorporated by reference.

[0451] In addition, the invention includes iRNA agents having a NMR and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a NMR.

[0452] An iRNA agent can include monomers which have been modified so as to inhibit degradation, e.g., by nucleases, e.g., endonucleases or exonucleases, found in the body of a subject. These monomers are referred to herein as NRM's, or nuclease resistance promoting monomers or modifications. In many cases these modifications will modulate other properties of the iRNA agent as well, e.g., the ability to interact with a protein, e.g., a transport protein, e.g., serum albumin, or a member of the RISC (RNA-induced Silencing Complex), or the ability of the first and second sequences to form a duplex with one another or to form a duplex with another sequence, e.g., a target molecule.

[0453] While not wishing to be bound by theory, it is believed that modifications of the sugar, base, and/or phosphate backbone in an iRNA agent can enhance endonuclease and exonuclease resistance, and can enhance interactions with transporter proteins and one or more of the functional components of the RISC complex. Preferred modifications are those that increase exonuclease and endonuclease resistance and thus prolong the halflife of the iRNA agent prior to interaction with the RISC complex, but at the same time do not render the iRNA agent resistant to endonuclease activity in the RISC complex. Again, while not wishing to be bound by any theory, it is believed that placement of the modifications at or near the 3′ and/or 5′ end of antisense strands can result in iRNA agents that meet the preferred nuclease resistance criteria delineated above. Again, still while not wishing to be bound by any theory, it is believed that placement of the modifications at e.g., the middle of a sense strand can result in iRNA agents that are relatively less likely to undergo off-targeting.

[0454] Modifications described herein can be incorporated into any double-stranded RNA and RNA-like molecule described herein, e.g., an iRNA agent. An iRNA agent may include a duplex comprising a hybridized sense and antisense strand, in which the antisense strand and/or the sense strand may include one or more of the modifications described herein. The anti sense strand may include modifications at the 3′ end and/or the 5′ end and/or at one or more positions that occur 1-6 (e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand. The sense strand may include modifications at the 3′ end and/or the 5′ end and/or at any one of the intervening positions between the two ends of the strand. The iRNA agent may also include a duplex comprising two hybridized antisense strands. The first and/or the second antisense strand may include one or more of the modifications described herein. Thus, one and/or both antisense strands may include modifications at the 3′ end and/or the 5′ end and/or at one or more positions that occur 1-6 (e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand. Particular configurations are discussed below.

[0455] Modifications that can be useful for producing iRNA agents that meet the preferred nuclease resistance criteria delineated above can include one or more of the following chemical and/or stereochemical modifications of the sugar, base, and/or phosphate backbone:

[0456] (i) chiral (S_P) thioates. Thus, preferred NRM's include nucleotide dimers with an enriched or pure for a particular chiral form of a modified phosphate group containing a heteroatom at the nonbridging position, e.g., Sp or Rp, at the position X, where this is the position normally occupied by the oxygen. The atom at X can also be S, Se, Nr₂, or Br₃. When X is S, enriched or chirally pure Sp linkage is preferred. Enriched means at least 70, 80, 90, 95, or 99% of the preferred form. Such NRM's are discussed in more detail below;

[0457] (ii) attachment of one or more cationic groups to the sugar, base, and/or the phosphorus atom of a phosphate or modified phosphate backbone moiety. Thus, preferred NRM's include monomers at the terminal position derivitized at a cationic group. As the 5′ end of an antisense sequence should have a terminal —OH or phosphate group this NRM is preferrably not used at the 5′ end of an anti-sense sequence. The group should be attached at a position on the base which minimizes interference with H bond formation and hybridization, e.g., away form the face which interacts with the complementary base on the other strand, e.g., at the 5′ position of a pyrimidine or a 7-position of a purine. These are discussed in more detail below;

[0458] (iii) nonphosphate linkages at the termini. Thus, preferred NRM's include Non-phosphate linkages, e.g., a linkage of 4 atoms which confers greater resistance to cleavage than does a phosphate bond. Examples include 3′ CH2-NCH₃—O—CH2-5′ and 3′ CH2-NH—(O═)—CH2-5′;

[0459] (iv) 3′-bridging thiophosphates and 5′-bridging thiophosphates. Thus, preferred NRM's can included these structures;

[0460] (v) L-RNA, 2′-5′ linkages, inverted linkages, a-nucleosides. Thus, other preferred NRM's include: L nucleosides and dimeric nucleotides derived from L-nucleosides; 2′-5′ phosphate, non-phosphate and modified phosphate linkages (e.g., thiophospahtes, phosphoramidates and boronophosphates); dimers having inverted linkages, e.g., 3′-3′ or 5′-5′ linkages; monomers having an alpha linkage at the 1′ site on the sugar, e.g., the structures described herein having an alpha linkage;

[0461] (vi) conjugate groups. Thus, preferred NRM's can include e.g., a targeting moiety or a conjugated ligand described herein conjugated with the monomer, e.g., through the sugar, base, or backbone;

[0462] (vi) abasic linkages. Thus, preferred NRM's can include an abasic monomer, e.g., an abasic monomer as described herein (e.g., a nucleobaseless monomer); an aromatic or heterocyclic or polyheterocyclic aromatic monomer as described herein; and

[0463] (vii) 5′-phosphonates and 5′-phosphate prodrugs. Thus, preferred NRM's include monomers, preferably at the terminal position, e.g., the 5′ position, in which one or more atoms of the phosphate group is derivatized with a protecting group, which protecting group or groups, are removed as a result of the action of a component in the subject's body, e.g., a carboxyesterase or an enzyme present in the subject's body. E.g., a phosphate prodrug in which a carboxy esterase cleaves the protected molecule resulting in the production of a thioate anion which attacks a carbon adjacent to the O of a phosphate and resulting in the production of an unprotected phosphate.

[0464] One or more different NRM modifications can be introduced into an iRNA agent or into a sequence of an iRNA agent. An NRM modification can be used more than once in a sequence or in an iRNA agent. As some NRM's interfere with hybridization the total number incorporated, should be such that acceptable levels of iRNA agent duplex formation are maintained.

[0465] In some embodiments NRM modifications are introduced into the terminal the cleavage site or in the cleavage region of a sequence (a sense strand or sequence) which does not target a desired sequence or gene in the subject. This can reduce off-target silencing.

[0466] Chiral S_P Thioates

[0467] A modification can include the alteration, e.g., replacement, of one or both of the non-linking (X and Y) phosphate oxygens and/or of one or more of the linking (W and Z) phosphate oxygens. Formula X below depicts a phosphate moiety linking two sugar/sugar surrogate-base moities, SB₁ and SB₂.

[0468]

[0469] In certain embodiments, one of the non-linking phosphate oxygens in the phosphate backbone moiety (X and Y) can be replaced by any one of the following: S, Se, BR₃ (R is hydrogen, alkyl, aryl, etc.), C (i.e., an alkyl group, an aryl group, etc.), H, NR₂ (R is hydrogen, alkyl, aryl, etc.), or OR (R is alkyl or aryl). The phosphorus atom in an unmodified phosphate group is achiral. However, replacement of one of the non-linking oxygens with one of the above atoms or groups of atoms renders the phosphorus atom chiral; in other words a phosphorus atom in a phosphate group modified in this way is a stereogenic center. The stereogenic phosphorus atom can possess either the “R” configuration (herein R_P) or the “S” configuration (herein S_P). Thus if 60% of a population of stereogenic phosphorus atoms have the R_P configuration, then the remaining 40% of the population of stereogenic phosphorus atoms have the S_P configuration.

[0470] In some embodiments, iRNA agents, having phosphate groups in which a phosphate non-linking oxygen has been replaced by another atom or group of atoms, may contain a population of stereogenic phosphorus atoms in which at least about 50% of these atoms (e.g., at least about 60% of these atoms, at least about 70% of these atoms, at least about 80% of these atoms, at least about 90% of these atoms, at least about 95% of these atoms, at least about 98% of these atoms, at least about 99% of these atoms) have the S_P configuration. Alternatively, iRNA agents having phosphate groups in which a phosphate non-linking oxygen has been replaced by another atom or group of atoms may contain a population of stereogenic phosphorus atoms in which at least about 50% of these atoms (e.g., at least about 60% of these atoms, at least about 70% of these atoms, at least about 80% of these atoms, at least about 90% of these atoms, at least about 95% of these atoms, at least about 98% of these atoms, at least about 99% of these atoms) have the R_P configuration. In other embodiments, the population of stereogenic phosphorus atoms may have the S_P configuration and may be substantially free of stereogenic phosphorus atoms having the RP configuration. In still other embodiments, the population of stereogenic phosphorus atoms may have the R_P configuration and may be substantially free of stereogenic phosphorus atoms having the S_P configuration. As used herein, the phrase “substantially free of stereogenic phosphorus atoms having the R_P configuration” means that moieties containing stereogenic phosphorus atoms having the R_P configuration cannot be detected by conventional methods known in the art (chiral HPLC, ¹H NMR analysis using chiral shift reagents, etc.). As used herein, the phrase “substantially free of stereogenic phosphorus atoms having the S_P configuration” means that moieties containing stereogenic phosphorus atoms having the S_P configuration cannot be detected by conventional methods known in the art (chiral HPLC, ¹H NMR analysis using chiral shift reagents, etc.).

[0471] In a preferred embodiment, modified iRNA agents contain a phosphorothioate group, i.e., a phosphate groups in which a phosphate non-linking oxygen has been replaced by a sulfur atom. In an especially preferred embodiment, the population of phosphorothioate stereogenic phosphorus atoms may have the S_P configuration and be substantially free of stereogenic phosphorus atoms having the R_P configuration.

[0472] Phosphorothioates may be incorporated into iRNA agents using dimers e.g., formulas X-1 and X-2. The former can be used to introduce phosphorothioate

[0473]
at the 3′ end of a strand, while the latter can be used to introduce this modification at the 5′ end or at a position that occurs e.g., 1, 2, 3, 4, 5, or 6 nucleotides from either end of the strand. In the above formulas, Y can be 2-cyanoethoxy, W and Z can be O, R₂, can be, e.g., a substituent that can impart the C-3 endo configuration to the sugar (e.g., OH, F, OCH₃), DMT is dimethoxytrityl, and “BASE” can be a natural, unusual, or a universal base.

[0474] X-1 and X-2 can be prepared using chiral reagents or directing groups that can result in phosphorothioate-containing dimers having a population of stereogenic phosphorus atoms having essentially only the R_P configuration (i.e., being substantially free of the S_P configuration) or only the S_P configuration (i.e., being substantially free of the RP configuration). Alternatively, dimers can be prepared having a population of stereogenic phosphorus atoms in which about 50% of the atoms have the R_P configuration and about 50% of the atoms have the S_P configuration. Dimers having stereogenic phosphorus atoms with the R_P configuration can be identified and separated from dimers having stereogenic phosphorus atoms with the S_P configuration using e.g., enzymatic degradation and/or conventional chromatography techniques.

[0475] Cationic Groups

[0476] Modifications can also include attachment of one or more cationic groups to the sugar, base, and/or the phosphorus atom of a phosphate or modified phosphate backbone moiety. A cationic group can be attached to any atom capable of substitution on a natural, unusual or universal base. A preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bonding interactions needed for base pairing. A cationic group can be attached e.g., through the C2′ position of a sugar or analogous position in a cyclic or acyclic sugar surrogate. Cationic groups can include e.g., protonated amino groups, derived from e.g., O-AMINE (AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino); aminoalkoxy, e.g., O(CH₂)AMINE, (e.g., AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino); amino (e.g. NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); or NH(CH₂CH₂NH)_nCH₂CH₂-AMNE (AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino).

[0477] Nonphosphate Linkages

[0478] Modifications can also include the incorporation of nonphosphate linkages at the 5′ and/or 3′ end of a strand. Examples of nonphosphate linkages which can replace the phosphate group include methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. Preferred replacements include the methyl phosphonate and hydroxylamino groups.

3′-Bridging Thiophosphates and 5′-Bridging Thiophosphates; Locked-RNA, 2′-5′ Linkages, Inverted Linkages, α-Nucleosides; Conjugate Groups; Abasic Linkages; and 5′-Phosphonates and 5′-Phosphate Prodrugs

[0479] Referring to formula X above, modifications can include replacement of one of the bridging or linking phosphate oxygens in the phosphate backbone moiety (W and Z). Unlike the situation where only one of X or Y is altered, the phosphorus center in the phosphorodithioates is achiral which precludes the formation of iRNA agents containing a stereogenic phosphorus atom.

[0480] Modifications can also include linking two sugars via a phosphate or modified phosphate group through the 2′ position of a first sugar and the 5′ position of a second sugar. Also contemplated are inverted linkages in which both a first and second sugar are each linked through the respective 3′ positions. Modified RNA's can also include “abasic” sugars, which lack a nucleobase at C-1′. The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified iRNA agent can include nucleotides containing e.g., arabinose, as the sugar. In another subset of this modification, the natural, unusual, or universal base may have the α-configuration. Modifications can also include L-RNA.

[0481] Modifications can also include 5′-phosphonates, e.g., P(O)(O⁻)₂—X—C⁵⁻-sugar (X═CH2, CF 2, CHF and 5′-phosphate prodrugs, e.g., P(O)[OCH2CH2SC(O)R]₂CH₂C^5′-sugar. In the latter case, the prodrug groups may be decomposed via reaction first with carboxy esterases. The remaining ethyl thiolate group via intramolecular S_N2 displacement can depart as episulfide to afford the underivatized phosphate group.

[0482] Modification can also include the addition of conjugating groups described else where herein, which are preferably attached to an iRNA agent through any amino group available for conjugation.

[0483] Nuclease resistant modifications include some which can be placed only at the terminus and others which can go at any position. Generally the modifications that can inhibit hybridization so it is preferably to use them only in terminal regions, and preferable to not use them at the cleavage site or in the cleavage region of an sequence which targets a subject sequence or gene. The can be used anywhere in a sense sequence, provided that sufficient hybridization between the two sequences of the iRNA agent is maintained. In some embodiments it is desirable to put the NRM at the cleavage site or in the cleavage region of a sequence which does not target a subject sequence or gene, as it can minimize off-target silencing.

[0484] In addition, an iRNA agent described herein can have an overhang which does not form a duplex structure with the other sequence of the iRNA agent—it is an overhang, but it does hybridize, either with itself, or with another nucleic acid, other than the other sequence of the iRNA agent.

[0485] In most cases, the nuclease-resistance promoting modifications will be distributed differently depending on whether the sequence will target a sequence in the subject (often referred to as an anti-sense sequence) or will not target a sequence in the subject (often referred to as a sense sequence). If a sequence is to target a sequence in the subject, modifications which interfere with or inhibit endonuclease cleavage should not be inserted in the region which is subject to RISC mediated cleavage, e.g., the cleavage site or the cleavage region (As described in Elbashir et al., 2001, Genes and Dev. 15: 188, hereby incorporated by reference, cleavage of the target occurs about in the middle of a 20 or 21 nt guide RNA, or about 10 or 11 nucleotides upstream of the first nucleotide which is complementary to the guide sequence. As used herein cleavage site refers to the nucleotide on either side of the cleavage site, on the target or on the iRNA agent strand which hybridizes to it. Cleavage region means an nucleotide with 1, 2, or 3 nucleotides of the cleave site, in either direction.)

[0486] Such modifications can be introduced into the terminal regions, e.g., at the terminal position or with 2, 3, 4, or 5 positions of the terminus, of a sequence which targets or a sequence which does not target a sequence in the subject.

[0487] An iRNA agent can have a first and a second strand chosen from the following:

[0488] a first strand which does not target a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

[0489] a first strand which does not target a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;

[0490] a first strand which does not target a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end and which has a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;

[0491] a first strand which does not target a sequence and which has an NRM modification at the cleavage site or in the cleavage region;

[0492] a first strand which does not target a sequence and which has an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4, 5, or 6 positions from both the 3′ and the 5′ end; and

[0493] a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

[0494] a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end (5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand);

[0495] a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end and which has a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;

[0496] a second strand which targets a sequence and which preferably does not have an NRM modification at the cleavage site or in the cleavage region;

[0497] a second strand which targets a sequence and which does not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4, 5, or 6 positions from both the 3′ and the 5′ end (5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand).

[0498] An iRNA agent can also target two sequences and can have a first and second strand chosen from:

[0499] a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

[0500] a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end (5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand);

[0501] a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end and which has a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;

[0502] a first strand which targets a sequence and which preferably does not have an NRM modification at the cleavage site or in the cleavage region;

[0503] a first strand which targets a sequence and which dose not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4, 5, or 6 positions from both the 3′ and the 5′ end (5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand) and

[0504] a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

[0505] a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end (5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand);

[0506] a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end and which has a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;

[0507] a second strand which targets a sequence and which preferably does not have an NRM modification at the cleavage site or in the cleavage region;

[0508] a second strand which targets a sequence and which dose not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4, 5, or 6 positions from both the 3′ and the 5′ end (5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand).

[0509] Ribose Mimics

[0510] In one aspect, the invention features a ribose mimic, or an iRNA agent which incorporates a ribose mimic, such as those described herein and those described in copending co-owned U.S. Provisional Application Ser. No. 60/454,962, filed on Mar. 13, 2003, which is hereby incorporated by reference.

[0511] In addition, the invention includes iRNA agents having a ribose mimic and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a ribose mimic.

[0512] Thus, an aspect of the invention features an iRNA agent that includes a secondary hydroxyl group, which can increase efficacy and/or confer nuclease resistance to the agent. Nucleases, e.g., cellular nucleases, can hydrolyze nucleic acid phosphodiester bonds, resulting in partial or complete degradation of the nucleic acid. The secondary hydroxy group confers nuclease resistance to an iRNA agent by rendering the iRNA agent less prone to nuclease degradation relative to an iRNA which lacks the modification. While not wishing to be bound by theory, it is believed that the presence of a secondary hydroxyl group on the iRNA agent can act as a structural mimic of a 3′ ribose hydroxyl group, thereby causing it to be less susceptible to degradation.

[0513] The secondary hydroxyl group refers to an “OH” radical that is attached to a carbon atom substituted by two other carbons and a hydrogen. The secondary hydroxyl group that confers nuclease resistance as described above can be part of any acyclic carbon-containing group. The hydroxyl may also be part of any cyclic carbon-containing group, and preferably one or more of the following conditions is met (1) there is no ribose moiety between the hydroxyl group and the terminal phosphate group or (2) the hydroxyl group is not on a sugar moiety which is coupled to a base. The hydroxyl group is located at least two bonds (e.g., at least three bonds away, at least four bonds away, at least five bonds away, at least six bonds away, at least seven bonds away, at least eight bonds away, at least nine bonds away, at least ten bonds away, etc.) from the terminal phosphate group phosphorus of the iRNA agent. In preferred embodiments, there are five intervening bonds between the terminal phosphate group phosphorus and the secondary hydroxyl group.

[0514] Preferred iRNA agent delivery modules with five intervening bonds between the terminal phosphate group phosphorus and the secondary hydroxyl group have the following structure (see formula Y below):

[0515]

[0516] Referring to formula Y, A is an iRNA agent, including any iRNA agent described herein. The iRNA agent may be connected directly or indirectly (e.g., through a spacer or linker) to “W” of the phosphate group. These spacers or linkers can include e.g., —(CH₂)_n—, —(CH₂)_nN—, —(CH₂)_nO—, —(CH₂)_nS—, O(CH₂CH₂O)_nCH₂CH₂OH (e.g., n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents.

[0517] The iRNA agents can have a terminal phosphate group that is unmodified (e.g., W, X, Y, and Z are O) or modified. In a modified phosphate group, W and Z can be independently NH, O, or S; and X and Y can be independently S, Se, BH₃⁻, C₁-C₆ alkyl, C₆-C₁₀ aryl, H, O, O⁻, alkoxy or amino (including alkylamino, arylamino, etc.). Preferably, W, X and Z are O and Y is S.

[0518] R₁ and R₃ are each, independently, hydrogen; or C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl.

[0519] R₂ is hydrogen; C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1, R₂ may be taken together with R₄ or R₆ to form a ring of 5-12 atoms.

[0520] R₄ is hydrogen; C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1, R₄ may be taken together with R₂ or R₅ to form a ring of 5-12 atoms.

[0521] R₅ is hydrogen, C₁-C₁₀₀ alkyl optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1, R₅ may be taken together with R₄ to form a ring of 5-12 atoms.

[0522] R₆ is hydrogen, C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl, or, when n is 1, R₆ may be taken together with R₂ to form a ring of 6-10 atoms;

[0523] R₇ is hydrogen, C₁-C₁₀₀ alkyl, or C(O)(CH₂)_qC(O)NHR₉; T is hydrogen or a functional group; n and q are each independently 1-100; R₈ is C₁-C₁₀ alkyl or C₆-C₁₀ aryl; and R₉ is hydrogen, C1-C10 alkyl, C6-C10 aryl or a solid support agent.

[0524] Preferred embodiments may include one of more of the following subsets of iRNA agent delivery modules.

[0525] In one subset of RNAi agent delivery modules, A can be connected directly or indirectly through a terminal 3′ or 5′ ribose sugar carbon of the RNA agent.

[0526] In another subset of RNAi agent delivery modules, X, W, and Z are O and Y is S.

[0527] In still yet another subset of RNAi agent delivery modules, n is 1, and R₂ and R₆ are taken together to form a ring containing six atoms and R₄ and R₅ are taken together to form a ring containing six atoms. Preferably, the ring system is a trans-decalin. For example, the RNAi agent delivery module of this subset can include a compound of Formula (Y-1):

[0528]

[0529] The functional group can be, for example, a targeting group (e.g., a steroid or a carbohydrate), a reporter group (e.g., a fluorophore), or a label (an isotopically labelled moiety). The targeting group can further include protein binding agents, endothelial cell targeting groups (e.g., RGD peptides and mimetics), cancer cell targeting groups (e.g., folate Vitamin B12, Biotin), bone cell targeting groups (e.g., bisphosphonates, polyglutamates, polyaspartates), multivalent mannose (for e.g., macrophage testing), lactose, galactose, N-acetyl-galactosamine, monoclonal antibodies, glycoproteins, lectins, melanotropin, or thyrotropin.

[0530] As can be appreciated by the skilled artisan, methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art. The synthesized compounds can be separated from a reaction mixture and further purified by a method such as column chromatography, high pressure liquid chromatography, or recrystallization. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

[0531] Ribose Replacement Monomer Subunits

[0532] iRNA agents can be modified in a number of ways which can optimize one or more characteristics of the iRNA agent. In one aspect, the invention features a ribose replacement monomer subunit (RRMS), or a an iRNA agent which incorporates a RRMS, such as those described herein and those described in one or more of U.S. Provisional Application Ser. No. 60/493,986, filed on Aug. 8, 2003, which is hereby incorporated by reference; U.S. Provisional Application Ser. No. 60/494,597, filed on Aug. 11, 2003, which is hereby incorporated by reference; U.S. Provisional Application Ser. No. 60/506,341, filed on Sep. 26, 2003, which is hereby incorporated by reference; and in U.S. Provisional Application Ser. No. 60/158,453, filed on Nov. 7, 2003, which is hereby incorporated by reference.

[0533] In addition, the invention includes iRNA agents having a RRMS and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a RRMS.

[0534] The ribose sugar of one or more ribonucleotide subunits of an iRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.

[0535] The carriers further include (i) at least two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” as used herein refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a ligand, e.g., a targeting or delivery moiety, or a moiety which alters a physical property, e.g., lipophilicity, of an iRNA agent. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, it will include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.

[0536] Incorporation of one or more RRMSs described herein into an RNA agent, e.g., an iRNA agent, particularly when tethered to an appropriate entity, can confer one or more new properties to the RNA agent and/or alter, enhance or modulate one or more existing properties in the RNA molecule. E.g., it can alter one or more of lipophilicity or nuclease resistance. Incorporation of one or more RRMSs described herein into an iRNA agent can, particularly when the RRMS is tethered to an appropriate entity, modulate, e.g., increase, binding affinity of an iRNA agent to a target mRNA, change the geometry of the duplex form of the iRNA agent, alter distribution or target the iRNA agent to a particular part of the body, or modify the interaction with nucleic acid binding proteins (e.g., during RISC formation and strand separation).

[0537] Accordingly, in one aspect, the invention features, an iRNA agent preferably comprising a first strand and a second strand, wherein at least one subunit having a formula (R-1) is incorporated into at least one of said strands.

[0538]

[0539] Referring to formula (R-1), X is N(CO)R⁷, NR⁷ or CH₂; Y is NR⁸, O, S, CR⁹R¹⁰, or absent; and Z is CR¹¹R¹² or absent.

[0540] Each of R¹, R², R³, R⁴, R⁹, and R¹⁰ is, independently, H, OR^a, OR^b, (CH₂)_nOR^a, or (CH₂)_nOR^b, provided that at least one of R¹, R², R³, R⁴, R⁹, and R¹⁰ is OR^a or OR^b and that at least one of R¹, R², R³, R⁴, R⁹, and R¹⁰ is (CH₂)_nOR^a, or (CH₂)_nOR^b (when the RRMS is terminal, one of R¹, R², R³, R⁴, R⁹, and R¹⁰ will include R^a and one will include R^b; when the RRMS is internal, two of R¹, R², R³, R⁴, R⁹, and R¹⁰ will each include an R^b); further provided that preferably OR^a may only be present with (CH₂)_nOR^b and (CH₂)_nOR^a may only be present with OR^b.

[0541] Each of R⁵, R⁶, R¹¹, and R¹² is, independently, H, C₁-C₆ alkyl optionally substituted with 1-3 R¹³, or C(O)NHR⁷; or R⁵ and R¹¹ together are C₃-C₈cycloalkyl optionally substituted with R¹⁴.

[0542] R⁷ is C₁-C₂₀ alkyl substituted with NR^cR^d; R⁸ is C₁-C₆ alkyl; R¹³ is hydroxy, C₁-C₄ alkoxy, or halo; and R¹⁴ is NR^cR⁷.

[0543] R^a is:

[0544]
and

[0545] R^b is:

[0546]

[0547] Each of A and C is, independently, O or S.

[0548] B is OH, O⁻, or

[0549]

[0550] R^c is H or C1-C6 alkyl; R^d is H or a ligand; and n is 1-4.

[0551] In a preferred embodiment the ribose is replaced with a pyrroline scaffold, and X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is absent.

[0552] In other preferred embodiments the ribose is replaced with a piperidine scaffold, and X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is CR¹¹R¹².

[0553] In other preferred embodiments the ribose is replaced with a piperazine scaffold, and X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CR¹¹R¹².

[0554] In other preferred embodiments the ribose is replaced with a morpholino scaffold, and X is N(CO)R⁷ or NR⁷, Y is O, and Z is CR¹¹R¹².

[0555] In other preferred embodiments the ribose is replaced with a decalin scaffold, and X is CH₂; Y is CR⁹R¹⁰; and Z is CR¹¹R¹²; and R⁵ and R¹¹ together are C⁶ cycloalkyl.

[0556] In other preferred embodiments the ribose is replaced with a decalin/indane scaffold and, and X is CH₂; Y is CR⁹R¹⁰; and Z is CR¹¹R¹²; and R⁵ and R¹¹ together are C⁵ cycloalkyl.

[0557] In other preferred embodiments, the ribose is replaced with a hydroxyproline scaffold.

[0558] RRMSs described herein may be incorporated into any double-stranded RNA-like molecule described herein, e.g., an iRNA agent. An iRNA agent may include a duplex comprising a hybridized sense and antisense strand, in which the antisense strand and/or the sense strand may include one or more of the RRMSs described herein. An RRMS can be introduced at one or more points in one or both strands of a double-stranded iRNA agent. An RRMS can be placed at or near (within 1, 2, or 3 positions) of the 3′ or 5′ end of the sense strand or at near (within 2 or 3 positions of) the 3′ end of the antisense strand. In some embodiments it is preferred to not have an RRMS at or near (within 1, 2, or 3 positions of) the 5′ end of the antisense strand. An RRMS can be internal, and will preferably be positioned in regions not critical for antisense binding to the target.

[0559] In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of the antisense strand. In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of the antisense strand and at (or within 1, 2, or 3 positions of) the 3′ end of the sense strand. In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of the antisense strand and an RRMS at the 5′ end of the sense strand, in which both ligands are located at the same end of the iRNA agent.

[0560] In certain embodiments, two ligands are tethered, preferably, one on each strand and are hydrophobic moieties. While not wishing to be bound by theory, it is believed that pairing of the hydrophobic ligands can stabilize the iRNA agent via intermolecular van der Waals interactions.

[0561] In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of the antisense strand and an RRMS at the 5′ end of the sense strand, in which both RRMSs may share the same ligand (e.g., cholic acid) via connection of their individual tethers to separate positions on the ligand. A ligand shared between two proximal RRMSs is referred to herein as a “hairpin ligand.”

[0562] In other embodiments, an iRNA agent may have an RRMS at the 3′ end of the sense strand and an RRMS at an internal position of the sense strand. An iRNA agent may have an RRMS at an internal position of the sense strand; or may have an RRMS at an internal position of the antisense strand; or may have an RRMS at an internal position of the sense strand and an RRMS at an internal position of the antisense strand.

[0563] In preferred embodiments the iRNA agent includes a first and second sequences, which are preferably two separate molecules as opposed to two sequences located on the same strand, have sufficient complementarity to each other to hybridize (and thereby form a duplex region), e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme.

[0564] It is preferred that the first and second sequences be chosen such that the ds iRNA agent includes a single strand or unpaired region at one or both ends of the molecule. Thus, a ds iRNA agent contains first and second sequences, preferable paired to contain an overhang, e.g., one or two 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3 nucleotides. Most embodiments will have a 3′ overhang. Preferred sRNA agents will have single-stranded overhangs, preferably 3′ overhangs, of 1 or preferably 2 or 3 nucleotides in length at each end. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. 5′ ends are preferably phosphorylated.

[0565] An RNA agent, e.g., an iRNA agent, containing a preferred, but nonlimiting RRMS is presented as formula (R-2) in FIG. 4. The carrier includes two “backbone attachment points” (hydroxyl groups), a “tethering attachment point,” and a ligand, which is connected indirectly to the carrier via an intervening tether. The RRMS may be the 5′ or 3′ terminal subunit of the RNA molecule, i.e., one of the two “W” groups may be a hydroxyl group, and the other “W” group may be a chain of two or more unmodified or modified ribonucleotides. Alternatively, the RRMS may occupy an internal position, and both “W” groups may be one or more unmodified or modified ribonucleotides. More than one RRMS may be present in a RNA molecule, e.g., an iRNA agent.

[0566] The modified RNA molecule of formula (R-2) can be obtained using oligonucleotide synthetic methods known in the art. In a preferred embodiment, the modified RNA molecule of formula (II) can be prepared by incorporating one or more of the corresponding RRMS monomer compounds (RRMS monomers, see, e.g., A, B, and C in FIG. 4) into a growing sense or antisense strand, utilizing, e.g., phosphoramidite or H-phosphonate coupling strategies.

[0567] The RRMS monomers generally include two differently functionalized hydroxyl groups (OFG¹ and OFG² above), which are linked to the carrier molecule (see A in FIG. 4), and a tethering attachment point. As used herein, the term “functionalized hydroxyl group” means that the hydroxyl proton has been replaced by another substituent. As shown in representative structures B and C, one hydroxyl group (OFG¹) on the carrier is functionalized with a protecting group (PG). The other hydroxyl group (OFG²) can be functionalized with either (1) a liquid or solid phase synthesis support reagent (solid circle) directly or indirectly through a linker, L, as in B, or (2) a phosphorus-containing moiety, e.g., a phosphoramidite as in C. The tethering attachment point may be connected to a hydrogen atom, a tether, or a tethered ligand at the time that the monomer is incorporated into the growing sense or antisense strand (see R in Scheme 1). Thus, the tethered ligand can be, but need not be attached to the monomer at the time that the monomer is incorporated into the growing strand. In certain embodiments, the tether, the ligand or the tethered ligand may be linked to a “precursor” RRMS after a “precursor” RRMS monomer has been incorporated into the strand.

[0568] The (OFG¹) protecting group may be selected as desired, e.g., from T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991). The protecting group is preferably stable under amidite synthesis conditions, storage conditions, and oligonucleotide synthesis conditions. Hydroxyl groups, —OH, are nucleophilic groups (i.e., Lewis bases), which react through the oxygen with electrophiles (i.e., Lewis acids). Hydroxyl groups in which the hydrogen has been replaced with a protecting group, e.g., a triarylmethyl group or a trialkylsilyl group, are essentially unreactive as nucleophiles in displacement reactions. Thus, the protected hydroxyl group is useful in preventing e.g., homocoupling of compounds exemplified by structure C during oligonucleotide synthesis. A preferred protecting group is the dimethoxytrityl group.

[0569] When the OFG² in B includes a linker, e.g., a long organic linker, connected to a soluble or insoluble support reagent, solution or solid phase synthesis techniques can be employed to build up a chain of natural and/or modified ribonucleotides once OFG¹ is deprotected and free to react as a nucleophile with another nucleoside or monomer containing an electrophilic group (e.g., an amidite group). Alternatively, a natural or modified ribonucleotide or oligoribonucleotide chain can be coupled to monomer C via an amidite group or H-phosphonate group at OFG². Subsequent to this operation, OFG¹ can be deblocked, and the restored nucleophilic hydroxyl group can react with another nucleoside or monomer containing an electrophilic group (see FIG. 1). R′ can be substituted or unsubstituted alkyl or alkenyl. In preferred embodiments, R′ is methyl, allyl or 2-cyanoethyl. R″ may a C₁-C₁₀alkyl group, preferably it is a branched group containing three or more carbons, e.g., isopropyl.

[0570] OFG² in B can be hydroxyl functionalized with a linker, which in turn contains a liquid or solid phase synthesis support reagent at the other linker terminus. The support reagent can be any support medium that can support the monomers described herein. The monomer can be attached to an insoluble support via a linker, L, which allows the monomer (and the growing chain) to be solubilized in the solvent in which the support is placed. The solubilized, yet immobilized, monomer can react with reagents in the surrounding solvent; unreacted reagents and soluble by-products can be readily washed away from the solid support to which the monomer or monomer-derived products is attached. Alternatively, the monomer can be attached to a soluble support moiety, e.g., polyethylene glycol (PEG) and liquid phase synthesis techniques can be used to build up the chain. Linker and support medium selection is within skill of the art. Generally the linker may be —C(O)(CH₂)_qC(O)—, or —C(O)(CH₂)_qS—, preferably, it is oxalyl, succinyl or thioglycolyl. Standard control pore glass solid phase synthesis supports can not be used in conjunction with fluoride labile 5′ silyl protecting groups because the glass is degraded by fluoride with a significant reduction in the amount of full-length product. Fluoride-stable polystyrene based supports or PEG are preferred.

[0571] Preferred carriers have the general formula (R-3) provided below. (In that structure preferred backbone attachment points can be chosen from R¹ or R²; R³ or R⁴; or R⁹ and R¹⁰ if Y is CR⁹R¹⁰ (two positions are chosen to give two backbone attachment points, e.g., R¹ and R⁴, or R⁴ and R⁹. Preferred tethering attachment points include R⁷; R⁵ or R⁶ when X is CH₂. The carriers are described below as an entity, which can be incorporated into a strand. Thus, it is understood that the structures also encompass the situations wherein one (in the case of a terminal position) or two (in the case of an internal position) of the attachment points, e.g., R¹ or R²; R³ or R⁴; or R⁹ or R¹⁰ (when Y is CR⁹R₁₀), is connected to the phosphate, or modified phosphate, e.g., sulfur containing, backbone. E.g., one of the above-named R groups can be —CH2-, wherein one bond is connected to the carrier and one to a backbone atom, e.g., a linking oxygen or a central phosphorus atom.)

[0572]

[0573] X is N(CO)R⁷, NR⁷ or CH₂; Y is NR⁸, O, S, CR⁹R¹⁰; and Z is CR¹¹R¹² or absent.

[0574] Each of R¹, R², R³, R⁴, R⁹, and R¹⁰ is, independently, H, OR^a, or (CH₂)_nOR^b, provided that at least two of R¹, R², R³, R⁴, R⁹, and R¹⁰ are OR^a and/or (CH₂)_nOR^b.

[0575] Each of R⁵, R⁶, R¹¹, and R¹² is, independently, a ligand, H, C₁-C₆ alkyl optionally substituted with 1-3 R¹³, or C(O)NHR⁷; or R⁵ and R¹¹ together are C₃-C₈ cycloalkyl optionally substituted with R¹⁴.

[0576] R⁷ is H, a ligand, or C1-C₂₀ alkyl substituted with NR^cR^d; R⁸ is H or C₁-C₆ alkyl; R¹³ is hydroxy, C₁-C₄ alkoxy, or halo; R¹⁴ is NR^cR⁷; R¹⁵ is C₁-C₆ alkyl optionally substituted with cyano, or C₂-C₆ alkenyl; R¹⁶ is C₁-C₁₀ alkyl; and R¹⁷ is a liquid or solid phase support reagent.

[0577] L is —C(O)(CH₂)_qC(O)—, or —C(O)(CH₂)_qS—; R^a is CAr₃; R^b is P(O)(O⁻)H, P(OR¹⁵)N(R¹⁶)₂ or L-R¹⁷; R^c is H or C₁-C₆ alkyl; and R^d is H or a ligand.

[0578] Each Ar is, independently, C₆-C₁₀ aryl optionally substituted with C₁-C₄ alkoxy; n is 1-4; and q is 0-4.

[0579] Exemplary carriers include those in which, e.g., X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is absent; or X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is CR¹¹R¹²; or X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CR¹¹R¹²; or X is N(CO)R⁷ or NR⁷, Y is O, and Z is CR¹¹R¹²; or X is CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹², and R⁵ and R¹¹ together form C₆ cycloalkyl (H, z=2), or the indane ring system, e.g., X is CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹², and R⁵ and R¹¹ together form C₅ cycloalkyl (H, z=1).

[0580] In certain embodiments, the carrier may be based on the pyrroline ring system or the 3-hydroxyproline ring system, e.g., X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is absent (D). OFG¹ is preferably attached to a primary carbon, e.g., an exocyclic alkylene

[0581]
group, e.g., a methylene group, connected to one of the carbons in the five-membered ring (—CH₂OFG¹ in D). OFG² is preferably attached directly to one of the carbons in the five-membered ring (—OFG² in D). For the pyrroline-based carriers, —CH₂OFG¹ may be attached to C-2 and OFG² may be attached to C-3; or —CH₂OFG¹ may be attached to C-3 and OFG² may be attached to C-4. In certain embodiments, CH₂OFG¹ and OFG² may be geminally substituted to one of the above-referenced carbons. For the 3-hydroxyproline-based carriers, —CH₂OFG¹ may be attached to C-2 and OFG² may be attached to C-4. The pyrroline- and 3-hydroxyproline-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, CH₂OFG¹ and OFG² may be cis or trans with respect to one another in any of the pairings delineated above Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. The tethering attachment point is preferably nitrogen.

[0582] In certain embodiments, the carrier may be based on the piperidine ring system (E), e.g., X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is CR¹¹R¹². OFG¹ is preferably

[0583]
attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group (n=1) or ethylene group (n=2), connected to one of the carbons in the six-membered ring [—(CH₂)_nOFG¹ in E]. OFG² is preferably attached directly to one of the carbons in the six-membered ring (—OFG² in E). —(CH₂)_nOFG¹ and OFG² may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, or C-4. Alternatively, —(CH₂)_nOFG¹ and OFG² may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., —(CH₂)_nOFG¹ may be attached to C-2 and OFG² may be attached to C-3; —(CH₂)_nOFG¹ may be attached to C-3 and OFG² may be attached to C-2; —(CH₂)_nOFG¹ may be attached to C-3 and OFG² may be attached to C-4; or —(CH₂)_nOFG¹ may be attached to C-4 and OFG² may be attached to C-3. The piperidine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, —(CH₂)_nOFG¹ and OFG² may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. The tethering attachment point is preferably nitrogen.

[0584] In certain embodiments, the carrier may be based on the piperazine ring system (F), e.g., X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CR¹¹R¹², or the morpholine ring system (G), e.g., X is N(CO)R⁷ or NR⁷, Y is O, and Z is CR¹¹R¹². OFG¹ is preferably

[0585]
attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, connected to one of the carbons in the six-membered ring (—CH₂OFG¹ in F or G). OFG² is preferably attached directly to one of the carbons in the six-membered rings (—OFG² in F or G). For both F and G, —CH₂OFG¹ may be attached to C-2 and OFG² may be attached to C-3; or vice versa. In certain embodiments, CH₂OFG¹ and OFG² may be geminally substituted to one of the above-referenced carbons. The piperazine- and morpholine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, CH₂OFG¹ and OFG² may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. R′″ can be, e.g., C₁-C₆ alkyl, preferably CH₃. The tethering attachment point is preferably nitrogen in both F and G.

[0586] In certain embodiments, the carrier may be based on the decalin ring system, e.g., X is CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹², and R⁵ and R¹¹ together form C₆ cycloalkyl (H, z=2), or the indane ring system, e.g., X is CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹², and R⁵ and R¹¹ together form C₅ cycloalkyl (H, z=1). OFG¹ is preferably attached to a primary carbon,

[0587]
e.g., an exocyclic methylene group (n=1) or ethylene group (n=2) connected to one of C-2, C-3, C-4, or C-5 [—(CH₂)_nOFG¹ in H]. OFG² is preferably attached directly to one of C-2, C-3, C-4, or C-5 (—OFG² in H). —(CH₂)_nOFG¹ and OFG² may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, C-4, or C-5. Alternatively, —(CH₂)_nOFG¹ and OFG² may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., —(CH₂)_nOFG¹ may be attached to C-2 and OFG² may be attached to C-3; —(CH₂)_nOFG¹ may be attached to C-3 and OFG² may be attached to C-2; —(CH₂)_nOFG¹ may be attached to C-3 and OFG² may be attached to C-4; or —(CH₂)_nOFG¹ may be attached to C-4 and OFG² may be attached to C-3; —(CH₂)_nOFG¹ may be attached to C-4 and OFG² may be attached to C-5; or —(CH₂)_nOFG¹ may be attached to C-5 and OFG² may be attached to C-4. The decalin or indane-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, —(CH₂)_nOFG¹ and OFG² may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. In a preferred embodiment, the substituents at C-1 and C-6 are trans with respect to one another. The tethering attachment point is preferably C-6 or C-7.

[0588] Other carriers may include those based on 3-hydroxyproline (J). Thus, —(CH₂)_nOFG¹ and OFG² may be cis or trans with respect to one another. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers

[0589]
and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. The tethering attachment point is preferably nitrogen.

[0590] Representative carriers are shown in FIG. 5.

[0591] In certain embodiments, a moiety, e.g., a ligand may be connected indirectly to the carrier via the intermediacy of an intervening tether. Tethers are connected to the carrier at the tethering attachment point (TAP) and may include any C₁-C₁₀₀ carbon-containing moiety, (e.g. C₁-C₇₅, C₁-C₅₀, C₁-C₂₀, C₁-C₁₀, C₁-C₆), preferably having at least one nitrogen atom. In preferred embodiments, the nitrogen atom forms part of a terminal amino group on the tether, which may serve as a connection point for the ligand. Preferred tethers (underlined) include TAP-(CH₂)_nNH₂; TAP-C(O)(CH₂)_nNH₂; or TAP-NR″″(CH₂)_nNH₂, in which n is 1-6 and R″″ is C₁-C₆ alkyl. and R^d is hydrogen or a ligand. In other embodiments, the nitrogen may form part of a terminal oxyamino group, e.g., —ONH₂, or hydrazino group, —NHNH₂. The tether may optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and/or optionally inserted with one or more additional heteroatoms, e.g., N, O, or S. Preferred tethered ligands may include, e.g., TAP-(CH₂)_nNH(LIGAND), TAP-C(O)(CH₂)_nNH(LIGAND), or TAP-NR″″(CH₂)_nNH(LIGAND); TAP-(CH₂)_nONH(LIGAND), TAP-C(O)(CH₂)_nONH(LIGAND), or TAP-NR″″(CH₂)_nONH(LIGAND); TAP-(CH₂)_nNHNH₂(LIGAND), TAP-C(O)(CH₂)_nNHNH₂(LIGAND), or TAP-NR″″(CH₂)_nNHNH₂(LIGAND).

[0592] In other embodiments the tether may include an electrophilic moiety, preferably at the terminal position of the tether. Preferred electrophilic moieties include, e.g., an aldehyde, alkyl halide, mesylate, tosylate, nosylate, or brosylate, or an activated carboxylic acid ester, e.g. an NHS ester, or a pentafluorophenyl ester. Preferred tethers (underlined) include TAP—(CH₂)_nCHO; TAP-C(O)(CH₂)_nCHO; or TAP-NR″″(CH₂)_nCHO, in which n is 1-6 and R″″ is C₁-C₆ alkyl; or TAP-(CH₂)_nC(O)ONHS; TAP-C(O)(CH₂)_nC(O)ONHS; or TAP-NR″″(CH₂)_nC(O)ONHS, in which n is 1-6 and R″″ is C₁-C₆ alkyl; TAP-(CH₂C(O)OC₆F₅; TAP-C(O)(CH₂)_nC(O) OC₆F₅; or TAP-NR″″(CH₂)_nC(O)OC₆F₅, in which n is 1-6 and R″″ is C₁-C₆ alkyl; or —(CH₂)_nCH₂LG; TAP-C(O)(CH₂)_nCH₂LG; or TAP-NR″″(CH₂)_nCH₂LG, in which n is 1-6 and R″ ″ is C₁-C₆ alkyl (LG can be a leaving group, e.g., halide, mesylate, tosylate, nosylate, brosylate). Tethering can be carried out by coupling a nucleophilic group of a ligand, e.g., a thiol or amino group with an electrophilic group on the tether.

[0593] Tethered Entities

[0594] A wide variety of entities can be tethered to an iRNA agent, e.g., to the carrier of an RRMS. Examples are described below in the context of an RRMS but that is only preferred, entities can be coupled at other points to an iRNA agent.

[0595] Preferred moieties are ligands, which are coupled, preferably covalently, either directly or indirectly via an intervening tether, to the RRMS carrier. In preferred embodiments, the ligand is attached to the carrier via an intervening tether. As discussed above, the ligand or tethered ligand may be present on the RRMS monomer when the RRMS monomer is incorporated into the growing strand. In some embodiments, the ligand may be incorporated into a “precursor” RRMS after a “precursor” RRMS monomer has been incorporated into the growing strand. For example, an RRMS monomer having, e.g., an amino-terminated tether (i.e., having no associated ligand), e.g., TAP-(CH₂)_nNH₂ may be incorporated into a growing sense or antisense strand. In a subsequent operation, i.e., after incorporation of the precursor monomer into the strand, a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can subsequently be attached to the precursor RRMS by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor RRMS tether.

[0596] In preferred embodiments, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid.

[0597] Preferred ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified oligoribonucleotide, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides.

[0598] Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; and nuclease-resistance conferring moieties. General examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.

[0599] Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

[0600] Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, bone cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic.

[0601] Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, bomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0602] Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

[0603] The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0604] The ligand can increase the uptake of the iRNA agent into the cell by activating an inflammatory response, for example. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, or gamma interferon.

[0605] In one aspect, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. Preferably, the target tissue is the liver, preferably parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a seru protein, e.g., HSA.

[0606] A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

[0607] In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

[0608] In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.

[0609] In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL).

[0610] In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

[0611] The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long (see Table 1, for example).

[0612]

TABLE 1
Exemplary Cell Permeation Peptides
Cell
Permeation
Peptide	Amino acid Sequence	Reference
Penetratin	RQIKIWFQNRRMKWKK	Derossi et al., J. Biol.
	(SEQ ID NO: 6737)	Chem. 269:10444,
		1994
Tat fragment	GRKKRRQRRRPPQC	Vives et al., J. Biol.
(48-60)	(SEQ ID NO: 6738)	Chem., 272:16010,
		1997
Signal	GALFLGWLGAAGSTMGAWSQPKKKRKV	Chaloin et al.,
Sequence-	(SEQ ID NO: 6738)	Biochem. Biophys.
based peptide		Res. Commun.,
		243:601, 1998
PVEC	LLIILRRRIRKQAHAHSK	Elmquist et al., Exp.
	(SEQ ID NO: 6739)	Cell Res., 269:237,
		2001
Transportan	GWTLNSAGYLLKINLKALAALAKKIL	Pooga et al., FASEB
	(SEQ ID NO: 6740)	J., 12:67, 1998
Amphiphilic	KLALKLALKALKAALKLA	Oehlke et al., Mol.
model peptide	(SEQ ID NO: 6741)	Ther., 2:339, 2000
Arg9	RRRRRRRRR (SEQ ID NO: 6742)	Mitchell et al., J.
		Pept. Res., 56:318,
		2000
Bacterial cell	KFFKFFKFFK (SEQ ID NO: 6743)
wall
permeating
LL-37	LLGDFFRKSKEKIGKEFKRIVQRIKDFLRN
	LVPRTES (SEQ ID NO: 6744)
Cecropin P1	SWLSKTAKKLENSAKKRISEGIAIAIQGGPR
	(SEQ ID NO: 6745)
α-defensin	ACYCRIPACIAGERRYGTCIYQGRLWAFCC
	(SEQ ID NO: 6746)
b-defensin	DHYNCVSSGGQCLYSACPIFTKIQGTCYR
	GKAKCCK (SEQ ID NO: 6747)
Bactenecin	RKCRIVVIRVCR (SEQ ID NO: 6748)
PR-39	RRRPRPPYLPRPRPPPFFPPRLPPRIPPGF
	PPRFPPRFPGKR-NH2
	(SEQ ID NO: 6749)
Indolicidin	ILPWKWPWWPWRR-NH2
	(SEQ ID NO: 6750)

[0613] A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 6751). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 6752)) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 6753)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 6754)) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Preferably the peptide or peptidomimetic tethered to an iRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

[0614] An RGD peptide moiety can be used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an iRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver an iRNA agent to a tumor cell expressing α_vβ₃ (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

[0615] Peptides that target markers enriched in proliferating cells can be used. E.g., RGD containing peptides and peptidomimetics can target cancer cells, in particular cells that exhibit an I_vθ₃ integrin. Thus, one could use RGD peptides, cyclic peptides containing RGD, RGD peptides that include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the I_v-θ₃ integrin ligand. Generally, such ligands can be used to control proliferating cells and angiogeneis. Preferred conjugates of this type include an iRNA agent that targets PECAM-1, VEGF, or other cancer gene, e.g., a cancer gene described herein.

[0616] A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, 3-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0617] In one embodiment, a targeting peptide tethered to an RRMS can be an amphipathic α-helical peptide. Exemplary amphipathic α-helical peptides include, but are not limited to, cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H₂A peptides, Xenopus peptides, esculentinis-1, and caerins. A number of factors will preferably be considered to maintain the integrity of helix stability. For example, a maximum number of helix stabilization residues will be utilized (e.g., leu, ala, or lys), and a minimum number helix destabilization residues will be utilized (e.g., proline, or cyclic monomeric units. The capping residue will be considered (for example Gly is an exemplary N-capping residue and/or C-terminal amidation can be used to provide an extra H-bond to stabilize the helix. Formation of salt bridges between residues with opposite charges, separated by i±3, or i±4 positions can provide stability. For example, cationic residues such as lysine, arginine, homo-arginine, omithine or histidine can form salt bridges with the anionic residues glutamate or aspartate.

[0618] Peptide and petidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; α, β, or γ peptides; N-methyl peptides; azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides.

[0619] Methods for Making iRNA Agents

[0620] iRNA agents can include modified or non-naturally occurring bases, e.g., bases described in copending and coowned U.S. Provisional Application Ser. No. 60/463,772, filed on Apr. 17, 2003, which is hereby incorporated by reference and/or in copending and coowned U.S. Provisional Application Ser. No. 60/465,802, filed on Apr. 25, 2003, which is hereby incorporated by reference. Monomers and iRNA agents which include such bases can be made by the methods found in U.S. Provisional Application Ser. No. 60/463,772, filed on Apr. 17, 2003, and/or in U.S. Provisional Application Ser. No. 60/465,802, filed on Apr. 25, 2003.

[0621] In addition, the invention includes iRNA agents having a modified or non-naturally occurring base and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a modified or non-naturally occurring base.

[0622] The synthesis and purification of oligonucleotide peptide conjugates can be performed by established methods. See, for example, Trufert et al., Tetrahedron, 52:3005, 1996; and Manoharan, “Oligonucleotide Conjugates in Antisense Technology,” in Antisense Drug Technology, ed. S. T. Crooke, Marcel Dekker, Inc., 2001.

[0623] In one embodiment of the invention, a peptidomimetic can be modified to create a constrained peptide that adopts a distinct and specific preferred conformation, which can increase the potency and selectivity of the peptide. For example, the constrained peptide can be an azapeptide (Gante, Synthesis, 405-413, 1989). An azapeptide is synthesized by replacing the α-carbon of an amino acid with a nitrogen atom without changing the structure of the amino acid side chain. For example, the azapeptide can be synthesized by using hydrazine in traditional peptide synthesis coupling methods, such as by reacting hydrazine with a “carbonyl donor,” e.g., phenylchloroformate.

[0624] In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be an N-methyl peptide. N-methyl peptides are composed of N-methyl amino acids, which provide an additional methyl group in the peptide backbone, thereby potentially providing additional means of resistance to proteolytic cleavage. N-methyl peptides can by synthesized by methods known in the art (see, for example, Lindgren et al., Trends Pharmacol. Sci. 21:99, 2000; Cell Penetrating Peptides: Processes and Applications, Langel, ed., CRC Press, Boca Raton, Fla., 2002; Fische et al., Bioconjugate. Chem. 12: 825, 2001; Wander et al., J. Am. Chem. Soc., 124:13382, 2002). For example, an Ant or Tat peptide can be an N-methyl peptide.

[0625] In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be a β-peptide. β-peptides form stable secondary structures such as helices, pleated sheets, turns and hairpins in solutions. Their cyclic derivatives can fold into nanotubes in the solid state. β-peptides are resistant to degradation by proteolytic enzymes. β-peptides can be synthesized by methods known in the art. For example, an Ant or Tat peptide can be a β-peptide.

[0626] In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be a oligocarbamate. Oligocarbamate peptides are internalized into a cell by a transport pathway facilitated by carbamate transporters. For example, an Ant or Tat peptide can be an oligocarbamate.

[0627] In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be an oligourea conjugate (or an oligothiourea conjugate), in which the amide bond of a peptidomimetic is replaced with a urea moiety. Replacement of the amide bond provides increased resistance to degradation by proteolytic enzymes, e.g., proteolytic enzymes in the gastrointestinal tract. In one embodiment, an oligourea conjugate is tethered to an iRNA agent for use in oral delivery. The backbone in each repeating unit of an oligourea peptidomimetic can be extended by one carbon atom in comparison with the natural amino acid. The single carbon atom extension can increase peptide stability and lipophilicity, for example. An oligourea peptide can therefore be advantageous when an iRNA agent is directed for passage through a bacterial cell wall, or when an iRNA agent must traverse the blood-brain barrier, such as for the treatment of a neurological disorder. In one embodiment, a hydrogen bonding unit is conjugated to the oligourea peptide, such as to create an increased affinity with a receptor. For example, an Ant or Tat peptide can be an oligourea conjugate (or an oligothiourea conjugate).

[0628] The siRNA peptide conjugates of the invention can be affiliated with, e.g., tethered to, RRMSs occurring at various positions on an iRNA agent. For example, a peptide can be terminally conjugated, on either the sense or the antisense strand, or a peptide can be bisconjugated (one peptide tethered to each end, one conjugated to the sense strand, and one conjugated to the antisense strand). In another option, the peptide can be internally conjugated, such as in the loop of a short hairpin iRNA agent. In yet another option, the peptide can be affiliated with a complex, such as a peptide-carrier complex.

[0629] A peptide-carrier complex consists of at least a carrier molecule, which can encapsulate one or more iRNA agents (such as for delivery to a biological system and/or a cell), and a peptide moiety tethered to the outside of the carrier molecule, such as for targeting the carrier complex to a particular tissue or cell type. A carrier complex can carry additional targeting molecules on the exterior of the complex, or fusogenic agents to aid in cell delivery. The one or more iRNA agents encapsulated within the carrier can be conjugated to lipophilic molecules, which can aid in the delivery of the agents to the interior of the carrier.

[0630] A carrier molecule or structure can be, for example, a micelle, a liposome (e.g., a cationic liposome), a nanoparticle, a microsphere, or a biodegradable polymer. A peptide moiety can be tethered to the carrier molecule by a variety of linkages, such as a disulfide linkage, an acid labile linkage, a peptide-based linkage, an oxyamino linkage or a hydrazine linkage. For example, a peptide-based linkage can be a GFLG peptide. Certain linkages will have particular advantages, and the advantages (or disadvantages) can be considered depending on the tissue target or intended use. For example, peptide based linkages are stable in the blood stream but are susceptible to enzymatic cleavage in the lysosomes.

[0631] Targeting

[0632] The iRNA agents of the invention are particularly useful when targeted to the liver. An iRNA agent can be targeted to the liver by incorporation of an RRMS containing a ligand that targets the liver. For example, a liver-targeting agent can be a lipophilic moiety. Preferred lipophilic moieties include lipid, cholesterols, oleyl, retinyl, or cholesteryl residues. Other lipophilic moieties that can function as liver-targeting agents include cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

[0633] An iRNA agent can also be targeted to the liver by association with a low-density lipoprotein (LDL), such as lactosylated LDL. Polymeric carriers complexed with sugar residues can also function to target iRNA agents to the liver.

[0634] A targeting agent that incorporates a sugar, e.g., galactose and/or analogues thereof, is particularly useful. These agents target, in particular, the parenchymal cells of the liver. For example, a targeting moiety can include more than one or preferably two or three galactose moieties, spaced about 15 angstroms from each other. The targeting moiety can alternatively be lactose (e.g., three lactose moieties), which is glucose coupled to a galactose. The targeting moiety can also be N-Acetyl-Galactosamine, N-Ac-Glucosamine. A mannose or mannose-6-phosphate targeting moiety can be used for macrophage targeting.

[0635] Conjugation of an iRNA agent with a serum albumin (SA), such as human serum albumin, can also be used to target the iRNA agent to the liver.

[0636] An iRNA agent targeted to the liver by an RRMS targeting moiety described herein can target a gene expressed in the liver. For example, the iRNA agent can target p21(WAF1/DIP1), P27(KIP1), the α-fetoprotein gene, beta-catenin, or c-MET, such as for treating a cancer of the liver. In another embodiment, the iRNA agent can target apoB-100, such as for the treatment of an HDL/LDL cholesterol imbalance; dyslipidemias, e.g., familial combined hyperlipidemia (FCHL), or acquired hyperlipidemia; hypercholesterolemia; statin-resistant hypercholesterolemia; coronary artery disease (CAD); coronary heart disease (CHD); or atherosclerosis. In another embodiment, the iRNA agent can target forkhead homologue in rhabdomyosarcoma (FKHR); glucagon; glucagon receptor; glycogen phosphorylase; PPAR-Gamma Coactivator (PGC-1); Fructose-1,6-bisphosphatase; glucose-6-phosphatase; glucose-6-phosphate translocator; glucokinase inhibitory regulatory protein; or phosphoenolpyruvate carboxykinase (PEPCK), such as to inhibit hepatic glucose production in a mammal, such as a human, such as for the treatment of diabetes. In another embodiment, an iRNA agent targeted to the liver can target Factor V, e.g., the Leiden Factor V allele, such as to reduce the tendency to form a blood clot. An iRNA agent targeted to the liver can include a sequence which targets hepatitis virus (e.g., Hepatitis A, B, C, D, E, F, G, or H). For example, an iRNA agent of the invention can target any one of the nonstructural proteins of HCV: NS3, 4A, 4B, 5A, or 5B. For the treatment of hepatitis B, an iRNA agent can target the protein X (HBx) gene, for example.

[0637] Preferred ligands on RRMSs include folic acid, glucose, cholesterol, cholic acid, Vitamin E, Vitamin K, or Vitamin A.

[0638] The term “halo” refers to any radical of fluorine, chlorine, bromine or iodine.

[0639] The term “alkyl” refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C₁-C₁₂ alkyl indicates that the group may have from 1 to 12 (inclusive) carbon atoms in it. The term “haloalkyl” refers to an alkyl in which one or more hydrogen atoms are replaced by halo, and includes alkyl moieties in which all hydrogens have been replaced by halo (e.g., perfluoroalkyl). Alkyl and haloalkyl groups may be optionally inserted with O, N, or S. The terms “aralkyl” refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of “aralkyl” include benzyl, 9-fluorenyl, benzhydryl, and trityl groups.

[0640] The term “alkenyl” refers to a straight or branched hydrocarbon chain containing 2-8 carbon atoms and characterized in having one or more double bonds. Examples of a typical alkenyl include, but not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups. The term “alkynyl” refers to a straight or branched hydrocarbon chain containing 2-8 carbon atoms and characterized in having one or more triple bonds. Some examples of a typical alkynyl are ethynyl, 2-propynyl, and 3-methylbutynyl, and propargyl. The sp² and sp³ carbons may optionally serve as the point of attachment of the alkenyl and alkynyl groups, respectively.

[0641] The term “alkoxy” refers to an —O-alkyl radical. The term “aminoalkyl” refers to an alkyl substituted with an amino The term “mercapto” refers to an —SH radical. The term “thioalkoxy” refers to an —S-alkyl radical.

[0642] The term “alkylene” refers to a divalent alkyl (i.e., —R—), e.g., —CH₂—, —CH₂CH₂—, and —CH₂CH₂CH₂—. The term “alkylenedioxo” refers to a divalent species of the structure —O—R—O—, in which R represents an alkylene.

[0643] The term “aryl” refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system, wherein any ring atom capable of substitution can be substituted by a substituent. Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, and anthracenyl.

[0644] The term “cycloalkyl” as employed herein includes saturated cyclic, bicyclic, tricyclic, or polycyclic hydrocarbon groups having 3 to 12 carbons, wherein any ring atom capable of substitution can be substituted by a substituent. The cycloalkyl groups herein described may also contain fused rings. Fused rings are rings that share a common carbon-carbon bond. Examples of cycloalkyl moieties include, but are not limited to, cyclohexyl, adamantyl, and norbornyl.

[0645] The term “heterocyclyl” refers to a nonaromatic 3-10 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein any ring atom capable of substitution can be substituted by a substituent. The heterocyclyl groups herein described may also contain fused rings. Fused rings are rings that share a common carbon-carbon bond. Examples of heterocyclyl include, but are not limited to tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino, pyrrolinyl and pyrrolidinyl.

[0646] The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein any ring atom capable of substitution can be substituted by a substituent.

[0647] The term “oxo” refers to an oxygen atom, which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.

[0648] The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted by substituents.

[0649] The term “substituents” refers to a group “substituted” on an alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group at any atom of that group. Suitable substituents include, without limitation, alkyl, alkenyl, alkynyl, alkoxy, halo, hydroxy, cyano, nitro, amino, SO₃H, sulfate, phosphate, perfluoroalkyl, perfluoroalkoxy, methylenedioxy, ethylenedioxy, carboxyl, oxo, thioxo, imino (alkyl, aryl, aralkyl), S(O)_nalkyl (where n is 0-2), S(O)_n aryl (where n is 0-2), S(O)_n heteroaryl (where n is 0-2), S(O)_n heterocyclyl (where n is 0-2), amine (mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, and combinations thereof), ester (alkyl, aralkyl, heteroaralkyl), amide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof), sulfonamide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof), unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heterocyclyl, and unsubstituted cycloalkyl. In one aspect, the substituents on a group are independently any one single, or any subset of the aforementioned substituents.

[0650] The terms “adeninyl, cytosinyl, guaninyl, thyminyl, and uracilyl” and the like refer to radicals of adenine, cytosine, guanine, thymine, and uracil.

[0651] As used herein, an “unusual” nucleobase can include any one of the following:

[0652] 2-methyladeninyl,

[0653] N6-methyladeninyl,

[0654] 2-methylthio-N6-methyladeninyl,

[0655] N6-isopentenyladeninyl,

[0656] 2-methylthio-N6-isopentenyladeninyl,

[0657] N6-(cis-hydroxyisopentenyl)adeninyl,

[0658] 2-methylthio-N6-(cis-hydroxyisopentenyl) adeninyl,

[0659] N6-glycinylcarbamoyladeninyl,

[0660] N6-threonylcarbamoyladeninyl,

[0661] 2-methylthio-N6-threonyl carbamoyladeninyl,

[0662] N6-methyl-N6-threonylcarbamoyladeninyl,

[0663] N6-hydroxynorvalylcarbamoyladeninyl,

[0664] 2-methylthio-N6-hydroxynorvalyl carbamoyladeninyl,

[0665] N6,N6-dimethyladeninyl,

[0666] 3-methylcytosinyl,

[0667] 5-methylcytosinyl,

[0668] 2-thiocytosinyl,

[0669] 5-formylcytosinyl,

[0670]

[0671] N4-methylcytosinyl,

[0672] 5-hydroxymethylcytosinyl,

[0673] 1-methylguaninyl,

[0674] N2-methylguaninyl,

[0675] 7-methylguaninyl,

[0676] N2,N2-dimethylguaninyl,

[0677]

[0678] N2,N2,7-trimethylguaninyl,

[0679] 1-methylguaninyl,

[0680] 7-cyano-7-deazaguaninyl,

[0681] 7-aminomethyl-7-deazaguaninyl,

[0682] pseudouracilyl,

[0683] dihydrouracilyl,

[0684] 5-methyluracilyl,

[0685] 1-methylpseudouracilyl,

[0686] 2-thiouracilyl,

[0687] 4-thiouracilyl,

[0688] 2-thiothyminyl

[0689] 5-methyl-2-thiouracilyl,

[0690] 3-(3-amino-3-carboxypropyl)uracilyl,

[0691] 5-hydroxyuracilyl,

[0692] 5-methoxyuracilyl,

[0693] uracilyl 5-oxyacetic acid,

[0694] uracilyl 5-oxyacetic acid methyl ester,

[0695] 5-(carboxyhydroxymethyl)uracilyl,

[0696] 5-(carboxyhydroxymethyl)uracilyl methyl ester,

[0697] 5-methoxycarbonylmethyluracilyl,

[0698] 5-methoxycarbonylmethyl-2-thiouracilyl,

[0699] 5-aminomethyl-2-thiouracilyl,

[0700] 5-methylaminomethyluracilyl,

[0701] 5-methylaminomethyl-2-thiouracilyl,

[0702] 5-methylaminomethyl-2-selenouracilyl,

[0703] 5-carbamoylmethyluracilyl,

[0704] 5-carboxymethylaminomethyluracilyl,

[0705] 5-carboxymethylaminomethyl-2-thiouracilyl,

[0706] 3-methyluracilyl,

[0707] 1-methyl-3-(3-amino-3-carboxypropyl) pseudouracilyl,

[0708] 5-carboxymethyluracilyl,

[0709] 5-methyldihydrouracilyl, or

[0710] 3-methylpseudouracilyl.

[0711] Asymmetrical Modifications

[0712] In one aspect, the invention features an iRNA agent which can be asymmetrically modified as described herein.

[0713] In addition, the invention includes iRNA agents having asymmetrical modifications and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates an asymmetrical modification.

[0714] iRNA agents of the invention can be asymmetrically modified. An asymmetrically modified iRNA agent is one in which a strand has a modification which is not present on the other strand. An asymmetrical modification is a modification found on one strand but not on the other strand. Any modification, e.g., any modification described herein, can be present as an asymmetrical modification. An asymmetrical modification can confer any of the desired properties associated with a modification, e.g., those properties discussed herein. E.g., an asymmetrical modification can: confer resistance to degradation, an alteration in half life; target the iRNA agent to a particular target, e.g., to a particular tissue; modulate, e.g., increase or decrease, the affinity of a strand for its complement or target sequence; or hinder or promote modification of a terminal moiety, e.g., modification by a kinase or other enzymes involved in the RISC mechanism pathway. The designation of a modification as having one property does not mean that it has no other property, e.g., a modification referred to as one which promotes stabilization might also enhance targeting.

[0715] While not wishing to be bound by theory or any particular mechanistic model, it is believed that asymmetrical modification allows an iRNA agent to be optimized in view of the different or “asymmetrical” functions of the sense and antisense strands. For example, both strands can be modified to increase nuclease resistance, however, since some changes can inhibit RISC activity, these changes can be chosen for the sense stand. In addition, since some modifications, e.g., targeting moieties, can add large bulky groups that, e.g., can interfere with the cleavage activity of the RISC complex, such modifications are preferably placed on the sense strand. Thus, targeting moieties, especially bulky ones (e.g. cholesterol), are preferentially added to the sense strand. In one embodiment, an asymmetrical modification in which a phosphate of the backbone is substituted with S, e.g., a phosphorothioate modification, is present in the antisense strand, and a 2′ modification, e.g., 2′ OMe is present in the sense strand. A targeting moiety can be present at either (or both) the 5′ or 3′ end of the sense strand of the iRNA agent. In a preferred example, a P of the backbone is replaced with S in the antisense strand, 2′OMe is present in the sense strand, and a targeting moiety is added to either the 5′ or 3′ end of the sense strand of the iRNA agent.

[0716] In a preferred embodiment an asymmetrically modified iRNA agent has a modification on the sense strand which modification is not found on the antisense strand and the antisense strand has a modification which is not found on the sense strand.

[0717] Each strand can include one or more asymmetrical modifications. By way of example: one strand can include a first asymmetrical modification which confers a first property on the iRNA agent and the other strand can have a second asymmetrical modification which confers a second property on the iRNA. E.g., one strand, e.g., the sense strand can have a modification which targets the iRNA agent to a tissue, and the other strand, e.g., the antisense strand, has a modification which promotes hybridization with the target gene sequence.

[0718] In some embodiments both strands can be modified to optimize the same property, e.g., to increase resistance to nucleolytic degradation, but different modifications are chosen for the sense and the antisense strands, e.g., because the modifications affect other properties as well. E.g., since some changes can affect RISC activity these modifications are chosen for the sense strand.

[0719] In an embodiment one strand has an asymmetrical 2′ modification, e.g., a 2′ OMe modification, and the other strand has an asymmetrical modification of the phosphate backbone, e.g., a phosphorothioate modification. So, in one embodiment the antisense strand has an asymmetrical 2′ OMe modification and the sense strand has an asymmetrical phosphorothioate modification (or vice versa). In a particularly preferred embodiment the RNAi agent will have asymmetrical 2′-O alkyl, preferably, 2′-OMe modifications on the sense strand and asymmetrical backbone P modification, preferably a phosphothioate modification in the antisense strand. There can be one or multiple 2′-OMe modifications, e.g., at least 2, 3, 4, 5, or 6, of the subunits of the sense strand can be so modified. There can be one or multiple phosphorothioate modifications, e.g., at least 2, 3, 4, 5, or 6, of the subunits of the antisense strand can be so modified. It is preferable to have an iRNA agent wherein there are multiple 2′-OMe modifications on the sense strand and multiple phophorothioate modifications on the antisense strand. All of the subunits on one or both strands can be so modified. A particularly preferred embodiment of multiple asymmetric modification on both strands has a duplex region about 20-21, and preferably 19, subunits in length and one or two 3′ overhangs of about 2 subunits in length.

[0720] Asymmetrical modifications are useful for promoting resistance to degradation by nucleases, e.g., endonucleases. iRNA agents can include one or more asymmetrical modifications which promote resistance to degradation. In preferred embodiments the modification on the antisense strand is one which will not interfere with silencing of the target, e.g., one which will not interfere with cleavage of the target. Most if not all sites on a strand are vulnerable, to some degree, to degradation by endonucleases. One can determine sites which are relatively vulnerable and insert asymmetrical modifications which inhibit degradation. It is often desirable to provide asymmetrical modification of a UA site in an iRNA agent, and in some cases it is desirable to provide the UA sequence on both strands with asymmetrical modification. Examples of modifications which inhibit endonucleolytic degradation can be found herein. Particularly favored modifications include: 2′ modification, e.g., provision of a 2′ OMe moiety on the U, especially on a sense strand; modification of the backbone, e.g., with the replacement of an O with an S, in the phosphate backbone, e.g., the provision of a phosphorothioate modification, on the U or the A or both, especially on an antisense strand; replacement of the U with a C5 amino linker; replacement of the A with a G (sequence changes are preferred to be located on the sense strand and not the antisense strand); and modification of the at the 2′, 6′, 7′, or 8′ position. Preferred embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications.

[0721] Asymmetrical modification can be used to inhibit degradation by exonucleases. Asymmetrical modifications can include those in which only one strand is modified as well as those in which both are modified. In preferred embodiments the modification on the antisense strand is one which will not interfere with silencing of the target, e.g., one which will not interfere with cleavage of the target. Some embodiments will have an asymmetrical modification on the sense strand, e.g., in a 3′ overhang, e.g., at the 3′ terminus, and on the antisense strand, e.g., in a 3′ overhang, e.g., at the 3′ terminus. If the modifications introduce moieties of different size it is preferable that the larger be on the sense strand. If the modifications introduce moieties of different charge it is preferable that the one with greater charge be on the sense strand.

[0722] Examples of modifications which inhibit exonucleolytic degradation can be found herein. Particularly favored modifications include: 2′ modification, e.g., provision of a 2′ OMe moiety in a 3′ overhang, e.g., at the 3′ terminus (3′ terminus means at the 3′ atom of the molecule or at the most 3′ moiety, e.g., the most 3′ P or 2′ position, as indicated by the context); modification of the backbone, e.g., with the replacement of a P with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P in a 3′ overhang, e.g., at the 3′ terminus; combination of a 2′ modification, e.g., provision of a 2′ O Me moiety and modification of the backbone, e.g., with the replacement of a P with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P, in a 3′ overhang, e.g., at the 3′ terminus; modification with a 3′ alkyl; modification with an abasic pyrolidine in a 3′ overhang, e.g., at the 3′ terminus; modification with naproxene, ibuprofen, or other moieties which inhibit degradation at the 3′ terminus. Preferred embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications.

[0723] Modifications, e.g., those described herein, which affect targeting can be provided as asymmetrical modifications. Targeting modifications which can inhibit silencing, e.g., by inhibiting cleavage of a target, can be provided as asymmetrical modifications of the sense strand. A biodistribution altering moiety, e.g., cholesterol, can be provided in one or more, e.g., two, asymmetrical modifications of the sense strand. Targeting modifications which introduce moieties having a relatively large molecular weight, e.g., a molecular weight of more than 400, 500, or 1000 daltons, or which introduce a charged moiety (e.g., having more than one positive charge or one negative charge) can be placed on the sense strand.

[0724] Modifications, e.g., those described herein, which modulate, e.g., increase or decrease, the affinity of a strand for its compliment or target, can be provided as asymmetrical modifications. These include: 5 methyl U; 5 methyl C; pseudouridine, Locked nucleic acids, 2 thio U and 2-amino-A. In some embodiments one or more of these is provided on the antisense strand.

[0725] iRNA agents have a defined structure, with a sense strand and an antisense strand, and in many cases short single strand overhangs, e.g., of 2 or 3 nucleotides are present at one or both 3′ ends. Asymmetrical modification can be used to optimize the activity of such a structure, e.g., by being placed selectively within the iRNA. E.g., the end region of the iRNA agent defined by the 5′ end of the sense strand and the 3′ end of the antisense strand is important for function. This region can include the terminal 2, 3, or 4 paired nucleotides and any 3′ overhang. In preferred embodiments asymmetrical modifications which result in one or more of the following are used: modifications of the 5′ end of the sense strand which inhibit kinase activation of the sense strand, including, e.g., attachments of conjugates which target the molecule or the use modifications which protect against 5′ exonucleolytic degradation; or modifications of either strand, but preferably the sense strand, which enhance binding between the sense and antisense strand and thereby promote a “tight” structure at this end of the molecule.

[0726] The end region of the iRNA agent defined by the 3′ end of the sense strand and the 5′ end of the antisense strand is also important for function. This region can include the terminal 2, 3, or 4 paired nucleotides and any 3′ overhang. Preferred embodiments include asymmetrical modifications of either strand, but preferably the sense strand, which decrease binding between the sense and antisense strand and thereby promote an “open” structure at this end of the molecule. Such modifications include placing conjugates which target the molecule or modifications which promote nuclease resistance on the sense strand in this region. Modification of the antisense strand which inhibit kinase activation are avoided in preferred embodiments.

[0727] Exemplary modifications for asymmetrical placement in the sense strand include the following:

[0728] (a) backbone modifications, e.g., modification of a backbone P, including replacement of P with S, or P substituted with alkyl or allyl, e.g., Me, and dithioates (S—P═S); these modifications can be used to promote nuclease resistance;

[0729] (b) 2′-O alkyl, e.g., 2′-OMe, 3′-O alkyl, e.g., 3′-OMe (at terminal and/or internal positions); these modifications can be used to promote nuclease resistance or to enhance binding of the sense to the antisense strand, the 3′ modifications can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC;

[0730] (c) 2′-5′ linkages (with 2′-H, 2′-OH and 2′-OMe and with P═O or P═S) these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC;

[0731] (d) L sugars (e.g., L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe); these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC;

[0732] (e) modified sugars (e.g., locked nucleic acids (LNA's), hexose nucleic acids (HNA's) and cyclohexene nucleic acids (CeNA's)); these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC;

[0733] (f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2 modified purines, N-7 modified purines, N-6 modified purines), these modifications can be used to promote nuclease resistance or to enhance binding of the sense to the antisense strand;

[0734] (g) cationic groups and Zwitterionic groups (preferably at a terminus), these modifications can be used to promote nuclease resistance;

[0735] (h) conjugate groups (preferably at terminal positions), e.g., naproxen, biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates; these modifications can be used to promote nuclease resistance or to target the molecule, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC.

[0736] Exemplary modifications for asymmetrical placement in the antisense strand include the following:

[0737] (a) backbone modifications, e.g., modification of a backbone P, including replacement of P with S, or P substituted with alkyl or allyl, e.g., Me, and dithioates (S—P═S);

[0738] (b) 2′-O alkyl, e.g., 2′-OMe, (at terminal positions);

[0739] (c) 2′-5′ linkages (with 2′-H, 2′-OH and 2′-OMe) e.g., terminal at the 3′ end); e.g., with P═O or P═S preferably at the 3′-end, these modifications are preferably excluded from the 5′ end region as they may interfere with RISC enzyme activity such as kinase activity;

[0740] (d) L sugars (e.g., L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe); e.g., terminal at the 3′ end; e.g., with P═O or P═S preferably at the 3′-end, these modifications are preferably excluded from the 5′ end region as they may interfere with kinase activity;

[0741] (e) modified sugars (e.g., LNA's, HNA's and CeNA's); these modifications are preferably excluded from the 5′ end region as they may contribute to unwanted enhancements of paring between the sense and antisense strands, it is often preferred to have a “loose” structure in the 5′ region, additionally, they may interfere with kinase activity;

[0742] (f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2 modified purines, N-7 modified purines, N-6 modified purines);

[0743] (g) cationic groups and Zwitterionic groups (preferably at a terminus);

[0744] conjugate groups (preferably at terminal positions), e.g., naproxen, biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates, but bulky groups or generally groups which inhibit RISC activity should are less preferred.

[0745] The 5′-OH of the antisense strand should be kept free to promote activity. In some preferred embodiments modifications that promote nuclease resistance should be included at the 3′ end, particularly in the 3′ overhang.

[0746] In another aspect, the invention features a method of optimizing, e.g., stabilizing, an iRNA agent. The method includes selecting a sequence having activity, introducing one or more asymmetric modifications into the sequence, wherein the introduction of the asymmetric modification optimizes a property of the iRNA agent but does not result in a decrease in activity.

[0747] The decrease in activity can be less than a preselected level of decrease. In preferred embodiments decrease in activity means a decrease of less than 5, 10, 20, 40, or 50% activity, as compared with an otherwise similar iRNA lacking the introduced modification. Activity can, e.g., be measured in vivo, or in vitro, with a result in either being sufficient to demonstrate the required maintenance of activity.

[0748] The optimized property can be any property described herein and in particular the properties discussed in the section on asymmetrical modifications provided herein. The modification can be any asymmetrical modification, e.g., an asymmetric modification described in the section on asymmetrical modifications described herein. Particularly preferred asymmetric modifications are 2′-O alkyl modifications, e.g., 2′-OMe modifications, particularly in the sense sequence, and modifications of a backbone O, particularly phosphorothioate modifications, in the antisense sequence.

[0749] In a preferred embodiment a sense sequence is selected and provided with an asymmetrical modification, while in other embodiments an antisense sequence is selected and provided with an asymmetrical modification. In some embodiments both sense and antisense sequences are selected and each provided with one or more asymmetrical modifications.

[0750] Multiple asymmetric modifications can be introduced into either or both of the sense and antisense sequence. A sequence can have at least 2, 4, 6, 8, or more modifications and all or substantially all of the monomers of a sequence can be modified.

[0751]

TABLE 2
Some examples of Asymmetric Modification
This table shows examples having strand I with a selected
modification and strand II with a selected modification.
Strand I	Strand II
Nuclease Resistance (e.g. 2′-OMe)	Biodistribution (e.g., P═S)
Biodistribution conjugate	Protein Binding Functionality
(e.g. Lipophile)	(e.g. Naproxen)
Tissue Distribution Functionality	Cell Targeting Functionality
(e.g. Carbohydrates)	(e.g. Folate for cancer cells)
Tissue Distribution Functionality	Fusogenic Functionality
(e.g. Liver Cell Targeting	(e.g. Polyethylene imines)
Carbohydrates)
Cancer Cell Targeting (e.g. RGD	Fusogenic Functionality
peptides and imines)	(e.g. peptides)
Nuclease Resistance (e.g. 2′-OMe)	Increase in binding Affinity
	(5-Me—C, 5-Me—U, 2-thio-U,
	2-amino-A, G-clamp, LNA)
Tissue Distribution Functionality	RISC activity improving
	Functionality
Helical conformation changing	Tissue Distribution Functionality
Functionalities	(P═S; lipophile, carbohydrates)

[0752] Z—X—Y Architecture

[0753] In one aspect, the invention features an iRNA agent which can have a Z—X—Y architecture or structure such as those described herein and those described in copending, co-owned U.S. Provisional Application Ser. No. 60/510,246, filed on Oct. 9, 2003, which is hereby incorporated by reference, and in copending, co-owned U.S. Provisional Application Ser. No. 60/510,318, filed on Oct. 10, 2003, which is hereby incorporated by reference.

[0754] In addition, the invention includes iRNA agents having a Z—X—Y structure and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a Z—X—Y architecture.

[0755] The invention provides an iRNA agent having a first segment, the Z region, a second segment, the X region, and optionally a third region, the Y region:

• • [0756] Z—X—Y.

[0757] It may be desirable to modify subunits in one or both of Z and/or Y on one hand and X on the other hand. In some cases they will have the same modification or the same class of modification but it will more often be the case that the modifications made in Z and/or Y will differ from those made in X.

[0758] The Z region typically includes a terminus of an iRNA agent. The length of the Z region can vary, but will typically be from 2-14, more preferably 2-10, subunits in length. It typically is single stranded, i.e., it will not base pair with bases of another strand, though it may in some embodiments self associate, e.g., to form a loop structure. Such structures can be formed by the end of a strand looping back and forming an intrastrand duplex. E.g., 2, 3, 4, 5 or more intra-strand bases pairs can form, having a looped out or connecting region, typically of 2 or more subunits which do not pair. This can occur at one or both ends of a strand. A typical embodiment of a Z region is a single strand overhang, e.g., an over hang of the length described elsewhere herein. The Z region can thus be or include a 3′ or 5′ terminal single strand. It can be sense or antisense strand but if it is antisense it is preferred that it is a 3-overhang. Typical inter-subunit bonds in the Z region include: P═O; P═S; S—P═S; P—NR₂; and P—BR₂. Chiral P═X, where X is S, N, or B) inter-subunit bonds can also be present. (These inter-subunit bonds are discussed in more detail elsewhere herein.) Other preferred Z region subunit modifications (also discussed elsewhere herein) can include: 3′-OR, 3′SR, 2′-OMe, 3′-OMe, and 2′OH modifications and moieties; alpha configuration bases; and 2′ arabino modifications.

[0759] The X region will in most cases be duplexed, in the case of a single strand iRNA agent, with a corresponding region of the single strand, or in the case of a double stranded iRNA agent, with the corresponding region of the other strand. The length of the X region can vary but will typically be between 10-45 and more preferably between 15 and 35 subunits. Particularly preferred region X's will include 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, though other suitable lengths are described elsewhere herein and can be used. Typical X region subunits include 2′-OH subunits. In typical embodiments phosphate inter-subunit bonds are preferred while phophorothioate or non-phosphate bonds are absent. Other modifications preferred in the X region include: modifications to improve binding, e.g., nucleobase modifications; cationic nucleobase modifications; and C-5 modified pyrimidines, e.g., allylamines. Some embodiments have 4 or more consecutive 2′OH subunits. While the use of phosphorothioate is sometimes non preferred they can be used if they connect less than 4 consecutive 2′OH subunits.

[0760] The Y region will generally conform to the parameters set out for the Z regions. However, the X and Z regions need not be the same, different types and numbers of modifications can be present, and in fact, one will usually be a 3′ overhang and one will usually be a 5′ overhang.

[0761] In a preferred embodiment the iRNA agent will have a Y and/or Z region each having ribonucleosides in which the 2′-OH is substituted, e.g., with 2′-OMe or other alkyl; and an X region that includes at least four consecutive ribonucleoside subunits in which the 2′-OH remains unsubstituted.

[0762] The subunit linkages (the linkages between subunits) of an iRNA agent can be modified, e.g., to promote resistance to degradation. Numerous examples of such modifications are disclosed herein, one example of which is the phosphorothioate linkage. These modifications can be provided between the subunits of any of the regions, Y, X, and Z. However, it is preferred that their occurrences minimized and in particular it is preferred that consecutive modified linkages be avoided.

[0763] In a preferred embodiment the iRNA agent will have a Y and Z region each having ribonucleosides in which the 2′-OH is substituted, e.g., with 2′-OMe; and an X region that includes at least four consecutive subunits, e.g., ribonucleoside subunits in which the 2′-OH remains unsubstituted.

[0764] As mentioned above, the subunit linkages of an iRNA agent can be modified, e.g., to promote resistance to degradation. These modifications can be provided between the subunits of any of the regions, Y, X, and Z. However, it is preferred that they are minimized and in particular it is preferred that consecutive modified linkages be avoided.

[0765] Thus, in a preferred embodiment, not all of the subunit linkages of the iRNA agent are modified and more preferably the maximum number of consecutive subunits linked by other than a phosphodiester bond will be 2, 3, or 4. Particularly preferred iRNA agents will not have four or more consecutive subunits, e.g., 2′-hydroxyl ribonucleoside subunits, in which each subunits is joined by modified linkages—i.e. linkages that have been modified to stabilize them from degradation as compared to the phosphodiester linkages that naturally occur in RNA and DNA.

[0766] It is particularly preferred to minimize the occurrence in region X. Thus, in preferred embodiments each of the nucleoside subunit linkages in X will be phosphodiester linkages, or if subunit linkages in region X are modified, such modifications will be minimized. E.g., although the Y and/or Z regions can include inter subunit linkages which have been stabilized against degradation, such modifications will be minimized in the X region, and in particular consecutive modifications will be minimized. Thus, in preferred embodiments the maximum number of consecutive subunits linked by other than a phosphodiester bond will be 2, 3, or 4. Particularly preferred X regions will not have four or more consecutive subunits, e.g., 2′-hydroxyl ribonucleoside subunits, in which each subunits is joined by modified linkages—i.e. linkages that have been modified to stabilize them from degradation as compared to the phosphodiester linkages that naturally occur in RNA and DNA.

[0767] In a preferred embodiment Y and/or Z will be free of phosphorothioate linkages, though either or both may contain other modifications, e.g., other modifications of the subunit linkages.

[0768] In a preferred embodiment region X, or in some cases, the entire iRNA agent, has no more than 3 or no more than 4 subunits having identical 2′ moieties.

[0769] In a preferred embodiment region X, or in some cases, the entire iRNA agent, has no more than 3 or no more than 4 subunits having identical subunit linkages.

[0770] In a preferred embodiment one or more phosphorothioate linkages (or other modifications of the subunit linkage) are present in Y and/or Z, but such modified linkages do not connect two adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., a 2′-O-alkyl moiety. E.g., any adjacent 2′-O-alkyl moieties in the Y and/or Z, are connected by a linkage other than a phosphorothioate linkage.

[0771] In a preferred embodiment each of Y and/or Z independently has only one phosphorothioate linkage between adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., 2′-O-alkyl nucleosides. If there is a second set of adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., 2′-O-alkyl nucleosides, in Y and/or Z that second set is connected by a linkage other than a phosphorothioate linkage, e.g., a modified linkage other than a phosphorothioate linkage.

[0772] In a preferred embodiment each of Y and/or Z independently has more than one phosphorothioate linkage connecting adjacent pairs of subunits, e.g., nucleosides, having a 2′ modification, e.g., 2′-O-alkyl nucleosides, but at least one pair of adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., 2′-O-alkyl nucleosides, are be connected by a linkage other than a phosphorothioate linkage, e.g., a modified linkage other than a phosphorothioate linkage.

[0773] In a preferred embodiment one of the above recited limitation on adjacent subunits in Y and or Z is combined with a limitation on the subunits in X. E.g., one or more phosphorothioate linkages (or other modifications of the subunit linkage) are present in Y and/or Z, but such modified linkages do not connect two adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., a 2′-O-alkyl moiety. E.g., any adjacent 2′-O-alkyl moieties in the Y and/or Z, are connected by a linkage other than a phosporothioate linkage. In addition, the X region has no more than 3 or no more than 4 identical subunits, e.g., subunits having identical 2′ moieties or the X region has no more than 3 or no more than 4 subunits having identical subunit linkages.

[0774] A Y and/or Z region can include at least one, and preferably 2, 3 or 4 of a modification disclosed herein. Such modifications can be chosen, independently, from any modification described herein, e.g., from nuclease resistant subunits, subunits with modified bases, subunits with modified intersubunit linkages, subunits with modified sugars, and subunits linked to another moiety, e.g., a targeting moiety. In a preferred embodiment more than 1 of such subunits can be present but in some embodiments it is preferred that no more than 1, 2, 3, or 4 of such modifications occur, or occur consecutively. In a preferred embodiment the frequency of the modification will differ between Y and/or Z and X, e.g., the modification will be present one of Y and/or Z or X and absent in the other.

[0775] An X region can include at least one, and preferably 2, 3 or 4 of a modification disclosed herein. Such modifications can be chosen, independently, from any modification described herein, e.g., from nuclease resistant subunits, subunits with modified bases, subunits with modified intersubunit linkages, subunits with modified sugars, and subunits linked to another moiety, e.g., a targeting moiety. In a preferred embodiment more than 1 of such subunits can b present but in some embodiments it is preferred that no more than 1, 2, 3, or 4 of such modifications occur, or occur consecutively.

[0776] An RRMS (described elsewhere herein) can be introduced at one or more points in one or both strands of a double-stranded iRNA agent. An RRMS can be placed in a Y and/or Z region, at or near (within 1, 2, or 3 positions) of the 3′ or 5′ end of the sense strand or at near (within 2 or 3 positions of) the 3′ end of the antisense strand. In some embodiments it is preferred to not have an RRMS at or near (within 1, 2, or 3 positions of) the 5′ end of the antisense strand. An RRMS can be positioned in the X region, and will preferably be positioned in the sense strand or in an area of the antisense strand not critical for antisense binding to the target.

[0777] Differential Modification of Terminal Duplex Stability

[0778] In one aspect, the invention features an iRNA agent which can have differential modification of terminal duplex stability (DMTDS).

[0779] In addition, the invention includes iRNA agents having DMTDS and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates DMTDS.

[0780] iRNA agents can be optimized by increasing the propensity of the duplex to disassociate or melt (decreasing the free energy of duplex association), in the region of the 5′ end of the antisense strand duplex. This can be accomplished, e.g., by the inclusion of subunits which increase the propensity of the duplex to disassociate or melt in the region of the 5′ end of the antisense strand. It can also be accomplished by the attachment of a ligand that increases the propensity of the duplex to disassociate of melt in the region of the 5′ end. While not wishing to be bound by theory, the effect may be due to promoting the effect of an enzyme such as helicase, for example, promoting the effect of the enzyme in the proximity of the 5′ end of the antisense strand.

[0781] The inventors have also discovered that iRNA agents can be optimized by decreasing the propensity of the duplex to disassociate or melt (increasing the free energy of duplex association), in the region of the 3′ end of the antisense strand duplex. This can be accomplished, e.g., by the inclusion of subunits which decrease the propensity of the duplex to disassociate or melt in the region of the 3′ end of the antisense strand. It can also be accomplished by the attachment of ligand that decreases the propensity of the duplex to disassociate of melt in the region of the 5′ end.

[0782] Modifications which increase the tendency of the 5′ end of the duplex to dissociate can be used alone or in combination with other modifications described herein, e.g., with modifications which decrease the tendency of the 3′ end of the duplex to dissociate. Likewise, modifications which decrease the tendency of the 3′ end of the duplex to dissociate can be used alone or in combination with other modifications described herein, e.g., with modifications which increase the tendency of the 5′ end of the duplex to dissociate.

Decreasing the stability of the AS 5′ end of the duplex

[0783] Subunit pairs can be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation:

• • [0784] A:U is preferred over G:C;
  • [0785] G:U is preferred over G:C;
  • [0786] I:C is preferred over G:C (I=inosine);
  • [0787] mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings;
  • [0788] pairings which include a universal base are preferred over canonical pairings.

[0789] A typical ds iRNA agent can be diagrammed as follows:

[0790] S 5′ R₁N₁N₂N₃N₄N₅ [N] N₋₅ N₋₄ N₋₃ N₋₂ N₋₁ R₂ 3′

[0791] AS 3′ R₃N₁N₂N₃N₄N₅ [N] N₋₅ N₋₄ N₋₃ N₋₂ N₋₁ R₄ 5′

[0792] S:AS P₁ P₂ P₃ P₄ P₅ [N] P₋₅P₋₄P₋₃P₋₂P₋₁ 5′

[0793] S indicates the sense strand; AS indicates antisense strand; R₁ indicates an optional (and nonpreferred) 5′ sense strand overhang; R₂ indicates an optional (though preferred) 3′ sense overhang; R₃ indicates an optional (though preferred) 3′ antisense sense overhang; R₄ indicates an optional (and nonpreferred) 5′ antisense overhang; N indicates subunits; [N] indicates that additional subunit pairs may be present; and P_x, indicates a paring of sense N_x and antisense N_x. Overhangs are not shown in the P diagram. In some embodiments a 3′ AS overhang corresponds to region Z, the duplex region corresponds to region X, and the 3′ S strand overhang corresponds to region Y, as described elsewhere herein. (The diagram is not meant to imply maximum or minimum lengths, on which guidance is provided elsewhere herein.)

[0794] It is preferred that pairings which decrease the propensity to form a duplex are used at 1 or more of the positions in the duplex at the 5′ end of the AS strand. The terminal pair (the most 5′ pair in terms of the AS strand) is designated as P₋₁, and the subsequent pairing positions (going in the 3′ direction in terms of the AS strand) in the duplex are designated, P₋₂, P₋₃, P₋₄, P₋₅, and so on. The preferred region in which to modify to modulate duplex formation is at P₋₅ through P₋₁, more preferably P₋₄ through P₋₁, more preferably P₋₃ through P₋₁. Modification at P₋₁, is particularly preferred, alone or with modification(s) other position(s), e.g., any of the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions be chosen independently from the group of:

• • [0795] A:U
  • [0796] G:U
  • [0797] I:C
  • [0798] mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base.

[0799] In preferred embodiments the change in subunit needed to achieve a pairing which promotes dissociation will be made in the sense strand, though in some embodiments the change will be made in the antisense strand.

[0800] In a preferred embodiment the at least 2, or 3, of the pairs in P₋₁, through P₋₄, are pairs which promote dissociation.

[0801] In a preferred embodiment the at least 2, or 3, of the pairs in P₋₁, through P₋₄, are A:U.

[0802] In a preferred embodiment the at least 2, or 3, of the pairs in P₋₁, through P₋₄, are G:U.

[0803] In a preferred embodiment the at least 2, or 3, of the pairs in P₋₁, through P₋₄, are I:C.

[0804] In a preferred embodiment the at least 2, or 3, of the pairs in P₋₁, through P₋₄, are mismatched pairs, e.g., non-canonical or other than canonical pairings.

[0805] In a preferred embodiment the at least 2, or 3, of the pairs in P₋₁, through P₋₄, are pairings which include a universal base.

Increasing the Stability of the AS 3′ End of the Duplex

[0806] Subunit pairs can be ranked on the basis of their propensity to promote stability and inhibit dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting duplex stability:

• • [0807] G:C is preferred over A:U
  • [0808] Watson-Crick matches (A:T, A:U, G:C) are preferred over non-canonical or other than canonical pairings
  • [0809] analogs that increase stability are preferred over Watson-Crick matches (A:T, A:U, G:C)
  • [0810] 2-amino-A:U is preferred over A:U

[0811] 2-thio U or 5 Me-thio-U:A are preferred over U:A

• • [0812] G-clamp (an analog of C having 4 hydrogen bonds):G is preferred over C:G
  • [0813] guanadinium-G-clamp:G is preferred over C:G
  • [0814] psuedo uridine:A is preferred over U:A
  • [0815] sugar modifications, e.g., 2′ modifications, e.g., 2′F, ENA, or LNA, which enhance binding are preferred over non-modified moieties and can be present on one or both strands to enhance stability of the duplex. It is preferred that pairings which increase the propensity to form a duplex are used at 1 or more of the positions in the duplex at the 3′ end of the AS strand. The terminal pair (the most 3′ pair in terms of the AS strand) is designated as P₁, and the subsequent pairing positions (going in the 5′ direction in terms of the AS strand) in the duplex are designated, P₂, P₃, P₄, P₅, and so on. The preferred region in which to modify to modulate duplex formation is at P₅ through P₁, more preferably P₄ through P₁, more preferably P₃ through P₁. Modification at P₁, is particularly preferred, alone or with modification(s) at other position(s), e.g., any of the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of the recited regions be chosen independently from the group of:
  • [0816] G:C
  • [0817] a pair having an analog that increases stability over Watson-Crick matches (A:T, A:U, G:C)
  • [0818] 2-amino-A:U

[0819] 2-thio U or 5 Me-thio-U:A

• • [0820] G-clamp (an analog of C having 4 hydrogen bonds):G
  • [0821] guanidinium-G-clamp:G
  • [0822] psuedo uridine:A
  • [0823] a pair in which one or both subunits has a sugar modification, e.g., a 2′ modification, e.g., 2′F, ENA, or LNA, which enhance binding.

[0824] In a preferred embodiment the at least 2, or 3, of the pairs in P₋₁, through P₋₄, are pairs which promote duplex stability.

[0825] In a preferred embodiment the at least 2, or 3, of the pairs in P₁, through P₄, are G:C.

[0826] In a preferred embodiment the at least 2, or 3, of the pairs in P1, through P₄, are a pair having an analog that increases stability over Watson-Crick matches.

[0827] In a preferred embodiment the at least 2, or 3, of the pairs in P₁, through P₄, are 2-amino-A:U.

[0828] In a preferred embodiment the at least 2, or 3, of the pairs in P1, through P₄, are 2-thio U or 5 Me-thio-U:A.

[0829] In a preferred embodiment the at least 2, or 3, of the pairs in P₁, through P₄, are G-clamp:G.

[0830] In a preferred embodiment the at least 2, or 3, of the pairs in P₁, through P₄, are guanidinium-G-clamp:G.

[0831] In a preferred embodiment the at least 2, or 3, of the pairs in P1, through P₄, are psuedo uridine:A.

[0832] In a preferred embodiment the at least 2, or 3, of the pairs in P₁, through P₄, are a pair in which one or both subunits has a sugar modification, e.g., a 2′ modification, e.g., 2′F, ENA, or LNA, which enhances binding.

[0833] G-clamps and guanidinium G-clamps are discussed in the following references: Holmes and Gait, “The Synthesis of 2′-O-Methyl G-Clamp Containing Oligonucleotides and Their Inhibition of the HIV-1 Tat-TAR Interaction,” Nucleosides, Nucleotides & Nucleic Acids, 22:1259-1262, 2003; Holmes et al., “Steric inhibition of human immunodeficiency virus type-1 Tat-dependent trans-activation in vitro and in cells by oligonucleotides containing 2′-O-methyl G-clamp ribonucleoside analogues,” Nucleic Acids Research, 31:2759-2768, 2003; Wilds, et al., “Structural basis for recognition of guanosine by a synthetic tricyclic cytosine analogue: Guanidinium G-clamp,” Helvetica Chimica Acta, 86:966-978, 2003; Rajeev, et al., “High-Affinity Peptide Nucleic Acid Oligomers Containing Tricyclic Cytosine Analogues,” Organic Letters, 4:4395-4398, 2002; Ausin, et al., “Synthesis of Amino- and Guanidino-G-Clamp PNA Monomers,” Organic Letters, 4:4073-4075, 2002; Maier et al., “Nuclease resistance of oligonucleotides containing the tricyclic cytosine analogues phenoxazine and 9-(2-aminoethoxy)-phenoxazine (“G-clamp”) and origins of their nuclease resistance properties,” Biochemistry, 41:1323-7, 2002; Flanagan, et al., “A cytosine analog that confers enhanced potency to antisense oligonucleotides,” Proceedings Of The National Academy Of Sciences Of The United States Of America, 96:3513-8, 1999.

[0834] Simultaneously Decreasing the Stability of the AS 5′ End of the Duplex and Increasing the Stability of the AS 3′ End of the Duplex

[0835] As is discussed above, an iRNA agent can be modified to both decrease the stability of the AS 5′ end of the duplex and increase the stability of the AS 3′ end of the duplex. This can be effected by combining one or more of the stability decreasing modifications in the AS 5′ end of the duplex with one or more of the stability increasing modifications in the AS 3′ end of the duplex. Accordingly a preferred embodiment includes modification in P₋₅ through P₋₁, more preferably P₋₄ through P₋₁ and more preferably P₋₃ through P₋₁. Modification at P₋₁, is particularly preferred, alone or with other position, e.g., the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions of the AS 5′ end of the duplex region be chosen independently from the group of:

• • [0836] A:U
  • [0837] G:U
  • [0838] I:C
  • [0839] mismatched pairs, e.g., non-canonical or other than canonical pairings which include a universal base; and

[0840] a modification in P₅ through P₁, more preferably P₄ through P₁ and more preferably P₃ through P₁. Modification at P₁, is particularly preferred, alone or with other position, e.g., the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions of the AS 3′ end of the duplex region be chosen independently from the group of:

• • [0841] G:C
  • [0842] a pair having an analog that increases stability over Watson-Crick matches (A:T, A:U, G:C)
  • [0843] 2-amino-A:U

[0844] 2-thio U or 5 Me-thio-U:A

• • [0845] G-clamp (an analog of C having 4 hydrogen bonds):G
  • [0846] guanidinium-G-clamp:G
  • [0847] psuedo uridine:A
  • [0848] a pair in which one or both subunits has a sugar modification, e.g., a 2′ modification, e.g., 2′F, ENA, or LNA, which enhance binding.

[0849] The invention also includes methods of selecting and making iRNA agents having DMTDS. E.g., when screening a target sequence for candidate sequences for use as iRNA agents one can select sequences having a DMTDS property described herein or one which can be modified, preferably with as few changes as possible, especially to the

[0850] AS strand, to provide a desired level of DMTDS.

[0851] The invention also includes, providing a candidate iRNA agent sequence, and modifying at least one P in P₋₅ through P₋₁ and/or at least one P in P₅ through P₁ to provide a DMTDS iRNA agent.

[0852] DMTDS iRNA agents can be used in any method described herein, e.g., to silence any gene disclosed herein, to treat any disorder described herein, in any formulation described herein, and generally in and/or with the methods and compositions described elsewhere herein. DMTDS iRNA agents can incorporate other modifications described herein, e.g., the attachment of targeting agents or the inclusion of modifications which enhance stability, e.g., the inclusion of nuclease resistant monomers or the inclusion of single strand overhangs (e.g., 3′ AS overhangs and/or 3′ S strand overhangs) which self associate to form intrastrand duplex structure.

[0853] Preferably these iRNA agents will have an architecture described herein.

[0854] In Vivo Delivery

[0855] An iRNA agent can be linked, e.g., noncovalently linked to a polymer for the efficient delivery of the iRNA agent to a subject, e.g., a mammal, such as a human. The iRNA agent can, for example, be complexed with cyclodextrin. Cyclodextrins have been used as delivery vehicles of therapeutic compounds. Cyclodextrins can form inclusion complexes with drugs that are able to fit into the hydrophobic cavity of the cyclodextrin. In other examples, cyclodextrins form non-covalent associations with other biologically active molecules such as oligonucleotides and derivatives thereof. The use of cyclodextrins creates a water-soluble drug delivery complex, that can be modified with targeting or other functional groups. Cyclodextrin cellular delivery system for oligonucleotides described in U.S. Pat. No. 5,691,316, which is hereby incorporated by reference, are suitable for use in methods of the invention. In this system, an oligonucleotide is noncovalently complexed with a cyclodextrin, or the oligonucleotide is covalently bound to adamantine which in turn is noncovalently associated with a cyclodextrin.

[0856] The delivery molecule can include a linear cyclodextrin copolymer or a linear oxidized cyclodextrin copolymer having at least one ligand bound to the cyclodextrin copolymer. Delivery systems, as described in U.S. Pat. No. 6,509,323, herein incorporated by reference, are suitable for use in methods of the invention. An iRNA agent can be bound to the linear cyclodextrin copolymer and/or a linear oxidized cyclodextrin copolymer. Either or both of the cyclodextrin or oxidized cyclodextrin copolymers can be crosslinked to another polymer and/or bound to a ligand.

[0857] A composition for iRNA delivery can employ an “inclusion complex,” a molecular compound having the characteristic structure of an adduct. In this structure, the “host molecule” spatially encloses at least part of another compound in the delivery vehicle. The enclosed compound (the “guest molecule”) is situated in the cavity of the host molecule without affecting the framework structure of the host. A “host” is preferably cyclodextrin, but can be any of the molecules suggested in U.S. Patent Publ. 2003/0008818, herein incorporated by reference.

[0858] Cyclodextrins can interact with a variety of ionic and molecular species, and the resulting inclusion compounds belong to the class of “host-guest” complexes. Within the host-guest relationship, the binding sites of the host and guest molecules should be complementary in the stereoelectronic sense. A composition of the invention can contain at least one polymer and at least one therapeutic agent, generally in the form of a particulate composite of the polymer and therapeutic agent, e.g., the iRNA agent. The iRNA agent can contain one or more complexing agents. At least one polymer of the particulate composite can interact with the complexing agent in a host-guest or a guest-host interaction to form an inclusion complex between the polymer and the complexing agent. The polymer and, more particularly, the complexing agent can be used to introduce functionality into the composition. For example, at least one polymer of the particulate composite has host functionality and forms an inclusion complex with a complexing agent having guest functionality. Alternatively, at least one polymer of the particulate composite has guest functionality and forms an inclusion complex with a complexing agent having host functionality. A polymer of the particulate composite can also contain both host and guest functionalities and form inclusion complexes with guest complexing agents and host complexing agents. A polymer with functionality can, for example, facilitate cell targeting and/or cell contact (e.g., targeting or contact to a liver cell), intercellular trafficking, and/or cell entry and release.

[0859] Upon forming the particulate composite, the iRNA agent may or may not retain its biological or therapeutic activity. Upon release from the therapeutic composition, specifically, from the polymer of the particulate composite, the activity of the iRNA agent is restored. Accordingly, the particulate composite advantageously affords the iRNA agent protection against loss of activity due to, for example, degradation and offers enhanced bioavailability. Thus, a composition may be used to provide stability, particularly storage or solution stability, to an iRNA agent or any active chemical compound. The iRNA agent may be further modified with a ligand prior to or after particulate composite or therapeutic composition formation. The ligand can provide further functionality. For example, the ligand can be a targeting moiety.

[0860] Physiological Effects

[0861] The iRNA agents described herein can be designed such that determining therapeutic toxicity is made easier by the complementarity of the iRNA agent with both a human and a non-human animal sequence. By these methods, an iRNA agent can consist of a sequence that is fully complementary to a nucleic acid sequence from a human and a nucleic acid sequence from at least one non-human animal, e.g., a non-human mammal, such as a rodent, ruminant or primate. For example, the non-human mammal can be a mouse, rat, dog, pig, goat, sheep, cow, monkey, Pan paniscus, Pan troglodytes, Macaca mulatto, or Cynomolgus monkey. The sequence of the iRNA agent could be complementary to sequences within homologous genes, e.g., oncogenes or tumor suppressor genes, of the non-human mammal and the human. By determining the toxicity of the iRNA agent in the non-human mammal, one can extrapolate the toxicity of the iRNA agent in a human. For a more strenuous toxicity test, the iRNA agent can be complementary to a human and more than one, e.g., two or three or more, non-human animals.

[0862] The methods described herein can be used to correlate any physiological effect of an iRNA agent on a human, e.g., any unwanted effect, such as a toxic effect, or any positive, or desired effect.

[0863] Delivery Module

[0864] In one aspect, the invention features a drug delivery conjugate or module, such as those described herein and those described in copending, co-owned U.S. Provisional Application Ser. No. 60/454,265, filed on Mar. 12, 2003, which is hereby incorporated by reference.

[0865] In addition, the invention includes iRNA agents described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having a chemical modification described herein, e.g., a modification which enhances resistance to degradation, an iRNA agent having an architecture or structure described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, combined with, associated with, and delivered by such a drug delivery conjugate or module.

[0866] The iRNA agents can be complexed to a delivery agent that features a modular complex. The complex can include a carrier agent linked to one or more of (preferably two or more, more preferably all three of): (a) a condensing agent (e.g., an agent capable of attracting, e.g., binding, a nucleic acid, e.g., through ionic or electrostatic interactions); (b) a fusogenic agent (e.g., an agent capable of fusing and/or being transported through a cell membrane, e.g., an endosome membrane); and (c) a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell or bone cell.

[0867] An iRNA agent, e.g., iRNA agent or sRNA agent described herein, can be linked, e.g., coupled or bound, to the modular complex. The iRNA agent can interact with the condensing agent of the complex, and the complex can be used to deliver an iRNA agent to a cell, e.g., in vitro or in vivo. For example, the complex can be used to deliver an iRNA agent to a subject in need thereof, e.g., to deliver an iRNA agent to a subject having a disorder, e.g., a disorder described herein, such as a disease or disorder of the liver.

[0868] The fusogenic agent and the condensing agent can be different agents or the one and the same agent. For example, a polyamino chain, e.g., polyethyleneimine (PEI), can be the fusogenic and/or the condensing agent.

[0869] The delivery agent can be a modular complex. For example, the complex can include a carrier agent linked to one or more of (preferably two or more, more preferably all three of):

[0870] (a) a condensing agent (e.g., an agent capable of attracting, e.g., binding, a nucleic acid, e.g., through ionic interaction),

[0871] (b) a fusogenic agent (e.g., an agent capable of fusing and/or being transported through a cell membrane, e.g., an endosome membrane), and

[0872] (c) a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, bone cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, Neproxin, or an RGD peptide or RGD peptide mimetic.

[0873] Carrier Agents

[0874] The carrier agent of a modular complex described herein can be a substrate for attachment of one or more of: a condensing agent, a fusogenic agent, and a targeting group. The carrier agent would preferably lack an endogenous enzymatic activity. The agent would preferably be a biological molecule, preferably a macromolecule. Polymeric biological carriers are preferred. It would also be preferred that the carrier molecule be biodegradable.

[0875] The carrier agent can be a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or lipid. The carrier molecule can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Other useful carrier molecules can be identified by routine methods.

[0876] A carrier agent can be characterized by one or more of: (a) is at least 1 Da in size; (b) has at least 5 charged groups, preferably between 5 and 5000 charged groups; (c) is present in the complex at a ratio of at least 1:1 carrier agent to fusogenic agent; (d) is present in the complex at a ratio of at least 1:1 carrier agent to condensing agent; (e) is present in the complex at a ratio of at least 1:1 carrier agent to targeting agent.

[0877] Fusogenic Agents

[0878] A fusogenic agent of a modular complex described herein can be an agent that is responsive to, e.g., changes charge depending on, the pH environment. Upon encountering the pH of an endosome, it can cause a physical change, e.g., a change in osmotic properties which disrupts or increases the permeability of the endosome membrane. Preferably, the fusogenic agent changes charge, e.g., becomes protonated, at pH lower than physiological range. For example, the fusogenic agent can become protonated at pH4.5-6.5. The fusogenic agent can serve to release the iRNA agent into the cytoplasm of a cell after the complex is taken up, e.g., via endocytosis, by the cell, thereby increasing the cellular concentration of the iRNA agent in the cell.

[0879] In one embodiment, the fusogenic agent can have a moiety, e.g., an amino group, which, when exposed to a specified pH range, will undergo a change, e.g., in charge, e.g., protonation. The change in charge of the fusogenic agent can trigger a change, e.g., an osmotic change, in a vesicle, e.g., an endocytic vesicle, e.g., an endosome. For example, the fusogenic agent, upon being exposed to the pH environment of an endosome, will cause a solubility or osmotic change substantial enough to increase the porosity of (preferably, to rupture) the endosomal membrane.

[0880] The fusogenic agent can be a polymer, preferably a polyamino chain, e.g., polyethyleneimine (PEI). The PEI can be linear, branched, synthetic or natural. The PEI can be, e.g., alkyl substituted PEI, or lipid substituted PEI.

[0881] In other embodiments, the fusogenic agent can be polyhistidine, polyimidazole, polypyridine, polypropyleneimine, mellitin, or a polyacetal substance, e.g., a cationic polyacetal. In some embodiment, the fusogenic agent can have an alpha helical structure. The fusogenic agent can be a membrane disruptive agent, e.g., mellittin.

[0882] A fusogenic agent can have one or more of the following characteristics: (a) is at least 1 Da in size; (b) has at least 10 charged groups, preferably between 10 and 5000 charged groups, more preferably between 50 and 1000 charged groups; (c) is present in the complex at a ratio of at least 1:1 fusogenic agent to carrier agent; (d) is present in the complex at a ratio of at least 1:1 fusogenic agent to condensing agent; (e) is present in the complex at a ratio of at least 1:1 fusogenic agent to targeting agent.

[0883] Other suitable fusogenic agents can be tested and identified by a skilled artisan. The ability of a compound to respond to, e.g., change charge depending on, the pH environment can be tested by routine methods, e.g., in a cellular assay. For example, a test compound is combined or contacted with a cell, and the cell is allowed to take up the test compound, e.g., by endocytosis. An endosome preparation can then be made from the contacted cells and the endosome preparation compared to an endosome preparation from control cells. A change, e.g., a decrease, in the endosome fraction from the contacted cell vs. the control cell indicates that the test compound can function as a fusogenic agent. Alternatively, the contacted cell and control cell can be evaluated, e.g., by microscopy, e.g., by light or electron microscopy, to determine a difference in endosome population in the cells. The test compound can be labeled. In another type of assay, a modular complex described herein is constructed using one or more test or putative fusogenic agents. The modular complex can be constructed using a labeled nucleic acid instead of the iRNA. The ability of the fusogenic agent to respond to, e.g., change charge depending on, the pH environment, once the modular complex is taken up by the cell, can be evaluated, e.g., by preparation of an endosome preparation, or by microscopy techniques, as described above. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to respond to, e.g., change charge depending on, the pH environment; and a second assay evaluates the ability of a modular complex that includes the test compound to respond to, e.g., change charge depending on, the pH environment.

[0884] Condensing Agent

[0885] The condensing agent of a modular complex described herein can interact with (e.g., attracts, holds, or binds to) an iRNA agent and act to (a) condense, e.g., reduce the size or charge of the iRNA agent and/or (b) protect the iRNA agent, e.g., protect the iRNA agent against degradation. The condensing agent can include a moiety, e.g., a charged moiety, that can interact with a nucleic acid, e.g., an iRNA agent, e.g., by ionic interactions. The condensing agent would preferably be a charged polymer, e.g., a polycationic chain. The condensing agent can be a polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

[0886] A condensing agent can have the following characteristics: (a) at least 1 Da in size; (b) has at least 2 charged groups, preferably between 2 and 100 charged groups; (c) is present in the complex at a ratio of at least 1:1 condensing agent to carrier agent; (d) is present in the complex at a ratio of at least 1:1 condensing agent to fusogenic agent; (e) is present in the complex at a ratio of at least 1:1 condensing agent to targeting agent.

[0887] Other suitable condensing agents can be tested and identified by a skilled artisan, e.g., by evaluating the ability of a test agent to interact with a nucleic acid, e.g., an iRNA agent. The ability of a test agent to interact with a nucleic acid, e.g., an iRNA agent, e.g., to condense or protect the iRNA agent, can be evaluated by routine techniques. In one assay, a test agent is contacted with a nucleic acid, and the size and/or charge of the contacted nucleic acid is evaluated by a technique suitable to detect changes in molecular mass and/or charge. Such techniques include non-denaturing gel electrophoresis, immunological methods, e.g., immunoprecipitation, gel filtration, ionic interaction chromatography, and the like. A test agent is identified as a condensing agent if it changes the mass and/or charge (preferably both) of the contacted nucleic acid, compared to a control. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to interact with, e.g., bind to, e.g., condense the charge and/or mass of, a nucleic cid; and a second assay evaluates the ability of a modular complex that includes the test compound to interact with, e.g., bind to, e.g., condense the charge and/or mass of, a nucleic acid.

[0888] Amphipathic Delivery Agents

[0889] In one aspect, the invention features an amphipathic delivery conjugate or module, such as those described herein and those described in copending, co-owned U.S. Provisional Application Ser. No. 60/455,050, filed on Mar. 13, 2003, which is hereby incorporated by reference.

[0890] In addition, the invention include an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having a chemical modification described herein, e.g., a modification which enhances resistance to degradation, an iRNA agent having an architecture or structure described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, combined with, associated with, and delivered by such an amphipathic delivery conjugate.

[0891] An amphipathic molecule is a molecule having a hydrophobic and a hydrophilic region. Such molecules can interact with (e.g., penetrate or disrupt) lipids, e.g., a lipid bilayer of a cell. As such, they can serve as delivery agent for an associated (e.g., bound) iRNA (e.g., an iRNA or sRNA described herein). A preferred amphipathic molecule to be used in the compositions described herein (e.g., the amphipathic iRNA constructs described herein) is a polymer. The polymer may have a secondary structure, e.g., a repeating secondary structure.

[0892] One example of an amphipathic polymer is an amphipathic polypeptide, e.g., a polypeptide having a secondary structure such that the polypeptide has a hydrophilic and a hydrophobic face. The design of amphipathic peptide structures (e.g., alpha-helical polypeptides) is routine to one of skill in the art. For example, the following references provide guidance: Grell et al. (2001) Protein design and folding: template trapping of self-assembled helical bundles J Pept Sci 7(3): 146-51; Chen et al. (2002) Determination of stereochemistry stability coefficients of amino acid side-chains in an amphipathic alpha-helix J Pept Res 59(1): 18-33; Iwata et al. (1994) Design and synthesis of amphipathic 3(10)-helical peptides and their interactions with phospholipid bilayers and ion channel formation J Biol Chem 269(7): 4928-33; Cornut et al. (1994) The amphipathic alpha-helix concept. Application to the de novo design of ideally amphipathic Leu, Lys peptides with hemolytic activity higher than that of melittin FEBS Lett 349(1): 29-33; Negrete et al. (1998) Deciphering the structural code for proteins: helical propensities in domain classes and statistical multiresidue information in alpha-helices. Protein Sci 7(6): 1368-79.

[0893] Another example of an amphipathic polymer is a polymer made up of two or more amphipathic subunits, e.g., two or more subunits containing cyclic moieties (e.g., a cyclic moiety having one or more hydrophilic groups and one or more hydrophobic groups). For example, the subunit may contain a steroid, e.g., cholic acid; or a aromatic moiety. Such moieties preferably can exhibit atropisomerism, such that they can form opposing hydrophobic and hydrophilic faces when in a polymer structure.

[0894] The ability of a putative amphipathic molecule to interact with a lipid membrane, e.g., a cell membrane, can be tested by routine methods, e.g., in a cell free or cellular assay. For example, a test compound is combined or contacted with a synthetic lipid bilayer, a cellular membrane fraction, or a cell, and the test compound is evaluated for its ability to interact with, penetrate or disrupt the lipid bilayer, cell membrane or cell. The test compound can labeled in order to detect the interaction with the lipid bilayer, cell membrane or cell. In another type of assay, the test compound is linked to a reporter molecule or an iRNA agent (e.g., an iRNA or sRNA described herein) and the ability of the reporter molecule or iRNA agent to penetrate the lipid bilayer, cell membrane or cell is evaluated. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to interact with a lipid bilayer, cell membrane or cell; and a second assay evaluates the ability of a construct (e.g., a construct described herein) that includes the test compound and a reporter or iRNA agent to interact with a lipid bilayer, cell membrane or cell.

[0895] An amphipathic polymer useful in the compositions described herein has at least 2, preferably at least 5, more preferably at least 10, 25, 50, 100, 200, 500, 1000, 2000, 50000 or more subunits (e.g., amino acids or cyclic subunits). A single amphipathic polymer can be linked to one or more, e.g., 2, 3, 5, 10 or more iRNA agents (e.g., iRNA or sRNA agents described herein). In some embodiments, an amphipathic polymer can contain both amino acid and cyclic subunits, e.g., aromatic subunits.

[0896] The invention features a composition that includes an iRNA agent (e.g., an iRNA or sRNA described herein) in association with an amphipathic molecule. Such compositions may be referred to herein as “amphipathic iRNA constructs.” Such compositions and constructs are useful in the delivery or targeting of iRNA agents, e.g., delivery or targeting of iRNA agents to a cell. While not wanting to be bound by theory, such compositions and constructs can increase the porosity of, e.g., can penetrate or disrupt, a lipid (e.g., a lipid bilayer of a cell), e.g., to allow entry of the iRNA agent into a cell.

[0897] In one aspect, the invention relates to a composition comprising an iRNA agent (e.g., an iRNA or sRNA agent described herein) linked to an amphipathic molecule. The iRNA agent and the amphipathic molecule may be held in continuous contact with one another by either covalent or noncovalent linkages.

[0898] The amphipathic molecule of the composition or construct is preferably other than a phospholipid, e.g., other than a micelle, membrane or membrane fragment.

[0899] The amphipathic molecule of the composition or construct is preferably a polymer. The polymer may include two or more amphipathic subunits. One or more hydrophilic groups and one or more hydrophobic groups may be present on the polymer. The polymer may have a repeating secondary structure as well as a first face and a second face. The distribution of the hydrophilic groups and the hydrophobic groups along the repeating secondary structure can be such that one face of the polymer is a hydrophilic face and the other face of the polymer is a hydrophobic face.

[0900] The amphipathic molecule can be a polypeptide, e.g., a polypeptide comprising an α-helical conformation as its secondary structure.

[0901] In one embodiment, the amphipathic polymer includes one or more subunits containing one or more cyclic moiety (e.g., a cyclic moiety having one or more hydrophilic groups and/or one or more hydrophobic groups). In one embodiment, the polymer is a polymer of cyclic moieties such that the moieties have alternating hydrophobic and hydrophilic groups. For example, the subunit may contain a steroid, e.g., cholic acid. In another example, the subunit may contain an aromatic moiety. The aromatic moiety may be one that can exhibit atropisomerism, e.g., a 2,2′-bis(substituted)-1-1′-binaphthyl or a 2,2′-bis(substituted) biphenyl. A subunit may include an aromatic moiety of Formula (M):

[0902]

[0903] The invention features a composition that includes an iRNA agent (e.g., an iRNA or sRNA described herein) in association with an amphipathic molecule. Such compositions may be referred to herein as “amphipathic iRNA constructs.” Such compositions and constructs are useful in the delivery or targeting of iRNA agents, e.g., delivery or targeting of iRNA agents to a cell. While not wanting to be bound by theory, such compositions and constructs can increase the porosity of, e.g., can penetrate or disrupt, a lipid (e.g., a lipid bilayer of a cell), e.g., to allow entry of the iRNA agent into a cell.

[0904] In one aspect, the invention relates to a composition comprising an iRNA agent (e.g., an iRNA or sRNA agent described herein) linked to an amphipathic molecule. The iRNA agent and the amphipathic molecule may be held in continuous contact with one another by either covalent or noncovalent linkages.

[0905] The amphipathic molecule of the composition or construct is preferably other than a phospholipid, e.g., other than a micelle, membrane or membrane fragment.

[0906] The amphipathic molecule of the composition or construct is preferably a polymer. The polymer may include two or more amphipathic subunits. One or more hydrophilic groups and one or more hydrophobic groups may be present on the polymer. The polymer may have a repeating secondary structure as well as a first face and a second face. The distribution of the hydrophilic groups and the hydrophobic groups along the repeating secondary structure can be such that one face of the polymer is a hydrophilic face and the other face of the polymer is a hydrophobic face.

[0907] The amphipathic molecule can be a polypeptide, e.g., a polypeptide comprising an α-helical conformation as its secondary structure.

[0908] In one embodiment, the amphipathic polymer includes one or more subunits containing one or more cyclic moiety (e.g., a cyclic moiety having one or more hydrophilic groups and/or one or more hydrophobic groups). In one embodiment, the polymer is a polymer of cyclic moieties such that the moieties have alternating hydrophobic and hydrophilic groups. For example, the subunit may contain a steroid, e.g., cholic acid. In another example, the subunit may contain an aromatic moiety. The aromatic moiety may be one that can exhibit atropisomerism, e.g., a 2,2′-bis(substituted)-1-1′-binaphthyl or a 2,2′-bis(substituted) biphenyl. A subunit may include an aromatic moiety of Formula (M):

[0909]

[0910] Referring to Formula M, R₁ is C₁-C₁₀₀ alkyl optionally substituted with aryl, alkenyl, alkynyl, alkoxy or halo and/or optionally inserted with O, S, alkenyl or alkynyl; C₁-C₁₀₀ perfluoroalkyl; or OR₅.

[0911] R₂ is hydroxy; nitro; sulfate; phosphate; phosphate ester; sulfonic acid; OR₆; or C₁-C₁₀₀ alkyl optionally substituted with hydroxy, halo, nitro, aryl or alkyl sulfinyl, aryl or alkyl sulfonyl, sulfate, sulfonic acid, phosphate, phosphate ester, substituted or unsubstituted aryl, carboxyl, carboxylate, amino carbonyl, or alkoxycarbonyl, and/or optionally inserted with O, NH, S, S(O), SO₂, alkenyl, or alkynyl.

[0912] R₃ is hydrogen, or when taken together with R₄ forms a fused phenyl ring.

[0913] R₄ is hydrogen, or when taken together with R₃ forms a fused phenyl ring.

[0914] R₅ is C₁-C₁₀₀ alkyl optionally substituted with aryl, alkenyl, alkynyl, alkoxy or halo and/or optionally inserted with O, S, alkenyl or alkynyl; or C₁-C₁₀₀ perfluoroalkyl; and R₆ is C₁-C₁₀₀ alkyl optionally substituted with hydroxy, halo, nitro, aryl or alkyl sulfinyl, aryl or alkyl sulfonyl, sulfate, sulfonic acid, phosphate, phosphate ester, substituted or unsubstituted aryl, carboxyl, carboxylate, amino carbonyl, or alkoxycarbonyl, and/or optionally inserted with O, NH, S, S(O), SO₂, alkenyl, or alkynyl.

[0915] Increasing Cellular Uptake of dsRNAs

[0916] A method of the invention that can include the administration of an iRNA agent and a drug that affects the uptake of the iRNA agent into the cell. The drug can be administered before, after, or at the same time that the iRNA agent is administered. The drug can be covalently linked to the iRNA agent. The drug can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB. The drug can have a transient effect on the cell.

[0917] The drug can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0918] The drug can also increase the uptake of the iRNA agent into the cell by activating an inflammatory response, for example. Exemplary drug's that would have such an effect include tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, or gamma interferon.

[0919] iRNA Conjugates

[0920] An iRNA agent can be coupled, e.g., covalently coupled, to a second agent. For example, an iRNA agent used to treat a particular disorder can be coupled to a second therapeutic agent, e.g., an agent other than the iRNA agent. The second therapeutic agent can be one which is directed to the treatment of the same disorder. For example, in the case of an iRNA used to treat a disorder characterized by unwanted cell proliferation, e.g., cancer, the iRNA agent can be coupled to a second agent which has an anti-cancer effect. For example, it can be coupled to an agent which stimulates the immune system, e.g., a CpG motif, or more generally an agent that activates a toll-like receptor and/or increases the production of gamma interferon.

iRNA Production

[0921] An iRNA can be produced, e.g., in bulk, by a variety of methods. Exemplary methods include: organic synthesis and RNA cleavage, e.g., in vitro cleavage.

[0922] Organic Synthesis

[0923] An iRNA can be made by separately synthesizing each respective strand of a double-stranded RNA molecule. The component strands can then be annealed.

[0924] A large bioreactor, e.g., the OligoPilot II from Pharmacia Biotec AB (Uppsala Sweden), can be used to produce a large amount of a particular RNA strand for a given iRNA. The OligoPilotlII reactor can efficiently couple a nucleotide using only a 1.5 molar excess of a phosphoramidite nucleotide. To make an RNA strand, ribonucleotides amidites are used. Standard cycles of monomer addition can be used to synthesize the 21 to 23 nucleotide strand for the iRNA. Typically, the two complementary strands are produced separately and then annealed, e.g., after release from the solid support and deprotection.

[0925] Organic synthesis can be used to produce a discrete iRNA species. The complementary of the species to a particular target gene can be precisely specified. For example, the species may be complementary to a region that includes a polymorphism, e.g., a single nucleotide polymorphism. Further the location of the polymorphism can be precisely defined. In some embodiments, the polymorphism is located in an internal region, e.g., at least 4, 5, 7, or 9 nucleotides from one or both of the termini.

[0926] dsRNA Cleavage

[0927] iRNAs can also be made by cleaving a larger ds iRNA. The cleavage can be mediated in vitro or in vivo. For example, to produce iRNAs by cleavage in vitro, the following method can be used:

[0928] In vitro transcription. dsRNA is produced by transcribing a nucleic acid (DNA) segment in both directions. For example, the HiScribe™ RNAi transcription kit (New England Biolabs) provides a vector and a method for producing a dsRNA for a nucleic acid segment that is cloned into the vector at a position flanked on either side by a T7 promoter. Separate templates are generated for T7 transcription of the two complementary strands for the dsRNA. The templates are transcribed in vitro by addition of T7 RNA polymerase and dsRNA is produced. Similar methods using PCR and/or other RNA polymerases (e.g., T3 or SP6 polymerase) can also be used. In one embodiment, RNA generated by this method is carefully purified to remove endotoxins that may contaminate preparations of the recombinant enzymes.

[0929] In vitro cleavage. dsRNA is cleaved in vitro into iRNAs, for example, using a Dicer or comparable RNAse III-based activity. For example, the dsRNA can be incubated in an in vitro extract from Drosophila or using purified components, e.g. a purified RNAse or RISC complex (RNA-induced silencing complex). See, e.g., Ketting et al. Genes Dev 2001 Oct. 15; 15(20): 2654-9. and Hammond Science 2001 Aug. 10; 293(5532): 1146-50.

[0930] dsRNA cleavage generally produces a plurality of iRNA species, each being a particular 21 to 23 nt fragment of a source dsRNA molecule. For example, iRNAs that include sequences complementary to overlapping regions and adjacent regions of a source dsRNA molecule may be present.

[0931] Regardless of the method of synthesis, the iRNA preparation can be prepared in a solution (e.g., an aqueous and/or organic solution) that is appropriate for formulation. For example, the iRNA preparation can be precipitated and redissolved in pure double-distilled water, and lyophilized. The dried iRNA can then be resuspended in a solution appropriate for the intended formulation process.

[0932] Synthesis of modified and nucleotide surrogate iRNA agents is discussed below.

[0933] Formulation

[0934] The iRNA agents described herein can be formulated for administration to a subject

[0935] For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention.

[0936] A formulated iRNA composition can assume a variety of states. In some examples, the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the iRNA is in an aqueous phase, e.g., in a solution that includes water.

[0937] The aqueous phase or the crystalline compositions can, e.g., be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase) or a particle (e.g., a microparticle as can be appropriate for a crystalline composition). Generally, the iRNA composition is formulated in a manner that is compatible with the intended method of administration (see, below).

[0938] In particular embodiments, the composition is prepared by at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication with a lipid, freeze-drying, condensation and other self-assembly.

[0939] A iRNA preparation can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes a iRNA, e.g., a protein that complexes with iRNA to form an iRNP. Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg²⁺), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.

[0940] In one embodiment, the iRNA preparation includes another iRNA agent, e.g., a second iRNA that can mediated RNAi with respect to a second gene, or with respect to the same gene. Still other preparation can include at least 3, 5, ten, twenty, fifty, or a hundred or more different iRNA species. Such iRNAs can mediated RNAi with respect to a similar number of different genes.

[0941] In one embodiment, the iRNA preparation includes at least a second therapeutic agent (e.g., an agent other than an RNA or a DNA). For example, a iRNA composition for the treatment of a viral disease, e.g. HIV, might include a known antiviral agent (e.g., a protease inhibitor or reverse transcriptase inhibitor). In another example, a iRNA composition for the treatment of a cancer might further comprise a chemotherapeutic agent.

[0942] Exemplary formulations are discussed below:

[0943] Liposomes

[0944] For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA s agents, and such practice is within the invention. An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) preparation can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the iRNA composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the iRNA are delivered into the cell where the iRNA can specifically bind to a target RNA and can mediate RNAi. In some cases the liposomes are also specifically targeted, e.g., to direct the iRNA to particular cell types.

[0945] A liposome containing a iRNA can be prepared by a variety of methods.

[0946] In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The iRNA preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the iRNA and condense around the iRNA to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of iRNA.

[0947] If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation.

[0948] Further description of methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are described in, e.g., WO 96/37194. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al. Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adapted to packaging iRNA preparations into liposomes.

[0949] Liposomes that are pH-sensitive or negatively-charged, entrap nucleic acid molecules rather than complex with them. Since both the nucleic acid molecules and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid molecules are entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 19, (1992)269-274).

[0950] One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0951] Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

[0952] In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver iRNAs to macrophages.

[0953] Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated iRNAs in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

[0954] A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of iRNA (see, e.g., Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

[0955] A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.

[0956] Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).

[0957] Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.

[0958] Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer iRNA, into the skin. In some implementations, liposomes are used for delivering iRNA to epidermal cells and also to enhance the penetration of iRNA into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

[0959] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with iRNA are useful for treating a dermatological disorder.

[0960] Liposomes that include iRNA can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include iRNA can be delivered, for example, subcutaneously by infection in order to deliver iRNA to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading. The iRNA agents can include an RRMS tethered to a moiety which improves association with a liposome.

[0961] Surfactants

[0962] For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes (see above). iRNA (or a precursor, e.g., a larger dsRNA which can be processed into a iRNA, or a DNA which encodes a iRNA or precursor) compositions can include a surfactant. In one embodiment, the iRNA is formulated as an emulsion that includes a surfactant. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0963] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0964] If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

[0965] If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

[0966] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

[0967] The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0968] Micelles and Other Membranous Formulations

[0969] For ease of exposition the micelles and other formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these micelles and other formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. The iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof)) composition can be provided as a micellar formulation. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

[0970] A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the iRNA composition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing is preferred in order to provide smaller size micelles.

[0971] In one method a first micellar composition is prepared which contains the iRNA composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the iRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.

[0972] Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.

[0973] For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e. there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g. through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.

[0974] The preferred propellants are hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Even more preferred is HFA 134a (1,1,1,2 tetrafluoroethane).

[0975] The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g. at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.

[0976] The iRNA agents can include an RRMS tethered to a moiety which improves association with a micelle or other membranous formulation.

[0977] Particles

[0978] For ease of exposition the particles, formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these particles, formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. In another embodiment, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) preparations may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques. See below for further description.

[0979] Sustained-Release Formulations. An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein can be formulated for controlled, e.g., slow release. Controlled release can be achieved by disposing the iRNA within a structure or substance which impedes its release. E.g., iRNA can be disposed within a porous matrix or in an erodible matrix, either of which allow release of the iRNA over a period of time.

[0980] Polymeric particles, e.g., polymeric in microparticles can be used as a sustained-release reservoir of iRNA that is taken up by cells only released from the microparticle through biodegradation. The polymeric particles in this embodiment should therefore be large enough to preclude phagocytosis (e.g., larger than 10 μm and preferably larger than 20 μm). Such particles can be produced by the same methods to make smaller particles, but with less vigorous mixing of the first and second emulsions. That is to say, a lower homogenization speed, vortex mixing speed, or sonication setting can be used to obtain particles having a diameter around 100 μm rather than 10 μm. The time of mixing also can be altered.

[0981] Larger microparticles can be formulated as a suspension, a powder, or an implantable solid, to be delivered by intramuscular, subcutaneous, intradermal, intravenous, or intraperitoneal injection; via inhalation (intranasal or intrapulmonary); orally; or by implantation. These particles are useful for delivery of any iRNA when slow release over a relatively long term is desired. The rate of degradation, and consequently of release, varies with the polymeric formulation.

[0982] Microparticles preferably include pores, voids, hollows, defects or other interstitial spaces that allow the fluid suspension medium to freely permeate or perfuse the particulate boundary. For example, the perforated microstructures can be used to form hollow, porous spray dried microspheres.

[0983] Polymeric particles containing iRNA (e.g., a sRNA) can be made using a double emulsion technique, for instance. First, the polymer is dissolved in an organic solvent. A preferred polymer is polylactic-co-glycolic acid (PLGA), with a lactic/glycolic acid weight ratio of 65:35, 50:50, or 75:25. Next, a sample of nucleic acid suspended in aqueous solution is added to the polymer solution and the two solutions are mixed to form a first emulsion. The solutions can be mixed by vortexing or shaking, and in a preferred method, the mixture can be sonicated. Most preferable is any method by which the nucleic acid receives the least amount of damage in the form of nicking, shearing, or degradation, while still allowing the formation of an appropriate emulsion. For example, acceptable results can be obtained with a Vibra-cell model VC-250 sonicator with a ⅛″ microtip probe, at setting #3.

[0984] Spray-Drying.

[0985] An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof)) can be prepared by spray drying. Spray dried iRNA can be administered to a subject or be subjected to further formulation. A pharmaceutical composition of iRNA can be prepared by spray drying a homogeneous aqueous mixture that includes a iRNA under conditions sufficient to provide a dispersible powdered composition, e.g., a pharmaceutical composition. The material for spray drying can also include one or more of: a pharmaceutically acceptable excipient, or a dispersibility-enhancing amount of a physiologically acceptable, water-soluble protein. The spray-dried product can be a dispersible powder that includes the iRNA.

[0986] Spray drying is a process that converts a liquid or slurry material to a dried particulate form. Spray drying can be used to provide powdered material for various administrative routes including inhalation. See, for example, M. Sacchetti and M. M. Van Oort in: Inhalation Aerosols: Physical and Biological Basis for Therapy, A. J. Hickey, ed. Marcel Dekkar, N.Y., 1996.

[0987] Spray drying can include atomizing a solution, emulsion, or suspension to form a fine mist of droplets and drying the droplets. The mist can be projected into a drying chamber (e.g., a vessel, tank, tubing, or coil) where it contacts a drying gas. The mist can include solid or liquid pore forming agents. The solvent and pore forming agents evaporate from the droplets into the drying gas to solidify the droplets, simultaneously forming pores throughout the solid. The solid (typically in a powder, particulate form) then is separated from the drying gas and collected.

[0988] Spray drying includes bringing together a highly dispersed liquid, and a sufficient volume of air (e.g., hot air) to produce evaporation and drying of the liquid droplets. The preparation to be spray dried can be any solution, course suspension, slurry, colloidal dispersion, or paste that may be atomized using the selected spray drying apparatus. Typically, the feed is sprayed into a current of warm filtered air that evaporates the solvent and conveys the dried product to a collector. The spent air is then exhausted with the solvent. Several different types of apparatus may be used to provide the desired product. For example, commercial spray dryers manufactured by Buchi Ltd. or Niro Corp. can effectively produce particles of desired size.

[0989] Spray-dried powdered particles can be approximately spherical in shape, nearly uniform in size and frequently hollow. There may be some degree of irregularity in shape depending upon the incorporated medicament and the spray drying conditions. In many instances the dispersion stability of spray-dried microspheres appears to be more effective if an inflating agent (or blowing agent) is used in their production. Particularly preferred embodiments may comprise an emulsion with an inflating agent as the disperse or continuous phase (the other phase being aqueous in nature). An inflating agent is preferably dispersed with a surfactant solution, using, for instance, a commercially available microfluidizer at a pressure of about 5000 to 15,000 psi. This process forms an emulsion, preferably stabilized by an incorporated surfactant, typically comprising submicron droplets of water immiscible blowing agent dispersed in an aqueous continuous phase. The formation of such dispersions using this and other techniques are common and well known to those in the art. The blowing agent is preferably a fluorinated compound (e.g. perfluorohexane, perfluorooctyl bromide, perfluorodecalin, perfluorobutyl ethane) which vaporizes during the spray-drying process, leaving behind generally hollow, porous aerodynamically light microspheres. As will be discussed in more detail below, other suitable blowing agents include chloroform, freons, and hydrocarbons. Nitrogen gas and carbon dioxide are also contemplated as a suitable blowing agent.

[0990] Although the perforated microstructures are preferably formed using a blowing agent as described above, it will be appreciated that, in some instances, no blowing agent is required and an aqueous dispersion of the medicament and surfactant(s) are spray dried directly. In such cases, the formulation may be amenable to process conditions (e.g., elevated temperatures) that generally lead to the formation of hollow, relatively porous microparticles. Moreover, the medicament may possess special physicochemical properties (e.g., high crystallinity, elevated melting temperature, surface activity, etc.) that make it particularly suitable for use in such techniques.

[0991] The perforated microstructures may optionally be associated with, or comprise, one or more surfactants. Moreover, miscible surfactants may optionally be combined with the suspension medium liquid phase. It will be appreciated by those skilled in the art that the use of surfactants may further increase dispersion stability, simplify formulation procedures or increase bioavailability upon administration. Of course combinations of surfactants, including the use of one or more in the liquid phase and one or more associated with the perforated microstructures are contemplated as being within the scope of the invention. By “associated with or comprise” it is meant that the structural matrix or perforated microstructure may incorporate, adsorb, absorb, be coated with or be formed by the surfactant.

[0992] Surfactants suitable for use include any compound or composition that aids in the formation and maintenance of the stabilized respiratory dispersions by forming a layer at the interface between the structural matrix and the suspension medium. The surfactant may comprise a single compound or any combination of compounds, such as in the case of co-surfactants. Particularly preferred surfactants are substantially insoluble in the propellant, nonfluorinated, and selected from the group consisting of saturated and unsaturated lipids, nonionic detergents, nonionic block copolymers, ionic surfactants, and combinations of such agents. It should be emphasized that, in addition to the aforementioned surfactants, suitable (i.e. biocompatible) fluorinated surfactants are compatible with the teachings herein and may be used to provide the desired stabilized preparations.

[0993] Lipids, including phospholipids, from both natural and synthetic sources may be used in varying concentrations to form a structural matrix. Generally, compatible lipids comprise those that have a gel to liquid crystal phase transition greater than about 40° C. Preferably, the incorporated lipids are relatively long chain (i.e. C₆-C₂₂) saturated lipids and more preferably comprise phospholipids. Exemplary phospholipids useful in the disclosed stabilized preparations comprise egg phosphatidylcholine, dilauroylphosphatidylcholine, dioleylphosphatidylcholine, dipalmitoylphosphatidyl-choline, disteroylphosphatidylcholine, short-chain phosphatidylcholines, phosphatidylethanolamine, dioleylphosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, glycolipids, ganglioside GM 1, sphingomyelin, phosphatidic acid, cardiolipin; lipids bearing polymer chains such as, polyethylene glycol, chitin, hyaluronic acid, or polyvinylpyrrolidone; lipids bearing sulfonated mono-, di-, and polysaccharides; fatty acids such as palmitic acid, stearic acid, and oleic acid; cholesterol, cholesterol esters, and cholesterol hemisuccinate. Due to their excellent biocompatibility characteristics, phospholipids and combinations of phospholipids and poloxamers are particularly suitable for use in the stabilized dispersions disclosed herein.

[0994] Compatible nonionic detergents comprise: sorbitan esters including sorbitan trioleate (Spans™ 85), sorbitan sesquioleate, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene (20) sorbitan monolaurate, and polyoxyethylene (20) sorbitan monooleate, oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, lauryl polyoxyethylene (4) ether, glycerol esters, and sucrose esters. Other suitable nonionic detergents can be easily identified using McCutcheon's Emulsifiers and Detergents (McPublishing Co., Glen Rock, N.J.). Preferred block copolymers include diblock and triblock copolymers of polyoxyethylene and polyoxypropylene, including poloxamer 188 (Pluronic® F68), poloxamer 407 (Pluronic® F-127), and poloxamer 338. Ionic surfactants such as sodium sulfosuccinate, and fatty acid soaps may also be utilized. In preferred embodiments, the microstructures may comprise oleic acid or its alkali salt.

[0995] In addition to the aforementioned surfactants, cationic surfactants or lipids are preferred especially in the case of delivery of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). Examples of suitable cationic lipids include: DOTMA, N-[-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium-chloride; DOTAP, 1,2-dioleyloxy-3-(trimethylammonio)propane; and DOTB, 1,2-dioleyl-3-(4′-trimethylammonio)butanoyl-sn-glycerol. Polycationic amino acids such as polylysine, and polyarginine are also contemplated.

[0996] For the spraying process, such spraying methods as rotary atomization, pressure atomization and two-fluid atomization can be used. Examples of the devices used in these processes include “Parubisu [phonetic rendering] Mini-Spray GA-32” and “Parubisu Spray Drier DL-41”, manufactured by Yamato Chemical Co., or “Spray Drier CL-8,” “Spray Drier L-8,” “Spray Drier FL-12,” “Spray Drier FL-16” or “Spray Drier FL-20,” manufactured by Okawara Kakoki Co., can be used for the method of spraying using rotary-disk atomizer.

[0997] While no particular restrictions are placed on the gas used to dry the sprayed material, it is recommended to use air, nitrogen gas or an inert gas. The temperature of the inlet of the gas used to dry the sprayed materials such that it does not cause heat deactivation of the sprayed material. The range of temperatures may vary between about 50° C. to about 200° C., preferably between about 50° C. and 100° C. The temperature of the outlet gas used to dry the sprayed material, may vary between about 0° C. and about 150° C., preferably between 0° C. and 90° C., and even more preferably between 0° C. and 60° C.

[0998] The spray drying is done under conditions that result in substantially amorphous powder of homogeneous constitution having a particle size that is respirable, a low moisture content and flow characteristics that allow for ready aerosolization. Preferably the particle size of the resulting powder is such that more than about 98% of the mass is in particles having a diameter of about 10 μm or less with about 90% of the mass being in particles having a diameter less than 5 μm. Alternatively, about 95% of the mass will have particles with a diameter of less than 10 μm with about 80% of the mass of the particles having a diameter of less than 5 μm.

[0999] The dispersible pharmaceutical-based dry powders that include the iRNA preparation may optionally be combined with pharmaceutical carriers or excipients which are suitable for respiratory and pulmonary administration. Such carriers may serve simply as bulking agents when it is desired to reduce the iRNA concentration in the powder which is being delivered to a patient, but may also serve to enhance the stability of the iRNA compositions and to improve the dispersibility of the powder within a powder dispersion device in order to provide more efficient and reproducible delivery of the iRNA and to improve handling characteristics of the iRNA such as flowability and consistency to facilitate manufacturing and powder filling.

[1000] Such carrier materials may be combined with the drug prior to spray drying, i.e., by adding the carrier material to the purified bulk solution. In that way, the carrier particles will be formed simultaneously with the drug particles to produce a homogeneous powder. Alternatively, the carriers may be separately prepared in a dry powder form and combined with the dry powder drug by blending. The powder carriers will usually be crystalline (to avoid water absorption), but might in some cases be amorphous or mixtures of crystalline and amorphous. The size of the carrier particles may be selected to improve the flowability of the drug powder, typically being in the range from 25 μm to 100 μm. A preferred carrier material is crystalline lactose having a size in the above-stated range.

[1001] Powders prepared by any of the above methods will be collected from the spray dryer in a conventional manner for subsequent use. For use as pharmaceuticals and other purposes, it will frequently be desirable to disrupt any agglomerates which may have formed by screening or other conventional techniques. For pharmaceutical uses, the dry powder formulations will usually be measured into a single dose, and the single dose sealed into a package. Such packages are particularly useful for dispersion in dry powder inhalers, as described in detail below. Alternatively, the powders may be packaged in multiple-dose containers.

[1002] Methods for spray drying hydrophobic and other drugs and components are described in U.S. Pat. Nos. 5,000,888; 5,026,550; 4,670,419, 4,540,602; and 4,486,435. Bloch and Speison (1983) Pharm. Acta Helv 58:14-22 teaches spray drying of hydrochlorothiazide and chlorthalidone (lipophilic drugs) and a hydrophilic adjuvant (pentaerythritol) in azeotropic solvents of dioxane-water and 2-ethoxyethanol-water. A number of Japanese Patent application Abstracts relate to spray drying of hydrophilic-hydrophobic product combinations, including JP806766; JP7242568; JP7101884; JP7101883; JP71018982; JP 7101881; and JP4036233. Other foreign patent publications relevant to spray drying hydrophilic-hydrophobic product combinations include FR 2594693; DE 2209477; and WO 88/07870.

[1003] Lyophilization.

[1004] An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) preparation can be made by lyophilization. Lyophilization is a freeze-drying process in which water is sublimed from the composition after it is frozen. The particular advantage associated with the lyophilization process is that biologicals and pharmaceuticals that are relatively unstable in an aqueous solution can be dried without elevated temperatures (thereby eliminating the adverse thermal effects), and then stored in a dry state where there are few stability problems. With respect to the instant invention such techniques are particularly compatible with the incorporation of nucleic acids in perforated microstructures without compromising physiological activity. Methods for providing lyophilized particulates are known to those of skill in the art and it would clearly not require undue experimentation to provide dispersion compatible microstructures in accordance with the teachings herein. Accordingly, to the extent that lyophilization processes may be used to provide microstructures having the desired porosity and size, they are conformance with the teachings herein and are expressly contemplated as being within the scope of the instant invention.

[1005] Targeting

[1006] For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNAs. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention.

[1007] In some embodiments, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) is targeted to a particular cell. For example, a liposome or particle or other structure that includes a iRNA can also include a targeting moiety that recognizes a specific molecule on a target cell. The targeting moiety can be a molecule with a specific affinity for a target cell. Targeting moieties can include antibodies directed against a protein found on the surface of a target cell, or the ligand or a receptor-binding portion of a ligand for a molecule found on the surface of a target cell. For example, the targeting moiety can recognize a cancer-specific antigen (e.g., CA 15-3, CA 19-9, CEA, or HER2/neu.) or a viral antigen, thus delivering the iRNA to a cancer cell or a virus-infected cell. Exemplary targeting moieties include antibodies (such as IgM, IgG, IgA, IgD, and the like, or a functional portions thereof), ligands for cell surface receptors (e.g., ectodomains thereof).

[1008] Table 3 provides a number of antigens which can be used to target selected cells.

[1009]

	TABLE 3
	ANTIGEN	Exemplary tumor tissue
	CEA (carcinoembryonic antigen)	colon, breast, lung
	PSA (prostate specific antigen)	prostate cancer
	CA-125	ovarian cancer
	CA 15-3	breast cancer
	CA 19-9	breast cancer
	HER2/neu	breast cancer
	α-feto protein	testicular cancer,
		hepatic cancer
	β-HCG (human chorionic	testicular cancer,
	gonadotropin)	choriocarcinoma
	MUC-1	breast cancer
	Estrogen receptor	breast cancer, uterine cancer
	Progesterone receptor	breast cancer, uterine cancer
	EGFr (epidermal growth	bladder cancer
	factor receptor)

[1010] In one embodiment, the targeting moiety is attached to a liposome. For example, U.S. Pat. No. 6,245,427 describes a method for targeting a liposome using a protein or peptide. In another example, a cationic lipid component of the liposome is derivatized with a targeting moiety. For example, WO 96/37194 describes converting N-glutaryldioleoylphosphatidyl ethanolamine to a N-hydroxysuccinimide activated ester. The product was then coupled to an RGD peptide.

[1011] Genes and Diseases

[1012] In one aspect, the invention features, a method of treating a subject at risk for or afflicted with unwanted cell proliferation, e.g., malignant or nonmalignant cell proliferation. The method includes:

[1013] providing an iRNA agent, e.g., an sRNA or iRNA agent described herein, e.g., an iRNA having a structure described herein, where the iRNA is homologous to and can silence, e.g., by cleavage, a gene which promotes unwanted cell proliferation;

[1014] administering an iRNA agent, e.g., an sRNA or iRNA agent described herein to a subject, preferably a human subject,

[1015] thereby treating the subject.

[1016] In a preferred embodiment the gene is a growth factor or growth factor receptor gene, a kinase, e.g., a protein tyrosine, serine or threonine kinase gene, an adaptor protein gene, a gene encoding a G protein superfamily molecule, or a gene encoding a transcription factor.

[1017] In a preferred embodiment the iRNA agent silences the PDGF beta gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PDGF beta expression, e.g., testicular and lung cancers.

[1018] In another preferred embodiment the iRNA agent silences the Erb-B gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Erb-B expression, e.g., breast cancer.

[1019] In a preferred embodiment the iRNA agent silences the Src gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Src expression, e.g., colon cancers.

[1020] In a preferred embodiment the iRNA agent silences the CRK gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted CRK expression, e.g., colon and lung cancers.

[1021] In a preferred embodiment the iRNA agent silences the GRB2 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted GRB2 expression, e.g., squamous cell carcinoma.

[1022] In another preferred embodiment the iRNA agent silences the RAS gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted RAS expression, e.g., pancreatic, colon and lung cancers, and chronic leukemia.

[1023] In another preferred embodiment the iRNA agent silences the MEKK gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MEKK expression, e.g., squamous cell carcinoma, melanoma or leukemia.

[1024] In another preferred embodiment the iRNA agent silences the JNK gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted JNK expression, e.g., pancreatic or breast cancers.

[1025] In a preferred embodiment the iRNA agent silences the RAF gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted RAF expression, e.g., lung cancer or leukemia.

[1026] In a preferred embodiment the iRNA agent silences the Erk1/2 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Erk1/2 expression, e.g., lung cancer.

[1027] In another preferred embodiment the iRNA agent silences the PCNA (p21) gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PCNA expression, e.g., lung cancer.

[1028] In a preferred embodiment the iRNA agent silences the MYB gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MYB expression, e.g., colon cancer or chronic myelogenous leukemia.

[1029] In a preferred embodiment the iRNA agent silences the c-MYC gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted c-MYC expression, e.g., Burkitt's lymphoma or neuroblastoma.

[1030] In another preferred embodiment the iRNA agent silences the JUN gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted JUN expression, e.g., ovarian, prostate or breast cancers.

[1031] In another preferred embodiment the iRNA agent silences the FOS gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted FOS expression, e.g., skin or prostate cancers.

[1032] In a preferred embodiment the iRNA agent silences the BCL-2 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted BCL-2 expression, e.g., lung or prostate cancers or Non-Hodgkin lymphoma.

[1033] In a preferred embodiment the iRNA agent silences the Cyclin D gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Cyclin D expression, e.g., esophageal and colon cancers.

[1034] In a preferred embodiment the iRNA agent silences the VEGF gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted VEGF expression, e.g., esophageal and colon cancers.

[1035] In a preferred embodiment the iRNA agent silences the EGFR gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted EGFR expression, e.g., breast cancer.

[1036] In another preferred embodiment the iRNA agent silences the Cyclin A gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Cyclin A expression, e.g., lung and cervical cancers.

[1037] In another preferred embodiment the iRNA agent silences the Cyclin E gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Cyclin E expression, e.g., lung and breast cancers.

[1038] In another preferred embodiment the iRNA agent silences the WNT-1 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted WNT-1 expression, e.g., basal cell carcinoma.

[1039] In another preferred embodiment the iRNA agent silences the beta-catenin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted beta-catenin expression, e.g., adenocarcinoma or hepatocellular carcinoma.

[1040] In another preferred embodiment the iRNA agent silences the c-MET gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted c-MET expression, e.g., hepatocellular carcinoma.

[1041] In another preferred embodiment the iRNA agent silences the PKC gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PKC expression, e.g., breast cancer.

[1042] In a preferred embodiment the iRNA agent silences the NFKB gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted NFKB expression, e.g., breast cancer.

[1043] In a preferred embodiment the iRNA agent silences the STAT3 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted STAT3 expression, e.g., prostate cancer.

[1044] In another preferred embodiment the iRNA agent silences the surviving gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted surviving expression, e.g., cervical or pancreatic cancers.

[1045] In another preferred embodiment the iRNA agent silences the Her 2/Neu gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Her 2/Neu expression, e.g., breast cancer.

[1046] In another preferred embodiment the iRNA agent silences the topoisomerase I gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted topoisomerase I expression, e.g., ovarian and colon cancers.

[1047] In a preferred embodiment the iRNA agent silences the topoisomerase II alpha gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted topoisomerase II expression, e.g., breast and colon cancers.

[1048] In a preferred embodiment the iRNA agent silences mutations in the p73 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p73 expression, e.g., colorectal adenocarcinoma.

[1049] In a preferred embodiment the iRNA agent silences mutations in the p21(WAF1/CIP1) gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p21(WAF1/CIP1) expression, e.g., liver cancer.

[1050] In a preferred embodiment the iRNA agent silences mutations in the p27(KIP1) gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p27(KIP1) expression, e.g., liver cancer.

[1051] In a preferred embodiment the iRNA agent silences mutations in the PPM 1D gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PPM 1D expression, e.g., breast cancer.

[1052] In a preferred embodiment the iRNA agent silences mutations in the RAS gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted RAS expression, e.g., breast cancer.

[1053] In another preferred embodiment the iRNA agent silences mutations in the caveolin I gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted caveolin I expression, e.g., esophageal squamous cell carcinoma.

[1054] In another preferred embodiment the iRNA agent silences mutations in the MIB I gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MIB I expression, e.g., male breast carcinoma (MBC).

[1055] In another preferred embodiment the iRNA agent silences mutations in the MTAI gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MTAI expression, e.g., ovarian carcinoma.

[1056] In another preferred embodiment the iRNA agent silences mutations in the M 68 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted M 68 expression, e.g., human adenocarcinomas of the esophagus, stomach, colon, and rectum.

[1057] In preferred embodiments the iRNA agent silences mutations in tumor suppressor genes, and thus can be used as a method to promote apoptotic activity in combination with chemotherapeutics.

[1058] In a preferred embodiment the iRNA agent silences mutations in the p53 tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p53 expression, e.g., gall bladder, pancreatic and lung cancers.

[1059] In a preferred embodiment the iRNA agent silences mutations in the p53 family member DN-p63, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted DN-p63 expression, e.g., squamous cell carcinoma

[1060] In a preferred embodiment the iRNA agent silences mutations in the pRb tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted pRb expression, e.g., oral squamous cell carcinoma

[1061] In a preferred embodiment the iRNA agent silences mutations in the APC1 tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted APC1 expression, e.g., colon cancer.

[1062] In a preferred embodiment the iRNA agent silences mutations in the BRCA1 tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted BRCA1 expression, e.g., breast cancer.

[1063] In a preferred embodiment the iRNA agent silences mutations in the PTEN tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PTEN expression, e.g., hamartomas, gliomas, and prostate and endometrial cancers.

[1064] In a preferred embodiment the iRNA agent silences MLL fusion genes, e.g., MLL-AF9, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MLL fusion gene expression, e.g., acute leukemias.

[1065] In another preferred embodiment the iRNA agent silences the BCR/ABL fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted BCR/ABL fusion gene expression, e.g., acute and chronic leukemias.

[1066] In another preferred embodiment the iRNA agent silences the TEL/AML1 fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted TEL/AML1 fusion gene expression, e.g., childhood acute leukemia.

[1067] In another preferred embodiment the iRNA agent silences the EWS/FLI1 fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted EWS/FLI1 fusion gene expression, e.g., Ewing Sarcoma.

[1068] In another preferred embodiment the iRNA agent silences the TLS/FUS1 fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted TLS/FUS1 fusion gene expression, e.g., Myxoid liposarcoma.

[1069] In another preferred embodiment the iRNA agent silences the PAX3/FKHR fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PAX3/FKHR fusion gene expression, e.g., Myxoid liposarcoma.

[1070] In another preferred embodiment the iRNA agent silences the AML1/ETO fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted AML1/ETO fusion gene expression, e.g., acute leukemia.

[1071] In another aspect, the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with a disease or disorder that may benefit by angiogenesis inhibition e.g., cancer. The method includes:

[1072] providing an iRNA agent, e.g., an iRNA agent having a structure described herein, which iRNA agent is homologous to and can silence, e.g., by cleavage, a gene which mediates angiogenesis;

[1073] administering the iRNA agent to a subject,

[1074] thereby treating the subject.

[1075] In a preferred embodiment the iRNA agent silences the alpha v-integrin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted alpha V integrin, e.g., brain tumors or tumors of epithelial origin.

[1076] In a preferred embodiment the iRNA agent silences the Flt-1 receptor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Flt-1 receptors, e.g. Cancer and rheumatoid arthritis.

[1077] In a preferred embodiment the iRNA agent silences the tubulin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted tubulin, e.g. Cancer and retinal neovascularization.

[1078] In a preferred embodiment the iRNA agent silences the tubulin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted tubulin, e.g. Cancer and retinal neovascularization.

[1079] In another aspect, the invention features a method of treating a subject infected with a virus or at risk for or afflicted with a disorder or disease associated with a viral infection. The method includes:

[1080] providing an iRNA agent, e.g., and iRNA agent having a structure described herein, which iRNA agent is homologous to and can silence, e.g., by cleavage, a viral gene of a cellular gene which mediates viral function, e.g., entry or growth;

[1081] administering the iRNA agent to a subject, preferably a human subject,

[1082] thereby treating the subject.

[1083] Thus, the invention provides for a method of treating patients infected by the Human Papilloma Virus (HPV) or at risk for or afflicted with a disorder mediated by HPV, e.g., cervical cancer. HPV is linked to 95% of cervical carcinomas and thus an antiviral therapy is an attractive method to treat these cancers and other symptoms of viral infection.

[1084] In a preferred embodiment, the expression of a HPV gene is reduced. In another preferred embodiment, the HPV gene is one of the group of E2, E6, or E7.

[1085] In a preferred embodiment the expression of a human gene that is required for HPV replication is reduced.

[1086] The invention also includes a method of treating patients infected by the Human Immunodeficiency Virus (HIV) or at risk for or afflicted with a disorder mediated by HIV, e.g., Acquired Immune Deficiency Syndrome (AIDS).

[1087] In a preferred embodiment, the expression of a HIV gene is reduced. In another preferred embodiment, the HIV gene is CCR5, Gag, or Rev.

[1088] In a preferred embodiment the expression of a human gene that is required for HIV replication is reduced. In another preferred embodiment, the gene is CD 4 or Tsg101.

[1089] The invention also includes a method for treating patients infected by the Hepatitis B Virus (HBV) or at risk for or afflicted with a disorder mediated by HBV, e.g., cirrhosis and heptocellular carcinoma.

[1090] In a preferred embodiment, the expression of a HBV gene is reduced. In another preferred embodiment, the targeted HBV gene encodes one of the group of the tail region of the HBV core protein, the pre-cregious (pre-c) region, or the cregious (c) region. In another preferred embodiment, a targeted HBV-RNA sequence is comprised of the poly(A) tail.

[1091] In preferred embodiment the expression of a human gene that is required for HBV replication is reduced.

[1092] The invention also provides for a method of treating patients infected by the Hepatitis A Virus (HAV), or at risk for or afflicted with a disorder mediated by HAV.

[1093] In a preferred embodiment the expression of a human gene that is required for HAV replication is reduced.

[1094] The present invention provides for a method of treating patients infected by the Hepatitis C Virus (HCV), or at risk for or afflicted with a disorder mediated by HCV, e.g., cirrhosis

[1095] In a preferred embodiment, the expression of a HCV gene is reduced.

[1096] In another preferred embodiment the expression of a human gene that is required for HCV replication is reduced.

[1097] The present invention also provides for a method of treating patients infected by the any of the group of Hepatitis Viral strains comprising hepatitis D, E, F, G, or H, or patients at risk for or afflicted with a disorder mediated by any of these strains of hepatitis.

[1098] In a preferred embodiment, the expression of a Hepatitis, D, E, F, G, or H gene is reduced.

[1099] In another preferred embodiment the expression of a human gene that is required for hepatitis D, E, F, G or H replication is reduced.

[1100] Methods of the invention also provide for treating patients infected by the Respiratory Syncytial Virus (RSV) or at risk for or afflicted with a disorder mediated by RSV, e.g., lower respiratory tract infection in infants and childhood asthma, pneumonia and other complications, e.g., in the elderly.

[1101] In a preferred embodiment, the expression of a RSV gene is reduced. In another preferred embodiment, the targeted HBV gene encodes one of the group of genes N, L, or P.

[1102] In a preferred embodiment the expression of a human gene that is required for RSV replication is reduced.

[1103] Methods of the invention provide for treating patients infected by the Herpes Simplex Virus (HSV) or at risk for or afflicted with a disorder mediated by HSV, e.g., genital herpes and cold sores as well as life-threatening or sight-impairing disease mainly in immunocompromised patients.

[1104] In a preferred embodiment, the expression of a HSV gene is reduced. In another preferred embodiment, the targeted HSV gene encodes DNA polymerase or the helicase-primase.

[1105] In a preferred embodiment the expression of a human gene that is required for HSV replication is reduced.

[1106] The invention also provides a method for treating patients infected by the herpes Cytomegalovirus (CMV) or at risk for or afflicted with a disorder mediated by CMV, e.g., congenital virus infections and morbidity in immunocompromised patients.

[1107] In a preferred embodiment, the expression of a CMV gene is reduced.

[1108] In a preferred embodiment the expression of a human gene that is required for CMV replication is reduced.

[1109] Methods of the invention also provide for a method of treating patients infected by the herpes Epstein Barr Virus (EBV) or at risk for or afflicted with a disorder mediated by EBV, e.g., NK/T-cell lymphoma, non-Hodgkin lymphoma, and Hodgkin disease.

[1110] In a preferred embodiment, the expression of a EBV gene is reduced.

[1111] In a preferred embodiment the expression of a human gene that is required for EBV replication is reduced.

[1112] Methods of the invention also provide for treating patients infected by Kaposi's Sarcoma-associated Herpes Virus (KSHV), also called human herpesvirus 8, or patients at risk for or afflicted with a disorder mediated by KSHV, e.g., Kaposi's sarcoma, multicentric Castleman's disease and AIDS-associated primary effusion lymphoma.

[1113] In a preferred embodiment, the expression of a KSHV gene is reduced.

[1114] In a preferred embodiment the expression of a human gene that is required for KSHV replication is reduced.

[1115] The invention also includes a method for treating patients infected by the JC Virus (JCV) or a disease or disorder associated with this virus, e.g., progressive multifocal leukoencephalopathy (PML).

[1116] In a preferred embodiment, the expression of a JCV gene is reduced.

[1117] In preferred embodiment the expression of a human gene that is required for JCV replication is reduced.

[1118] Methods of the invention also provide for treating patients infected by the myxovirus or at risk for or afflicted with a disorder mediated by myxovirus, e.g., influenza.

[1119] In a preferred embodiment, the expression of a myxovirus gene is reduced.

[1120] In a preferred embodiment the expression of a human gene that is required for myxovirus replication is reduced.

[1121] Methods of the invention also provide for treating patients infected by the rhinovirus or at risk for of afflicted with a disorder mediated by rhinovirus, e.g., the common cold.

[1122] In a preferred embodiment, the expression of a rhinovirus gene is reduced.

[1123] In preferred embodiment the expression of a human gene that is required for rhinovirus replication is reduced.

[1124] Methods of the invention also provide for treating patients infected by the coronavirus or at risk for of afflicted with a disorder mediated by coronavirus, e.g., the common cold.

[1125] In a preferred embodiment, the expression of a coronavirus gene is reduced.

[1126] In preferred embodiment the expression of a human gene that is required for coronavirus replication is reduced.

[1127] Methods of the invention also provide for treating patients infected by the flavivirus West Nile or at risk for or afflicted with a disorder mediated by West Nile Virus.

[1128] In a preferred embodiment, the expression of a West Nile Virus gene is reduced. In another preferred embodiment, the West Nile Virus gene is one of the group comprising E, NS3, or NS5.

[1129] In a preferred embodiment the expression of a human gene that is required for West Nile Virus replication is reduced.

[1130] Methods of the invention also provide for treating patients infected by the St. Louis Encephalitis flavivirus, or at risk for or afflicted with a disease or disorder associated with this virus, e.g., viral haemorrhagic fever or neurological disease.

[1131] In a preferred embodiment, the expression of a St. Louis Encephalitis gene is reduced.

[1132] In a preferred embodiment the expression of a human gene that is required for St. Louis Encephalitis virus replication is reduced.

[1133] Methods of the invention also provide for treating patients infected by the Tick-borne encephalitis flavivirus, or at risk for or afflicted with a disorder mediated by Tick-borne encephalitis virus, e.g., viral haemorrhagic fever and neurological disease.

[1134] In a preferred embodiment, the expression of a Tick-borne encephalitis virus gene is reduced.

[1135] In a preferred embodiment the expression of a human gene that is required for Tick-borne encephalitis virus replication is reduced.

[1136] Methods of the invention also provide for methods of treating patients infected by the Murray Valley encephalitis flavivirus, which commonly results in viral haemorrhagic fever and neurological disease.

[1137] In a preferred embodiment, the expression of a Murray Valley encephalitis virus gene is reduced.

[1138] In a preferred embodiment the expression of a human gene that is required for Murray Valley encephalitis virus replication is reduced.

[1139] The invention also includes methods for treating patients infected by the dengue flavivirus, or a disease or disorder associated with this virus, e.g., dengue haemorrhagic fever.

[1140] In a preferred embodiment, the expression of a dengue virus gene is reduced.

[1141] In a preferred embodiment the expression of a human gene that is required for dengue virus replication is reduced.

[1142] Methods of the invention also provide for treating patients infected by the Simian Virus 40 (SV40) or at risk for or afflicted with a disorder mediated by SV40, e.g., tumorigenesis.

[1143] In a preferred embodiment, the expression of a SV40 gene is reduced.

[1144] In a preferred embodiment the expression of a human gene that is required for SV40 replication is reduced.

[1145] The invention also includes methods for treating patients infected by the Human T Cell Lymphotropic Virus (HTLV), or a disease or disorder associated with this virus, e.g., leukemia and myelopathy.

[1146] In a preferred embodiment, the expression of a HTLV gene is reduced. In another preferred embodiment the HTLV 1 gene is the Tax transcriptional activator.

[1147] In a preferred embodiment the expression of a human gene that is required for HTLV replication is reduced.

[1148] Methods of the invention also provide for treating patients infected by the Moloney-Murine Leukemia Virus (Mo-MuLV) or at risk for or afflicted with a disorder mediated by Mo-MuLV, e.g., T-cell leukemia.

[1149] In a preferred embodiment, the expression of a Mo-MuLV gene is reduced.

[1150] In a preferred embodiment the expression of a human gene that is required for Mo-MuLV replication is reduced.

[1151] Methods of the invention also provide for treating patients infected by the encephalomyocarditis virus (EMCV) or at risk for or afflicted with a disorder mediated by EMCV, e.g. myocarditis. EMCV leads to myocarditis in mice and pigs and is capable of infecting human myocardial cells. This virus is therefore a concern for patients undergoing xenotransplantation.

[1152] In a preferred embodiment, the expression of a EMCV gene is reduced.

[1153] In a preferred embodiment the expression of a human gene that is required for EMCV replication is reduced.

[1154] The invention also includes a method for treating patients infected by the measles virus (MV) or at risk for or afflicted with a disorder mediated by MV, e.g. measles.

[1155] In a preferred embodiment, the expression of a MV gene is reduced.

[1156] In a preferred embodiment the expression of a human gene that is required for MV replication is reduced.

[1157] The invention also includes a method for treating patients infected by the Vericella zoster virus (VZV) or at risk for or afflicted with a disorder mediated by VZV, e.g. chicken pox or shingles (also called zoster).

[1158] In a preferred embodiment, the expression of a VZV gene is reduced.

[1159] In a preferred embodiment the expression of a human gene that is required for VZV replication is reduced.

[1160] The invention also includes a method for treating patients infected by an adenovirus or at risk for or afflicted with a disorder mediated by an adenovirus, e.g. respiratory tract infection.

[1161] In a preferred embodiment, the expression of an adenovirus gene is reduced.

[1162] In a preferred embodiment the expression of a human gene that is required for adenovirus replication is reduced.

[1163] The invention includes a method for treating patients infected by a yellow fever virus (YFV) or at risk for or afflicted with a disorder mediated by a YFV, e.g. respiratory tract infection.

[1164] In a preferred embodiment, the expression of a YFV gene is reduced. In another preferred embodiment, the preferred gene is one of a group that includes the E, NS2A, or NS3 genes.

[1165] In a preferred embodiment the expression of a human gene that is required for YFV replication is reduced.

[1166] Methods of the invention also provide for treating patients infected by the poliovirus or at risk for or afflicted with a disorder mediated by poliovirus, e.g., polio.

[1167] In a preferred embodiment, the expression of a poliovirus gene is reduced.

[1168] In a preferred embodiment the expression of a human gene that is required for poliovirus replication is reduced.

[1169] Methods of the invention also provide for treating patients infected by a poxvirus or at risk for or afflicted with a disorder mediated by a poxvirus, e.g., smallpox

[1170] In a preferred embodiment, the expression of a poxvirus gene is reduced.

[1171] In a preferred embodiment the expression of a human gene that is required for poxvirus replication is reduced.

[1172] In another, aspect the invention features methods of treating a subject infected with a pathogen, e.g., a bacterial, amoebic, parasitic, or fungal pathogen. The method includes:

[1173] providing a iRNA agent, e.g., a siRNA having a structure described herein, where siRNA is homologous to and can silence, e.g., by cleavage of a pathogen gene;

[1174] administering the iRNA agent to a subject, preferably a human subject,

[1175] thereby treating the subject.

[1176] The target gene can be one involved in growth, cell wall synthesis, protein synthesis, transcription, energy metabolism, e.g., the Krebs cycle, or toxin production.

[1177] Thus, the present invention provides for a method of treating patients infected by a plasmodium that causes malaria.

[1178] In a preferred embodiment, the expression of a plasmodium gene is reduced. In another preferred embodiment, the gene is apical membrane antigen 1 (AMA 1).

[1179] In a preferred embodiment the expression of a human gene that is required for plasmodium replication is reduced.

[1180] The invention also includes methods for treating patients infected by the Mycobacterium ulcerans, or a disease or disorder associated with this pathogen, e.g. Buruli ulcers.

[1181] In a preferred embodiment, the expression of a Mycobacterium ulcerans gene is reduced.

[1182] In a preferred embodiment the expression of a human gene that is required for Mycobacterium ulcerans replication is reduced.

[1183] The invention also includes methods for treating patients infected by the Mycobacterium tuberculosis, or a disease or disorder associated with this pathogen, e.g. tuberculosis.

[1184] In a preferred embodiment, the expression of a Mycobacterium tuberculosis gene is reduced.

[1185] In a preferred embodiment the expression of a human gene that is required for Mycobacterium tuberculosis replication is reduced.

[1186] The invention also includes methods for treating patients infected by the Mycobacterium leprae, or a disease or disorder associated with this pathogen, e.g. leprosy.

[1187] In a preferred embodiment, the expression of a Mycobacterium leprae gene is reduced.

[1188] In a preferred embodiment the expression of a human gene that is required for Mycobacterium leprae replication is reduced.

[1189] The invention also includes methods for treating patients infected by the bacteria Staphylococcus aureus, or a disease or disorder associated with this pathogen, e.g. infections of the skin and muscous membranes.

[1190] In a preferred embodiment, the expression of a Staphylococcus aureus gene is reduced.

[1191] In a preferred embodiment the expression of a human gene that is required for Staphylococcus aureus replication is reduced.

[1192] The invention also includes methods for treating patients infected by the bacteria Streptococcus pneumoniae, or a disease or disorder associated with this pathogen, e.g. pneumonia or childhood lower respiratory tract infection.

[1193] In a preferred embodiment, the expression of a Streptococcus pneumoniae gene is reduced.

[1194] In a preferred embodiment the expression of a human gene that is required for Streptococcus pneumoniae replication is reduced.

[1195] The invention also includes methods for treating patients infected by the bacteria Streptococcus pyogenes, or a disease or disorder associated with this pathogen, e.g. Strep throat or Scarlet fever.

[1196] In a preferred embodiment, the expression of a Streptococcus pyogenes gene is reduced.

[1197] In a preferred embodiment the expression of a human gene that is required for Streptococcus pyogenes replication is reduced.

[1198] The invention also includes methods for treating patients infected by the bacteria Chlamydia pneumoniae, or a disease or disorder associated with this pathogen, e.g. pneumonia or childhood lower respiratory tract infection

[1199] In a preferred embodiment, the expression of a Chlamydia pneumoniae gene is reduced.

[1200] In a preferred embodiment the expression of a human gene that is required for Chlamydia pneumoniae replication is reduced.

[1201] The invention also includes methods for treating patients infected by the bacteria Mycoplasma pneumoniae, or a disease or disorder associated with this pathogen, e.g. pneumonia or childhood lower respiratory tract infection

[1202] In a preferred embodiment, the expression of a Mycoplasma pneumoniae gene is reduced.

[1203] In a preferred embodiment the expression of a human gene that is required for Mycoplasma pneumoniae replication is reduced.

[1204] In one aspect, the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with a disease or disorder characterized by an unwanted immune response, e.g., an inflammatory disease or disorder, or an autoimmune disease or disorder. The method includes:

[1205] providing an iRNA agent, e.g., an iRNA agent having a structure described herein, which iRNA agent is homologous to and can silence, e.g., by cleavage, a gene which mediates an unwanted immune response;

[1206] administering the iRNA agent to a subject,

[1207] thereby treating the subject.

[1208] In a preferred embodiment the disease or disorder is an ischemia or reperfusion injury, e.g., ischemia or reperfusion injury associated with acute myocardial infarction, unstable angina, cardiopulmonary bypass, surgical intervention e.g., angioplasty, e.g., percutaneous transluminal coronary angioplasty, the response to a transplanted organ or tissue, e.g., transplanted cardiac or vascular tissue; or thrombolysis.

[1209] In a preferred embodiment the disease or disorder is restenosis, e.g., restenosis associated with surgical intervention e.g., angioplasty, e.g., percutaneous transluminal coronary angioplasty.

[1210] In a preferred embodiment the disease or disorder is Inflammatory Bowel Disease, e.g., Crohn Disease or Ulcerative Colitis.

[1211] In a preferred embodiment the disease or disorder is inflammation associated with an infection or injury.

[1212] In a preferred embodiment the disease or disorder is asthma, lupus, multiple sclerosis, diabetes, e.g., type II diabetes, arthritis, e.g., rheumatoid or psoriatic.

[1213] In particularly preferred embodiments the iRNA agent silences an integrin or co-ligand thereof, e.g., VLA4, VCAM, ICAM.

[1214] In particularly preferred embodiments the iRNA agent silences a selectin or co-ligand thereof, e.g., P-selectin, E-selectin (ELAM), I-selectin, P-selectin glycoprotein-1 (PSGL-1).

[1215] In particularly preferred embodiments the iRNA agent silences a component of the complement system, e.g., C3, C5, C3aR, C5aR, C3 convertase, C5 convertase.

[1216] In particularly preferred embodiments the iRNA agent silences a chemokine or receptor thereof, e.g., TNFI, TNFJ, IL-1I, IL-1J, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-6, IL-8, TNFRI, TNFRII, IgE, SCYA11, CCR3.

[1217] In other embodiments the iRNA agent silences GCSF, Gro1, Gro2, Gro3, PF4, MIG, Pro-Platelet Basic Protein (PPBP), MIP-1I, MIP-1J, RANTES, MCP-1, MCP-2, MCP-3, CMBKR1, CMBKR2, CMBKR3, CMBKR5, AIF-1, I-309.

[1218] In one aspect, the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with acute pain or chronic pain. The method includes:

[1219] providing an iRNA agent, which iRNA is homologous to and can silence, e.g., by cleavage, a gene which mediates the processing of pain;

[1220] administering the iRNA to a subject,

[1221] thereby treating the subject.

[1222] In particularly preferred embodiments the iRNA agent silences a component of an ion channel.

[1223] In particularly preferred embodiments the iRNA agent silences a neurotransmitter receptor or ligand.

[1224] In one aspect, the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with a neurological disease or disorder. The method includes:

[1225] providing an iRNA agent which iRNA is homologous to and can silence, e.g., by cleavage, a gene which mediates a neurological disease or disorder;

[1226] administering the to a subject,

[1227] thereby treating the subject.

[1228] In a preferred embodiment the disease or disorder is Alzheimer Disease or Parkinson Disease.

[1229] In particularly preferred embodiments the iRNA agent silences an amyloid-family gene, e.g., APP; a presenilin gene, e.g., PSEN1 and PSEN2, or I-synuclein.

[1230] In a preferred embodiment the disease or disorder is a neurodegenerative trinucleotide repeat disorder, e.g., Huntington disease, dentatorubral pallidoluysian atrophy or a spinocerebellar ataxia, e.g., SCA1, SCA2, SCA3 (Machado-Joseph disease), SCA7 or SCA8.

[1231] In particularly preferred embodiments the iRNA agent silences HD, DRPLA, SCA1, SCA2, MJD 1, CACNL1A4, SCA7, SCA8.

[1232] The loss of heterozygosity (LOH) can result in hemizygosity for sequence, e.g., genes, in the area of LOH. This can result in a significant genetic difference between normal and disease-state cells, e.g., cancer cells, and provides a useful difference between normal and disease-state cells, e.g., cancer cells. This difference can arise because a gene or other sequence is heterozygous in euploid cells but is hemizygous in cells having LOH. The regions of LOH will often include a gene, the loss of which promotes unwanted proliferation, e.g., a tumor suppressor gene, and other sequences including, e.g., other genes, in some cases a gene which is essential for normal function, e.g., growth. Methods of the invention rely, in part, on the specific cleavage or silencing of one allele of an essential gene with an iRNA agent of the invention. The iRNA agent is selected such that it targets the single allele of the essential gene found in the cells having LOH but does not silence the other allele, which is present in cells which do not show LOH. In essence, it discriminates between the two alleles, preferentially silencing the selected allele. In essence polymorphisms, e.g., SNPs of essential genes that are affected by LOH, are used as a target for a disorder characterized by cells having LOH, e.g., cancer cells having LOH.

[1233] E.g., one of ordinary skill in the art can identify essential genes which are in proximity to tumor suppressor genes, and which are within a LOH region which includes the tumor suppressor gene. The gene encoding the large subunit of human RNA polymerase II, POLR2A, a gene located in close proximity to the tumor suppressor gene p53, is such a gene. It frequently occurs within a region of LOH in cancer cells. Other genes that occur within LOH regions and are lost in many cancer cell types include the group comprising replication protein A 70-kDa subunit, replication protein A 32-kD, ribonucleotide reductase, thymidilate synthase, TATA associated factor 2H, ribosomal protein S 14, eukaryotic initiation factor 5A, alanyl tRNA synthetase, cysteinyl tRNA synthetase, NaK ATPase, alpha-1 subunit, and transferrin receptor.

[1234] Accordingly, the invention features, a method of treating a disorder characterized by LOH, e.g., cancer. The method includes:

[1235] optionally, determining the genotype of the allele of a gene in the region of LOH and preferably determining the genotype of both alleles of the gene in a normal cell;

[1236] providing an iRNA agent which preferentially cleaves or silences the allele found in the LOH cells;

[1237] administering the iRNA to the subject,

[1238] thereby treating the disorder.

[1239] The invention also includes a iRNA agent disclosed herein, e.g., an iRNA agent which can preferentially silence, e.g., cleave, one allele of a polymorphic gene

[1240] In another aspect, the invention provides a method of cleaving or silencing more than one gene with an iRNA agent. In these embodiments the iRNA agent is selected so that it has sufficient homology to a sequence found in more than one gene. For example, the sequence AAGCTGGCCCTGGACATGGAGAT (SEQ ID NO: 6736) is conserved between mouse lamin B1, lamin B2, keratin complex 2-gene 1 and lamin A/C. Thus an iRNA agent targeted to this sequence would effectively silence the entire collection of genes.

[1241] The invention also includes an iRNA agent disclosed herein, which can silence more than one gene.

[1242] Route of Delivery

[1243] For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. A composition that includes a iRNA can be delivered to a subject by a variety of routes. Exemplary routes include: intravenous, topical, rectal, anal, vaginal, nasal, pulmonary, ocular.

[1244] The iRNA molecules of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include one or more species of iRNA and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[1245] The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.

[1246] The route and site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice. Lung cells might be targeted by administering the iRNA in aerosol form. The vascular endothelial cells could be targeted by coating a balloon catheter with the iRNA and mechanically introducing the DNA.

[1247] Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

[1248] Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches. In the case of tablets, carriers that can be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the nucleic acid compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added.

[1249] Compositions for intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

[1250] Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic.

[1251] For ocular administration, ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers. Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives such as sorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers.

[1252] Topical Delivery

[1253] For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. In a preferred embodiment, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) is delivered to a subject via topical administration. “Topical administration” refers to the delivery to a subject by contacting the formulation directly to a surface of the subject. The most common form of topical delivery is to the skin, but a composition disclosed herein can also be directly applied to other surfaces of the body, e.g., to the eye, a mucous membrane, to surfaces of a body cavity or to an internal surface. As mentioned above, the most common topical delivery is to the skin. The term encompasses several routes of administration including, but not limited to, topical and transdermal. These modes of administration typically include penetration of the skin's permeability barrier and efficient delivery to the target tissue or stratum. Topical administration can be used as a means to penetrate the epidermis and dermis and ultimately achieve systemic delivery of the composition. Topical administration can also be used as a means to selectively deliver oligonucleotides to the epidermis or dermis of a subject, or to specific strata thereof, or to an underlying tissue.

[1254] The term “skin,” as used herein, refers to the epidermis and/or dermis of an animal. Mammalian skin consists of two major, distinct layers. The outer layer of the skin is called the epidermis. The epidermis is comprised of the stratum corneum, the stratum granulosum, the stratum spinosum, and the stratum basale, with the stratum comeum being at the surface of the skin and the stratum basale being the deepest portion of the epidermis. The epidermis is between 50 μm and 0.2 mm thick, depending on its location on the body.

[1255] Beneath the epidermis is the dermis, which is significantly thicker than the epidermis. The dermis is primarily composed of collagen in the form of fibrous bundles. The collagenous bundles provide support for, inter alia, blood vessels, lymph capillaries, glands, nerve endings and immunologically active cells.

[1256] One of the major functions of the skin as an organ is to regulate the entry of substances into the body. The principal permeability barrier of the skin is provided by the stratum corneum, which is formed from many layers of cells in various states of differentiation. The spaces between cells in the stratum comeum is filled with different lipids arranged in lattice-like formations that provide seals to further enhance the skins permeability barrier.

[1257] The permeability barrier provided by the skin is such that it is largely impermeable to molecules having molecular weight greater than about 750 Da. For larger molecules to cross the skin's permeability barrier, mechanisms other than normal osmosis must be used.

[1258] Several factors determine the permeability of the skin to administered agents. These factors include the characteristics of the treated skin, the characteristics of the delivery agent, interactions between both the drug and delivery agent and the drug and skin, the dosage of the drug applied, the form of treatment, and the post treatment regimen. To selectively target the epidermis and dermis, it is sometimes possible to formulate a composition that comprises one or more penetration enhancers that will enable penetration of the drug to a preselected stratum.

[1259] Transdermal delivery is a valuable route for the administration of lipid soluble therapeutics. The dermis is more permeable than the epidermis and therefore absorption is much more rapid through abraded, burned or denuded skin. Inflammation and other physiologic conditions that increase blood flow to the skin also enhance transdermal adsorption. Absorption via this route may be enhanced by the use of an oily vehicle (injunction) or through the use of one or more penetration enhancers. Other effective ways to deliver a composition disclosed herein via the transdermal route include hydration of the skin and the use of controlled release topical patches. The transdermal route provides a potentially effective means to deliver a composition disclosed herein for systemic and/or local therapy.

[1260] In addition, iontophoresis (transfer of ionic solutes through biological membranes under the influence of an electric field) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 163), phonophoresis or sonophoresis (use of ultrasound to enhance the absorption of various therapeutic agents across biological membranes, notably the skin and the cornea) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 166), and optimization of vehicle characteristics relative to dose position and retention at the site of administration (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 168) may be useful methods for enhancing the transport of topically applied compositions across skin and mucosal sites.

[1261] The compositions and methods provided may also be used to examine the function of various proteins and genes in vitro in cultured or preserved dermal tissues and in animals. The invention can be thus applied to examine the function of any gene. The methods of the invention can also be used therapeutically or prophylactically. For example, for the treatment of animals that are known or suspected to suffer from diseases such as psoriasis, lichen planus, toxic epidermal necrolysis, ertythema multiforme, basal cell carcinoma, squamous cell carcinoma, malignant melanoma, Paget's disease, Kaposi's sarcoma, pulmonary fibrosis, Lyme disease and viral, fungal and bacterial infections of the skin.

[1262] Pulmonary Delivery

[1263] For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. A composition that includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) can be administered to a subject by pulmonary delivery. Pulmonary delivery compositions can be delivered by inhalation by the patient of a dispersion so that the composition, preferably iRNA, within the dispersion can reach the lung where it can be readily absorbed through the alveolar region directly into blood circulation. Pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs.

[1264] Pulmonary delivery can be achieved by different approaches, including the use of nebulized, aerosolized, micellular and dry powder-based formulations. Delivery can be achieved with liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered-dose devices are preferred. One of the benefits of using an atomizer or inhaler is that the potential for contamination is minimized because the devices are self contained. Dry powder dispersion devices, for example, deliver drugs that may be readily formulated as dry powders. A iRNA composition may be stably stored as lyophilized or spray-dried powders by itself or in combination with suitable powder carriers. The delivery of a composition for inhalation can be mediated by a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.

[1265] The term “powder” means a composition that consists of finely dispersed solid particles that are free flowing and capable of being readily dispersed in an inhalation device and subsequently inhaled by a subject so that the particles reach the lungs to permit penetration into the alveoli. Thus, the powder is said to be “respirable.” Preferably the average particle size is less than about 10 μm in diameter preferably with a relatively uniform spheroidal shape distribution. More preferably the diameter is less than about 7.5 μm and most preferably less than about 5.0 μm. Usually the particle size distribution is between about 0.1 μm and about 5 μm in diameter, particularly about 0.3 μm to about 5 μm.

[1266] The term “dry” means that the composition has a moisture content below about 10% by weight (% w) water, usually below about 5% w and preferably less it than about 3% w. A dry composition can be such that the particles are readily dispersible in an inhalation device to form an aerosol.

[1267] The term “therapeutically effective amount” is the amount present in the composition that is needed to provide the desired level of drug in the subject to be treated to give the anticipated physiological response.

[1268] The term “physiologically effective amount” is that amount delivered to a subject to give the desired palliative or curative effect.

[1269] The term “pharmaceutically acceptable carrier” means that the carrier can be taken into the lungs with no significant adverse toxicological effects on the lungs.

[1270] The types of pharmaceutical excipients that are useful as carrier include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two.

[1271] Bulking agents that are particularly valuable include compatible carbohydrates, polypeptides, amino acids or combinations thereof. Suitable carbohydrates include monosaccharides such as galactose, D-mannose, sorbose, and the like; disaccharides, such as lactose, trehalose, and the like; cyclodextrins, such as 2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such as raffinose, maltodextrins, dextrans, and the like; alditols, such as mannitol, xylitol, and the like. A preferred group of carbohydrates includes lactose, threhalose, raffinose maltodextrins, and mannitol. Suitable polypeptides include aspartame. Amino acids include alanine and glycine, with glycine being preferred.

[1272] Additives, which are minor components of the composition of this invention, may be included for conformational stability during spray drying and for improving dispersibility of the powder. These additives include hydrophobic amino acids such as tryptophan, tyrosine, leucine, phenylalanine, and the like.

[1273] Suitable pH adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred.

[1274] Pulmonary administration of a micellar iRNA formulation may be achieved through metered dose spray devices with propellants such as tetrafluoroethane, heptafluoroethane, dimethylfluoropropane, tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFC and CFC propellants.

[1275] Oral or Nasal Delivery

[1276] For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. Both the oral and nasal membranes offer advantages over other routes of administration. For example, drugs administered through these membranes have a rapid onset of action, provide therapeutic plasma levels, avoid first pass effect of hepatic metabolism, and avoid exposure of the drug to the hostile gastrointestinal (GI) environment. Additional advantages include easy access to the membrane sites so that the drug can be applied, localized and removed easily.

[1277] In oral delivery, compositions can be targeted to a surface of the oral cavity, e.g., to sublingual mucosa which includes the membrane of ventral surface of the tongue and the floor of the mouth or the buccal mucosa which constitutes the lining of the cheek. The sublingual mucosa is relatively permeable thus giving rapid absorption and acceptable bioavailability of many drugs. Further, the sublingual mucosa is convenient, acceptable and easily accessible.

[1278] The ability of molecules to permeate through the oral mucosa appears to be related to molecular size, lipid solubility and peptide protein ionization. Small molecules, less than 1000 daltons appear to cross mucosa rapidly. As molecular size increases, the permeability decreases rapidly. Lipid soluble compounds are more permeable than non-lipid soluble molecules. Maximum absorption occurs when molecules are un-ionized or neutral in electrical charges. Therefore charged molecules present the biggest challenges to absorption through the oral mucosae.

[1279] A pharmaceutical composition of iRNA may also be administered to the buccal cavity of a human being by spraying into the cavity, without inhalation, from a metered dose spray dispenser, a mixed micellar pharmaceutical formulation as described above and a propellant. In one embodiment, the dispenser is first shaken prior to spraying the pharmaceutical formulation and propellant into the buccal cavity.

[1280] Devices

[1281] For ease of exposition the devices, formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these devices, formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) can be disposed on or in a device, e.g., a device which implanted or otherwise placed in a subject. Exemplary devices include devices which are introduced into the vasculature, e.g., devices inserted into the lumen of a vascular tissue, or which devices themselves form a part of the vasculature, including stents, catheters, heart valves, and other vascular devices. These devices, e.g., catheters or stents, can be placed in the vasculature of the lung, heart, or leg.

[1282] Other devices include non-vascular devices, e.g., devices implanted in the peritoneum, or in organ or glandular tissue, e.g., artificial organs. The device can release a therapeutic substance in addition to a iRNA, e.g., a device can release insulin.

[1283] Other devices include artificial joints, e.g., hip joints, and other orthopedic implants.

[1284] In one embodiment, unit doses or measured doses of a composition that includes iRNA are dispensed by an implanted device. The device can include a sensor that monitors a parameter within a subject. For example, the device can include pump, e.g., and, optionally, associated electronics.

[1285] Tissue, e.g., cells or organs can be treated with An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) ex vivo and then administered or implanted in a subject.

[1286] The tissue can be autologous, allogeneic, or xenogeneic tissue. E.g., tissue can be treated to reduce graft v. host disease. In other embodiments, the tissue is allogeneic and the tissue is treated to treat a disorder characterized by unwanted gene expression in that tissue. E.g., tissue, e.g., hematopoietic cells, e.g., bone marrow hematopoietic cells, can be treated to inhibit unwanted cell proliferation.

[1287] Introduction of treated tissue, whether autologous or transplant, can be combined with other therapies.

[1288] In some implementations, the iRNA treated cells are insulated from other cells, e.g., by a semi-permeable porous barrier that prevents the cells from leaving the implant, but enables molecules from the body to reach the cells and molecules produced by the cells to enter the body. In one embodiment, the porous barrier is formed from alginate.

[1289] In one embodiment, a contraceptive device is coated with or contains an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). Exemplary devices include condoms, diaphragms, IUD (implantable uterine devices, sponges, vaginal sheaths, and birth control devices. In one embodiment, the iRNA is chosen to inactive sperm or egg. In another embodiment, the iRNA is chosen to be complementary to a viral or pathogen RNA, e.g., an RNA of an STD. In some instances, the iRNA composition can include a spermicide.

[1290] Dosage

[1291] In one aspect, the invention features a method of administering an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, to a subject (e.g., a human subject). The method includes administering a unit dose of the iRNA agent, e.g., a sRNA agent, e.g., double stranded sRNA agent that (a) the double-stranded part is 19-25 nucleotides (nt) long, preferably 21-23 nt, (b) is complementary to a target RNA (e.g., an endogenous or pathogen target RNA), and, optionally, (c) includes at least one 3′ overhang 1-5 nucleotide long. In one embodiment, the unit dose is less than 1.4 mg per kg of bodyweight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight, and less than 200 nmole of RNA agent (e.g. about 4.4×10¹⁶ copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of RNA agent per kg of bodyweight.

[1292] The defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with the target RNA. The unit dose, for example, can be administered by injection (e.g., intravenous or intramuscular), an inhaled dose, or a topical application. Particularly preferred dosages are less than 2, 1, or 0.1 mg/kg of body weight.

[1293] In a preferred embodiment, the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered a single time.

[1294] In one embodiment, the effective dose is administered with other traditional therapeutic modalities. In one embodiment, the subject has a viral infection and the modality is an antiviral agent other than an iRNA agent, e.g., other than a double-stranded iRNA agent, or sRNA agent. In another embodiment, the subject has atherosclerosis and the effective dose of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, is administered in combination with, e.g., after surgical intervention, e.g., angioplasty.

[1295] In one embodiment, a subject is administered an initial dose and one or more maintenance doses of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). The maintenance dose or doses are generally lower than the initial dose, e.g., one-half less of the initial dose. A maintenance regimen can include treating the subject with a dose or doses ranging from 0.01 μg to 1.4 mg/kg of body weight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of bodyweight per day. The maintenance doses are preferably administered no more than once every 5, 10, or 30 days. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient. In preferred embodiments the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g., no more than once for every 5 or 8 days. Following treatment, the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state. The dosage of the compound may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.

[1296] The effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.

[1297] In one embodiment, the iRNA agent pharmaceutical composition includes a plurality of iRNA agent species. In another embodiment, the iRNA agent species has sequences that are non-overlapping and non-adjacent to another species with respect to a naturally occurring target sequence. In another embodiment, the plurality of iRNA agent species is specific for different naturally occurring target genes. In another embodiment, the iRNA agent is allele specific.

[1298] In some cases, a patient is treated with a iRNA agent in conjunction with other therapeutic modalities. For example, a patient being treated for a viral disease, e.g. an HIV associated disease (e.g., AIDS), may be administered a iRNA agent specific for a target gene essential to the virus in conjunction with a known antiviral agent (e.g., a protease inhibitor or reverse transcriptase inhibitor). In another example, a patient being treated for cancer may be administered a iRNA agent specific for a target essential for tumor cell proliferation in conjunction with a chemotherapy.

[1299] Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the compound of the invention is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight (see U.S. Pat. No. 6,107,094).

[1300] The concentration of the iRNA agent composition is an amount sufficient to be effective in treating or preventing a disorder or to regulate a physiological condition in humans. The concentration or amount of iRNA agent administered will depend on the parameters determined for the agent and the method of administration, e.g. nasal, buccal, pulmonary. For example, nasal formulations tend to require much lower concentrations of some ingredients in order to avoid irritation or burning of the nasal passages. It is sometimes desirable to dilute an oral formulation up to 10-100 times in order to provide a suitable nasal formulation.

[1301] Certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) can include a single treatment or, preferably, can include a series of treatments. It will also be appreciated that the effective dosage of a iRNA agent such as a sRNA agent used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein. For example, the subject can be monitored after administering a iRNA agent composition. Based on information from the monitoring, an additional amount of the iRNA agent composition can be administered.

[1302] Dosing is dependent on severity and responsiveness of the disease condition to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual compounds, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. In some embodiments, the animal models include transgenic animals that express a human gene, e.g. a gene that produces a target RNA. The transgenic animal can be deficient for the corresponding endogenous RNA. In another embodiment, the composition for testing includes a iRNA agent that is complementary, at least in an internal region, to a sequence that is conserved between the target RNA in the animal model and the target RNA in a human.

[1303] The inventors have discovered that iRNA agents described herein can be administered to mammals, particularly large mammals such as nonhuman primates or humans in a number of ways.

[1304] In one embodiment, the administration of the iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, composition is parenteral, e.g. intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.

[1305] The invention provides methods, compositions, and kits, for rectal administration or delivery of iRNA agents described herein.

[1306] Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes a an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of a iRNA agent described herein, e.g., a iRNA agent having a double stranded region of less than 40, and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered rectally, e.g., introduced through the rectum into the lower or upper colon. This approach is particularly useful in the treatment of, inflammatory disorders, disorders characterized by unwanted cell proliferation, e.g., polyps, or colon cancer.

[1307] The medication can be delivered to a site in the colon by introducing a dispensing device, e.g., a flexible, camera-guided device similar to that used for inspection of the colon or removal of polyps, which includes means for delivery of the medication.

[1308] The rectal administration of the iRNA agent is by means of an enema. The iRNA agent of the enema can be dissolved in a saline or buffered solution. The rectal administration can also by means of a suppository, which can include other ingredients, e.g., an excipient, e.g., cocoa butter or hydropropylmethylcellulose.

[1309] Any of the iRNA agents described herein can be administered orally, e.g., in the form of tablets, capsules, gel capsules, lozenges, troches or liquid syrups. Further, the composition can be applied topically to a surface of the oral cavity.

[1310] Any of the iRNA agents described herein can be administered buccally. For example, the medication can be sprayed into the buccal cavity or applied directly, e.g., in a liquid, solid, or gel form to a surface in the buccal cavity. This administration is particularly desirable for the treatment of inflammations of the buccal cavity, e.g., the gums or tongue, e.g., in one embodiment, the buccal administration is by spraying into the cavity, e.g., without inhalation, from a dispenser, e.g., a metered dose spray dispenser that dispenses the pharmaceutical composition and a propellant.

[1311] Any of the iRNA agents described herein can be administered to ocular tissue. For example, the medications can be applied to the surface of the eye or nearby tissue, e.g., the inside of the eyelid. They can be applied topically, e.g., by spraying, in drops, as an eyewash, or an ointment. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. The medication can also be administered to the interior of the eye, and can be introduced by a needle or other delivery device which can introduce it to a selected area or structure. Ocular treatment is particularly desirable for treating inflammation of the eye or nearby tissue.

[1312] Any of the iRNA agents described herein can be administered directly to the skin. For example, the medication can be applied topically or delivered in a layer of the skin, e.g., by the use of a microneedle or a battery of microneedles which penetrate into the skin, but preferably not into the underlying muscle tissue. Administration of the iRNA agent composition can be topical. Topical applications can, for example, deliver the composition to the dermis or epidermis of a subject. Topical administration can be in the form of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids or powders. A composition for topical administration can be formulated as a liposome, micelle, emulsion, or other lipophilic molecular assembly. The transdermal administration can be applied with at least one penetration enhancer, such as iontophoresis, phonophoresis, and sonophoresis.

[1313] Any of the iRNA agents described herein can be administered to the pulmonary system. Pulmonary administration can be achieved by inhalation or by the introduction of a delivery device into the pulmonary system, e.g., by introducing a delivery device which can dispense the medication. A preferred method of pulmonary delivery is by inhalation. The medication can be provided in a dispenser which delivers the medication, e.g., wet or dry, in a form sufficiently small such that it can be inhaled. The device can deliver a metered dose of medication. The subject, or another person, can administer the medication.

[1314] Pulmonary delivery is effective not only for disorders which directly affect pulmonary tissue, but also for disorders which affect other tissue.

[1315] iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder, crystal, or aerosol for pulmonary delivery.

[1316] Any of the iRNA agents described herein can be administered nasally. Nasal administration can be achieved by introduction of a delivery device into the nose, e.g., by introducing a delivery device which can dispense the medication. Methods of nasal delivery include spray, aerosol, liquid, e.g., by drops, or by topical administration to a surface of the nasal cavity. The medication can be provided in a dispenser with delivery of the medication, e.g., wet or dry, in a form sufficiently small such that it can be inhaled. The device can deliver a metered dose of medication. The subject, or another person, can administer the medication.

[1317] Nasal delivery is effective not only for disorders which directly affect nasal tissue, but also for disorders which affect other tissue

[1318] iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder, crystal, or for nasal delivery.

[1319] An iRNA agent can be packaged in a viral natural capsid or in a chemically or enzymatically produced artificial capsid or structure derived therefrom.

[1320] The dosage of a pharmaceutical composition including a iRNA agent can be administered in order to alleviate the symptoms of a disease state, e.g., cancer or a cardiovascular disease. A subject can be treated with the pharmaceutical composition by any of the methods mentioned above.

[1321] Gene expression in a subject can be modulated by administering a pharmaceutical composition including an iRNA agent.

[1322] A subject can be treated by administering a defined amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent) composition that is in a powdered form, e.g., a collection of microparticles, such as crystalline particles. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

[1323] A subject can be treated by administering a defined amount of an iRNA agent composition that is prepared by a method that includes spray-drying, i.e. atomizing a liquid solution, emulsion, or suspension, immediately exposing the droplets to a drying gas, and collecting the resulting porous powder particles. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

[1324] The iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), can be provided in a powdered, crystallized or other finely divided form, with or without a carrier, e.g., a micro- or nano-particle suitable for inhalation or other pulmonary delivery. This can include providing an aerosol preparation, e.g., an aerosolized spray-dried composition. The aerosol composition can be provided in and/or dispensed by a metered dose delivery device.

[1325] The subject can be treated for a condition treatable by inhalation, e.g., by aerosolizing a spray-dried iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) composition and inhaling the aerosolized composition. The iRNA agent can be an sRNA. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

[1326] A subject can be treated by, for example, administering a composition including an effective/defined amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), wherein the composition is prepared by a method that includes spray-drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques

[1327] In another aspect, the invention features a method that includes: evaluating a parameter related to the abundance of a transcript in a cell of a subject; comparing the evaluated parameter to a reference value; and if the evaluated parameter has a preselected relationship to the reference value (e.g., it is greater), administering a iRNA agent (or a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes a iRNA agent or precursor thereof) to the subject. In one embodiment, the iRNA agent includes a sequence that is complementary to the evaluated transcript. For example, the parameter can be a direct measure of transcript levels, a measure of a protein level, a disease or disorder symptom or characterization (e.g., rate of cell proliferation and/or tumor mass, viral load)

[1328] In another aspect, the invention features a method that includes: administering a first amount of a composition that comprises an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) to a subject, wherein the iRNA agent includes a strand substantially complementary to a target nucleic acid; evaluating an activity associated with a protein encoded by the target nucleic acid; wherein the evaluation is used to determine if a second amount should be administered. In a preferred embodiment the method includes administering a second amount of the composition, wherein the timing of administration or dosage of the second amount is a function of the evaluating. The method can include other features described herein.

[1329] In another aspect, the invention features a method of administering a source of a double-stranded iRNA agent (ds iRNA agent) to a subject. The method includes administering or implanting a source of a ds iRNA agent, e.g., a sRNA agent, that (a) includes a double-stranded region that is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to a target RNA (e.g., an endogenous RNA or a pathogen RNA), and, optionally, (c) includes at least one 3′ overhang 1-5 nt long. In one embodiment, the source releases ds iRNA agent over time, e.g. the source is a controlled or a slow release source, e.g., a microparticle that gradually releases the ds iRNA agent. In another embodiment, the source is a pump, e.g., a pump that includes a sensor or a pump that can release one or more unit doses.

[1330] In one aspect, the invention features a pharmaceutical composition that includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) including a nucleotide sequence complementary to a target RNA, e.g., substantially and/or exactly complementary. The target RNA can be a transcript of an endogenous human gene. In one embodiment, the iRNA agent (a) is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to an endogenous target RNA, and, optionally, (c) includes at least one 3′ overhang 1-5 nt long. In one embodiment, the pharmaceutical composition can be an emulsion, microemulsion, cream, jelly, or liposome.

[1331] In one example the pharmaceutical composition includes an iRNA agent mixed with a topical delivery agent. The topical delivery agent can be a plurality of microscopic vesicles. The microscopic vesicles can be liposomes. In a preferred embodiment the liposomes are cationic liposomes.

[1332] In another aspect, the pharmaceutical composition includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) admixed with a topical penetration enhancer. In one embodiment, the topical penetration enhancer is a fatty acid. The fatty acid can be arachidonic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C_1-10 alkyl ester, monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.

[1333] In another embodiment, the topical penetration enhancer is a bile salt. The bile salt can be cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate, polyoxyethylene-9-lauryl ether or a pharmaceutically acceptable salt thereof.

[1334] In another embodiment, the penetration enhancer is a chelating agent. The chelating agent can be EDTA, citric acid, a salicyclate, a N-acyl derivative of collagen, laureth-9, an N-amino acyl derivative of a beta-diketone or a mixture thereof.

[1335] In another embodiment, the penetration enhancer is a surfactant, e.g., an ionic or nonionic surfactant. The surfactant can be sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether, a perfluorchemical emulsion or mixture thereof.

[1336] In another embodiment, the penetration enhancer can be selected from a group consisting of unsaturated cyclic ureas, 1-alkyl-alkones, 1-alkenylazacyclo-alakanones, steroidal anti-inflammatory agents and mixtures thereof. In yet another embodiment the penetration enhancer can be a glycol, a pyrrol, an azone, or a terpenes.

[1337] In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a form suitable for oral delivery. In one embodiment, oral delivery can be used to deliver an iRNA agent composition to a cell or a region of the gastro-intestinal tract, e.g., small intestine, colon (e.g., to treat a colon cancer), and so forth. The oral delivery form can be tablets, capsules or gel capsules. In one embodiment, the iRNA agent of the pharmaceutical composition modulates expression of a cellular adhesion protein, modulates a rate of cellular proliferation, or has biological activity against eukaryotic pathogens or retroviruses. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablets, capsules or gel capsules in a mammalian stomach. In a preferred embodiment the enteric material is a coating. The coating can be acetate phthalate, propylene glycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxy propyl methylcellulose phthalate or cellulose acetate phthalate.

[1338] In another embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer. The penetration enhancer can be a bile salt or a fatty acid. The bile salt can be ursodeoxycholic acid, chenodeoxycholic acid, and salts thereof. The fatty acid can be capric acid, lauric acid, and salts thereof.

[1339] In another embodiment, the oral dosage form of the pharmaceutical composition includes an excipient. In one example the excipient is polyethyleneglycol. In another example the excipient is precirol.

[1340] In another embodiment, the oral dosage form of the pharmaceutical composition includes a plasticizer. The plasticizer can be diethyl phthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethyl citrate.

[1341] In one aspect, the invention features a pharmaceutical composition including an iRNA agent and a delivery vehicle. In one embodiment, the iRNA agent is (a) is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to an endogenous target RNA, and, optionally, (c) includes at least one 3′ overhang 1-5 nucleotides long.

[1342] In one embodiment, the delivery vehicle can deliver an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) to a cell by a topical route of administration. The delivery vehicle can be microscopic vesicles. In one example the microscopic vesicles are liposomes. In a preferred embodiment the liposomes are cationic liposomes. In another example the microscopic vesicles are micelles. In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in an injectable dosage form. In one embodiment, the injectable dosage form of the pharmaceutical composition includes sterile aqueous solutions or dispersions and sterile powders. In a preferred embodiment the sterile solution can include a diluent such as water; saline solution; fixed oils, polyethylene glycols, glycerin, or propylene glycol.

[1343] In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in oral dosage form. In one embodiment, the oral dosage form is selected from the group consisting of tablets, capsules and gel capsules. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablets, capsules or gel capsules in a mammalian stomach. In a preferred embodiment the enteric material is a coating. The coating can be acetate phthalate, propylene glycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxy propyl methyl cellulose phthalate or cellulose acetate phthalate. In one embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer, e.g., a penetration enhancer described herein.

[1344] In another embodiment, the oral dosage form of the pharmaceutical composition includes an excipient. In one example the excipient is polyethyleneglycol. In another example the excipient is precirol.

[1345] In another embodiment, the oral dosage form of the pharmaceutical composition includes a plasticizer. The plasticizer can be diethyl phthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethyl citrate.

[1346] In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a rectal dosage form. In one embodiment, the rectal dosage form is an enema. In another embodiment, the rectal dosage form is a suppository.

[1347] In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a vaginal dosage form. In one embodiment, the vaginal dosage form is a suppository. In another embodiment, the vaginal dosage form is a foam, cream, or gel.

[1348] In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a pulmonary or nasal dosage form. In one embodiment, the iRNA agent is incorporated into a particle, e.g., a macroparticle, e.g., a microsphere. The particle can be produced by spray drying, lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination thereof. The microsphere can be formulated as a suspension, a powder, or an implantable solid.

[1349] In one aspect, the invention features a spray-dried iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) composition suitable for inhalation by a subject, including: (a) a therapeutically effective amount of a iRNA agent suitable for treating a condition in the subject by inhalation; (b) a pharmaceutically acceptable excipient selected from the group consisting of carbohydrates and amino acids; and (c) optionally, a dispersibility-enhancing amount of a physiologically-acceptable, water-soluble polypeptide.

[1350] In one embodiment, the excipient is a carbohydrate. The carbohydrate can be selected from the group consisting of monosaccharides, disaccharides, trisaccharides, and polysaccharides. In a preferred embodiment the carbohydrate is a monosaccharide selected from the group consisting of dextrose, galactose, mannitol, D-mannose, sorbitol, and sorbose. In another preferred embodiment the carbohydrate is a disaccharide selected from the group consisting of lactose, maltose, sucrose, and trehalose.

[1351] In another embodiment, the excipient is an amino acid. In one embodiment, the amino acid is a hydrophobic amino acid. In a preferred embodiment the hydrophobic amino acid is selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. In yet another embodiment the amino acid is a polar amino acid. In a preferred embodiment the amino acid is selected from the group consisting of arginine, histidine, lysine, cysteine, glycine, glutamine, serine, threonine, tyrosine, aspartic acid and glutamic acid.

[1352] In one embodiment, the dispersibility-enhancing polypeptide is selected from the group consisting of human serum albumin, α-lactalbumin, trypsinogen, and polyalanine.

[1353] In one embodiment, the spray-dried iRNA agent composition includes particles having a mass median diameter (MMD) of less than 10 microns. In another embodiment, the spray-dried iRNA agent composition includes particles having a mass median diameter of less than 5 microns. In yet another embodiment the spray-dried iRNA agent composition includes particles having a mass median aerodynamic diameter (MMAD) of less than 5 microns.

[1354] In certain other aspects, the invention provides kits that include a suitable container containing a pharmaceutical formulation of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). In certain embodiments the individual components of the pharmaceutical formulation may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical formulation separately in two or more containers, e.g., one container for an iRNA agent preparation, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition. The kit can also include a delivery device.

[1355] In another aspect, the invention features a device, e.g., an implantable device, wherein the device can dispense or administer a composition that includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), e.g., a iRNA agent that silences an endogenous transcript. In one embodiment, the device is coated with the composition. In another embodiment the iRNA agent is disposed within the device. In another embodiment, the device includes a mechanism to dispense a unit dose of the composition. In other embodiments the device releases the composition continuously, e.g., by diffusion. Exemplary devices include stents, catheters, pumps, artificial organs or organ components (e.g., artificial heart, a heart valve, etc.), and sutures.

[1356] As used herein, the term “crystalline” describes a solid having the structure or characteristics of a crystal, i.e., particles of three-dimensional structure in which the plane faces intersect at definite angles and in which there is a regular internal structure. The compositions of the invention may have different crystalline forms. Crystalline forms can be prepared by a variety of methods, including, for example, spray drying.

[1357] The invention is further illustrated by the following examples, which should not be construed as further limiting.

[1358] Apolipoprotein M (ApoM) is a human apolipoprotein predominantly present in high-density lipoprotein (HDL) in plasma. ApoM is reported to be expressed exclusively in liver and in kidney (Xu N et al., Biochem J Biol Chem 1999 Oct. 29; 274(44): 31286-90). Mouse ApoM is a 21 kD membrane associated protein, and, in serum, the protein is associated with HDL particles. ApoM gene expression is regulated by the transcription factor hepatocyte nuclear factor 1 alpha (Hnf-1α), as Hnf-1α^−/− mice are ApoM deficient. In humans, mutations in the HNF-1 alpha gene represent a common cause of maturity-onset diabetes of the young (MODY).

[1359] A variety of test iRNAs were synthesized to target the mouse ApoM gene. This gene was chosen in part because of its high expression levels and exclusive activity in the liver and kidney.

[1360] Three different classes of dsRNA agents were synthesized, each class having different modifications and features at the 5′ and 3′ ends, see Table 4.

[1361]

TABLE 4
Targeted ORF's
5	The23mer: AAGTTTGGGCAGCTCTGCTCT (SEQ ID NO: 6708)
19	The23mer: AAGTGGACATACCGATTGACT (SEQ ID NO: 6709)
25	The23mer: AACTCAGAACTGAAGGGCGCC (SEQ ID NO: 6710)
27	The23mer: AAGGGCGCCCAGACATGAAAA (SEQ ID NO: 6711)
3′-UTR (beginning at 645)
42: AAGATAGGAGCCCAGCTTCGA (SEQ ID NO: 6712)
Class I
21-nt iRNAs, t, deoxythymidine; p, phosphate
pGUUUGGGCAGCUCUGCUCUtt (SEQ ID NO: 6712) #1
pAGAGCAGAGCUGCCCAAACtt (SEQ ID NO: 6713)
pGUGGACAUACCGAUUGACUtt (SEQ ID NO: 6714) #2
pAGUCAAUCGGUAUGUCCACtt (SEQ ID NO: 6715)
pCUCAGAACUGAAGGGCGCCtt (SEQ ID NO: 6716) #3
pGGCGCCCUUCAGUUCUGAGtt (SEQ ID NO: 6717)
pGAUAGGAGCCCAGCUUCGAtt (SEQ ID NO: 6718) #4
pUCGAAGCUGGGCUCCUAUCtt (SEQ ID NO: 6719)
Class II
21-nt iRNAs, t, deoxythymidine; p, phosphate; ps, thiophosphate
pGUUUGGGCAGCUCUGCUCpsUpstpst (SEQ ID NO:6720) #11
pAGAGCAGAGCUGCCCAAApsCpstpst (SEQ ID NO:6721)
pGUGGACAUACCGAUUGACpsUpstpst (SEQ ID NO:6722) #13
pAGUCAAUCGGUAUGUCCApsCpstpst (SEQ ID NO:6723)
pCUCAGAACUGAAGGGCGCpsCpstpst (SEQ ID NO:6724) #15
pGGCGCCCUUCAGUUCUGApsGpstpst (SEQ ID NO:6725)
pGAUAGGAGCCCAGCUUCGpsApstpst (SEQ ID NO:6726) #17
pUCGAAGCUGGGCUCCUAUpsCpstpst (SEQ ID NO:6727)
Class III
23-nt antisense, 21-nt sense, blunt-ended 5′-as
GUUUGGGCAGCUCUGCUCUCU	(SEQ ID NO:6728) #19
AGAGAGCAGAGCUGCCCAAACUU	(SEQ ID NO:6729)
GUGGACAUACCGAUUGACUGA	(SEQ ID NO:6730) #21
UCAGUCAAUCGGUAUGUCCACUU	(SEQ ID NO:6731)
CUCAGAACUGAAGGGCGCCCA	(SEQ ID NO:6732) #23
PUGGGCGCCCUUCAGUUCUGAGUU	(SEQ ID NO:6733)
GAUAGGAGCCCAGCUUCGAGU	(SEQ ID NO:6734) #25
ACUCGAAGCUGGGCUCCUAUCUU	(SEQ ID NO:6735)

[1362] Class I dsRNAs consisted of 21 nucleotide paired sense and antisense strands. The sense and antisense strands were each phosphorylated at their 5′ ends. The double stranded region was 19 nucleotides long and consisted of ribonucleotides. The 3′ end of each strand created a two nucleotide overhang consisting of two deoxyribonucleotide thymidines. See constructs #1-4 in Table 4.

[1363] Class II dsRNAs were also 21 nucleotides long, with a 19 nucleotide double strand region. The sense and antisense strands were each phosphorylated at their 5′ ends. The three 3′ terminal nucleotides of the sense and antisense strands were phosphorothioate deoxyribonucleotides, and the two terminal phosphorothioate thymidines were unpaired, creating a 3′ overhang region at each end of the iRNA molecule. See constructs 11, 13, 15, and 17 in Table 4.

[1364] Class III dsRNAs included a 23 ribonucleotide antisense strand and a 21 ribonucleotide sense strand, to form a construct having a blunt 5′ and a 3′ overhang region. See constructs 19, 21, 23, and 25 in Table 4.

[1365] Within each of the three classes of iRNAs, the four dsRNA molecules were designed to target four different regions of the ApoM transcript. dsRNAs 1, 11, and 19 targeted the 5′ end of the open reading frame (ORF). dsRNAs 2, 13, and 21, and 3, 15, and 23, targeted two internal regions (one 5′ proximal and one 3′ proximal) of the ORF, and the 4, 17, and 25 iRNA constructs targeted to a region of the 3′ untranslated sequence (3′ UTS) of the ApoM mRNA. This is summarized in Table 5.

[1366]

TABLE 5
iRNA molecules targeted to mouse ApoM
	iRNA targeted	iRNA targeted	iRNA targeted
	to 5′ end	to middle ORF	to middle ORF	iRNA targeted
	of ORF	(5′ proximal)	(3′ proximal)	to 3′UTS
Class	1	2	3	4
I
Class	11	13	15	17
II
Class	19	21	23	25
III

[1367] CD 1 mice (6-8 weeks old, ˜35 g) were administered one of the test iRNAs in PBS solution. Two hundred micrograms of iRNA in a volume of solution equal to 10% body weight (˜5.7 mg iRNA/kg mouse) was administered by the method of high pressure tail vein injection, over a 10-20 sec. time interval. After a 24 h recovery period, a second injection was performed using the same dose and mode of administration as the first injection, and following another 24 h, a third and final injection was administered, also using the same dose and mode of administration. After a final 24 h recovery, the mouse was sacrificed, serum was collected and the liver and kidney harvested to assay for an affect on ApoM gene expression. Expression was monitored by quantitative RT-PCR and Western blot analyses. This experiment was repeated for each of the iRNAs listed in table 4.

[1368] Class I iRNAs did not alter ApoM RNA levels in mice, as indicated by quantitative RT-PCR. This is in contrast to the effect of these iRNAs in cultured HepG2 cells. Cells cotransfected with a plasmid expressing exogenous ApoM RNA under a CMV promoter and a class I iRNA demonstrated a 25% or greater reduction in ApoM RNA concentrations as compared to control transfections. The iRNA molecules 1, 2 and 3 each caused a 75% decrease in exogenous ApoM mRNA levels.

[1369] Class II iRNAs reduced liver and kidney ApoM mRNA levels by ˜30-85%. The iRNA molecule “13” elicited the most dramatic reduction in mRNA levels; quantitative RT-PCR indicated a decrease of about 85% in liver tissue. Serum ApoM protein levels were also reduced as was evidenced by Western blot analysis. The iRNAs 11, 13 and 15, reduced protein levels by about 50%, while iRNA 17 had the mildest effect, reducing levels only by ˜15-20%.

[1370] Class III iRNAs (constructs 19, 21, and 23) reduced serum Apo levels by ˜40-50%.

[1371] To determine the effect of dosage on iRNA mediated ApoM inhibition, the experiment described above was repeated with three injections of 50 μg iRNA “11” (˜1.4 mg iRNA/kg mouse). This lower dosage of iRNA resulted in a reduction of serum ApoM levels of about 50%. This is compared with the reduction seen with the 200 μg injections, which reduced serum levels by 25-45%. These results indicated the lower dosage amounts of iRNAs were effective.

[1372] In an effort to increase iRNA uptake by cells, iRNAs were precomplexed with lipofectamine prior to tail vein injections. ApoM protein levels were about 50% of wildtype levels in mice injected with iRNA “11” when the molecules were preincubated with lipofectamine; ApoM levels were also about 50% of wildtype when mice were injected with iRNA “11” that was not precomplexed with lipofectamine.

[1373] These experiments revealed that modified iRNAs can greatly influence RNAi-mediated gene silencing. As demonstrated herein, modifications including phosphorothioate nucleotides are particularly effective at decreasing target protein levels.

[1374] Apolipoprotein B (apoB) is a candidate target gene for the development of novel therapies for lipid-based diseases.

[1375] Methods described herein can be used to evaluate the efficacy of a particular siRNA as a therapeutic tool for treating lipid metabolism disorders resulting elevated apoB levels. Use of siRNA duplexes to selectively bind and inactivate the target apoB mRNA is an approach to treat these disorders.

[1376] Two approaches:

[1377] i) Inhibition of apoB in ex-vivo models by transfecting siRNA duplexes homologous to human apoB mRNA in a human hepatoma cell line (Hep G2) and monitor the level of the protein and the RNA using the Western blotting and RT-PCR methods, respectively. siRNA molecules that efficiently inhibit apoB expression will be tested for similar effects in vivo.

[1378] ii) In vivo trials using an apoB transgenic mouse model (apoB100 Transgenic Mice, C57BL/6NTac-TgN (APOB100), Order Model #'s: 1004-T (hemizygotes), B6 (control)). siRNA duplexes are designed to target apoB-100 or CETP/apoB double transgenic mice which express both cholesteryl ester transfer protein (CETP) and apoB. The effect of the siRNA on gene expression in vivo can be measured by monitoring the HDL/LDL cholesterol level in serum. The results of these experiments would indicate the therapeutic potential of siRNAs to treat lipid-based diseases, including hypercholesterolemia, HDL/LDL cholesterol imbalance, familial combined hyperlipidemia, and acquired hyperlipidemia.

Background

[1379] Fats, in the form of triglycerides, are ideal for energy storage because they are highly reduced and anhydrous. An adipocyte (or fat cell) consists of a nucleus, a cell membrane, and triglycerides, and its function is to store triglycerides.

[1380] The lipid portion of the human diet consists largely of triglycerides and cholesterol (and its esters). These must be emulsified and digested to be absorbed. Specifically, fats (triacylglycerols) are ingested. Bile (bile acids, salts, and cholesterol), which is made in the liver, is secreted by the gall bladder. Pancreatic lipase digests the triglycerides to fatty acids, and also digests di-, and mono-acylglycerols, which are absorbed by intestinal epithelial cells and then are resynthesized into triacylglycerols once inside the cells. These triglycerides and some cholesterols are combined with apolipoproteins to produce chylomicrons. Chylomicrons consist of approximately 95% triglycerides. The chylomicrons transport fatty acids to peripheral tissues. Any excess fat is stored in adipose tissue.

[1381] Lipid transport and clearance from the blood into cells, and from the cells into the blood and the liver, is mediated by the lipoprotein transport proteins. This class of approximately 17 proteins can be divided into three groups: Apolipoproteins, lipoprotein processing proteins, and lipoprotein receptors.

[1382] Apolipoproteins coat lipoprotein particles, and include the A-I, A-II, A-IV, B, CI, CII, CIII, D, E, Apo(a) proteins. Lipoprotein processing proteins include lipoprotein lipase, hepatic lipase, lecithin cholesterol acyltransferase and cholesterol ester transfer protein. Lipoprotein receptors include the low density lipoprotein (LDL) receptor, chylomicron-remnant receptor (the LDL receptor like protein or LDL receptor related protein—LRP) and the scavenger receptor.

Lipoprotein Metabolism

[1383] Since the triglycerides, cholesterol esters, and cholesterol absorbed into the small intestine are not soluble in aqueous medium, they must be combined with suitable proteins (apolipoproteins) in order to prevent them from forming large oil droplets. The resulting lipoproteins undergo a type of metabolism as they pass through the bloodstream and certain organs (notably the liver).

[1384] Also synthesized in the liver is high density lipoprotein (HDL), which contains the apoproteins A-1, A-2, C-1, and D; HDL collects cholesterol from peripheral tissues and blood vessels and returns it to the liver. LDL is taken up by specific cell surface receptors into an endosome, which fuses with a lysosome where cholesterol ester is converted to free cholesterol. The apoproteins (including apo B-100) are digested to amino acids. The receptor protein is recycled to the cell membrane.

[1385] The free cholesterol formed by this process has two fates. First, it can move to the endoplasmic reticulum (ER), where it can inhibit HMG-CoA reductase, the synthesis of HMG-CoA reductase, and the synthesis of cell surface receptors for LDL. Also in the ER, cholesterol can speed up the degradation of HMG-CoA reductase. The free cholesterol can also be converted by acyl-CoA and acyl transferase (ACAT) to cholesterol esters, which form oil droplets.

[1386] ApoB is the major apolipoprotein of chylomicrons of very low density lipoproteins (VLDL, which carry most of the plasma triglyceride) and low density lipoprotein (LDL, which carry most of the plasma cholesterol). ApoB exists in human plasma in two isoforms, apoB-48 and apoB-100.

[1387] ApoB-100 is the major physiological ligand for the LDL receptor. The ApoB precursor has 4563 amino acids, and the mature apoB-100 has 4536 amino acid residues. The LDL-binding domain of ApoB-100 is proposed to be located between residues 3129 and 3532. ApoB-100 is synthesized in the liver and is required for the assembly of very low density lipoproteins VLDL and for the preparation of apoB-100 to transport triglycerides (TG) and cholesterol from the liver to other tissues. ApoB-100 does not interchange between lipoprotein particles, as do the other lipoproteins, and it is found in IDL and LDL particles. After the removal of apolipoproteins A, E and C, apoB is incorporation into VLDL by hepatocytes. ApoB-48 is present in chylomicrons and plays an essential role in the intestinal absorption of dietary fats. ApoB-48 is synthesized in the small intestine. It comprises the N-terminal 48% of apoB-100 and is produced by a posttranscriptional apoB-100 mRNA editing event at codon 2153 (C to U). This editing event is a product of the apoBEC-1b enzyme, which is expressed in the intestine. This editing event creates a stop codon instead of a glutamine codon, and therefore apoB-48, instead of apoB-100 is expressed in the intestine (apoB-100 is expressed in the liver).

[1388] There is also strong evidence that plasma apoB levels may be a better index of the risk of coronary artery disease (CAD) than total or LDL cholesterol levels. Clinical studies have demonstrated the value of measuring apoB in hypertriglyceridemic, hypercholesterolemic and normalipidemic subjects.

[1389]

TABLE 6
Reference Range Lipid level in the Blood
	Lipid	Range (mmols/L)
	Plasma Cholesterol	3.5-6.5
	Low density lipoprotein	1.55-4.4
	Very low density lipoprotein	0.128-0.645
	High density lipoprotein/triglycerides	0.5-2.1
	Total lipid	4.0-10 g/L

Molecular Genetics of Lipid Metabolism in Both Humans and Induced Mutant Mouse Models

[1390] Elevated plasma levels of LDL and apoB are associated with a higher risk for atherosclerosis and coronary heart disease, a leading cause of mortality. ApoB is the mandatory constituent of LDL particles. In addition to its role in lipoprotein metabolism, apoB has also been implicated as a factor in male infertility and fetal development. Furthermore, two quantitative trait loci regulating plasma apoB levels have been discovered, through the use of transgenic mouse models. Future experiments will facilitate the identification of human orthologous genes encoding regulators of plasma apoB levels. These loci are candidate therapeutic targets for human disorders characterized by altered plasma apoB levels. Such disorders include non-apoB linked hypobetalipoproteinemia and familial combined hyperlipidemia. The identification of these genetic loci would also reveal possible new pathways involved in the regulation of apoB secretion, potentially providing novel sites for pharmacological therapy.

Diseases and Clinical Pharmacology

[1391] Familial combined hyperlipemia (FCHL) affects an estimated one in 10 Americans. FCHL can cause premature heart disease.

Familial Hypercholesterolemia (High Level of Apo B)

[1392] A common genetic disorder of lipid metabolism. Familial hypercholesterolemia is characterized by elevated serum TC in association with xanthelasma, tendon and tuberous xanthomas, accelerated atherosclerosis, and early death from myocardial infarction (MI). It is caused by absent or defective LDL cell receptors, resulting in delayed LDL clearance, an increase in plasma LDL levels, and an accumulation of LDL cholesterol in macrophages over joints and pressure points, and in blood vessels.

Atherosclerosis (High Level of Apo B)

[1393] Atherosclerosis develops as a deposition of cholesterol and fat in the arterial wall due to disturbances in lipid transport and clearance from the blood into cells and from the cells to blood and the liver.

[1394] Clinical studies have demonstrated that elevation of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and apoB-100 promote human atherosclerosis. Similarly, decreased levels of high-density lipoprotein cholesterol (HDL-C) are associated with the development of atherosclerosis.

[1395] ApoB may be factor in the genetic cause of high cholesterol.

The Risk of Coronary Artery Disease (CAD) (High Level of Apo B)

[1396] Cardiovascular disease, including coronary heart disease and stroke, is a leading cause of death and disability. The major risk factors include age, gender, elevated low-density lipoprotein cholesterol blood levels, decreased high-density lipoprotein cholesterol levels, cigarette smoking, hypertension, and diabetes. Emerging risk factors include elevated lipoprotein (a), remnant lipoproteins, and C reactive protein. Dietary intake, physical activity and genetics also impact cardiovascular risk. Hypertension and age are the major risk factors for stroke.

[1397] Abetalipoproteinemia, an inherited human disease characterized by a near-complete absence of apoB-containing lipoproteins in the plasma, is caused by mutations in the gene for microsomal triglyceride transfer protein (MTP).

Model for Human Atherosclerosis (Lipoprotein a Transgenic Mouse)

[1398] Numerous studies have demonstrated that an elevated plasma level of lipoprotein(a) (Lp(a)) is a major independent risk factor for coronary heart disease (CHD). Current therapies, however, have little or no effect on apo(a) levels and the homology between apo(a) and plasminogen presents barriers to drug development. Lp(a) particles consist of apo(a) and apoB-100 proteins, and they are found only in primates and the hedgehog. The development of LPA transgenic mouse requires the creation of animals that express both human apoB and apo(a) transgenes to achieve assembly of LP(a). An atherosclerosis mouse model would facilitate the study of the disease process and factors influencing it, and further would facilitate the development of therapeutic or preventive agents. There are several strategies for gene-oriented therapy. For example, the missing or non-functional gene can be replaced, or unwanted gene activity can be inhibited.

Model for Lipid Metabolism and Atherosclerosis

[1399] DNX Transgenic Sciences has demonstrated that both CETP/ApoB and ApoB transgenic mice develop atherosclerotic plaques.

Model for apoB-100 Overexpression

[1400] The apoB-100 transgenic mice express high levels of human apoB-100. They consequently demonstrate elevated serum levels of LDL cholesterol. After 6 months on a high-fat diet, the mice develop significant foam cell accumulation under the endothelium and within the media, as well as cholesterol crystals and fibrotic lesions.

Model for Cholesteryl Ester Transfer Protein Over Expression

[1401] The apoB-100 transgenic mice express the human enzyme, CETP, and consequently demonstrate a dramatically reduced level of serum HDL cholesterol.

Model for apoB-100 and CETP Overexpression

[1402] The apoB-100 transgenic mice express both CETP and apoB-100, resulting in mice with a human like serum HDL/LDL distribution. Following 6 months on a high-fat diet these mice develop significant foam cell accumulation underlying the endothelium and within the media, as well as cholesterol crystals and fibrotic lesions.

ApoB100 Transgenic Mice (Order Model #'s: 1004-T (Hemizygotes), B6 (Control))

[1403] These mice express high levels of human apoB-100, resulting in mice with elevated serum levels of LDL cholesterol. These mice are useful in identifying and evaluating compounds to reduce elevated levels of LDL cholesterol and the risk of atherosclerosis. When fed a high fat cholesterol diet, these mice develop significant foam cell accumulation underlay the endothelium and within the media, and have significantly more complex atherosclerotic lesions than control animals.

Double Transgenic Mice, CETP/ApoB100 (Order Model #: 1007-TT)

[1404] These mice express both CETP and apoB-100, resulting in a human-like serum HDL/LDL distribution. These mice are useful for evaluating compounds to treat hypercholesterolemia or HDL/LDL cholesterol imbalance to reduce the risk of developing atherosclerosis. When fed a high fat high cholesterol diet, these mice develop significant foam cell accumulation underlying the endothelium and within the media, and have significantly more complex atherosclerotic lesions than control animals.

ApoE Gene Knockout Mouse

[1405] Homozygous apoE knockout mice exhibit strong hypercholesterolemia, primarily due to elevated levels of VLDL and IDL caused by a defect in lipoprotein clearance from plasma. These mice develop atherosclerotic lesions which progress with age and resemble human lesions (Zhang et al., Science 258:46-71, 1992; Plump et al., Cell 71:343-353, 1992; Nakashima et al., Arterioscler Thromp. 14:133-140, 1994; Reddick et al., Arterioscler Tromb. 14:141-147, 1994). These mice are a promising model for studying the effect of diet and drugs on atherosclerosis.

[1406] Low density lipoprotein receptor (LDLR) mediates lipoprotein clearance from plasma through the recognition of apoB and apoE on the surface of lipoprotein particles. Humans, who lack or have a decreased number of the LDL receptors, have familial hypercholesterolemia and develop CHD at an early age.

ApoE Knockout Mice (Order Model #: APOE-M)

[1407] The apoE knockout mouse was created by gene targeting in embryonic stem cells to disrupt the apoE gene. ApoE, a glycoprotein, is a structural component of very low density lipoprotein (VLDL) synthesized by the liver and intestinally synthesized chylomicrons. It is also a constituent of a subclass of high density lipoproteins (HDLs) involved in cholesterol transport activity among cells. One of the most important roles of apoE is to mediate high affinity binding of chylomicrons and VLDL particles that contain apoE to the low density lipoprotein (LDL) receptor. This allows for the specific uptake of these particles by the liver which is necessary for transport preventing the accumulation in plasma of cholesterol-rich remnants. The homozygous inactivation of the apoE gene results in animals that are devoid of apoE in their sera. The mice appear to develop normally, but they exhibit five times the normal serum plasma cholesterol and spontaneous atherosclerotic lesions. This is similar to a disease in people who have a variant form of the apoE gene that is defective in binding to the LDL receptor and are at risk for early development of atherosclerosis and increased plasma triglyceride and cholesterol levels. There are indications that apoE is also involved in immune system regulation, nerve regeneration and muscle differentiation. The apoE knockout mice can be used to study the role of apoE in lipid metabolism, atherogenesis, and nerve injury, and to investigate intervention therapies that modify the atherogenic process.

Apoe4 Targeted Replacement Mouse (Order Model #: 001549-M)

[1408] ApoE is a plasma protein involved in cholesterol transport, and the three human isoforms (E2, E3, and E4) have been associated with atherosclerosis and Alzheimer's disease. Gene targeting of 129 ES cells was used to replace the coding sequence of mouse apoE with human APOE 4 without disturbing the murine regulatory sequences. The E4 isoform occurs in approximately 14% of the human population and is associated with increased plasma cholesterol and a greater risk of coronary artery disease. The Taconic apoE4 Targeted Replacement model has normal plasma cholesterol and triglyceride levels, but altered quantities of different plasma lipoprotein particles. This model also has delayed plasma clearance of cholesterol-rich lipoprotein particles (VLDL), with only half the clearance rate seen in the apoE3 Targeted Replacement model. Like the apoE3 model, the apoE4 mice develop altered plasma lipoprotein values and atherosclerotic plaques on an atherogenic diet. However, the atherosclerosis is more severe in the apoE4 model, with larger plaques and cholesterol apoE and apoB-48 levels twice that seen in the apoE3 model. The Taconic apoE4 Targeted Replacement model, along with the apoE2 and apoE3 Targeted Replacement Mice, provide an excellent tool for in vivo study of the human apoE isoforms.

CETP Transgenic Mice (Order Model #: 1003-T)

[1409] These animals express the human plasma enzyme, CETP, resulting in mice with a dramatic reduction in serum HDL cholesterol. The mice can be useful in identifying and evaluating compounds that increase the levels of HDL cholesterol for reducing the risk of developing atherosclerosis

Transgene/Promoter: Human Apolipoprotein A-I

[1410] These mice produce mouse HDL cholesterol particles that contain human apolipoprotein A-I. Transgenic expression is life-long in both sexes (Biochemical Genetics and Metabolism Laboratory, Rockefeller University, NY City).

A Mouse Model for Abetalipoproteinemia

[1411] Abetalipoproteinemia, an inherited human disease characterized by a near-complete absence of apoB-containing lipoproteins in the plasma, is caused by mutations in the gene for microsomal triglyceride transfer protein (MTP). Gene targeting was used to knock out the mouse MTP gene (Mttp). In heterozygous knockout mice (Mttp^+/−), the MTP mRNA, protein, and activity levels were reduced by 50% in both liver and intestine. Recent studies with heterozygous MTP knockout mice have suggested that half-normal levels of MTP in the liver reduce apoB secretion. They hypothesized that reduced apoB secretion in the setting of half-normal MTP levels might be caused by a reduced MTP:apoB ratio in the endoplasmic reticulum, which would reduce the number of apoB-MTP interactions. If this hypothesis were true, half-normal levels of MTP might have little impact on lipoprotein secretion in the setting of half-normal levels of apoB synthesis (since the ratio of MTP to apoB would not be abnormally low) and might cause an exaggerated reduction in lipoprotein secretion in the setting of apoB overexpression (since the ratio of MTP to apoB would be even lower). To test this hypothesis, they examined the effects of heterozygous MTP deficiency on apoB metabolism in the setting of normal levels of apoB synthesis, half-normal levels of apoB synthesis (heterozygous Apob deficiency), and increased levels of apoB synthesis (transgenic overexpression of human apoB). Contrary to their expectations, half-normal levels of MTP reduced plasma apoB-100 levels to the same extent (˜25-35%) at each level of apoB synthesis. In addition, apoB secretion from primary hepatocytes was reduced to a comparable extent at each level of apoB synthesis. Thus, these results indicate that the concentration of MTP within the endoplasmic reticulum, rather than the MTP:apoB ratio, is the critical determinant of lipoprotein secretion. Finally, heterozygosity for an apoB knockout mutation was found to lower plasma apoB-100 levels more than heterozygosity for an MTP knockout allele. Consistent with that result, hepatic triglyceride accumulation was greater in heterozygous apoB knockout mice than in heterozygous MTP knockout mice. Cre/loxP tissue-specific recombination techniques were also used to generate liver-specific Mttp knockout mice. Inactivation of the Mttp gene in the liver caused a striking reduction in very low density lipoprotein (VLDL) triglycerides and large reductions in both VLDL/low density lipoproteins (LDL) and high density lipoprotein cholesterol levels. Histologic studies in liver-specific knockout mice revealed moderate hepatic steatosis. Currently being tested is the hypothesis that accumulation of triglycerides in the liver renders the liver more susceptible to injury by a second insult (e.g., lipopolysaccharide).

Human Apo B (Apolipoprotein B) Transgene Mice Show Apo B Locus May have a Causative Role Male Infertility

[1412] The fertility of apoB (apolipoprotein B) (+/−) mice was recorded during the course of backcrossing (to C57BL/6J mice) and test mating. No apparent fertility problem was observed in female apoB (+/−) and wild-type female mice, as was documented by the presence of vaginal plugs in female mice. Although apoB (+/−) mice mated normally, only 40% of the animals from the second backcross generation produced any offspring within the 4-month test period. Of the animals that produced progeny, litters resulted from <50% of documented matings. In contrast, all wild-type mice (6/6—i. e., 100%) tested were fertile. These data suggest genetic influence on the infertility phenotype, as a small number of male heterozygotes were not sterile. Fertilization in vivo was dramatically impaired in male apoB (+/−) mice. 74% of eggs examined were fertilized by the sperm from wild-type mice, whereas only 3% of eggs examined were fertilized by the sperm from apoB (+/−) mice. The sperm counts of apoB (+/−) mice were mildly but significantly reduced compared with controls. However, the percentage of motile sperm was markedly reduced in the apoB (+/−) animals compared with that of the wild-type controls. Of the sperm from apoB (+/−) mice, 20% (i.e., 4.9% of the initial 20% motile sperm) remained motile after 6 hr of incubation, whereas 45% (i.e., 33.6% of the initial 69.5%) of the motile sperm retained motility in controls after this time. In vitro fertilization yielded no fertilized eggs in three attempts with apo B (+/−) mice, while wild-type controls showed a fertilization rate of 53%. However, sperm from apoB (+/−) mice fertilized 84% of eggs once the zona pellucida had been removed. Numerous sperm from apoB (+/−) mice were seen binding to zona-intact eggs. However, these sperm lost their motility when observed 4-6 hours after binding, showing that sperm from apoB (+/−) mice were unable to penetrate the zona pellucida but that the interaction between sperm and egg was probably not direct. Sperm binding to zona-free oocytes was abnormal. In the apoB (+/−) mice, sperm binding did not attenuate, even after pronuclei had clearly formed, suggesting that apoB deficiency results in abnormal surface interaction between the sperm and egg.

[1413] Knockout of the mouse apoB gene resulted in embryonic lethality in homozygotes, protection against diet-induced hypercholesterolemia in heterozygotes, and developmental abnormalities in mice.

Model of Insulin Resistance, Dyslipidemia & Overexpression of Human apoB

[1414] It was shown that the livers of apoB mice assemble and secrete increased numbers of VLDL particles.

Introduction

[1415] The regulation of hepatic gluconeogenesis is an important process in the adjustment of the blood glucose level. Pathological changes in the glucose production of the liver are a central characteristic in type-2-diabetes. For example, the fasting hyperglycemia observed in patients with type-2-diabetes reflects the lack of inhibition of hepatic gluconeogenesis and glycogenolysis due to the underlying insulin resistance in this disease. Extreme conditions of insulin resistance can be observed for example in mice with a liver-specific insulin receptor knockout (‘LIRKO’). These mice have an increased expression of the two rate-limiting gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and the glucose-6-phosphatase catalytic subunit (G6Pase). Insulin is known to repress both PEPCK and G6Pase gene expression at the transcriptional level and the signal transduction involved in the regulation of G6Pase and PEPCK gene expression by insulin is only partly understood. While PEPCK is involved in a very early step of hepatic gluconeogenesis (synthesis of phosphoenolpyruvate from oxaloacetate), G6Pase catalyzes the terminal step of both, gluconeogenesis and glycogenolysis, the cleavage of glucose-6-phosphate into phosphate and free glucose, which is then delivered into the blood stream.

[1416] The pharmacological intervention in the regulation of expression of PEPCK and G6Pase can be used for the treatment of the metabolic aberrations associated with diabetes. Hepatic glucose production can be reduced by an iRNA-based reduction of PEPCK and G6Pase enzymatic activity in subjects with type-2-diabetes.

Targets for iRNA

[1417] Glucose-6-Phosphatase (G6Pase)

[1418] G6Pase mRNA is expressed principally in liver and kidney, and in lower amounts in the small intestine. Membrane-bound G6Pase is associated with the endoplasmic reticulum. Low activities have been detected in skeletal muscle and in astrocytes as well.

[1419] G6Pase catalyzes the terminal step in gluconeogenesis and glycogenolysis. The activity of the enzyme is several fold higher in diabetic animals and probably in diabetic humans. Starvation and diabetes cause a 2-3-fold increase in G6Pase activity in the liver and a 2-4-fold increase in G6Pase mRNA.

[1420] Phosphoenolpyruvate Carboxykinase (PEPCK)

[1421] Overexpression of PEPCK in mice results in symptoms of type-2-diabetes mellitus. PEPCK overexpression results in a metabolic pattern that increases G6Pase mRNA and results in a selective decrease in insulin receptor substrate (IRS)-2 protein, decreased phosphatidylinositol 3-kinase activity, and reduced ability of insulin to suppress gluconeogenic gene expression.

[1422]

TABLE 7
Other targets to inhibit hepatic glucose production
Target	Comment
FKHR	good evidence for antidiabetic
	phenotype (Nakae et al.,
	Nat Genetics 32: 245(2002)
Glucagon
Glucagon receptor
Glycogen phosphorylase
PGC-1 (PPAR-Gamma	regulates the cAMP response
Coactivator)	(and probably the PKB/FKHR-
	regulation) on PEPCK/G6Pase
Fructose-1,6-bisphosphatase
Glucose-6-phospate translocator
Glucokinase inhibitory
regulatory protein

Materials and Methods
Animals:

[1423] BKS.Cg-m+/+ Lepr db mice, which contain a point mutation in the leptin receptor gene are used to examine the efficacy of iRNA for the targets listed above.

[1424] BKS.Cg-m+/+ Lepr db are available from the Jackson Laboratory (Stock Number 000642). These animals are obese at 3-4 weeks after birth, show elevation of plasma insulin at 10 to 14 days, elevation of blood sugar at 4 to 8 weeks, and uncontrolled rise in blood sugar. Exogenous insulin fails to control blood glucose levels and gluconeogenic activity increases.

[1425] The following numbers of male animals (age>12 weeks) would ideally be tested with the following iRNAs:

• • [1426] PEPCK, 2 sequences, 5 animals per sequence
    • [1427] G6Pase, 2 sequences, 5 animals per sequence
    • [1428] 1 nonspecific sequence, 5 animals
    • [1429] 1 control group (only injected, no siRNA), 5 animals
    • [1430] 1 control group (not injected, no siRNA), 5 animals
      Reagents:

[1431] Necessary reagents would ideally include a Glucometer Elite XL (Bayer, Pittsburgh, Pa.) for glucose quantification, and an Insulin Radioimmunoassay (RIA) kit (Amersham, Piscataway, N.J.) for insulin quanitation

Assays:

[1432] G6P enzyme assays and PEPCK enzyme assays are used to measure the activity of the enzymes. Northern blotting is used to detect levels of G6Pase and PEPCK mRNA. Antibody-based techniques (e.g., immunoblotting, immunofluorescence) are used to detect levels of G6Pase and PEPCK protein. Glycogen staining is used to detect levels of glycogen in the liver. Histological analysis is performed to analyze tissues.

Gene Information:

G6Pase

GenBank® No.: NM_008061, Mus musculus glucose-6-phosphatase, catalytic (G6pc), mRNA 1 . . . 2259, ORF 83 . . . 1156;

GenBank® No: U00445, Mus musculus glucose-6-phosphatase mRNA, complete cds 1 . . . 2259, ORF 83 . . . 1156

GenBank® No: BC013448

PEPCK

GenBank® No: NM_011044, Mus musculus phosphoenolpyruvate carboxykinase 1, cytosolic (Pck1), mRNA. 1 . . . 2618, ORF 141 . . . 2009

GenBank® No: AF009605.1

Administration of iRNA:

[1433] iRNA corresponding to the genes described above would be administered to mice with hydrodynamic injection. One control group of animals would be treated with Metformin as a positive control for reduction in hepatic glucose levels.

Experimental Protocol

[1434] Mice would be housed in a facility in which there is light from 7:00 AM to 7:00 PM. Mice would be fed ad libidum from 7:00 PM to 7:00 AM and fast from 7:00 AM to 7:00 PM.

Day 0: 7:00 PM: Approximately 100 μl blood would be drawn from the tail. Serum would be isolated to measure glucose, insulin, HbA1c (EDTA-blood), glucagon, FFAs, lactate, corticosterone, serum triglycerides.

• [1435] Day 1-7: Blood glucose would be measured daily at 8:00 AM and 6:00 PM (approx. 3-5 μl; measured with a Haemoglucometer)
  Day 8: Blood glucose would be measured daily at 8:00 AM and 6:00 PM. iRNA would be injected between 10:00 AM and 2:00 PM
  Day 9-20: Blood glucose would be measured daily at 8:00 AM and 6:00 PM.
  Day 21: Mice would be sacrificed after 10 hours of fasting.

[1436] Blood would be isolated. Glucose, insulin, HbA1c (EDTA-blood), glucagon, FFAs, lactate, corticosterone, serum triglycerides would be measured. Liver tissue would be isolated for histology, protein assays, RNA assays, glycogen quantitation, and enzyme assays.

[1437] iRNA targeted to the Glucose-6-Phosphatase (G6P) gene was used to examine the effects of inhibition of G6P expression on glucose metabolism in vivo.

[1438] Female mice, 10 weeks of age, strain BKS.Cg-m+/+ Lepr db (The Jackson Laboratory) were used for in vivo analysis of enzymes of the hepatic glucose production. Mice were housed under conditions where it was light from 6:30 am to 6:30 μm. Mice were fed (ad libidum) during the night period and fasted during the day period.

[1439] On day 1, approximately 100 μl of blood was collected from test animals by puncturing the retroorbital plexus. On days 1-7, blood glucose was measured in blood obtained from tail veins (approximately 3-5 μl) using a Glucometer (Elite XL, Bayer). Blood glucose was sampled daily at 8 am and 6 μm.

[1440] On day 7 at approximately 2 μm, GL3 plasmid (10 μg) and siRNAs (100 μg G6Pase specific, Renilla nonspecific or no siRNA control) were delivered to animals using hydrodynamic coinjection.

[1441] On day 8, GL3 expression was analyzed by injection of luceferin (3 mg) after anaesthesia with avertin and imaging. This was done to control for successful hydrodynamic delivery.

[1442] On days 8-10, blood glucose was measured in blood obtained from tail veins (approximately 3-5 ml) using a Glucometer (Elite XL, Bayer).

[1443] On day 10, mice were sacrificed after 10 hours of fasting. Blood and liver were isolated from sacrificed animals.

[1444] Results: Coinjection of GL3 plasmid and G6Pase iRNA (G6P4) reduced blood glucose levels for the short term. Coinjection of GL3 plasmid and Renilla nonspecific iRNA had no effect on blood glucose levels.

[1445] Tables 8-13 below provide selected palindromic sequences from the following genes: human ApoB, human glucose-6-phosphatase, rat glucose-6-phosphatase, β-catenin, and hepatitis C virus (HCV).

[1446]

TABLE 8
Selected palindromic sequences from human ApoB
		Start	End
	Source	Index	Index
SEQ ID NO: 1	ggccattccagaagggaag	509	528	SEQ ID NO: 1004
SEQ ID NO: 2	tgccatctcgagagttcca	4099	4118	SEQ ID NO: 1005
SEQ ID NO: 3	catgtcaaacactttgtta	7056	7075	SEQ ID NO: 1006
SEQ ID NO: 4	tttgttataaatcttattg	7068	7087	SEQ ID NO: 1007
SEQ ID NO: 5	tctggaaaagggtcatgga	8880	8699	SEQ ID NO: 1008
SEQ ID NO: 6	cagctcttgttcaggtcca	10900	10919	SEQ ID NO: 1009
SEQ ID NO: 7	ggaggttccccagctctgc	356	375	SEQ ID NO: 1010
SEQ ID NO: 8	ctgttttgaagactctcca	1081	1100	SEQ ID NO: 1011
SEQ ID NO: 9	agtggctgaaacgtgtgca	1297	1316	SEQ ID NO: 1012
SEQ ID NO: 10	ccaaaatagaagggaatct	2068	2087	SEQ ID NO: 1013
SEQ ID NO: 11	tgaagagaagattgaattt	3620	3639	SEQ ID NO: 1014
SEQ ID NO: 12	agtggtggcaacaccagca	4230	4249	SEQ ID NO: 1015
SEQ ID NO: 13	aaggctccacaagtcatca	5950	5969	SEQ ID NO: 1016
SEQ ID NO: 14	gtcagccaggtttatagca	7725	7744	SEQ ID NO: 1017
SEQ ID NO: 15	tgatatctggaaccttgaa	10727	10746	SEQ ID NO: 1018
SEQ ID NO: 16	gtcaagttgagcaatttct	13423	13442	SEQ ID NO: 1019
SEQ ID NO: 17	atccagatggaaaagggaa	13480	13499	SEQ ID NO: 1020
SEQ ID NO: 18	atttgtttgtcaaagaagt	4543	4562	SEQ ID NO: 1021
SEQ ID NO: 19	ctggaaaatgtcagcctgg	204	223	SEQ ID NO: 1022
SEQ ID NO: 20	accaggaggttcttcttca	1729	1748	SEQ ID NO: 1023
SEQ ID NO: 21	aaagaagttctgaaagaat	1956	1975	SEQ ID NO: 1024
SEQ ID NO: 22	gctacagcttatggctcca	3570	3589	SEQ ID NO: 1025
SEQ ID NO: 23	atcaatattgatcaatttg	6414	6433	SEQ ID NO: 1026
SEQ ID NO: 24	gaattatcttttaaaacat	7326	7345	SEQ ID NO: 1027
SEQ ID NO: 25	cgaggcccgcgctgctggc	130	149	SEQ ID NO: 1027
SEQ ID NO: 26	acaactatgaggctgagag	271	290	SEQ ID NO: 1029
SEQ ID NO: 27	gctgagagttccagtggag	282	301	SEQ ID NO: 1030
SEQ ID NO: 28	tgaagaaaaccaagaactc	448	467	SEQ ID NO: 1031
SEQ ID NO: 29	cctacttacatcctgaaaa	558	577	SEQ ID NO: 1032
SEQ ID NO: 30	ctacttacatcctgaacat	559	578	SEQ ID NO: 1033
SEQ ID NO: 31	gagacagaagaagccaagc	615	634	SEQ ID NO: 1034
SEQ ID NO: 32	cactcactttaccgtcaag	671	690	SEQ ID NO: 1035
SEQ ID NO: 33	ctgatcagcagcagccagt	822	841	SEQ ID NO: 1036
SEQ ID NO: 34	actggacgctaagaggaag	854	873	SEQ ID NO: 1037
SEQ ID NO: 35	agaggaagcatgtggcaga	865	884	SEQ ID NO: 1038
SEQ ID NO: 36	tgaagactctccaggaact	1087	1106	SEQ ID NO: 1039
SEQ ID NO: 37	ctctgagcaaaatatccag	1121	1140	SEQ ID NO: 1040
SEQ ID NO: 38	atgaagcagtcacatctct	1189	1208	SEQ ID NO: 1041
SEQ ID NO: 39	ttgccacagctgattgagg	1209	1228	SEQ ID NO: 1042
SEQ ID NO: 40	agctgattgaggtgtccag	1216	1235	SEQ ID NO: 1043
SEQ ID NO: 41	tgctccactcacatcctcc	1278	1297	SEQ ID NO: 1044
SEQ ID NO: 42	tgaaacgtgtgcatgccaa	1303	1322	SEQ ID NO: 1045
SEQ ID NO: 43	gacattgctaattacctga	1503	1522	SEQ ID NO: 1046
SEQ ID NO: 44	ttcttcttcagactttcct	1738	1757	SEQ ID NO: 1047
SEQ ID NO: 45	ccaatatcttgaactcaga	1903	1922	SEQ ID NO: 1048
SEQ ID NO: 46	aaagttagtgaaagaagtt	1946	1965	SEQ ID NO: 1049
SEQ ID NO: 47	aagttagtgaaagaagttc	1947	1966	SEQ ID NO: 1050
SEQ ID NO: 48	aaagaagttctgaaagaat	1956	1975	SEQ ID NO: 1051
SEQ ID NO: 49	tttggctataccaaagatg	2322	2341	SEQ ID NO: 1052
SEQ ID NO: 50	tgttgagaagctgattaaa	2381	2400	SEQ ID NO: 1053
SEQ ID NO: 51	caggaagggctcaaagaat	2561	2580	SEQ ID NO: 1054
SEQ ID NO: 52	aggaagggctcaaagaatg	2562	2581	SEQ ID NO: 1055
SEQ ID NO: 53	gaagggctcaaagaatgac	2564	2583	SEQ ID NO: 1056
SEQ ID NO: 54	caaagaatgacttttttct	2572	2591	SEQ ID NO: 1057
SEQ ID NO: 55	catggagaatgcctttgaa	2603	2622	SEQ ID NO: 1058
SEQ ID NO: 56	ggagccaaggctggagtaa	2679	2698	SEQ ID NO: 1059
SEQ ID NO: 57	tcattccttccccaaagag	2884	2903	SEQ ID NO: 1060
SEQ ID NO: 58	acctatgagctccagagag	3165	3184	SEQ ID NO: 1061
SEQ ID NO: 59	gggcaaaacgtcttacaga	3365	3384	SEQ ID NO: 1062
SEQ ID NO: 60	accctggacattcagaaca	3387	3406	SEQ ID NO: 1063
SEQ ID NO: 61	atgggcgacctaagttgtg	3429	3448	SEQ ID NO: 1064
SEQ ID NO: 62	gatgaagagaagattgaat	3618	3637	SEQ ID NO: 1065
SEQ ID NO: 63	caatgtagataccaaaaaa	3656	3675	SEQ ID NO: 1066
SEQ ID NO: 64	gtagataccaaaaaaatga	3660	3679	SEQ ID NO: 1067
SEQ ID NO: 65	gcttcagttcatttggact	4509	4528	SEQ ID NO: 1068
SEQ ID NO: 66	tttgtttgtcaaagaagtc	4544	4563	SEQ ID NO: 1069
SEQ ID NO: 67	ttgtttgtcaaagaagtca	4545	4564	SEQ ID NO: 1070
SEQ ID NO: 68	tggcaatgggaaactcgct	5846	5865	SEQ ID NO: 1071
SEQ ID NO: 69	aacctctggcatttacttt	5917	5936	SEQ ID NO: 1072
SEQ ID NO: 70	catttactttctctcatga	5926	5945	SEQ ID NO: 1073
SEQ ID NO: 71	aaagtcagtgccctgctta	6009	6028	SEQ ID NO: 1074
SEQ ID NO: 72	tcccattttttgagacctt	6322	6341	SEQ ID NO: 1075
SEQ ID NO: 73	catcaatattgatcaattt	6413	6432	SEQ ID NO: 1076
SEQ ID NO: 74	taaagatagttatgattta	6665	6684	SEQ ID NO: 1077
SEQ ID NO: 75	tattgatgaaatcattgaa	6713	6732	SEQ ID NO: 1078
SEQ ID NO: 76	atgatctacatttgtttat	6790	6809	SEQ ID NO: 1079
SEQ ID NO: 77	agagacacatacagaatat	6919	6938	SEQ ID NO: 1080
SEQ ID NO: 78	gacacatacagaatataga	6922	6941	SEQ ID NO: 1081
SEQ ID NO: 79	agcatgtcaaacactttgt	7054	7073	SEQ ID NO: 1082
SEQ ID NO: 80	tttttagaggaaaccaagg	7515	7534	SEQ ID NO: 1083
SEQ ID NO: 81	ttttagaggaaaccaaggc	7516	7535	SEQ ID NO: 1084
SEQ ID NO: 82	ggaagatagacttcctgaa	9307	9326	SEQ ID NO: 1085
SEQ ID NO: 83	cactgtttctgagtcccag	9334	9353	SEQ ID NO: 1086
SEQ ID NO: 84	cacaaatcctttggctgtg	9668	9687	SEQ ID NO: 1087
SEQ ID NO: 85	ttcctggatacactgttcc	9853	9872	SEQ ID NO: 1088
SEQ ID NO: 86	gaaatctcaagctttctct	10042	10061	SEQ ID NO: 1089
SEQ ID NO: 87	tttcttcatcttcatctgt	10210	10229	SEQ ID NO: 1090
SEQ ID NO: 88	tctaccgctaaaggagcag	10521	10540	SEQ ID NO: 1091
SEQ ID NO: 89	ctaccgctaaaggagcagt	10522	10541	SEQ ID NO: 1092
SEQ ID NO: 90	agggcctctttttcaccaa	10831	10850	SEQ ID NO: 1093
SEQ ID NO: 91	ttctccatccctgtaaaag	11265	11284	SEQ ID NO: 1094
SEQ ID NO: 92	gaaaaacaaagcagattat	11816	11835	SEQ ID NO: 1095
SEQ ID NO: 93	actcactcattgattttct	12682	12701	SEQ ID NO: 1096
SEQ ID NO: 94	taaactaatagatgtaatc	12890	12909	SEQ ID NO: 1097
SEQ ID NO: 95	caaaacgagcttcaggaag	13200	13219	SEQ ID NO: 1098
SEQ ID NO: 96	tggaataatgctcagtgtt	2366	2385	SEQ ID NO: 1099
SEQ ID NO: 97	gatttgaaatccaaagaag	2400	2419	SEQ ID NO: 1100
SEQ ID NO: 98	atttgaaatccaaagaagt	2401	2420	SEQ ID NO: 1101
SEQ ID NO: 99	atcaacagccgcttctttg	990	1009	SEQ ID NO: 1102
SEQ ID NO: 100	tgttttgaagactctccag	1082	1101	SEQ ID NO: 1103
SEQ ID NO: 101	cccttctgatagatgtggt	1324	1343	SEQ ID NO: 1104
SEQ ID NO: 102	tgagcaagtgaagaacttt	1868	1887	SEQ ID NO: 1105
SEQ ID NO: 103	atttgaaatccaaagaagt	2401	2420	SEQ ID NO: 1106
SEQ ID NO: 104	atccaaagaagtcccggaa	2408	2427	SEQ ID NO: 1107
SEQ ID NO: 105	agagcctacctccgcatct	2430	2449	SEQ ID NO: 1108
SEQ ID NO: 106	aatgcctttgaactcccca	2610	2629	SEQ ID NO: 1109
SEQ ID NO: 107	gaagtccaaattccggatt	3297	3316	SEQ ID NO: 1110
SEQ ID NO: 108	tgcaagcagaagccagaag	3496	3515	SEQ ID NO: 1111
SEQ ID NO: 109	gaagagaagattgaatttg	3621	3640	SEQ ID NO: 1112
SEQ ID NO: 110	atgctaaaggcacatatgg	4597	4616	SEQ ID NO: 1113
SEQ ID NO: 111	tccctcacctccacctctg	4737	4756	SEQ ID NO: 1114
SEQ ID NO: 112	atttacagctctgacaagt	5427	5446	SEQ ID NO: 1115
SEQ ID NO: 113	aggagcctaccaaaataat	5594	5613	SEQ ID NO: 1116
SEQ ID NO: 114	aaagctgaagcacatcaat	6401	6420	SEQ ID NO: 1117
SEQ ID NO: 115	ctgctggaaacaacgagaa	9418	9437	SEQ ID NO: 1118
SEQ ID NO: 116	ttgaaggaattcttgaaaa	9582	9601	SEQ ID NO: 1119
SEQ ID NO: 117	gaagtaaaagaaaattttg	10743	10762	SEQ ID NO: 1120
SEQ ID NO: 118	tgaagaagatggcaaattt	11984	12003	SEQ ID NO: 1121
SEQ ID NO: 119	aggatctgagttattttgc	14035	14054	SEQ ID NO: 1122
SEQ ID NO: 120	gtgcccttctcggttgctg	18	37	SEQ ID NO: 1123
SEQ ID NO: 121	ggcgctgcctgcgctgctg	146	165	SEQ ID NO: 1124
SEQ ID NO: 122	ctgcgctgctgctgctgct	154	173	SEQ ID NO: 1125
SEQ ID NO: 123	gctgctggcgggcgccagg	170	189	SEQ ID NO: 1126
SEQ ID NO: 124	aagaggaaatgctggaaaa	193	212	SEQ ID NO: 1127
SEQ ID NO: 125	ctggaaaatgtcagcctgg	204	223	SEQ ID NO: 1128
SEQ ID NO: 126	tggagtccctgggactgct	296	315	SEQ ID NO: 1129
SEQ ID NO: 127	ggagtccctgggactgctg	297	316	SEQ ID NO: 1130
SEQ ID NO: 128	tgggactgctgattcaaga	305	324	SEQ ID NO: 1131
SEQ ID NO: 129	ctgctgattcaagaagtgc	310	329	SEQ ID NO: 1132
SEQ ID NO: 130	tgccaccaggatcaactgc	326	345	SEQ ID NO: 1133
SEQ ID NO: 131	gccaccaggatcaactgca	327	346	SEQ ID NO: 1134
SEQ ID NO: 132	tgcaaggttgagctggagg	342	361	SEQ ID NO: 1135
SEQ ID NO: 133	caaggttgagctggaggtt	344	363	SEQ ID NO: 1136
SEQ ID NO: 134	ctctgcagcttcatcctga	369	388	SEQ ID NO: 1137
SEQ ID NO: 135	cagcttcatcctgaagacc	374	393	SEQ ID NO: 1138
SEQ ID NO: 136	gcttcatcctgaagaccag	376	395	SEQ ID NO: 1139
SEQ ID NO: 137	tcatcctgaagaccagcca	379	398	SEQ ID NO: 1140
SEQ ID NO: 138	gaaaaccaagaactctgag	452	471	SEQ ID NO: 1141
SEQ ID NO: 139	agaactctgaggagtttgc	460	479	SEQ ID NO: 1142
SEQ ID NO: 140	tctgaggagtttgctgcag	465	484	SEQ ID NO: 1143
SEQ ID NO: 141	tttgctgcagccatgtcca	474	493	SEQ ID NO: 1144
SEQ ID NO: 142	caagaggggcatcatttct	578	597	SEQ ID NO: 1145
SEQ ID NO: 143	tcactttaccgtcaagacg	674	693	SEQ ID NO: 1146
SEQ ID NO: 144	tttaccgtcaagacgagga	678	697	SEQ ID NO: 1147
SEQ ID NO: 145	cactggacgctaagaggaa	853	872	SEQ ID NO: 1148
SEQ ID NO: 146	aggaagcatgtggcagaag	867	886	SEQ ID NO: 1149
SEQ ID NO: 147	caaggagcaacacctcttc	893	912	SEQ ID NO: 1150
SEQ ID NO: 148	acagactttgaaacttgaa	959	978	SEQ ID NO: 1151
SEQ ID NO: 149	tgatgaagcagtcacatct	1187	1206	SEQ ID NO: 1152
SEQ ID NO: 150	agcagtcacatctctcttg	1193	1212	SEQ ID NO: 1153
SEQ ID NO: 151	ccagccccatcactttaca	1231	1250	SEQ ID NO: 1154
SEQ ID NO: 152	ctccactcacatcctccag	1280	1299	SEQ ID NO: 1155
SEQ ID NO: 153	catgccaacccccttctga	1314	1333	SEQ ID NO: 1156
SEQ ID NO: 154	gagagatcttcaacatggc	1390	1409	SEQ ID NO: 1157
SEQ ID NO: 155	tcaacatggcgagggatca	1399	1418	SEQ ID NO: 1158
SEQ ID NO: 156	ccaccttgtatgcgctgag	1429	1448	SEQ ID NO: 1159
SEQ ID NO: 157	gtcaacaactatcataaga	1455	1474	SEQ ID NO: 1160
SEQ ID NO: 158	tggacattgctaattacct	1501	1520	SEQ ID NO: 1161
SEQ ID NO: 159	ggacattgctaattacctg	1502	1521	SEQ ID NO: 1162
SEQ ID NO: 160	ttctgcgggtcattggaaa	1573	1592	SEQ ID NO: 1163
SEQ ID NO: 161	ccagaactcaagtcttcaa	1620	1639	SEQ ID NO: 1164
SEQ ID NO: 162	agtcttcaatcctgaaatg	1630	1649	SEQ ID NO: 1165
SEQ ID NO: 163	tgagcaagtgaagaacttt	1868	1887	SEQ ID NO: 1166
SEQ ID NO: 164	agcaagtgaagaactttgt	1870	1889	SEQ ID NO: 1167
SEQ ID NO: 165	tctgaaagaatctcaactt	1964	1983	SEQ ID NO: 1168
SEQ ID NO: 166	actgtcatggacttcagaa	1986	2005	SEQ ID NO: 1169
SEQ ID NO: 167	acttgacccagcctcagcc	2051	2070	SEQ ID NO: 1170
SEQ ID NO: 168	tccaaataactaccttcct	2096	2115	SEQ ID NO: 1171
SEQ ID NO: 169	actaccctcactgcctttg	2133	2152	SEQ ID NO: 1172
SEQ ID NO: 170	ttggatttgcttcagctga	2149	2168	SEQ ID NO: 1173
SEQ ID NO: 171	ttggaagctctttttggaa	2211	2230	SEQ ID NO: 1174
SEQ ID NO: 172	ggaagctctttttgggaag	2213	2232	SEQ ID NO: 1175
SEQ ID NO: 173	tttttcccagacagtgtca	2238	2257	SEQ ID NO: 1176
SEQ ID NO: 174	agacagtgtcaacaaagct	2246	2265	SEQ ID NO: 1177
SEQ ID NO: 175	ctttggctataccaaagat	2321	2340	SEQ ID NO: 1178
SEQ ID NO: 176	caaagatgataaacatgag	2333	2352	SEQ ID NO: 1179
SEQ ID NO: 177	gatatggtaaatggaataa	2355	2374	SEQ ID NO: 1180
SEQ ID NO: 178	ggaataatgctcagtgttg	2367	2386	SEQ ID NO: 1181
SEQ ID NO: 179	tttgaaatccaaagaagtc	2402	2421	SEQ ID NO: 1182
SEQ ID NO: 180	gatcccccagatgattgga	2534	2553	SEQ ID NO: 1183
SEQ ID NO: 181	cagatgattggagaggtca	2541	2560	SEQ ID NO: 1184
SEQ ID NO: 182	agaatgacttttttcttca	2575	2594	SEQ ID NO: 1185
SEQ ID NO: 183	gaactccccactggagctg	2619	2638	SEQ ID NO: 1186
SEQ ID NO: 184	atatcttcatctggagtca	2652	2671	SEQ ID NO: 1187
SEQ ID NO: 185	gtcattgctcccggagcca	2667	2686	SEQ ID NO: 1188
SEQ ID NO: 186	gctgaagtttatcattcca	2873	2892	SEQ ID NO: 1189
SEQ ID NO: 187	ttccttccccaaagagact	2886	2905	SEQ ID NO: 1190
SEQ ID NO: 188	ctcattgagaacaggcagt	2976	2995	SEQ ID NO: 1191
SEQ ID NO: 189	ttgagcagtattctgtcag	3142	3161	SEQ ID NO: 1192
SEQ ID NO: 190	accttgtccagtgaagtcc	3285	3304	SEQ ID NO: 1193
SEQ ID NO: 191	ccagtgaagtccaaattcc	3292	3311	SEQ ID NO: 1194
SEQ ID NO: 192	acattcagaacaagaaaat	3394	3413	SEQ ID NO: 1195
SEQ ID NO: 193	gaaaaatcaagggtgttat	3463	3482	SEQ ID NO: 1196
SEQ ID NO: 194	aaatcaagggtgttatttc	3466	3485	SEQ ID NO: 1197
SEQ ID NO: 195	tggcattatgatgaagaga	3609	3628	SEQ ID NO: 1198
SEQ ID NO: 196	aagagaagattgaatttga	3622	3641	SEQ ID NO: 1199
SEQ ID NO: 197	aaatgacttccaatttccc	3673	3692	SEQ ID NO: 1200
SEQ ID NO: 198	atgacttccaatttccctg	3675	3694	SEQ ID NO: 1201
SEQ ID NO: 199	acttccaatttccctgtgg	3678	3697	SEQ ID NO: 1202
SEQ ID NO: 200	agttgcaatgagctcatgg	3803	3822	SEQ ID NO: 1203
SEQ ID NO: 201	tttgcaagaccacctcaat	3860	3879	SEQ ID NO: 1204
SEQ ID NO: 202	gaaggagttcaacctccag	3884	3903	SEQ ID NO: 1205
SEQ ID NO: 203	acttccacatcccagaaaa	3919	3938	SEQ ID NO: 1206
SEQ ID NO: 204	ctcttcttaaaaagcgatg	3939	3958	SEQ ID NO: 1207
SEQ ID NO: 205	aaaagcgatggccgggtca	3948	3967	SEQ ID NO: 1208
SEQ ID NO: 206	ttcctttgccttttggtgg	4003	4022	SEQ ID NO: 1209
SEQ ID NO: 207	caagtctgtgggattccat	4079	4098	SEQ ID NO: 1210
SEQ ID NO: 208	aagtccctacttttaccat	4117	4136	SEQ ID NO: 1211
SEQ ID NO: 209	tgcctctcctgggtgttct	4159	4178	SEQ ID NO: 1212
SEQ ID NO: 210	accagcacagaccatttca	4242	4261	SEQ ID NO: 1213
SEQ ID NO: 211	accagcacagaccatttca	4243	4262	SEQ ID NO: 1214
SEQ ID NO: 212	ccagcacagaccatttcag	4367	4386	SEQ ID NO: 1215
SEQ ID NO: 213	actatcatgtgatgggtct	4496	4515	SEQ ID NO: 1216
SEQ ID NO: 214	ccacagatgtctgcttcag	4497	4516	SEQ ID NO: 1217
SEQ ID NO: 215	tttggactccaaaaagaaa	4520	4539	SEQ ID NO: 1218
SEQ ID NO: 216	tcaaagaagtcaagattga	4552	4571	SEQ ID NO: 1219
SEQ ID NO: 217	atgagaactacgagctgac	4798	4817	SEQ ID NO: 1220
SEQ ID NO: 218	ttaaaatctgacaccaatg	4818	4837	SEQ ID NO: 1221
SEQ ID NO: 219	gaagtataagaactttgcc	4838	4857	SEQ ID NO: 1222
SEQ ID NO: 220	aagtataagaactttgcca	4839	4858	SEQ ID NO: 1223
SEQ ID NO: 221	ttcttcagcctgctttctg	4941	4960	SEQ ID NO: 1224
SEQ ID NO: 222	ctggatcactaaattccca	4957	4976	SEQ ID NO: 1225
SEQ ID NO: 223	aaattaatagtggtgctca	5014	5033	SEQ ID NO: 1226
SEQ ID NO: 224	agtgcaacgaccaacttga	5073	5092	SEQ ID NO: 1227
SEQ ID NO: 225	ctgggaagtgcttatcagg	5238	5257	SEQ ID NO: 1228
SEQ ID NO: 226	gcaaaaacattttcaactt	5278	5297	SEQ ID NO: 1229
SEQ ID NO: 227	aaaaacattttcaacttca	5280	5299	SEQ ID NO: 1230
SEQ ID NO: 228	tcagtcaagaaggacttaa	5302	5321	SEQ ID NO: 1231
SEQ ID NO: 229	tcaaatgacatgatgggct	5325	5344	SEQ ID NO: 1232
SEQ ID NO: 230	cacacaaacagtctgaaca	5367	5386	SEQ ID NO: 1233
SEQ ID NO: 231	tcttcaaaacttgacaaca	5409	5428	SEQ ID NO: 1234
SEQ ID NO: 232	caagttttataagcaaact	5441	5460	SEQ ID NO: 1235
SEQ ID NO: 233	tggtaactactttaaacag	5488	5507	SEQ ID NO: 1236
SEQ ID NO: 234	aacagtgacctgaaataca	5502	5521	SEQ ID NO: 1237
SEQ ID NO: 235	gggaaactacggctagaac	5544	5563	SEQ ID NO: 1238
SEQ ID NO: 236	aacacatctatgccatctc	5620	5639	SEQ ID NO: 1239
SEQ ID NO: 237	tcagcaagctataaagcag	5652	5671	SEQ ID NO: 1240
SEQ ID NO: 238	gcagacactgttgctaagg	5667	5686	SEQ ID NO: 1241
SEQ ID NO: 239	tctggggagaacatactgg	5866	5885	SEQ ID NO: 1242
SEQ ID NO: 240	ttctctcatgattacaaag	5934	5953	SEQ ID NO: 1243
SEQ ID NO: 241	ctgagcagacaggcacctg	6034	6053	SEQ ID NO: 1244
SEQ ID NO: 242	caatttaacaacaatgaat	6066	6085	SEQ ID NO: 1245
SEQ ID NO: 243	tggacgaactctggctgac	6140	6159	SEQ ID NO: 1246
SEQ ID NO: 244	cttttactcagtgagccca	6192	6211	SEQ ID NO: 1247
SEQ ID NO: 245	cttttactcagtgagccca	6217	6236	SEQ ID NO: 1248
SEQ ID NO: 246	tcattgatgctttagagat	6295	6314	SEQ ID NO: 1249
SEQ ID NO: 247	aaaaccaagatgttcactc	6357	6376	SEQ ID NO: 1250
SEQ ID NO: 248	aggaatcgacaaaccatta	6376	6395	SEQ ID NO: 1251
SEQ ID NO: 249	tagttgtactggaaaacgt	6386	6405	SEQ ID NO: 1252
SEQ ID NO: 250	ggaaaacgtacagagaaag	6387	6406	SEQ ID NO: 1253
SEQ ID NO: 251	gaaaacgtacagagaaagc	6401	6420	SEQ ID NO: 1254
SEQ ID NO: 252	aaagctgaagcacatcaat	6402	6421	SEQ ID NO: 1255
SEQ ID NO: 253	aagctgaagcacatcaata	6406	6425	SEQ ID NO: 1256
SEQ ID NO: 254	atcaatattgatcaatttg	6414	6433	SEQ ID NO: 1257
SEQ ID NO: 255	taatgattatctgaattca	6476	6495	SEQ ID NO: 1258
SEQ ID NO: 256	gattatctgaattcattca	6480	6499	SEQ ID NO: 1259
SEQ ID NO: 257	aattgggagagacaagttt	6498	6517	SEQ ID NO: 1260
SEQ ID NO: 258	aaaatagctattgctaata	6693	6712	SEQ ID NO: 1261
SEQ ID NO: 259	aaaattaaaaagtcttgat	6731	6750	SEQ ID NO: 1262
SEQ ID NO: 260	ttgaaaatattgattttaa	6808	6827	SEQ ID NO: 1263
SEQ ID NO: 261	agacatccagcacctagct	6938	6957	SEQ ID NO: 1264
SEQ ID NO: 262	caatttcatttgaaagaat	7021	7040	SEQ ID NO: 1265
SEQ ID NO: 263	aggttttaatggataaatt	7174	7193	SEQ ID NO: 1266
SEQ ID NO: 264	cagaagctaagcaatgtcc	7233	7252	SEQ ID NO: 1267
SEQ ID NO: 265	taagataaaagattacttt	7262	7281	SEQ ID NO: 1268
SEQ ID NO: 266	aaagattactttgagaaat	7269	7288	SEQ ID NO: 1269
SEQ ID NO: 267	gagaaattagttggattta	7281	7300	SEQ ID NO: 1270
SEQ ID NO: 268	atttattgatgatgctgtc	7295	7314	SEQ ID NO: 1271
SEQ ID NO: 269	gaattatcttttaaaacat	7326	7345	SEQ ID NO: 1272
SEQ ID NO: 270	ttaccaccagtttgtagat	7403	7422	SEQ ID NO: 1273
SEQ ID NO: 271	ttgcagtgtatctggaaag	7540	7559	SEQ ID NO: 1274
SEQ ID NO: 272	cattcagcaggaacttcaa	7691	7710	SEQ ID NO: 1275
SEQ ID NO: 273	acacctgattttatagtcc	7950	7969	SEQ ID NO: 1276
SEQ ID NO: 274	ggattccatcagttcagat	7984	8003	SEQ ID NO: 1277
SEQ ID NO: 275	ttgtagaaatgaaagtaaa	8104	8123	SEQ ID NO: 1278
SEQ ID NO: 276	ctgaacagtgagctgcagt	8148	8167	SEQ ID NO: 1279
SEQ ID NO: 277	aatccaatctcctcttttc	8399	8418	SEQ ID NO: 1280
SEQ ID NO: 278	attttgattttcaagcaaa	8524	8543	SEQ ID NO: 1281
SEQ ID NO: 279	ttttgattttcaagcaaat	8525	8544	SEQ ID NO: 1282
SEQ ID NO: 280	tgattttcaagcaaatgca	8528	8547	SEQ ID NO: 1283
SEQ ID NO: 281	atgctgttttttggaaatg	8637	8656	SEQ ID NO: 1284
SEQ ID NO: 282	tgctgttttttggaaatgc	8638	8657	SEQ ID NO: 1285
SEQ ID NO: 283	aaaaaaatacactggagct	8698	8717	SEQ ID NO: 1286
SEQ ID NO: 284	actggagcttagtaatgga	8708	8727	SEQ ID NO: 1287
SEQ ID NO: 285	cttctggaaaagggtcatg	8876	8897	SEQ ID NO: 1288
SEQ ID NO: 286	ggaaaagggtcatggaaat	8883	8902	SEQ ID NO: 1289
SEQ ID NO: 287	gggcctgccccagattctc	8902	8921	SEQ ID NO: 1290
SEQ ID NO: 288	ttctcagatgagggaacac	8916	8935	SEQ ID NO: 1291
SEQ ID NO: 289	gatgagggaacacatgaat	8922	8941	SEQ ID NO: 1292
SEQ ID NO: 290	ctttggactgtccaataag	8978	8997	SEQ ID NO: 1293
SEQ ID NO: 291	gcatccacaaacaatgaag	9252	9271	SEQ ID NO: 1294
SEQ ID NO: 292	cacaaacaatgaagggaat	9257	9276	SEQ ID NO: 1295
SEQ ID NO: 293	ccaaaatttctctgctgga	9407	9426	SEQ ID NO: 1296
SEQ ID NO: 294	caaaatttctctgctggaa	9408	9427	SEQ ID NO: 1297
SEQ ID NO: 295	tctgctggaaacaacgaga	9417	9436	SEQ ID NO: 1298
SEQ ID NO: 296	tctgctggaaacaacgaga	9418	9437	SEQ ID NO: 1299
SEQ ID NO: 297	agaacattatggaggccca	9433	9452	SEQ ID NO: 1300
SEQ ID NO: 298	agaagcaaatctggatttc	9467	9486	SEQ ID NO: 1301
SEQ ID NO: 299	tttctctctatgggaaaaa	9557	9576	SEQ ID NO: 1302
SEQ ID NO: 300	tcagagcatcaaatccttt	9704	9723	SEQ ID NO: 1303
SEQ ID NO: 301	cagaaacaatgcattagat	9743	9762	SEQ ID NO: 1304
SEQ ID NO: 302	tacacattaatcctgccat	9993	10012	SEQ ID NO: 1305
SEQ ID NO: 303	agtcagatattgttgctca	10186	10205	SEQ ID NO: 1306
SEQ ID NO: 304	ggagggtagtcataacagt	10328	10347	SEQ ID NO: 1307
SEQ ID NO: 305	caaaagccgaaattccaat	10396	10415	SEQ ID NO: 1308
SEQ ID NO: 306	aaaagccgaaattccaatt	10397	10416	SEQ ID NO: 1309
SEQ ID NO: 307	ttcaagcaagaacttaatg	10428	10447	SEQ ID NO: 1310
SEQ ID NO: 308	cctcttacttttccattga	10570	10589	SEQ ID NO: 1311
SEQ ID NO: 309	tgaggccaacacttacttg	10655	10674	SEQ ID NO: 1312
SEQ ID NO: 310	cacttacttgaattccaag	10664	10683	SEQ ID NO: 1313
SEQ ID NO: 311	gaagtaaaagaaaattttg	10743	10762	SEQ ID NO: 1314
SEQ ID NO: 312	cctggaactctctccatgg	10874	10893	SEQ ID NO: 1315
SEQ ID NO: 313	agctggatgtaaccaccag	11176	11195	SEQ ID NO: 1316
SEQ ID NO: 314	aaaattccctgaagttgat	11477	11496	SEQ ID NO: 1317
SEQ ID NO: 315	cagatggcattgctgcttt	11605	11624	SEQ ID NO: 1318
SEQ ID NO: 316	agatggcattgctgctttg	11606	11625	SEQ ID NO: 1319
SEQ ID NO: 317	tgttgaaacagtcctggat	11834	11853	SEQ ID NO: 1320
SEQ ID NO: 318	catattcaaaactgagttg	12221	12240	SEQ ID NO: 1321
SEQ ID NO: 319	aaagatttatcaaaagaag	12930	12949	SEQ ID NO: 1322
SEQ ID NO: 320	attttccaactaatagaag	13026	13045	SEQ ID NO: 1323
SEQ ID NO: 321	aattatatccaagatgaga	13089	13108	SEQ ID NO: 1324
SEQ ID NO: 322	ttcaggaagcttctcaaga	13210	13229	SEQ ID NO: 1325
SEQ ID NO: 323	ttgagcaatttctgcacag	13429	13448	SEQ ID NO: 1326
SEQ ID NO: 324	ctgatatacatcacggagt	13704	13723	SEQ ID NO: 1327
SEQ ID NO: 325	acatcacggagttactgaa	13711	13730	SEQ ID NO: 1328
SEQ ID NO: 326	actgcctatattgataaaa	13874	13893	SEQ ID NO: 1329
SEQ ID NO: 327	aggatggcattttttgcaa	14003	14022	SEQ ID NO: 1330
SEQ ID NO: 328	ttttttgcaagttaaagaa	14012	14031	SEQ ID NO: 1331
SEQ ID NO: 329	tccagaactcaagtcttca	1619	1638	SEQ ID NO: 1332
SEQ ID NO: 330	agttagtgaaagaagttct	1948	1967	SEQ ID NO: 1333
SEQ ID NO: 331	atttacagctctgacaagt	5427	5446	SEQ ID NO: 1334
SEQ ID NO: 332	gattatctgaattcattca	6480	6499	SEQ ID NO: 1335
SEQ ID NO: 333	gtgcccttctcggttgctg	18	37	SEQ ID NO: 1336
SEQ ID NO: 334	attcaagcacctccggaag	245	264	SEQ ID NO: 1337
SEQ ID NO: 335	gactgctgattcaagaagt	308	327	SEQ ID NO: 1338
SEQ ID NO: 336	ttgctgcagccatgtccag	475	494	SEQ ID NO: 1339
SEQ ID NO: 337	agaaagatgaacctactta	547	566	SEQ ID NO: 1340
SEQ ID NO: 338	tgaagactctccaggaact	1087	1106	SEQ ID NO: 1341
SEQ ID NO: 339	atctctcttgccacagctg	1202	1221	SEQ ID NO: 1342
SEQ ID NO: 340	tctctcttgccacagctga	1203	1222	SEQ ID NO: 1343
SEQ ID NO: 341	tgaggtgtccagccccatc	1223	1242	SEQ ID NO: 1344
SEQ ID NO: 342	ccagaactcaagtcttcaa	1620	1639	SEQ ID NO: 1345
SEQ ID NO: 343	ctgaaaaagttagtgaaag	1941	1960	SEQ ID NO: 1346
SEQ ID NO: 344	tttttcccagacagtgtca	2238	2257	SEQ ID NO: 1347
SEQ ID NO: 345	ttttcccagacagtgtcaa	2239	2258	SEQ ID NO: 1348
SEQ ID NO: 346	cattcagaacaagaaaatt	3395	3414	SEQ ID NO: 1349
SEQ ID NO: 347	tgaagagaagattgaattt	3620	3639	SEQ ID NO: 1350
SEQ ID NO: 348	tttgaatggaacacaggca	3636	3655	SEQ ID NO: 1351
SEQ ID NO: 349	ttctagattcgaatatcaa	4399	4418	SEQ ID NO: 1352
SEQ ID NO: 350	gattcgaatatcaaattca	4404	4423	SEQ ID NO: 1353
SEQ ID NO: 351	tgcaacgaccaacttgaag	5075	5094	SEQ ID NO: 1354
SEQ ID NO: 352	ttaagctctcaaatgacat	5317	5336	SEQ ID NO: 1355
SEQ ID NO: 353	caatttaacaacaatgaat	6066	6085	SEQ ID NO: 1356
SEQ ID NO: 354	tgaatacagccaggacttg	6080	6099	SEQ ID NO: 1357
SEQ ID NO: 355	catcaatattgatcaattt	6413	6432	SEQ ID NO: 1358
SEQ ID NO: 356	ttgagcatgtcaaacactt	7051	7070	SEQ ID NO: 1359
SEQ ID NO: 357	tgaaggagactattcagaa	7219	7238	SEQ ID NO: 1360
SEQ ID NO: 358	ttcaggctcttcagaaagc	7921	7940	SEQ ID NO: 1361
SEQ ID NO: 359	tccacaaattgaacatccc	8779	8798	SEQ ID NO: 1362
SEQ ID NO: 360	tgaataccaatgctgaact	10159	10178	SEQ ID NO: 1363
SEQ ID NO: 361	taaactaatagatgtaatc	12890	12909	SEQ ID NO: 1364
SEQ ID NO: 362	ttgacctgtccattcaaaa	13672	13691	SEQ ID NO: 1365
SEQ ID NO: 363	gggctgagtgcccttctcg	11	30	SEQ ID NO: 1366
SEQ ID NO: 364	ggctgagtgcccttctcgg	12	31	SEQ ID NO: 1367
SEQ ID NO: 365	ctgagtgcccttctcggtt	14	33	SEQ ID NO: 1368
SEQ ID NO: 366	tctcggttgctgccgctga	25	44	SEQ ID NO: 1369
SEQ ID NO: 367	caggccgcagcccaggagc	82	101	SEQ ID NO: 1370
SEQ ID NO: 368	gctggcgctgcctgcgctg	143	162	SEQ ID NO: 1371
SEQ ID NO: 369	tgctgctggcgggcgccag	169	188	SEQ ID NO: 1372
SEQ ID NO: 370	ctggtctgtccaaaagatg	219	238	SEQ ID NO: 1373
SEQ ID NO: 371	ctgagagttccagtggagt	283	302	SEQ ID NO: 1374
SEQ ID NO: 372	tccagtggagtccctggga	291	310	SEQ ID NO: 1375
SEQ ID NO: 373	aggttgagctggaggttcc	346	365	SEQ ID NO: 1376
SEQ ID NO: 374	tgagctggaggttccccag	350	369	SEQ ID NO: 1377
SEQ ID NO: 375	tctgcagcttcatcctgaa	370	389	SEQ ID NO: 1378
SEQ ID NO: 376	gccagtgcaccctgaaaga	394	413	SEQ ID NO: 1379
SEQ ID NO: 377	ctctgaggagtttgctgca	464	483	SEQ ID NO: 1380
SEQ ID NO: 378	aggtatgagctcaagctgg	492	511	SEQ ID NO: 1381
SEQ ID NO: 379	tcctttacccggagaaaga	535	554	SEQ ID NO: 1382
SEQ ID NO: 380	catcaagaggggcatcatt	575	594	SEQ ID NO: 1383
SEQ ID NO: 381	tcctggttcccccagagac	601	620	SEQ ID NO: 1384
SEQ ID NO: 382	aagaagccaagcaagtgtg	622	641	SEQ ID NO: 1385
SEQ ID NO: 383	aagcaagtgttgtttctgg	630	649	SEQ ID NO: 1386
SEQ ID NO: 384	tctggataccgtgtatgga	644	663	SEQ ID NO: 1387
SEQ ID NO: 385	ccactcactttaccgtcaa	670	689	SEQ ID NO: 1388
SEQ ID NO: 386	aggaagggcaatgtggcaa	693	712	SEQ ID NO: 1389
SEQ ID NO: 387	gcaatgtggcaacagaaat	700	719	SEQ ID NO: 1390
SEQ ID NO: 388	caatgtggcaacagaaata	701	720	SEQ ID NO: 1391
SEQ ID NO: 389	tggcaacagaaatatccac	706	725	SEQ ID NO: 1392
SEQ ID NO: 390	agagacctgggccagtgtg	729	748	SEQ ID NO: 1393
SEQ ID NO: 391	tgtgatcgcttcaagccca	744	763	SEQ ID NO: 1394
SEQ ID NO: 392	gtgatcgcttcaagcccat	745	764	SEQ ID NO: 1395
SEQ ID NO: 393	cagcccacttgctctcatc	776	795	SEQ ID NO: 1396
SEQ ID NO: 394	gctctcatcaaaggcatga	786	805	SEQ ID NO: 1397
SEQ ID NO: 395	ccttgtcaactctgatcag	811	830	SEQ ID NO: 1398
SEQ ID NO: 396	cttgtcaactctgatcagc	812	831	SEQ ID NO: 1399
SEQ ID NO: 397	agccatctgcaaggagcaa	884	903	SEQ ID NO: 1400
SEQ ID NO: 398	gccatctgcaaggagcaac	885	904	SEQ ID NO: 1401
SEQ ID NO: 399	cttcctgcctttctcctac	908	927	SEQ ID NO: 1402
SEQ ID NO: 400	ctttctcctacaagaataa	916	935	SEQ ID NO: 1403
SEQ ID NO: 401	gatcaacagccgcttcttt	989	1008	SEQ ID NO: 1404
SEQ ID NO: 402	atcaacagccgcttctttg	990	1009	SEQ ID NO: 1405
SEQ ID NO: 403	acagccgcttctttggtga	994	1013	SEQ ID NO: 1406
SEQ ID NO: 404	aagatgggcctcgcatttg	1023	1042	SEQ ID NO: 1407
SEQ ID NO: 405	tgttttgaagactctccag	1082	1101	SEQ ID NO: 1408
SEQ ID NO: 406	ttgaagactctccaggaac	1086	1105	SEQ ID NO: 1409
SEQ ID NO: 407	aactgaaaaaactaaccat	1102	1121	SEQ ID NO: 1410
SEQ ID NO: 408	ctgaaaaaactaaccatct	1104	1123	SEQ ID NO: 1411
SEQ ID NO: 409	aaaactaaccatctctgag	1109	1128	SEQ ID NO: 1412
SEQ ID NO: 410	tgagcaaaatatccagaga	1124	1143	SEQ ID NO: 1413
SEQ ID NO: 411	caataagctggttactgag	1154	1173	SEQ ID NO: 1414
SEQ ID NO: 412	tactgagctgagaggcctc	1166	1185	SEQ ID NO: 1415
SEQ ID NO: 413	gcctcagtgatgaagcagt	1180	1199	SEQ ID NO: 1416
SEQ ID NO: 414	agtcacatctctcttgcca	1196	1215	SEQ ID NO: 1417
SEQ ID NO: 415	atctctcttgccacagctg	1202	1221	SEQ ID NO: 1418
SEQ ID NO: 416	tctctcttgccacagctga	1203	1222	SEQ ID NO: 1419
SEQ ID NO: 417	tgccacagctgattgaggt	1210	1229	SEQ ID NO: 1420
SEQ ID NO: 418	gccacagctgattgaggtg	1211	1230	SEQ ID NO: 1421
SEQ ID NO: 419	tcactttacaagccttggt	1240	1259	SEQ ID NO: 1422
SEQ ID NO: 420	cccttctgatagatgtggt	1324	1343	SEQ ID NO: 1423
SEQ ID NO: 421	gtcacctacctggtggccc	1341	1360	SEQ ID NO: 1424
SEQ ID NO: 422	ccttgtatgcgctgagcca	1432	1451	SEQ ID NO: 1425
SEQ ID NO: 423	gacaaaccctacagggacc	1472	1491	SEQ ID NO: 1426
SEQ ID NO: 424	tgctaattacctgatggaa	1508	1527	SEQ ID NO: 1427
SEQ ID NO: 425	tgactgcactggggatgaa	1538	1557	SEQ ID NO: 1428
SEQ ID NO: 426	actgcactggggatgaaga	1540	1559	SEQ ID NO: 1429
SEQ ID NO: 427	atgaagattacacctattt	1552	1571	SEQ ID NO: 1430
SEQ ID NO: 428	accatggagcagttaactc	1602	1621	SEQ ID NO: 1431
SEQ ID NO: 429	gcagttaactccagaactc	1610	1629	SEQ ID NO: 1432
SEQ ID NO: 430	cagaactcaagtcttcaat	1621	1640	SEQ ID NO: 1433
SEQ ID NO: 431	caggctctgcggaaaatgg	1695	1714	SEQ ID NO: 1434
SEQ ID NO: 432	ccaggaggttcttcttcag	1730	1749	SEQ ID NO: 1435
SEQ ID NO: 433	ggttcttcttcagactttc	1736	1755	SEQ ID NO: 1436
SEQ ID NO: 434	tttccttgatgatgcttct	1751	1770	SEQ ID NO: 1437
SEQ ID NO: 435	ggagataagcgactggctg	1773	1792	SEQ ID NO: 1438
SEQ ID NO: 436	gctgcctatcttatgttga	1788	1807	SEQ ID NO: 1439
SEQ ID NO: 437	actttgtggcttcccatat	1882	1901	SEQ ID NO: 1440
SEQ ID NO: 438	gccaatatcttgaactcag	1902	1921	SEQ ID NO: 1441
SEQ ID NO: 439	aatatcttgaactcagaag	1905	1924	SEQ ID NO: 1442
SEQ ID NO: 440	ctcagaagaattggatatc	1916	1935	SEQ ID NO: 1443
SEQ ID NO: 441	aagaattggatatccaaga	1921	1940	SEQ ID NO: 1444
SEQ ID NO: 442	agaattggatatccaagat	1922	1941	SEQ ID NO: 1445
SEQ ID NO: 443	tggatatccaagatctgaa	1927	1946	SEQ ID NO: 1446
SEQ ID NO: 444	atatccaagatctgaaaaa	1930	1949	SEQ ID NO: 1447
SEQ ID NO: 445	tatccaagatctgaaaaag	1931	1950	SEQ ID NO: 1448
SEQ ID NO: 446	caagatctgaaaaagttag	1935	1954	SEQ ID NO: 1449
SEQ ID NO: 447	aagatctgaaaaagttagt	1936	1955	SEQ ID NO: 1450
SEQ ID NO: 448	tgaaaaagttagtgaaaga	1942	1961	SEQ ID NO: 1451
SEQ ID NO: 449	tccaactgtcatggacttc	1982	2001	SEQ ID NO: 1452
SEQ ID NO: 450	tcagaaaattctctcggaa	1999	2018	SEQ ID NO: 1453
SEQ ID NO: 451	ttccatcacttgacccagc	2044	2063	SEQ ID NO: 1454
SEQ ID NO: 452	cccagcctcagccaaaata	2057	2076	SEQ ID NO: 1455
SEQ ID NO: 453	agcctcagccaaaatagaa	2060	2079	SEQ ID NO: 1456
SEQ ID NO: 454	tcttattatttgatccaaa	2083	2102	SEQ ID NO: 1457
SEQ ID NO: 455	tcttatatttgatccaaat	2084	2103	SEQ ID NO: 1458
SEQ ID NO: 456	cttcctaaagaaagcatgc	2109	2128	SEQ ID NO: 1459
SEQ ID NO: 457	ctaaagaaagcatgctgaa	2113	2132	SEQ ID NO: 1460
SEQ ID NO: 458	taaagaaagcatgctgaaa	2114	2133	SEQ ID NO: 1461
SEQ ID NO: 459	gagattggcttggaaggaa	2175	2194	SEQ ID NO: 1462
SEQ ID NO: 460	ctttgagccaacattggaa	2198	2217	SEQ ID NO: 1463
SEQ ID NO: 461	cagacagtgtcaacaaagc	2245	2264	SEQ ID NO: 1464
SEQ ID NO: 462	cagtgtcaacaaagctttg	2249	2268	SEQ ID NO: 1465
SEQ ID NO: 463	agtgtcaacaaagctttgt	2250	2269	SEQ ID NO: 1466
SEQ ID NO: 464	ctgatggtgtctctaaggt	2290	2309	SEQ ID NO: 1467
SEQ ID NO: 465	tgatggtgtctctaaggtc	2291	2310	SEQ ID NO: 1468
SEQ ID NO: 466	aaacatgagcaggatatgg	2343	2362	SEQ ID NO: 1469
SEQ ID NO: 467	gaagctgattaaagatttg	2387	2406	SEQ ID NO: 1470
SEQ ID NO: 468	aaagatttgaaatccaaag	2397	2416	SEQ ID NO: 1471
SEQ ID NO: 469	gatgggtgcccgcactctg	2510	2529	SEQ ID NO: 1472
SEQ ID NO: 470	gggatcccccagatgattg	2532	2551	SEQ ID NO: 1473
SEQ ID NO: 471	ttttcttcactactacttc	2585	2604	SEQ ID NO: 1474
SEQ ID NO: 472	tcttcactacatcttcatg	2588	2607	SEQ ID NO: 1475
SEQ ID NO: 473	tacatcttcatggagaatg	2595	2614	SEQ ID NO: 1476
SEQ ID NO: 474	ttcatggagaatgcctttg	2601	2620	SEQ ID NO: 1477
SEQ ID NO: 475	tcatggagaatgcctttga	2602	2621	SEQ ID NO: 1478
SEQ ID NO: 476	tttgaactccccactggag	2616	2635	SEQ ID NO: 1479
SEQ ID NO: 477	ttgaactccccactggagc	2617	2636	SEQ ID NO: 1480
SEQ ID NO: 478	tgaactccccactggagct	2618	2637	SEQ ID NO: 1481
SEQ ID NO: 479	cactggagctggattacag	2627	2646	SEQ ID NO: 1482
SEQ ID NO: 480	actggagctggattacagt	2628	2647	SEQ ID NO: 1483
SEQ ID NO: 481	agttgcaaatatcttcatc	2644	2663	SEQ ID NO: 1484
SEQ ID NO: 482	gttgcaaatatcttcatct	2645	2664	SEQ ID NO: 1485
SEQ ID NO: 483	aaatatcttcatctggagt	2650	2669	SEQ ID NO: 1486
SEQ ID NO: 484	taaaactggaagtagccaa	2695	2714	SEQ ID NO: 1487
SEQ ID NO: 485	ggctgaactggtggcaaaa	2720	2739	SEQ ID NO: 1488
SEQ ID NO: 486	tgtggagtttgtgacaaat	2750	2769	SEQ ID NO: 1489
SEQ ID NO: 487	ttgtgacaaatatgggcat	2758	2777	SEQ ID NO: 1490
SEQ ID NO: 488	atgaacaccaacttcttcc	2811	2830	SEQ ID NO: 1491
SEQ ID NO: 489	cttccacgagtcgggtctg	2825	2844	SEQ ID NO: 1492
SEQ ID NO: 490	gagtcgggtctggaggctc	2832	2851	SEQ ID NO: 1493
SEQ ID NO: 491	cctaaaagctgggaagctg	2858	2877	SEQ ID NO: 1494
SEQ ID NO: 492	agctgggaagctgaagttt	2864	2883	SEQ ID NO: 1495
SEQ ID NO: 493	ccagattagagctggaact	3106	3125	SEQ ID NO: 1496
SEQ ID NO: 494	ggataccctgaagtttgta	3200	3219	SEQ ID NO: 1497
SEQ ID NO: 495	ctgaggctaccatgacatt	3244	3263	SEQ ID NO: 1498
SEQ ID NO: 496	tgtccagtgaagtccaaat	3289	3308	SEQ ID NO: 1499
SEQ ID NO: 497	aattccggattttgatgtt	3305	3324	SEQ ID NO: 1500
SEQ ID NO: 498	ttccggattttgatgttga	3307	3326	SEQ ID NO: 1501
SEQ ID NO: 499	cggaacaatcctcagagtt	3329	3348	SEQ ID NO: 1502
SEQ ID NO: 500	tcctcagagttaatgatga	3337	3356	SEQ ID NO: 1503
SEQ ID NO: 501	ctcaccctggacattcaga	3384	3403	SEQ ID NO: 1504
SEQ ID NO: 502	cattcagaacaagaaaatt	3395	3414	SEQ ID NO: 1505
SEQ ID NO: 503	actgaggtcgccctcatgg	3414	3433	SEQ ID NO: 1506
SEQ ID NO: 504	ttatttccataccccgttt	3478	3497	SEQ ID NO: 1507
SEQ ID NO: 505	gtttgcaagcagaagccag	3493	3512	SEQ ID NO: 1508
SEQ ID NO: 506	tttgcaagcagaagccaga	3494	3513	SEQ ID NO: 1509
SEQ ID NO: 507	ttgcaagcagaagccagaa	3495	3514	SEQ ID NO: 1510
SEQ ID NO: 508	ctgcttctccaaatggact	3546	3565	SEQ ID NO: 1511
SEQ ID NO: 509	tgctacagcttatggctcc	3569	3588	SEQ ID NO: 1512
SEQ ID NO: 510	acagcttatggctccacag	3573	3592	SEQ ID NO: 1513
SEQ ID NO: 511	tttccaagagggtggcatg	3592	3611	SEQ ID NO: 1514
SEQ ID NO: 512	ccaagagggtggcatggca	3595	3514	SEQ ID NO: 1515
SEQ ID NO: 513	gtggcatggcattatgatg	3603	3622	SEQ ID NO: 1516
SEQ ID NO: 514	tgatgaagagaagattgaa	3617	3636	SEQ ID NO: 1517
SEQ ID NO: 515	gaagagaagattgaatttg	3621	3640	SEQ ID NO: 1518
SEQ ID NO: 516	gagaagattgaatttgaat	3624	3643	SEQ ID NO: 1519
SEQ ID NO: 517	tttgaatggaacacaggca	3636	3655	SEQ ID NO: 1520
SEQ ID NO: 518	aggcaccaatgtagatacc	3650	3669	SEQ ID NO: 1521
SEQ ID NO: 519	caaaaaaatgacttccaat	3668	3687	SEQ ID NO: 1522
SEQ ID NO: 520	aaaaaaatgacttccaatt	3669	3688	SEQ ID NO: 1523
SEQ ID NO: 521	aaaaaatgacttccaattt	3670	3689	SEQ ID NO: 1524
SEQ ID NO: 522	cagagtccctcaaacagac	3572	3771	SEQ ID NO: 1525
SEQ ID NO: 523	aaattaatagttgcaatga	3795	3814	SEQ ID NO: 1526
SEQ ID NO: 524	ttcaacctccagaacatgg	3891	3910	SEQ ID NO: 1527
SEQ ID NO: 525	tgggattgccagacttcca	3907	3926	SEQ ID NO: 1528
SEQ ID NO: 526	cagtttgaaaattgagatt	3986	4005	SEQ ID NO: 1529
SEQ ID NO: 527	gaaaattgagattcctttg	3992	4011	SEQ ID NO: 1530
SEQ ID NO: 528	tttgccttttggtggcaaa	4007	4026	SEQ ID NO: 1531
SEQ ID NO: 529	ctccagagatctaaagatg	4028	4047	SEQ ID NO: 1532
SEQ ID NO: 530	tctaaagatgttagagact	4037	4056	SEQ ID NO: 1533
SEQ ID NO: 531	ctgtgggattccatctgcc	4084	4103	SEQ ID NO: 1534
SEQ ID NO: 532	atctgccatctcgagagtt	4096	4115	SEQ ID NO: 1535
SEQ ID NO: 533	tctcgagagttccaagtcc	4104	4123	SEQ ID NO: 1536
SEQ ID NO: 534	agtccctacttttaccatt	4118	4137	SEQ ID NO: 1537
SEQ ID NO: 535	acttttaccattcccaagt	4125	4144	SEQ ID NO: 1538
SEQ ID NO: 536	cattcccaagttgtatcaa	4133	4152	SEQ ID NO: 1539
SEQ ID NO: 537	accacatgaaggctgactc	4276	4295	SEQ ID NO: 1540
SEQ ID NO: 538	tttcctacaatgtgcaagg	4309	4328	SEQ ID NO: 1541
SEQ ID NO: 539	ctggagaaacaacatatga	4330	4349	SEQ ID NO: 1542
SEQ ID NO: 540	atcatgtgatgggtctcta	4370	4389	SEQ ID NO: 1543
SEQ ID NO: 541	catgtgatgggtctctacg	4372	4391	SEQ ID NO: 1544
SEQ ID NO: 542	ttctagattcgaatatcaa	4399	4418	SEQ ID NO: 1545
SEQ ID NO: 543	tggggaccacagatgtctg	4491	4510	SEQ ID NO: 1546
SEQ ID NO: 544	ctaacactggccggctcaa	4636	4655	SEQ ID NO: 1547
SEQ ID NO: 545	taacactggccggctcaat	4637	4656	SEQ ID NO: 1548
SEQ ID NO: 546	aacactggccggctcaatg	4638	4657	SEQ ID NO: 1549
SEQ ID NO: 547	ctggccggctcaatggaga	4642	4661	SEQ ID NO: 1550
SEQ ID NO: 548	agataacaggaagatatga	4705	4724	SEQ ID NO: 1551
SEQ ID NO: 549	tccctcacctccacctctg	4737	4756	SEQ ID NO: 1552
SEQ ID NO: 550	agctgactttaaaatctga	4810	4829	SEQ ID NO: 1553
SEQ ID NO: 551	ctgactttaaaatctgaca	4812	4831	SEQ ID NO: 1554
SEQ ID NO: 552	caagatggatatgaccttc	4865	4884	SEQ ID NO: 1555
SEQ ID NO: 553	gctgcgttctgaatatcag	4901	4920	SEQ ID NO: 1556
SEQ ID NO: 554	cgttctgaatatcaggctg	4905	4924	SEQ ID NO: 1557
SEQ ID NO: 555	aattcccatggtcttgagt	4968	4987	SEQ ID NO: 1558
SEQ ID NO: 556	tggtcttgagttaaatgct	4976	4995	SEQ ID NO: 1559
SEQ ID NO: 557	cttgagttaaatgctgaca	4980	4999	SEQ ID NO: 1560
SEQ ID NO: 558	ttgagttaaatgctgacat	4961	5000	SEQ ID NO: 1561
SEQ ID NO: 559	tgagttaaatgctgacatc	4982	5001	SEQ ID NO: 1562
SEQ ID NO: 560	acttgaagtgtagtctcct	5086	5105	SEQ ID NO: 1563
SEQ ID NO: 561	agtgtagtctcctggtgct	5092	5111	SEQ ID NO: 1564
SEQ ID NO: 562	gtgctggagaatgagctga	5106	5125	SEQ ID NO: 1565
SEQ ID NO: 563	ctggggcatctatgaaatt	5143	5162	SEQ ID NO: 1566
SEQ ID NO: 564	atggccgcttcagggaaca	5170	5189	SEQ ID NO: 1567
SEQ ID NO: 565	ttcagtctggatgggaaag	5199	5218	SEQ ID NO: 1568
SEQ ID NO: 566	ccatgattctgggtgtcga	5257	5276	SEQ ID NO: 1569
SEQ ID NO: 567	aaaacattttcaacttcaa	5281	5300	SEQ ID NO: 1570
SEQ ID NO: 568	cttaagctctcaaatgaca	5316	5335	SEQ ID NO: 1571
SEQ ID NO: 569	ttaagctctcaaatgacat	5317	5336	SEQ ID NO: 1572
SEQ ID NO: 570	catgatgggctcatatgct	5333	5352	SEQ ID NO: 1573
SEQ ID NO: 571	tgggctcatatgctgaaat	5338	5357	SEQ ID NO: 1574
SEQ ID NO: 572	actggacttctcttcaaaa	5399	5418	SEQ ID NO: 1575
SEQ ID NO: 573	acttctcttcaaaacttga	5404	5423	SEQ ID NO: 1576
SEQ ID NO: 574	ctgacaagttttataagca	5437	5456	SEQ ID NO: 1577
SEQ ID NO: 575	aagttttataagcaaactg	5442	5461	SEQ ID NO: 1578
SEQ ID NO: 576	ctgttaatttacagctaca	5458	5477	SEQ ID NO: 1579
SEQ ID NO: 577	ttacagctacagccctatt	5466	5485	SEQ ID NO: 1580
SEQ ID NO: 578	tctggtaactactttaaac	5486	5505	SEQ ID NO: 1581
SEQ ID NO: 579	tttaaacagtgacctgaaa	5498	5517	SEQ ID NO: 1582
SEQ ID NO: 580	ttaaacagtgacctgaaat	5499	5518	SEQ ID NO: 1583
SEQ ID NO: 581	cagtgacctgaaatacaat	5504	5523	SEQ ID NO: 1584
SEQ ID NO: 582	tgtggctggtaacctaaaa	5576	5595	SEQ ID NO: 1585
SEQ ID NO: 583	ttatcagcaagctataaag	5649	5668	SEQ ID NO: 1586
SEQ ID NO: 584	ggttcagggtgtggagttt	5684	5703	SEQ ID NO: 1587
SEQ ID NO: 585	attcagactcactgcattt	5767	5786	SEQ ID NO: 1588
SEQ ID NO: 586	ttcagactcactgcatttc	5768	5787	SEQ ID NO: 1589
SEQ ID NO: 587	tacaaatggcaatgggaaa	5840	5859	SEQ ID NO: 1590
SEQ ID NO: 588	gctgtatagcaaattcctg	5888	5907	SEQ ID NO: 1591
SEQ ID NO: 589	tgagcagacaggcacctgg	6035	6054	SEQ ID NO: 1592
SEQ ID NO: 590	ggcacctggaaactcaaga	6045	6064	SEQ ID NO: 1593
SEQ ID NO: 591	tgaatacagccaggacttg	6080	6099	SEQ ID NO: 1594
SEQ ID NO: 592	taatacagccaggacttgg	6081	6100	SEQ ID NO: 1595
SEQ ID NO: 593	ctggacgaactctggctga	6139	6158	SEQ ID NO: 1596
SEQ ID NO: 594	ttttactcagtgagcccat	6193	6212	SEQ ID NO: 1597
SEQ ID NO: 595	gatgagagatgccgttgag	6233	6252	SEQ ID NO: 1598
SEQ ID NO: 596	aattgttgcttttgtaaag	6269	6288	SEQ ID NO: 1599
SEQ ID NO: 597	cttttgtaaagtatgataa	6277	6296	SEQ ID NO: 1600
SEQ ID NO: 598	tttgtaaagtatgataaaa	6279	6298	SEQ ID NO: 1601
SEQ ID NO: 599	tccattaacctcccatttt	6312	6331	SEQ ID NO: 1602
SEQ ID NO: 600	ccattaacctcccattttt	6313	6332	SEQ ID NO: 1603
SEQ ID NO: 601	cttgcaagaatattttgag	5338	6357	SEQ ID NO: 1604
SEQ ID NO: 602	agaatattttgagaggaat	6344	6363	SEQ ID NO: 1605
SEQ ID NO: 603	attatagttgtactggaaa	6372	6391	SEQ ID NO: 1606
SEQ ID NO: 604	gaagcacatcaatattgat	6407	6426	SEQ ID NO: 1607
SEQ ID NO: 605	acatcaatattgatcaatt	6412	6431	SEQ ID NO: 1608
SEQ ID NO: 606	gaaaactcccacagcaagc	6457	6476	SEQ ID NO: 1609
SEQ ID NO: 607	ctgaattcattcaattggg	6486	6505	SEQ ID NO: 1610
SEQ ID NO: 608	tgaattcattcaattggga	6487	6506	SEQ ID NO: 1611
SEQ ID NO: 609	aactgactgctctcacaaa	6532	6551	SEQ ID NO: 1612
SEQ ID NO: 610	aaaagtatagaattacaga	6550	6569	SEQ ID NO: 1613
SEQ ID NO: 611	atcaactttaatgaaaaac	6603	6622	SEQ ID NO: 1614
SEQ ID NO: 612	tgatttgaaaatagctatt	6686	6705	SEQ ID NO: 1615
SEQ ID NO: 613	atttgaaaatagctattgc	6688	6707	SEQ ID NO: 1616
SEQ ID NO: 614	attgctaatattattgatg	6702	6721	SEQ ID NO: 1617
SEQ ID NO: 615	gaaaaattaaaaagtcttg	6729	6748	SEQ ID NO: 1618
SEQ ID NO: 616	actatcatatccgtgtaat	6754	6773	SEQ ID NO: 1619
SEQ ID NO: 617	tattgattttaacaaaagt	6815	6834	SEQ ID NO: 1620
SEQ ID NO: 618	ctgcagcagcttaagagac	6906	6925	SEQ ID NO: 1621
SEQ ID NO: 619	aaaacaacacattgaggct	6965	6984	SEQ ID NO: 1622
SEQ ID NO: 620	ttgagcatgtcaaacactt	7051	7070	SEQ ID NO: 1623
SEQ ID NO: 621	tttgaagtagctgagaaaa	7092	7111	SEQ ID NO: 1624
SEQ ID NO: 622	ttagtagagttggcccacc	7191	7210	SEQ ID NO: 1625
SEQ ID NO: 623	tgaaggagactattcagaa	7219	7238	SEQ ID NO: 1626
SEQ ID NO: 624	gagactattcagaagctaa	7224	7243	SEQ ID NO: 1627
SEQ ID NO: 625	aattagttggatttattga	7285	7304	SEQ ID NO: 1628
SEQ ID NO: 626	gcttaatgaattatctttt	7319	7338	SEQ ID NO: 1629
SEQ ID NO: 627	ttaacaaattccttgacat	7357	7376	SEQ ID NO: 1630
SEQ ID NO: 628	aaattaaagtcatttgatt	7386	7405	SEQ ID NO: 1631
SEQ ID NO: 629	gactcaatggtgaaattca	7456	7475	SEQ ID NO: 1632
SEQ ID NO: 630	gaaattcaggctctggaac	7467	7486	SEQ ID NO: 1633
SEQ ID NO: 631	actaccacaaaaagctgaa	7484	7503	SEQ ID NO: 1634
SEQ ID NO: 632	ccaaaataaccttaatcat	7570	7589	SEQ ID NO: 1635
SEQ ID NO: 633	aaataaccttaatcatcaa	7573	7592	SEQ ID NO: 1636
SEQ ID NO: 634	tttaagttcagcatctttg	7607	7626	SEQ ID NO: 1637
SEQ ID NO: 635	caggtttatagcacacttg	7731	7750	SEQ ID NO: 1638
SEQ ID NO: 636	gttcactgttcctgaaatc	7862	7881	SEQ ID NO: 1639
SEQ ID NO: 637	cactgttcctgaaatcaag	7865	7884	SEQ ID NO: 1640
SEQ ID NO: 638	actgttcctgaaatcaaga	7866	7885	SEQ ID NO: 1641
SEQ ID NO: 639	gcctgcctttgaagtcagt	7901	7920	SEQ ID NO: 1642
SEQ ID NO: 640	taacagatttgaggattcc	7972	7991	SEQ ID NO: 1643
SEQ ID NO: 641	gttttccacaccagaattt	8042	8061	SEQ ID NO: 1644
SEQ ID NO: 642	tcagaaccattgaccagat	8128	8147	SEQ ID NO: 1645
SEQ ID NO: 643	tagcgagaatcaccctgcc	8218	8237	SEQ ID NO: 1646
SEQ ID NO: 644	ccttaatgattttcaagtt	8291	8310	SEQ ID NO: 1647
SEQ ID NO: 645	acataccagaattccagct	8320	8339	SEQ ID NO: 1648
SEQ ID NO: 646	aatgctgacatagggaatg	8430	8449	SEQ ID NO: 1649
SEQ ID NO: 647	atgctgacatagggaatgg	8431	8450	SEQ ID NO: 1650
SEQ ID NO: 648	aaccacctcagcaaacgaa	8450	8469	SEQ ID NO: 1651
SEQ ID NO: 649	agcaggtatcgcagcttcc	8468	8487	SEQ ID NO: 1652
SEQ ID NO: 650	tgcacaactctcaaaccct	8543	8562	SEQ ID NO: 1653
SEQ ID NO: 651	aggagtcagtgaagttctc	8584	8603	SEQ ID NO: 1654
SEQ ID NO: 652	tttttggaaatgccattga	8644	8663	SEQ ID NO: 1655
SEQ ID NO: 653	aatggagtgattgtcaaga	8721	8740	SEQ ID NO: 1656
SEQ ID NO: 654	gtcaagataaacaatcagc	8733	8752	SEQ ID NO: 1657
SEQ ID NO: 655	tccacaaattgaacatccc	8779	8798	SEQ ID NO: 1658
SEQ ID NO: 656	ttgaacatccccaaactgg	8787	8806	SEQ ID NO: 1659
SEQ ID NO: 657	acatccccaaactggactt	8791	8810	SEQ ID NO: 1660
SEQ ID NO: 658	acttctctagtcaggctga	8806	8825	SEQ ID NO: 1661
SEQ ID NO: 659	tgaatcacaaattagtttc	8936	8955	SEQ ID NO: 1662
SEQ ID NO: 660	agaaggacccctcacttcc	8960	8979	SEQ ID NO: 1663
SEQ ID NO: 661	ttggactgtccaataagat	8980	8999	SEQ ID NO: 1664
SEQ ID NO: 662	actgtccaataagatcaat	8984	9003	SEQ ID NO: 1665
SEQ ID NO: 663	ctgtccaataagatcaata	8985	9004	SEQ ID NO: 1666
SEQ ID NO: 664	gtttatgaatctggctccc	9033	9052	SEQ ID NO: 1667
SEQ ID NO: 665	atgaatctggctccctcaa	9037	9056	SEQ ID NO: 1668
SEQ ID NO: 666	ctcaacttttctaaacttg	9051	9070	SEQ ID NO: 1669
SEQ ID NO: 667	ctaaaggcatggcactgtt	9121	9140	SEQ ID NO: 1670
SEQ ID NO: 668	aaggcatggcactgtttgg	9124	9143	SEQ ID NO: 1671
SEQ ID NO: 669	atccacaaacaatgaaggg	9254	9273	SEQ ID NO: 1672
SEQ ID NO: 670	ggaatttgaaagttcgttt	9271	9290	SEQ ID NO: 1673
SEQ ID NO: 671	aataactatgcactgtttc	9324	9343	SEQ ID NO: 1674
SEQ ID NO: 672	gaaacaacgagaacattat	9424	9443	SEQ ID NO: 1675
SEQ ID NO: 673	ttcttgaaaacgacaaagc	9591	9610	SEQ ID NO: 1676
SEQ ID NO: 674	ataagaaaaacaaacacag	9640	9659	SEQ ID NO: 1677
SEQ ID NO: 675	aaaacaaacacaggcattc	9646	9665	SEQ ID NO: 1678
SEQ ID NO: 676	gcattccatcacaaatcct	9659	9678	SEQ ID NO: 1679
SEQ ID NO: 677	tttgaaaaaaacagaaaca	6732	9751	SEQ ID NO: 1680
SEQ ID NO: 678	caatgcattagattttgtc	9749	9768	SEQ ID NO: 1681
SEQ ID NO: 679	caaagctgaaaaatctcag	9809	9828	SEQ ID NO: 1682
SEQ ID NO: 680	cctggatacactgttccag	9855	9874	SEQ ID NO: 1683
SEQ ID NO: 681	gttgaagtgtctccattca	9882	9901	SEQ ID NO: 1684
SEQ ID NO: 682	tttctccatcctaggttct	9956	9975	SEQ ID NO: 1685
SEQ ID NO: 683	ttctccatcctaggttctg	9957	9976	SEQ ID NO: 1686
SEQ ID NO: 684	tcattagagctgccagtcc	10011	10030	SEQ ID NO: 1687
SEQ ID NO: 685	tgctgaactttttaaccag	10169	10188	SEQ ID NO: 1688
SEQ ID NO: 686	ctcctttcttcatcttcat	10206	10225	SEQ ID NO: 1689
SEQ ID NO: 687	tgtcattgatgcactgcag	10226	10245	SEQ ID NO: 1690
SEQ ID NO: 688	tgatgcactgcagtacaaa	10232	10251	SEQ ID NO: 1691
SEQ ID NO: 689	agctctgtctctgagcaac	10301	10320	SEQ ID NO: 1692
SEQ ID NO: 690	agccgaaattccaattttg	10400	10419	SEQ ID NO: 1693
SEQ ID NO: 691	ttgagaatgaatttcaagc	10416	10435	SEQ ID NO: 1694
SEQ ID NO: 692	aaacctactgtctcttcct	10461	10480	SEQ ID NO: 1695
SEQ ID NO: 693	tacttttccattgagtcat	10575	10594	SEQ ID NO: 1696
SEQ ID NO: 694	tcaggtccatgcaagtcag	10910	10929	SEQ ID NO: 1697
SEQ ID NO: 695	atgcaagtcagcccagttc	10918	10937	SEQ ID NO: 1698
SEQ ID NO: 696	tgaatgctaacactaagaa	10975	10994	SEQ ID NO: 1699
SEQ ID NO: 697	agaagatcagatggaaaaa	10996	11015	SEQ ID NO: 1700
SEQ ID NO: 698	ggctattcattctccatcc	11256	11275	SEQ ID NO: 1701
SEQ ID NO: 699	aaagttttggctgataaat	11280	11299	SEQ ID NO: 1702
SEQ ID NO: 700	agttttggctgataaattc	11282	11301	SEQ ID NO: 1703
SEQ ID NO: 701	ctgggctgaaactaaatga	11308	11327	SEQ ID NO: 1704
SEQ ID NO: 702	cagagaaatacaaatctat	11405	11424	SEQ ID NO: 1705
SEQ ID NO: 703	gaggtaaaattccctgaag	11472	11491	SEQ ID NO: 1706
SEQ ID NO: 704	cttttttgagataaccgtg	11537	11556	SEQ ID NO: 1707
SEQ ID NO: 705	gctggaattgtcattcctt	11727	11746	SEQ ID NO: 1708
SEQ ID NO: 706	gtgtataatgccacttgga	11787	11806	SEQ ID NO: 1709
SEQ ID NO: 707	attccacatgcagctcaac	11851	11870	SEQ ID NO: 1710
SEQ ID NO: 708	tgaagaagatggcaaattt	11984	12003	SEQ ID NO: 1711
SEQ ID NO: 709	atcaaaagcccagcgttca	12042	12061	SEQ ID NO: 1712
SEQ ID NO: 710	gtgggcatggatatggatg	12135	12154	SEQ ID NO: 1713
SEQ ID NO: 711	aaatggaacttctactaca	12171	12190	SEQ ID NO: 1714
SEQ ID NO: 712	aaaaactcaccatattcaa	12211	12230	SEQ ID NO: 1715
SEQ ID NO: 713	ctgagaagaaatctgcaga	12420	12439	SEQ ID NO: 1716
SEQ ID NO: 714	acaatgctgagtgggttta	12439	12458	SEQ ID NO: 1717
SEQ ID NO: 715	caatgctgagtgggtttat	12440	12459	SEQ ID NO: 1718
SEQ ID NO: 716	ttaggcaaattgatgatat	12469	12488	SEQ ID NO: 1719
SEQ ID NO: 717	ataaactaatagatgtaat	12889	12908	SEQ ID NO: 1720
SEQ ID NO: 718	ccaactaatagaagataac	13031	13050	SEQ ID NO: 1721
SEQ ID NO: 719	ttaattatatccaagatga	13087	13106	SEQ ID NO: 1722
SEQ ID NO: 720	tttaaattgttgaaagaaa	13143	13162	SEQ ID NO: 1723
SEQ ID NO: 721	aagttcaatgaatttattc	13182	13201	SEQ ID NO: 1724
SEQ ID NO: 722	ttgaagaaaagatagtcag	13318	13337	SEQ ID NO: 1725
SEQ ID NO: 723	acttccattctgaatatat	13369	13388	SEQ ID NO: 1726
SEQ ID NO: 724	cacagaaatattcaggaat	13443	13462	SEQ ID NO: 1727
SEQ ID NO: 725	ccattgcgacgaagaaaat	13552	13571	SEQ ID NO: 1728
SEQ ID NO: 726	tataaactgcaagattttt	13599	13618	SEQ ID NO: 1729
SEQ ID NO: 727	tctgattacctatgaaaat	13629	13648	SEQ ID NO: 1730
SEQ ID NO: 728	ggagttactgaaaaagctg	13718	13737	SEQ ID NO: 1731
SEQ ID NO: 729	tgaagcttgctccaggaga	13765	13784	SEQ ID NO: 1732
SEQ ID NO: 730	tgaactggacctgcaccaa	13947	13966	SEQ ID NO: 1733
SEQ ID NO: 731	ttgctaaacttgggggagg	14050	14069	SEQ ID NO: 1734
SEQ ID NO: 732	gattcgaatatcaaattca	4404	4423	SEQ ID NO: 1735
SEQ ID NO: 733	atttgtttgtcaaagaagt	4543	4562	SEQ ID NO: 1736
SEQ ID NO: 734	tctcggttgctgccgctga	25	44	SEQ ID NO: 1737
SEQ ID NO: 735	gctgaggagcccgcccagc	39	58	SEQ ID NO: 1738
SEQ ID NO: 736	ctggtctgtccaaaagatg	219	238	SEQ ID NO: 1739
SEQ ID NO: 737	ctgagagttccagtggagt	283	302	SEQ ID NO: 1740
SEQ ID NO: 738	cagtgcaccctgaaagagg	396	415	SEQ ID NO: 1741
SEQ ID NO: 739	ctctgaggagtttgctgca	464	483	SEQ ID NO: 1742
SEQ ID NO: 740	acatcaagaggggcatcat	574	593	SEQ ID NO: 1743
SEQ ID NO: 741	ctgatcagcagcagccagt	822	841	SEQ ID NO: 1744
SEQ ID NO: 742	ggacgctaagaggaagcat	857	876	SEQ ID NO: 1745
SEQ ID NO: 743	agctgttttgaagactctc	1079	1098	SEQ ID NO: 1746
SEQ ID NO: 744	tgaaaaaactaaccatctc	1105	1124	SEQ ID NO: 1747
SEQ ID NO: 745	ctgagctgagaggcctcag	1168	1187	SEQ ID NO: 1748
SEQ ID NO: 746	tgaaacgtgtgcatgccaa	1303	1322	SEQ ID NO: 1749
SEQ ID NO: 747	ccttgtatgcgctgagcca	1432	1451	SEQ ID NO: 1750
SEQ ID NO: 748	aggagctgctggacattgc	1492	1511	SEQ ID NO: 1751
SEQ ID NO: 749	atttgattctgcgggtcat	1567	1586	SEQ ID NO: 1752
SEQ ID NO: 750	tccagaactcaagtcttca	1619	1638	SEQ ID NO: 1753
SEQ ID NO: 751	ggttcttcttcagactttc	1736	1755	SEQ ID NO: 1754
SEQ ID NO: 752	gttgatgaggagtccttca	1802	1821	SEQ ID NO: 1755
SEQ ID NO: 753	tccaagatctgaaaaagtt	1933	1952	SEQ ID NO: 1756
SEQ ID NO: 754	agttagtgaaagaagttct	1948	1967	SEQ ID NO: 1757
SEQ ID NO: 755	gaagggaatcttatatttg	2076	2095	SEQ ID NO: 1758
SEQ ID NO: 756	ggaagctctttttgggaag	2213	2232	SEQ ID NO: 1759
SEQ ID NO: 757	tggaatattgctcagtgtt	2366	2385	SEQ ID NO: 1760
SEQ ID NO: 758	gatttgaaatccaaagaag	2400	2419	SEQ ID NO: 1761
SEQ ID NO: 759	tccaaagaagtcccggaag	2409	2428	SEQ ID NO: 1762
SEQ ID NO: 760	aggaagggctcaaagaatg	2562	2581	SEQ ID NO: 1763
SEQ ID NO: 761	agaatgacttttttcttca	2575	2594	SEQ ID NO: 1764
SEQ ID NO: 762	tttgtgacaaatatgggca	2757	2776	SEQ ID NO: 1765
SEQ ID NO: 763	ctgaggctaccatgacatt	3244	3263	SEQ ID NO: 1766
SEQ ID NO: 764	gtagataccaaaaaaatga	3660	3676	SEQ ID NO: 1767
SEQ ID NO: 765	aaatgacttccaatttccc	3673	3692	SEQ ID NO: 1768
SEQ ID NO: 766	atgacttccaatttccctg	3675	3694	SEQ ID NO: 1769
SEQ ID NO: 767	atctgccatctcgagagtt	4096	4115	SEQ ID NO: 1770
SEQ ID NO: 768	atttgtttgtcaaagaagt	4543	4562	SEQ ID NO: 1771
SEQ ID NO: 769	gcagagcttggcctctctg	5127	5146	SEQ ID NO: 1772
SEQ ID NO: 770	atatgctgaaatgaaattt	5345	5364	SEQ ID NO: 1773
SEQ ID NO: 771	tcaaaacttgacaacattt	5412	5431	SEQ ID NO: 1774
SEQ ID NO: 772	cagtgacctgaaatacaat	5504	5523	SEQ ID NO: 1775
SEQ ID NO: 773	tacaaatggcaatgggaaa	5840	5859	SEQ ID NO: 1776
SEQ ID NO: 774	cttttgtaaagtatgataa	6277	6296	SEQ ID NO: 1777
SEQ ID NO: 775	ttgtaaagtatgataaaaa	6280	6299	SEQ ID NO: 1778
SEQ ID NO: 776	tccattaacctcccatttt	6312	6331	SEQ ID NO: 1779
SEQ ID NO: 777	gattatctgaattcattca	6480	6499	SEQ ID NO: 1780
SEQ ID NO: 778	aattgggagagacaagttt	6498	6517	SEQ ID NO: 1781
SEQ ID NO: 779	atttgaaaatagctattgc	6688	6707	SEQ ID NO: 1782
SEQ ID NO: 780	tgagcatgtcaaacacttt	7052	7071	SEQ ID NO: 1783
SEQ ID NO: 781	ttgaagatgttaacaaatt	7348	7367	SEQ ID NO: 1784
SEQ ID NO: 782	acttgtcacctacatttct	7745	7764	SEQ ID NO: 1785
SEQ ID NO: 783	gttttccacaccagaattt	8042	8061	SEQ ID NO: 1786
SEQ ID NO: 784	ataagtacaaccaaaattt	9397	9416	SEQ ID NO: 1787
SEQ ID NO: 785	cgggacctgcggggctgag	0	19	SEQ ID NO: 1788
SEQ ID NO: 786	agtgcccttctcggttgct	17	36	SEQ ID NO: 1789
SEQ ID NO: 787	gctgaggagcccgcccagc	39	58	SEQ ID NO: 1790
SEQ ID NO: 788	gaggagcccgcccagccag	42	61	SEQ ID NO: 1791
SEQ ID NO: 789	gggccgcgaggccgaggcc	64	83	SEQ ID NO: 1792
SEQ ID NO: 790	ccaggccgcagcccaggag	81	100	SEQ ID NO: 1793
SEQ ID NO: 791	ggagccgccccaccgcagc	96	115	SEQ ID NO: 1794
SEQ ID NO: 792	gaagaggaaatgctggaaa	192	211	SEQ ID NO: 1795
SEQ ID NO: 793	caaaagatgcgacccgatt	229	248	SEQ ID NO: 1796
SEQ ID NO: 794	attcaagcacctccggaag	245	264	SEQ ID NO: 1797
SEQ ID NO: 795	gttccagtggagtccctgg	289	308	SEQ ID NO: 1798
SEQ ID NO: 796	gactgctgattcaagaagt	308	327	SEQ ID NO: 1799
SEQ ID NO: 797	gtgccaccaggatcaactg	325	344	SEQ ID NO: 1800
SEQ ID NO: 798	gatcaactgcaaggttgag	335	354	SEQ ID NO: 1801
SEQ ID NO: 799	actgcaaggttgagctgga	340	359	SEQ ID NO: 1802
SEQ ID NO: 800	ccagctctgcagcttcatc	365	384	SEQ ID NO: 1803
SEQ ID NO: 801	agcttcatcctgaagacca	375	394	SEQ ID NO: 1804
SEQ ID NO: 802	cttcatcctgaagaccagc	377	396	SEQ ID NO: 1805
SEQ ID NO: 803	ccagccagtgcaccctgaa	391	410	SEQ ID NO: 1806
SEQ ID NO: 804	cagtgcaccctgaaagagg	396	415	SEQ ID NO: 1807
SEQ ID NO: 805	tggcttcaaccctgagggc	419	438	SEQ ID NO: 1808
SEQ ID NO: 806	cttcaaccctgagggcaaa	422	441	SEQ ID NO: 1809
SEQ ID NO: 807	ttcaaccctgagggcaaag	423	442	SEQ ID NO: 1810
SEQ ID NO: 808	cttgctgaagaaaaccaag	443	462	SEQ ID NO: 1811
SEQ ID NO: 809	tgctgaagaaaaccaagaa	445	464	SEQ ID NO: 1812
SEQ ID NO: 810	ttgctgcagccatgtccag	475	494	SEQ ID NO: 1813
SEQ ID NO: 811	tgctgcagccatgtccagg	476	495	SEQ ID NO: 1814
SEQ ID NO: 812	agccatgtccaggtatgag	482	501	SEQ ID NO: 1815
SEQ ID NO: 813	agctcaagctggccattcc	499	518	SEQ ID NO: 1816
SEQ ID NO: 814	agaagggaagcaggttttc	518	537	SEQ ID NO: 1817
SEQ ID NO: 815	aagggaagcaggttttcct	520	539	SEQ ID NO: 1818
SEQ ID NO: 816	agaaagatgaacctactta	547	566	SEQ ID NO: 1819
SEQ ID NO: 817	atcctgaacatcaagaggg	567	586	SEQ ID NO: 1820
SEQ ID NO: 818	tcctgaacatcaagagggg	568	587	SEQ ID NO: 1821
SEQ ID NO: 819	ctgaacatcaagaggggca	570	589	SEQ ID NO: 1822
SEQ ID NO: 820	aacatcaagaggggcatca	573	592	SEQ ID NO: 1823
SEQ ID NO: 821	acatcaagaggggcatcat	574	593	SEQ ID NO: 1824
SEQ ID NO: 822	tcatttctgccctcctggt	589	608	SEQ ID NO: 1825
SEQ ID NO: 823	ttcccccagagacagaaga	607	626	SEQ ID NO: 1826
SEQ ID NO: 824	gaagaagccaagcaagtgt	621	640	SEQ ID NO: 1827
SEQ ID NO: 825	ttgtttctggataccggtg	639	658	SEQ ID NO: 1828
SEQ ID NO: 826	tgtatggaaactgctccac	655	674	SEQ ID NO: 1829
SEQ ID NO: 827	aaactgctccactcacttt	662	681	SEQ ID NO: 1830
SEQ ID NO: 828	actcactttaccgtcaaga	672	691	SEQ ID NO: 1831
SEQ ID NO: 829	ctttaccgtcaagacgagg	677	696	SEQ ID NO: 1832
SEQ ID NO: 830	ttaccgtcaagacgaggaa	679	698	SEQ ID NO: 1833
SEQ ID NO: 831	acgaggaagggcaatgtgg	690	709	SEQ ID NO: 1834
SEQ ID NO: 832	cgaggaagggcaatgtggc	691	710	SEQ ID NO: 1835
SEQ ID NO: 833	gaggaagggcaatgtggca	692	711	SEQ ID NO: 1836
SEQ ID NO: 834	ggaagggcaatgtggcaac	694	713	SEQ ID NO: 1837
SEQ ID NO: 835	gaagggcaatgtggcaaca	695	714	SEQ ID NO: 1838
SEQ ID NO: 836	caggcatcagcccacttgc	769	788	SEQ ID NO: 1839
SEQ ID NO: 837	aggcatcagcccacttgct	770	789	SEQ ID NO: 1840
SEQ ID NO: 838	tcagcccacttgctctcat	775	794	SEQ ID NO: 1841
SEQ ID NO: 839	gtcaactctgatcagcagc	815	834	SEQ ID NO: 1842
SEQ ID NO: 840	ggacgctaagaggaagcat	857	876	SEQ ID NO: 1843
SEQ ID NO: 841	aaggagcaacacctcttcc	894	913	SEQ ID NO: 1844
SEQ ID NO: 842	aggagcaacacctcttcct	895	914	SEQ ID NO: 1845
SEQ ID NO: 843	caacacctcttcctgcctt	900	919	SEQ ID NO: 1846
SEQ ID NO: 844	aacacctcttcctgccttt	901	920	SEQ ID NO: 1847
SEQ ID NO: 845	acaagaataagtatgggat	925	944	SEQ ID NO: 1848
SEQ ID NO: 846	caagaataagtatgggatg	926	945	SEQ ID NO: 1849
SEQ ID NO: 847	tagcacaagtgacacagac	946	965	SEQ ID NO: 1850
SEQ ID NO: 848	agcacaagtgacacagact	947	966	SEQ ID NO: 1851
SEQ ID NO: 849	gcacaagtgacacagactt	948	967	SEQ ID NO: 1852
SEQ ID NO: 850	aacttgaagacacaccaaa	970	989	SEQ ID NO: 1853
SEQ ID NO: 851	gcttctttggtgaaggtac	1000	1019	SEQ ID NO: 1854
SEQ ID NO: 852	ctttggtgaaggtactaag	1004	1023	SEQ ID NO: 1855
SEQ ID NO: 853	tactaagaagatgggcctc	1016	1038	SEQ ID NO: 1856
SEQ ID NO: 854	tttgagagcaccaaatcca	1038	1057	SEQ ID NO: 1857
SEQ ID NO: 855	agagcaccaaatccacatc	1042	1061	SEQ ID NO: 1858
SEQ ID NO: 856	agctgttttgaagactctc	1079	1098	SEQ ID NO: 1859
SEQ ID NO: 857	tgaaaaaactaaccatctc	1105	1124	SEQ ID NO: 1860
SEQ ID NO: 858	tctgagcaaaatatccaga	1106	1125	SEQ ID NO: 1861
SEQ ID NO: 859	tctcttcaataagctggtt	1122	1141	SEQ ID NO: 1862
SEQ ID NO: 860	ctgagctgagaggcctcag	1148	1167	SEQ ID NO: 1863
SEQ ID NO: 861	tgaagcagtcacatctctc	1168	1187	SEQ ID NO: 1864
SEQ ID NO: 862	aagcagtcacatctctctt	1190	1209	SEQ ID NO: 1865
SEQ ID NO: 863	ctctcttgccacagctgat	1192	1211	SEQ ID NO: 1866
SEQ ID NO: 864	tcttgccacagctgattga	1204	1223	SEQ ID NO: 1867
SEQ ID NO: 865	cttgccacagctgattgag	1207	1226	SEQ ID NO: 1868
SEQ ID NO: 866	tgaggtgtccagccccatc	1208	1227	SEQ ID NO: 1869
SEQ ID NO: 867	tgaggtgtccagccccatc	1223	1242	SEQ ID NO: 1870
SEQ ID NO: 868	tcagtgtggacagcctcag	1259	1278	SEQ ID NO: 1871
SEQ ID NO: 869	acatcctccagtggctgaa	1288	1307	SEQ ID NO: 1872
SEQ ID NO: 870	gcacagcagctgcgagaga	1377	1396	SEQ ID NO: 1873
SEQ ID NO: 871	cagcagctgcgagagatct	1380	1399	SEQ ID NO: 1874
SEQ ID NO: 872	gcgagggatcagcgcagcc	1407	1426	SEQ ID NO: 1875
SEQ ID NO: 873	aagacaaaccctacaggga	1470	1489	SEQ ID NO: 1876
SEQ ID NO: 874	caggagctgctggacattg	1491	1510	SEQ ID NO: 1877
SEQ ID NO: 875	aggagctgctggacattgc	1492	1511	SEQ ID NO: 1878
SEQ ID NO: 876	ctgctggacattgctaatt	1497	1516	SEQ ID NO: 1879
SEQ ID NO: 877	gattacacctatttgattc	1557	1576	SEQ ID NO: 1880
SEQ ID NO: 878	atttgattctgcgggtcat	1567	1586	SEQ ID NO: 1881
SEQ ID NO: 879	tctgcgggtcattggaaat	1574	1593	SEQ ID NO: 1882
SEQ ID NO: 880	aaccatggagcagttaact	1601	1620	SEQ ID NO: 1883
SEQ ID NO: 881	ggagcagttaactccagaa	1607	1626	SEQ ID NO: 1884
SEQ ID NO: 882	actccagaactcaagtctt	1617	1636	SEQ ID NO: 1885
SEQ ID NO: 883	tccagaactcaagtcttca	1619	1638	SEQ ID NO: 1886
SEQ ID NO: 884	aagtacaaagccatcactg	1655	1674	SEQ ID NO: 1887
SEQ ID NO: 885	gccatcactgatgatccag	1664	1683	SEQ ID NO: 1888
SEQ ID NO: 886	ccatcactgatgatccaga	1665	1684	SEQ ID NO: 1889
SEQ ID NO: 887	atccagaaagctgccatcc	1677	1696	SEQ ID NO: 1890
SEQ ID NO: 888	cagaaagctgccatccagg	1680	1699	SEQ ID NO: 1891
SEQ ID NO: 889	acaaggaccaggaggttct	1723	1742	SEQ ID NO: 1892
SEQ ID NO: 890	aggaccaggaggttcttct	1726	1745	SEQ ID NO: 1893
SEQ ID NO: 891	accaggaggttcttcttca	1729	1748	SEQ ID NO: 1894
SEQ ID NO: 892	tcttcagactttccttgat	1742	1761	SEQ ID NO: 1895
SEQ ID NO: 893	ttcagactttccttgatga	1744	1763	SEQ ID NO: 1896
SEQ ID NO: 894	gttgatgaggagtccttca	1802	1821	SEQ ID NO: 1897
SEQ ID NO: 895	cttcacaggcagatattaa	1816	1835	SEQ ID NO: 1898
SEQ ID NO: 896	ttcacaggcagatattaac	1817	1836	SEQ ID NO: 1899
SEQ ID NO: 897	ggcagatattaacaaaatt	1823	1842	SEQ ID NO: 1900
SEQ ID NO: 898	atattaacaaaattgtcca	1828	1847	SEQ ID NO: 1901
SEQ ID NO: 899	acaaaattgtccaaattct	1834	1853	SEQ ID NO: 1902
SEQ ID NO: 900	gagcaagtgaagaactttg	1869	1888	SEQ ID NO: 1903
SEQ ID NO: 901	gtgaagaactttgtggctt	1875	1894	SEQ ID NO: 1904
SEQ ID NO: 902	agaactttgtggcttccca	1876	1898	SEQ ID NO: 1905
SEQ ID NO: 903	tttgtggcttcccatattg	1884	1903	SEQ ID NO: 1906
SEQ ID NO: 904	tggcttcccatattgccaa	1888	1907	SEQ ID NO: 1907
SEQ ID NO: 905	ttcccatattgccaatatc	1892	1911	SEQ ID NO: 1908
SEQ ID NO: 906	tcccatattgccaatatct	1893	1912	SEQ ID NO: 1909
SEQ ID NO: 907	ttgccaatatcttgaactc	1900	1919	SEQ ID NO: 1910
SEQ ID NO: 908	ttggatatccaagatctga	1926	1945	SEQ ID NO: 1911
SEQ ID NO: 909	tccaagatctgaaaaagtt	1933	1952	SEQ ID NO: 1912
SEQ ID NO: 910	ctgaaaaagttagtgaaag	1941	1960	SEQ ID NO: 1913
SEQ ID NO: 911	agttagtgaaagaagttct	1948	1967	SEQ ID NO: 1914
SEQ ID NO: 912	aatctcaacttccaactgt	1972	1991	SEQ ID NO: 1915
SEQ ID NO: 913	gtcatggacttcagaaat	1989	2008	SEQ ID NO: 1916
SEQ ID NO: 914	tcaactctacaaatctgtt	2021	2040	SEQ ID NO: 1917
SEQ ID NO: 915	aactctacaaatctgtttc	2023	2042	SEQ ID NO: 1918
SEQ ID NO: 916	aaatagaagggaatcttat	2071	2090	SEQ ID NO: 1919
SEQ ID NO: 917	agaagggaatcttatattt	2075	2094	SEQ ID NO: 1920
SEQ ID NO: 918	gaagggaatcttatatttg	2076	2095	SEQ ID NO: 1921
SEQ ID NO: 919	tgatccaaataactacctt	2093	2112	SEQ ID NO: 1922
SEQ ID NO: 920	tggatttgcttcagctgac	2150	2169	SEQ ID NO: 1923
SEQ ID NO: 921	tttgcttcagctgacctca	2154	2173	SEQ ID NO: 1924
SEQ ID NO: 922	cttggaaggaaaaggcttt	2183	2202	SEQ ID NO: 1925
SEQ ID NO: 923	tggaaggaaaaggctttga	2185	2204	SEQ ID NO: 1926
SEQ ID NO: 924	ggctttgagccaacattgg	2196	2215	SEQ ID NO: 1927
SEQ ID NO: 925	tgagccaacattggaagct	2201	2220	SEQ ID NO: 1928
SEQ ID NO: 926	gagccaacattggaagctc	2202	2221	SEQ ID NO: 1929
SEQ ID NO: 927	aacattggaagctcttttt	2207	2226	SEQ ID NO: 1930
SEQ ID NO: 928	tggaagctctttttgggaa	2212	2231	SEQ ID NO: 1931
SEQ ID NO: 929	ctctttttgggaagcaagg	2218	2237	SEQ ID NO: 1932
SEQ ID NO: 930	tttttgggaagcaaggatt	2221	2240	SEQ ID NO: 1933
SEQ ID NO: 931	ttttcccagacagtgtcaa	2239	2258	SEQ ID NO: 1934
SEQ ID NO: 932	ttggctataccaaagatga	2323	2342	SEQ ID NO: 1935
SEQ ID NO: 933	ataccaaagatgataaaca	2329	2348	SEQ ID NO: 1936
SEQ ID NO: 934	gagcaggatatggtaaatg	2349	2368	SEQ ID NO: 1937
SEQ ID NO: 935	atggtaaatggaataatgc	2358	2377	SEQ ID NO: 1938
SEQ ID NO: 936	tggtaaatggaataatgct	2359	2378	SEQ ID NO: 1939
SEQ ID NO: 937	taaatggaataatgctcag	2352	2381	SEQ ID NO: 1940
SEQ ID NO: 938	tggaataatgctcagtgtt	2366	2385	SEQ ID NO: 1941
SEQ ID NO: 939	tcagtgttgagaagctgat	2377	2396	SEQ ID NO: 1942
SEQ ID NO: 940	cagtgttgagaagctgatt	2378	2397	SEQ ID NO: 1943
SEQ ID NO: 941	agtgttgagaagctgatta	2379	2398	SEQ ID NO: 1944
SEQ ID NO: 942	gattaaagatttgaaatcc	2393	2412	SEQ ID NO: 1945
SEQ ID NO: 943	gatttgaaatccaaagaag	2400	2419	SEQ ID NO: 1946
SEQ ID NO: 944	atttgaaatccaaagaagt	2401	2420	SEQ ID NO: 1947
SEQ ID NO: 945	atccaaagaagtcccggaa	2408	2427	SEQ ID NO: 1948
SEQ ID NO: 946	tccaaagaagtcccggaag	2409	2428	SEQ ID NO: 1949
SEQ ID NO: 947	agagcctacctccgcatct	2430	2449	SEQ ID NO: 1950
SEQ ID NO: 948	gagcctacctccgcatctt	2431	2450	SEQ ID NO: 1951
SEQ ID NO: 949	cttgggagaggagcttggt	2447	2466	SEQ ID NO: 1952
SEQ ID NO: 950	ggagcttggttttgccagt	2456	2475	SEQ ID NO: 1953
SEQ ID NO: 951	ttggttttgccagtctcca	2461	2480	SEQ ID NO: 1954
SEQ ID NO: 952	cagtctccatgacctccag	2471	2490	SEQ ID NO: 1955
SEQ ID NO: 953	ctccatgacctccagctcc	2475	2494	SEQ ID NO: 1956
SEQ ID NO: 954	ctgggaaagctgcttctga	2493	2512	SEQ ID NO: 1957
SEQ ID NO: 955	gaggtcatcaggaagggct	2553	2572	SEQ ID NO: 1958
SEQ ID NO: 956	aagaatgacttttttcctc	2574	2593	SEQ ID NO: 1959
SEQ ID NO: 957	cttttttcttcactacatc	2582	2601	SEQ ID NO: 1960
SEQ ID NO: 958	catcttcatggagaatgcc	2597	2616	SEQ ID NO: 1961
SEQ ID NO: 959	cttcatggagaatgccttt	2600	2619	SEQ ID NO: 1962
SEQ ID NO: 960	aatgcctttgaactcccca	2610	2629	SEQ ID NO: 1963
SEQ ID NO: 961	gcctttgaactccccactg	2613	2632	SEQ ID NO: 1964
SEQ ID NO: 962	caaggctggagtaaaactg	2684	2703	SEQ ID NO: 1965
SEQ ID NO: 963	tggagtaaaactggaagta	2690	2709	SEQ ID NO: 1966
SEQ ID NO: 964	ggaagtagccaacatgcag	2702	2721	SEQ ID NO: 1967
SEQ ID NO: 965	tttgtgacaaatatgggca	2757	2776	SEQ ID NO: 1968
SEQ ID NO: 966	tgtgacaaatatgggcatc	2759	2778	SEQ ID NO: 1969
SEQ ID NO: 967	ggacttcgctaggagtggg	2786	2805	SEQ ID NO: 1970
SEQ ID NO: 968	gtggggtccagatgaacac	2800	2819	SEQ ID NO: 1971
SEQ ID NO: 969	ttccacgagtcgggtctgg	2826	2845	SEQ ID NO: 1972
SEQ ID NO: 970	agtcgggtctggaggctca	2833	2852	SEQ ID NO: 1973
SEQ ID NO: 971	tcgggtctggaggctcatg	2835	2854	SEQ ID NO: 1974
SEQ ID NO: 972	aaaagctgggaagctgaag	2861	2880	SEQ ID NO: 1975
SEQ ID NO: 973	aagctgaagtttatcattc	2871	2890	SEQ ID NO: 1976
SEQ ID NO: 974	gagaccagtcaagtcgctc	2900	2919	SEQ ID NO: 1977
SEQ ID NO: 975	gcaacacattacatttggt	2926	2945	SEQ ID NO: 1978
SEQ ID NO: 976	acattacatttggtctcta	2931	2950	SEQ ID NO: 1979
SEQ ID NO: 977	cattacatttggtctctac	2932	2951	SEQ ID NO: 1980
SEQ ID NO: 978	aaacggaggtgatcccacc	2956	2975	SEQ ID NO: 1981
SEQ ID NO: 979	attgagaacaggcagtcct	2979	2998	SEQ ID NO: 1982
SEQ ID NO: 980	tgagaacaggcagtcctgg	2981	3000	SEQ ID NO: 1983
SEQ ID NO: 981	ctgcacctcaggcgcttac	3035	3054	SEQ ID NO: 1984
SEQ ID NO: 982	tccacagactccgcctcct	3066	3085	SEQ ID NO: 1985
SEQ ID NO: 983	ctgaccggggacaccagat	3093	3112	SEQ ID NO: 1986
SEQ ID NO: 984	tagagctggaactgaggcc	3131	3131	SEQ ID NO: 1987
SEQ ID NO: 985	ctatgagctccagagagag	3167	3186	SEQ ID NO: 1988
SEQ ID NO: 986	cttggtggataccctgaag	3194	3213	SEQ ID NO: 1989
SEQ ID NO: 987	ttgtaactcaagcagaagg	3214	3233	SEQ ID NO: 1990
SEQ ID NO: 988	taactcaagcagaaggtgc	3217	3236	SEQ ID NO: 1991
SEQ ID NO: 989	gcagaaggtgcgaagcaga	3225	3244	SEQ ID NO: 1992
SEQ ID NO: 990	cagaaggtgcgaagcagac	3226	3245	SEQ ID NO: 1993
SEQ ID NO: 991	gtatgaccttgtccagtga	3280	3299	SEQ ID NO: 1994
SEQ ID NO: 992	tatgaccttgtccagtgaa	3281	3300	SEQ ID NO: 1995
SEQ ID NO: 993	gaagtccaaattccggatt	3297	3316	SEQ ID NO: 1996
SEQ ID NO: 994	gagggcaaaacgtcttaca	3363	3382	SEQ ID NO: 1997
SEQ ID NO: 995	agggcaaaacgtcttacag	3364	3383	SEQ ID NO: 1998
SEQ ID NO: 996	gactcaccctggacattca	3382	3401	SEQ ID NO: 1999
SEQ ID NO: 997	ctggacattcagaacaaga	3390	3409	SEQ ID NO: 2000
SEQ ID NO: 998	tcatgggcgacctaagttg	3427	3446	SEQ ID NO: 2001
SEQ ID NO: 999	tgggcgacctaagttgtga	3430	3449	SEQ ID NO: 2002
SEQ ID NO: 1000	agttgtgacacaaaggaag	3441	3460	SEQ ID NO: 2003
SEQ ID NO: 1001	tgacacaaaggaagaaaga	3446	3465	SEQ ID NO: 2004
SEQ ID NO: 1002	gacacaaaggaagaaagaa	3447	3466	SEQ ID NO: 2005
SEQ ID NO: 1003	ggaagaaagaaaaatcaag	3455	3474	SEQ ID NO: 2006
SEQ ID NO: 2007	aaaaagcgatggccgggtc	3947	3966	SEQ ID NO: 2313
SEQ ID NO: 2008	gtcaaatataccttgaaca	3963	3982	SEQ ID NO: 2314
SEQ ID NO: 2009	tgaacaagaacagtttgaa	6976	3995	SEQ ID NO: 2315
SEQ ID NO: 2010	agtttgaaaattgagattc	3987	4006	SEQ ID NO: 2316
SEQ ID NO: 2011	gtttgaaaattgagattcc	3988	4007	SEQ ID NO: 2317
SEQ ID NO: 2012	ttgaaaattgagattcctt	3990	4009	SEQ ID NO: 2318
SEQ ID NO: 2013	ctaaagatgttagagactg	4038	4057	SEQ ID NO: 2319
SEQ ID NO: 2014	atgttagagactgttagga	4044	4063	SEQ ID NO: 2320
SEQ ID NO: 2015	cagccctccacttcaagtc	4066	4085	SEQ ID NO: 2321
SEQ ID NO: 2016	agccctccacttcaagtct	4067	4086	SEQ ID NO: 2322
SEQ ID NO: 2017	ccatctgccatctcgagat	4094	4113	SEQ ID NO: 2323
SEQ ID NO: 2018	attcccaagttgtatcaac	4134	4153	SEQ ID NO: 2324
SEQ ID NO: 2019	tcaactgcaagtgcctctc	4148	4167	SEQ ID NO: 2325
SEQ ID NO: 2020	ggtgttctagacctctcca	4170	4189	SEQ ID NO: 2326
SEQ ID NO: 2021	ctccacgaatgtctacagc	4184	4203	SEQ ID NO: 2327
SEQ ID NO: 2022	cacgaatgtctacagcaac	4187	4206	SEQ ID NO: 2328
SEQ ID NO: 2023	acgaatgtctacagcaact	4188	4207	SEQ ID NO: 2329
SEQ ID NO: 2024	tcctacagtggtggcaaca	4224	4243	SEQ ID NO: 2330
SEQ ID NO: 2025	cgttaccacatgaaggctg	4272	4291	SEQ ID NO: 2331
SEQ ID NO: 2026	gaaggctgactctgtggtt	4283	4302	SEQ ID NO: 2332
SEQ ID NO: 2027	tgtggttgacctgctttcc	4295	4314	SEQ ID NO: 2333
SEQ ID NO: 2028	cctgctttcctacaatgtg	4304	4323	SEQ ID NO: 2334
SEQ ID NO: 2029	ctgctttcctacaatgtgc	4305	4324	SEQ ID NO: 2335
SEQ ID NO: 2030	tcctacaatgtgcaaggat	4311	4330	SEQ ID NO: 2336
SEQ ID NO: 2031	tatgaccacaagaatacgt	4344	4363	SEQ ID NO: 2337
SEQ ID NO: 2032	atgaccacaagaatacgtc	4345	4364	SEQ ID NO: 2338
SEQ ID NO: 2033	gaatacgtctacactatca	4355	4374	SEQ ID NO: 2339
SEQ ID NO: 2034	tttctagattcgaatatca	4398	4417	SEQ ID NO: 2340
SEQ ID NO: 2035	gattcgaaatacaaattca	4404	4423	SEQ ID NO: 2341
SEQ ID NO: 2036	gaaacaacccagtctcaaa	4441	4460	SEQ ID NO: 2342
SEQ ID NO: 2037	cccagtctcaaaaggttta	4448	4467	SEQ ID NO: 2343
SEQ ID NO: 2038	ctcaaaaggtttactaata	4454	4473	SEQ ID NO: 2344
SEQ ID NO: 2039	tcaaaaggtttactaatat	4455	4474	SEQ ID NO: 2345
SEQ ID NO: 2040	aaaaggtttactaatattc	4457	4476	SEQ ID NO: 2346
SEQ ID NO: 2041	gaaacagcatttgtttgtc	4535	4554	SEQ ID NO: 2347
SEQ ID NO: 2042	atttgtttgtcaaagaagt	4543	4562	SEQ ID NO: 2348
SEQ ID NO: 2043	tcaagattgatgggcagtt	4561	4580	SEQ ID NO: 2349
SEQ ID NO: 2044	ttcagagtctcttcgttct	4578	4597	SEQ ID NO: 2350
SEQ ID NO: 2045	cagagtctcttcgttctat	4580	4599	SEQ ID NO: 2351
SEQ ID NO: 2046	atgctaaaggcacatatgg	4597	4616	SEQ ID NO: 2352
SEQ ID NO: 2047	gcacatatggcctgtcttg	4606	4625	SEQ ID NO: 2353
SEQ ID NO: 2048	gagtccaacctgaggttta	4659	4678	SEQ ID NO: 2354
SEQ ID NO: 2049	agtccaacctgaggtttaa	4660	4679	SEQ ID NO: 2355
SEQ ID NO: 2050	cctacctccaaggcaccaa	4684	4703	SEQ ID NO: 2356
SEQ ID NO: 2051	gaagatggaaccctctccc	4722	4741	SEQ ID NO: 2357
SEQ ID NO: 2052	tgatctgcaaagtggcatc	4754	4773	SEQ ID NO: 2358
SEQ ID NO: 2053	gatctgcaaagtggcatca	4755	4774	SEQ ID NO: 2359
SEQ ID NO: 2054	gcttccctaaagtatgaga	4785	4804	SEQ ID NO: 2360
SEQ ID NO: 2055	gtatgagaactacgagctg	4796	4815	SEQ ID NO: 2361
SEQ ID NO: 2056	tctaacaagatggatatga	4860	4879	SEQ ID NO: 2362
SEQ ID NO: 2057	ctgctgcgttctgaatatc	4899	4918	SEQ ID NO: 2363
SEQ ID NO: 2058	tcattgaggttcttcagcc	4932	4951	SEQ ID NO: 2364
SEQ ID NO: 2059	ttctggatcactaaattcc	4955	4974	SEQ ID NO: 2365
SEQ ID NO: 2060	ccatggtcttgagttaaat	4973	4992	SEQ ID NO: 2366
SEQ ID NO: 2061	tcttaggcactgacaaaat	4999	5018	SEQ ID NO: 2367
SEQ ID NO: 2062	acaaggcgacactaaggat	5032	5051	SEQ ID NO: 2368
SEQ ID NO: 2063	tgcaacgaccaacttgaag	5075	5094	SEQ ID NO: 2369
SEQ ID NO: 2064	caacttgaagtgtagtctc	5084	5103	SEQ ID NO: 2370
SEQ ID NO: 2065	gctggagaatgagctgaat	5108	5127	SEQ ID NO: 2371
SEQ ID NO: 2066	gcagagcttggcctctctg	5127	5146	SEQ ID NO: 2372
SEQ ID NO: 2067	tctctggggcatctatgaa	5140	5159	SEQ ID NO: 2373
SEQ ID NO: 2068	tctggggcatctatgaaat	5142	5161	SEQ ID NO: 2374
SEQ ID NO: 2069	aacacaatgcaaaattcag	5185	5204	SEQ ID NO: 2375
SEQ ID NO: 2070	ctcacagagctatcactgg	5223	5242	SEQ ID NO: 2376
SEQ ID NO: 2071	tgggaagtgcttatcaggc	5239	5258	SEQ ID NO: 2377
SEQ ID NO: 2072	ttcaaggtcagtcaagaag	5295	5314	SEQ ID NO: 2378
SEQ ID NO: 2073	aatgacatgatgggctcat	5328	5347	SEQ ID NO: 2379
SEQ ID NO: 2074	gctcatatgctgaaatgaa	5341	5360	SEQ ID NO: 2380
SEQ ID NO: 2075	atatgctgaaatgaaattt	5345	5364	SEQ ID NO: 2381
SEQ ID NO: 2076	tctgaacattgcaggctta	5378	5397	SEQ ID NO: 2382
SEQ ID NO: 2077	gaacattgcaggcttatca	5381	5400	SEQ ID NO: 2383
SEQ ID NO: 2078	tgcaggcttatcactggac	5387	5406	SEQ ID NO: 2384
SEQ ID NO: 2079	tcaaacttgacaaacattt	5412	5431	SEQ ID NO: 2385
SEQ ID NO: 2080	atttacagctctgacaagt	5427	5446	SEQ ID NO: 2386
SEQ ID NO: 2081	ctctgacaagttttattag	5438	5454	SEQ ID NO: 2387
SEQ ID NO: 2082	gttaatttacagctacagc	5460	5479	SEQ ID NO: 2388
SEQ ID NO: 2083	ttctctggtaactacttta	5483	5502	SEQ ID NO: 2389
SEQ ID NO: 2084	cctaaaaggagcctaccaa	5586	5607	SEQ ID NO: 2390
SEQ ID NO: 2085	aaaaggagcctaccaaaat	5591	5610	SEQ ID NO: 2391
SEQ ID NO: 2086	aggagcctaccaaaataat	5594	5613	SEQ ID NO: 2392
SEQ ID NO: 2087	ataatgaaataaaacacat	5608	5627	SEQ ID NO: 2393
SEQ ID NO: 2088	aaaacacatctatgccatc	5618	5637	SEQ ID NO: 2394
SEQ ID NO: 2089	tgctaaggttcagggtgtg	5678	5697	SEQ ID NO: 2395
SEQ ID NO: 2090	gagtttagccatcggctca	5697	5716	SEQ ID NO: 2396
SEQ ID NO: 2091	gctggcttcagccattgac	5732	5751	SEQ ID NO: 2397
SEQ ID NO: 2092	atttcagcaatgtcttccg	5782	5801	SEQ ID NO: 2398
SEQ ID NO: 2093	tttcagcaatgtcttccgt	5783	5802	SEQ ID NO: 2399
SEQ ID NO: 2094	ttcagcaatgtcttccgtt	5784	5803	SEQ ID NO: 2400
SEQ ID NO: 2095	cagcaatgtcttccgttct	5786	5805	SEQ ID NO: 2401
SEQ ID NO: 2096	tgtcttccgttctgtaatg	5792	5811	SEQ ID NO: 2402
SEQ ID NO: 2097	gtcttccgttctgtaatgg	5793	5812	SEQ ID NO: 2403
SEQ ID NO: 2098	atgggaaactcgctctgtg	5851	5870	SEQ ID NO: 2404
SEQ ID NO: 2099	ggagaacatactgggcagc	5871	5890	SEQ ID NO: 2405
SEQ ID NO: 2100	gttgaaagcagaacctctg	5906	5925	SEQ ID NO: 2406
SEQ ID NO: 2101	gtctaggaaaagcatcagt	5975	5994	SEQ ID NO: 2407
SEQ ID NO: 2102	agcatcagtgcagctcttg	5985	6004	SEQ ID NO: 2408
SEQ ID NO: 2103	ttgaacacaaagtcagtgc	6001	6020	SEQ ID NO: 2409
SEQ ID NO: 2104	gcagacaggcacctggaaa	6038	6057	SEQ ID NO: 2410
SEQ ID NO: 2105	gaaactcaagacccaattt	6053	6072	SEQ ID NO: 2411
SEQ ID NO: 2106	acaatgaatacagccagga	6076	6095	SEQ ID NO: 2412
SEQ ID NO: 2107	cttggatgcttacaacact	6095	6114	SEQ ID NO: 2413
SEQ ID NO: 2108	ttggcgtggagcttactgg	6124	6143	SEQ ID NO: 2414
SEQ ID NO: 2109	cacttttactcagtgagcc	6190	6209	SEQ ID NO: 2415
SEQ ID NO: 2110	tttagagatgagagatgcc	6227	6246	SEQ ID NO: 2416
SEQ ID NO: 2111	gagaagccccaagaattta	6249	6268	SEQ ID NO: 2417
SEQ ID NO: 2112	caattgttgcttttgtaaa	6268	6287	SEQ ID NO: 2418
SEQ ID NO: 2113	ttttgtaaagtatgataaa	6278	6297	SEQ ID NO: 2419
SEQ ID NO: 2114	ttgtaaagtatgataaaas	6280	6299	SEQ ID NO: 2420
SEQ ID NO: 2115	ttcactccattaacctccc	6307	6326	SEQ ID NO: 2421
SEQ ID NO: 2116	ttttgagaccttgcaagaa	6329	6348	SEQ ID NO: 2422
SEQ ID NO: 2117	accttgcaagaatattttg	6336	6355	SEQ ID NO: 2423
SEQ ID NO: 2118	tcaatattgatcaatttgt	6415	6434	SEQ ID NO: 2424
SEQ ID NO: 2119	cagagcagccctgggaaaa	6443	6462	SEQ ID NO: 2425
SEQ ID NO: 2120	cctgggaaaactcccacag	6452	6471	SEQ ID NO: 2426
SEQ ID NO: 2121	actcccacagcaagctaat	6461	6480	SEQ ID NO: 2427
SEQ ID NO: 2122	aattcattcaattgggaga	6486	6508	SEQ ID NO: 2428
SEQ ID NO: 2123	ttcaattgggagagacaag	6495	6514	SEQ ID NO: 2429
SEQ ID NO: 2124	aggagaaactgactgctct	6526	6545	SEQ ID NO: 2430
SEQ ID NO: 2125	actgactgctctcacaaaa	6533	6552	SEQ ID NO: 2431
SEQ ID NO: 2126	gactgctctcacaaaaaag	6536	6555	SEQ ID NO: 2432
SEQ ID NO: 2127	cagacatatatgatacaat	6633	6652	SEQ ID NO: 2433
SEQ ID NO: 2128	aatttgatcagtatattaa	6649	6668	SEQ ID NO: 2434
SEQ ID NO: 2129	tatgatttacatgatttga	6675	6694	SEQ ID NO: 2435
SEQ ID NO: 2130	tttgaaaatagctattgct	6689	6708	SEQ ID NO: 2436
SEQ ID NO: 2131	ttgaaaatagctattgcta	6690	6709	SEQ ID NO: 2437
SEQ ID NO: 2132	aatagctattgctaatatt	6695	6714	SEQ ID NO: 2438
SEQ ID NO: 2133	attattgatgaaatcattg	6711	6730	SEQ ID NO: 2439
SEQ ID NO: 2134	aaagtcttgatgagcacta	6739	6758	SEQ ID NO: 2440
SEQ ID NO: 2135	aagtcttgatgagcactat	6740	6759	SEQ ID NO: 2441
SEQ ID NO: 2136	ttgatgagcactatcatat	6745	6764	SEQ ID NO: 2442
SEQ ID NO: 2137	taattttagtaaaaacaat	6769	6788	SEQ ID NO: 2443
SEQ ID NO: 2138	ttttagtaaaaacaatcca	6772	6791	SEQ ID NO: 2444
SEQ ID NO: 2139	acatttgtttattgaaaat	6797	6816	SEQ ID NO: 2445
SEQ ID NO: 2140	attgattttaacaaaagtg	6816	3835	SEQ ID NO: 2446
SEQ ID NO: 2141	attttaacaaaagtggaag	6820	3839	SEQ ID NO: 2447
SEQ ID NO: 2142	aaatcagaatccagataca	6880	6899	SEQ ID NO: 2448
SEQ ID NO: 2143	gaatccagatacaagaaaa	6886	6905	SEQ ID NO: 2449
SEQ ID NO: 2144	ttaagagacacatacagaa	6916	6935	SEQ ID NO: 2450
SEQ ID NO: 2145	atccagcacctagctggaa	6942	6961	SEQ ID NO: 2451
SEQ ID NO: 2146	tgagcatgtcaaacacttt	7052	7071	SEQ ID NO: 2452
SEQ ID NO: 2147	gagcatgtcaaacactttg	7053	7072	SEQ ID NO: 2453
SEQ ID NO: 2148	aaacactttgttataaatc	7062	7061	SEQ ID NO: 2454
SEQ ID NO: 2149	tgagaaaatcaatgccttc	7103	7122	SEQ ID NO: 2455
SEQ ID NO: 2150	tatgaagtagaccaacaaa	7152	7171	SEQ ID NO: 2456
SEQ ID NO: 2151	aagtagaccaacaaatcca	7156	7175	SEQ ID NO: 2457
SEQ ID NO: 2152	aagttgaaggagactattc	7215	7234	SEQ ID NO: 2458
SEQ ID NO: 2153	acaagttaagataaaagat	7256	7275	SEQ ID NO: 2459
SEQ ID NO: 2154	aagataaaagattactttg	7263	7282	SEQ ID NO: 2460
SEQ ID NO: 5255	gattactttgagaaattag	7272	7291	SEQ ID NO: 2461
SEQ ID NO: 2156	tgagaaattagttggattt	7280	7299	SEQ ID NO: 2462
SEQ ID NO: 2157	aaattagttggatttattg	7284	7303	SEQ ID NO: 2463
SEQ ID NO: 5158	tggatttattgatgatgct	7292	7311	SEQ ID NO: 2464
SEQ ID NO: 2159	tcattgaagatgttaacaa	7345	7364	SEQ ID NO: 2465
SEQ ID NO: 2160	cattgaagatgttaacaaa	7346	7365	SEQ ID NO: 2466
SEQ ID NO: 2161	attgaagatgttaacaaat	7347	7366	SEQ ID NO: 2467
SEQ ID NO: 2162	ttgaagatgttaacaaatt	7348	7367	SEQ ID NO: 2468
SEQ ID NO: 2163	tgaagatgttaacaaattc	7349	7368	SEQ ID NO: 2469
SEQ ID NO: 2164	acatgttgataaagaaatt	7372	7391	SEQ ID NO: 2470
SEQ ID NO: 2165	tttgattaccaccagtttg	7398	7417	SEQ ID NO: 2471
SEQ ID NO: 2166	caaaatccgtgaggtgact	7433	7452	SEQ ID NO: 2472
SEQ ID NO: 2167	aaaatccgtgaggtgactc	7434	7453	SEQ ID NO: 2473
SEQ ID NO: 2168	aggtgactcagagactcaa	7444	7463	SEQ ID NO: 2474
SEQ ID NO: 2169	gtgaaattcaggctctgga	7465	7484	SEQ ID NO: 2475
SEQ ID NO: 2170	gttgcagtgtatctggaaa	7539	7558	SEQ ID NO: 2476
SEQ ID NO: 2171	ttaagttcagcatctttgg	7608	7627	SEQ ID NO: 2477
SEQ ID NO: 2172	tgaaggccaaattccgaga	7633	7652	SEQ ID NO: 2478
SEQ ID NO: 2173	aatgtatcaaatggacatt	7676	7695	SEQ ID NO: 2479
SEQ ID NO: 2174	attcagcaggaacttcaac	7692	7711	SEQ ID NO: 2480
SEQ ID NO: 2175	acctgtctctggtcagcca	7714	7733	SEQ ID NO: 2481
SEQ ID NO: 2176	cctgtctctggtcagccag	7715	7734	SEQ ID NO: 2482
SEQ ID NO: 2177	ggtcagccaggtttatagc	7724	7743	SEQ ID NO: 2483
SEQ ID NO: 2178	ccaggtttatagcacactt	7730	7749	SEQ ID NO: 2484
SEQ ID NO: 2179	gtttatagcacacttgtca	7734	7753	SEQ ID NO: 2485
SEQ ID NO: 2180	acttgtcacctacatttct	7745	7764	SEQ ID NO: 2486
SEQ ID NO: 2181	ctgattggtggactcttgc	7762	7781	SEQ ID NO: 2487
SEQ ID NO: 2182	atgaaagcattggtagagc	7839	7858	SEQ ID NO: 2488
SEQ ID NO: 2183	tgaaagcattggtagagca	7840	7859	SEQ ID NO: 2489
SEQ ID NO: 2184	gggttcactgttcctgaaa	7860	7879	SEQ ID NO: 2490
SEQ ID NO: 2185	tcaagaccatccttgggac	7879	7898	SEQ ID NO: 2491
SEQ ID NO: 2186	ccttgggaccatgcctgcc	7889	7908	SEQ ID NO: 2492
SEQ ID NO: 2187	ttcaggctcttcagaaagc	7921	7940	SEQ ID NO: 2493
SEQ ID NO: 2188	ttcagataaacttcaaaga	7996	8015	SEQ ID NO: 2494
SEQ ID NO: 2189	acttcaaagacttaaaaaa	8005	8024	SEQ ID NO: 2495
SEQ ID NO: 2190	atcccatccaggttttcca	8031	8050	SEQ ID NO: 2496
SEQ ID NO: 2191	gaatttaccatccttaaca	8055	8074	SEQ ID NO: 2497
SEQ ID NO: 2192	cattccttcctttacaatt	8081	8100	SEQ ID NO: 2498
SEQ ID NO: 2193	ttgaccagatgctgaacag	8137	8156	SEQ ID NO: 2499
SEQ ID NO: 2194	aatcaccctgccagacttc	8225	8244	SEQ ID NO: 2500
SEQ ID NO: 2195	tgaccttcacataccagaa	8312	8331	SEQ ID NO: 2501
SEQ ID NO: 2196	ttccagcttcaccacatct	8331	8350	SEQ ID NO: 2502
SEQ ID NO: 2197	aagctatacagtattctga	8379	8398	SEQ ID NO: 2503
SEQ ID NO: 2198	attctgaaaatccaatctc	8391	8410	SEQ ID NO: 2504
SEQ ID NO: 2199	tttcacattagatgcaaat	8414	8433	SEQ ID NO: 2505
SEQ ID NO: 2200	caaatgctgacatagggaa	8428	8447	SEQ ID NO: 2506
SEQ ID NO: 2201	gagagtccaaattagaagt	8500	8519	SEQ ID NO: 2507
SEQ ID NO: 2202	agagtccaaattagaagtt	8501	8520	SEQ ID NO: 2508
SEQ ID NO: 2203	tctcaattttgattttcaa	8519	8538	SEQ ID NO: 2509
SEQ ID NO: 2204	caattttgattttcaagca	8522	8541	SEQ ID NO: 2510
SEQ ID NO: 2205	aatgcacaactctcaaacc	8541	8560	SEQ ID NO: 2511
SEQ ID NO: 2206	agttctccagcaagtacct	8596	8615	SEQ ID NO: 2512
SEQ ID NO: 2207	agtacctgagaacggagca	8608	8627	SEQ ID NO: 2513
SEQ ID NO: 2208	tcaaacacagtggcaagtt	8670	8689	SEQ ID NO: 2514
SEQ ID NO: 2209	acaatcagcttaccctgga	8743	8762	SEQ ID NO: 2515
SEQ ID NO: 2210	ctggatagcaacactaaat	8757	8776	SEQ ID NO: 2516
SEQ ID NO: 2211	ctgacctgcgcaacgagat	8821	8840	SEQ ID NO: 2517
SEQ ID NO: 2212	agatgagggaacacatgaa	8921	8940	SEQ ID NO: 2518
SEQ ID NO: 2213	tcaacttttctaaacttga	9052	9071	SEQ ID NO: 2519
SEQ ID NO: 2214	ttctaaacttgaaattcaa	9059	9078	SEQ ID NO: 2520
SEQ ID NO: 2215	gaaattcaatcacaagtcg	9069	9088	SEQ ID NO: 2521
SEQ ID NO: 2216	cactgtttggagaagggaa	9133	9152	SEQ ID NO: 2522
SEQ ID NO: 2217	actgtttggagaagggaag	9134	9153	SEQ ID NO: 2523
SEQ ID NO: 2218	aattctcttttcttttcag	9213	9232	SEQ ID NO: 2524
SEQ ID NO: 2219	ttcttttcagcccagccat	9222	9241	SEQ ID NO: 2525
SEQ ID NO: 2220	tttgaaagttcgttttcca	9275	9294	SEQ ID NO: 2526
SEQ ID NO: 2221	cagggaagatagacttcct	9304	9323	SEQ ID NO: 2527
SEQ ID NO: 2222	ataagtacaaccaaaattt	9397	9416	SEQ ID NO: 2528
SEQ ID NO: 2223	acaacgagaacattatgga	9427	9446	SEQ ID NO: 2529
SEQ ID NO: 2224	aggaataaatggagaagca	9455	9474	SEQ ID NO: 2530
SEQ ID NO: 2225	agcaaatctggatttctta	9470	9489	SEQ ID NO: 2531
SEQ ID NO: 2226	tcctttaacaattggtgaa	9494	9513	SEQ ID NO: 2532
SEQ ID NO: 2227	tttaacaattcctgaaatg	9497	9516	SEQ ID NO: 2533
SEQ ID NO: 2228	acacaataatcacaactcc	9526	9545	SEQ ID NO: 2534
SEQ ID NO: 2229	aagatttctctctatggga	9553	9572	SEQ ID NO: 2535
SEQ ID NO: 2230	gaaaaaacaggcttgaagg	9570	9589	SEQ ID NO: 2536
SEQ ID NO: 2231	ttgaaggaattcttgaaaa	9582	9601	SEQ ID NO: 2537
SEQ ID NO: 2232	tgaaggaattcttgaaaac	9583	9602	SEQ ID NO: 2538
SEQ ID NO: 2233	agctcagtataagaaaaac	9632	9651	SEQ ID NO: 2539
SEQ ID NO: 2234	tcaaatcctttgacaggca	9712	9731	SEQ ID NO: 2540
SEQ ID NO: 2235	atgaaacaaaaattaagtt	9781	9800	SEQ ID NO: 2541
SEQ ID NO: 2236	aattcctggatacactgtt	9851	9870	SEQ ID NO: 2542
SEQ ID NO: 2237	ttccagttgtcaatgttga	9868	9887	SEQ ID NO: 2543
SEQ ID NO: 2238	aagtgtctccattcaccat	9886	9905	SEQ ID NO: 2544
SEQ ID NO: 2239	gtcagcatgcctagtttct	9942	9961	SEQ ID NO: 2545
SEQ ID NO: 2240	ctgccatgggcaatattac	10105	10124	SEQ ID NO: 2546
SEQ ID NO: 2241	tgaataccaatgctgaact	10159	10178	SEQ ID NO: 2547
SEQ ID NO: 2242	tattgttgctcatctcctt	10193	10212	SEQ ID NO: 2548
SEQ ID NO: 2243	tgttgctcatctcctttct	10196	10215	SEQ ID NO: 2549
SEQ ID NO: 2244	tctgtcattgatgcactgc	10224	10243	SEQ ID NO: 2550
SEQ ID NO: 2245	ccacagctctgtctctgag	10297	10316	SEQ ID NO: 2551
SEQ ID NO: 2246	atttgtggagggtagtcat	10322	10341	SEQ ID NO: 2552
SEQ ID NO: 2247	atatggaagtgtcagtggc	10369	10388	SEQ ID NO: 2553
SEQ ID NO: 2248	tggaaataccaagtcaaaa	10445	10464	SEQ ID NO: 2554
SEQ ID NO: 2249	aagtcaaaacctactgtct	10455	10474	SEQ ID NO: 2555
SEQ ID NO: 2250	actgtctcttcctccatgg	10467	10486	SEQ ID NO: 2556
SEQ ID NO: 2251	cttcctccatggaatttaa	10474	10493	SEQ ID NO: 2557
SEQ ID NO: 2252	attcttcaatgctgtactc	10504	10523	SEQ ID NO: 2558
SEQ ID NO: 2253	ttgaccacaagcttagctt	10540	10559	SEQ ID NO: 2559
SEQ ID NO: 2254	cctcacctcttacttttcc	10565	10584	SEQ ID NO: 2560
SEQ ID NO: 2255	agctgcagggcacttccaa	10702	10721	SEQ ID NO: 2561
SEQ ID NO: 2256	ttccaaaattgatgatatc	10715	10734	SEQ ID NO: 2562
SEQ ID NO: 2257	gagaacatacaagcaaagc	10852	10871	SEQ ID NO: 2563
SEQ ID NO: 2258	atggcaaatgtcagctctt	10889	10908	SEQ ID NO: 2564
SEQ ID NO: 2259	tggcaaatgtcagctcttg	10890	10909	SEQ ID NO: 2565
SEQ ID NO: 2260	ttgttcaggtccatgcaag	10906	10925	SEQ ID NO: 2566
SEQ ID NO: 2261	tgttcaggtccatgcaagt	10907	10926	SEQ ID NO: 2567
SEQ ID NO: 2262	agttccttccatgatttcc	10932	10951	SEQ ID NO: 2568
SEQ ID NO: 2263	tgctaacactaagaaccag	10979	10998	SEQ ID NO: 2569
SEQ ID NO: 2264	actaagaaccagaagatca	10986	11005	SEQ ID NO: 2570
SEQ ID NO: 2265	ctaagaaccagaagatcag	10987	11006	SEQ ID NO: 2571
SEQ ID NO: 2266	cagaagatcagatggaaaa	10995	11014	SEQ ID NO: 2572
SEQ ID NO: 2267	aaaaatgaagtccggattc	11010	11029	SEQ ID NO: 2573
SEQ ID NO: 2268	gattcattctgggtctttc	11024	11043	SEQ ID NO: 2574
SEQ ID NO: 2269	aagaaaaggcacaccttga	11071	11090	SEQ ID NO: 2575
SEQ ID NO: 2270	aaggacacctaaggttcct	11107	11126	SEQ ID NO: 2576
SEQ ID NO: 2271	ccagcattggtaggagaca	11191	11210	SEQ ID NO: 2577
SEQ ID NO: 2272	ctttgtgtacaccaaaaac	11231	11250	SEQ ID NO: 2578
SEQ ID NO: 2273	ccatccctgtaaaagtttt	11269	11288	SEQ ID NO: 2579
SEQ ID NO: 2274	tgatctaaattcagttctt	11324	11343	SEQ ID NO: 2580
SEQ ID NO: 2275	aagaagctgagaacttcat	11424	11443	SEQ ID NO: 2581
SEQ ID NO: 2276	tttgccctcaacctaccaa	11445	11464	SEQ ID NO: 2582
SEQ ID NO: 2277	cttgattcccttttttgag	11528	11547	SEQ ID NO: 2583
SEQ ID NO: 2278	ttcacgcttccaaaaagtg	11583	11602	SEQ ID NO: 2584
SEQ ID NO: 2279	tgtttcagatggcattgct	11600	11619	SEQ ID NO: 2585
SEQ ID NO: 2280	aatgcagtagccaacaaga	11631	11650	SEQ ID NO: 2586
SEQ ID NO: 2281	ctgagcagaccattgagat	11683	11702	SEQ ID NO: 2587
SEQ ID NO: 2282	tgagcagaccattgagatt	11684	11703	SEQ ID NO: 2588
SEQ ID NO: 2283	ttgagattccctccattaa	11695	11714	SEQ ID NO: 2589
SEQ ID NO: 2284	acttggagtgccagtttga	11799	11818	SEQ ID NO: 2590
SEQ ID NO: 2285	caaatttgaaggacttcag	11996	12015	SEQ ID NO: 2591
SEQ ID NO: 2286	agcccagcgttcaccgatc	12048	12067	SEQ ID NO: 2592
SEQ ID NO: 2287	cagcgttcaccgatctcca	12052	12071	SEQ ID NO: 2593
SEQ ID NO: 2288	ctccatctgcgctaccaga	12066	12085	SEQ ID NO: 2594
SEQ ID NO: 2289	atgaggaaactcagatcaa	12256	12275	SEQ ID NO: 2595
SEQ ID NO: 2290	aggcagcttctggcttgct	12292	12311	SEQ ID NO: 2596
SEQ ID NO: 2291	tgaaagacaacgtgcccaa	12319	12338	SEQ ID NO: 2597
SEQ ID NO: 2292	tatgattatgtcaacaagt	12354	12373	SEQ ID NO: 2598
SEQ ID NO: 2293	cattaggcaaattgatgat	12467	12486	SEQ ID NO: 2599
SEQ ID NO: 2294	ttgactcaggaaggccaag	12576	12595	SEQ ID NO: 2600
SEQ ID NO: 2295	gaaacctgggatatacact	12728	12747	SEQ ID NO: 2601
SEQ ID NO: 2296	tcctttcgagttaaggaaa	12869	12888	SEQ ID NO: 2602
SEQ ID NO: 2297	gccattcagtctctcaaga	12966	12985	SEQ ID NO: 2603
SEQ ID NO: 2298	gtgctacgtaatcttcagg	12993	13012	SEQ ID NO: 2604
SEQ ID NO: 2299	agctgaaagagatgaaatt	13057	13076	SEQ ID NO: 2605
SEQ ID NO: 2300	aatttacttatcttattaa	13072	13091	SEQ ID NO: 2606
SEQ ID NO: 2301	ttttaaattgttgaaagaa	13142	13161	SEQ ID NO: 2607
SEQ ID NO: 2302	taatcttcataagttcaat	13172	13191	SEQ ID NO: 2608
SEQ ID NO: 2303	atattttgatccaagtata	13271	13290	SEQ ID NO: 2609
SEQ ID NO: 2304	tgaaatattatgaacttga	13303	13322	SEQ ID NO: 2610
SEQ ID NO: 2305	caatttctgcacagaaata	13434	13453	SEQ ID NO: 2611
SEQ ID NO: 2306	agaagattgcagagctttc	13501	13520	SEQ ID NO: 2612
SEQ ID NO: 2307	gaagaaaataatttctgat	13562	13581	SEQ ID NO: 2613
SEQ ID NO: 2308	ttgacctgtccattcaaaa	13672	13691	SEQ ID NO: 2614
SEQ ID NO: 2309	tcaaaactaccacacattt	13685	13704	SEQ ID NO: 2615
SEQ ID NO: 2310	ttttttaaaagaaatcttc	13803	13822	SEQ ID NO: 2616
SEQ ID NO: 2311	aggatctgagttattttgc	14035	14054	SEQ ID NO: 2617
SEQ ID NO: 2312	tttgctaaacttgggggag	14049	14068	SEQ ID NO: 2618
		Start	End
	Match	Index	Index	#	B
SEQ ID NO: 1	cttccgttctgtaatggcc	5795	5814	1	9
SEQ ID NO: 2	tggaactctctccatggca	10876	10895	1	8
SEQ ID NO: 3	taacaaattccttgacatg	7358	7377	1	8
SEQ ID NO: 4	caataagatcaatagcaaa	8990	9009	1	8
SEQ ID NO: 5	tccatgtcccatttacaga	11356	11375	1	8
SEQ ID NO: 6	tggacctgcaccaaagctg	13952	13971	1	8
SEQ ID NO: 7	gcagccctgggaaaactcc	6447	6466	1	7
SEQ ID NO: 8	tggagggtagtcataacag	10327	10346	1	7
SEQ ID NO: 9	tgcagagctttctgccact	13508	13527	1	7
SEQ ID NO: 10	agattcctttgccttttgg	4000	4019	1	7
SEQ ID NO: 11	aaattctcttttcttttca	9212	9231	1	7
SEQ ID NO: 12	tgctagtgaggccaacact	10649	10668	1	7
SEQ ID NO: 13	tgatgatatctggaacctt	10724	10743	1	7
SEQ ID NO: 14	tgctaagaaccttactgac	7781	7800	1	7
SEQ ID NO: 15	ttcactgttcctgaaatca	7863	7882	1	7
SEQ ID NO: 16	agaaaaggcacaccttgac	11072	11091	1	7
SEQ ID NO: 17	ttccaatttccctgtggat	3680	3699	1	7
SEQ ID NO: 18	acttcagagaaatacaaat	11401	11420	4	6
SEQ ID NO: 19	ccagacttccgtttaccag	8235	8254	2	6
SEQ ID NO: 20	tgaagtgtagtctcctggt	5089	5108	2	6
SEQ ID NO: 21	attccatcacaaatccttt	9661	9680	2	6
SEQ ID NO: 22	tggatctaaatgcagtagc	11623	11642	2	6
SEQ ID NO: 23	caaagaagtcaagattgat	4553	4572	2	6
SEQ ID NO: 24	atgtgttaacaaaatattc	11494	11513	2	6
SEQ ID NO: 25	gccagaagtgagatcctcg	3507	3526	1	6
SEQ ID NO: 26	ctctgagcaacaaatttgt	10309	10328	1	6
SEQ ID NO: 27	ctccatggcaaatgtcagc	10885	10904	1	6
SEQ ID NO: 28	gagtcattgaggttcttca	4929	4948	1	6
SEQ ID NO: 29	tgttcataagggaggtagg	12766	12785	1	6
SEQ ID NO: 30	atgttcataagggaggtag	12765	12784	1	6
SEQ ID NO: 31	gcttggttttgccagtctc	2459	2478	1	6
SEQ ID NO: 32	cttgaacacaaagtcagtg	6000	6019	1	6
SEQ ID NO: 33	actgggaagtgcttatcag	5237	5256	1	6
SEQ ID NO: 34	cttccccaaagagaccagt	2890	2909	1	6
SEQ ID NO: 35	tctggcatttactttctct	5921	5940	1	6
SEQ ID NO: 36	agttgaaggagactattca	7216	7235	1	6
SEQ ID NO: 37	ctggttactgagctgagag	1161	1180	1	6
SEQ ID NO: 38	agagctgccagtccttcat	10016	11035	1	6
SEQ ID NO: 39	cctcctacagtggtggcaa	4222	4241	1	6
SEQ ID NO: 40	ctggattccacatgcagct	11847	11866	1	6
SEQ ID NO: 41	ggaggctttaagttcagca	7601	7620	1	6
SEQ ID NO: 42	ttgggagagacaagtttca	6500	6519	1	6
SEQ ID NO: 43	tcagaagctaagcaatgtc	7232	7251	1	6
SEQ ID NO: 44	aggagagtccaaattagaa	8498	8517	1	6
SEQ ID NO: 45	tctgaattcattcaattgg	6485	6504	1	6
SEQ ID NO: 46	aactaccctcactgccttt	2132	2151	1	6
SEQ ID NO: 47	gaacctctggcatttactt	5916	5935	1	6
SEQ ID NO: 48	attctctggtaactacttt	5482	5501	1	6
SEQ ID NO: 49	catcttaggcactgacaaa	4997	5016	1	6
SEQ ID NO: 50	tttagccatcggctcaaca	5700	5719	1	6
SEQ ID NO: 51	attcctttaacaattcctg	9492	9511	1	6
SEQ ID NO: 52	cattcctttaacaattcct	9491	9510	1	6
SEQ ID NO: 53	gtcagtcttcaggctcttc	7914	7933	1	6
SEQ ID NO: 54	agaaggatggcattttttg	14000	14019	1	6
SEQ ID NO: 55	ttcagagccaaagtccatg	7119	7138	1	6
SEQ ID NO: 56	ttactccaacgccagctcc	3050	3069	1	6
SEQ ID NO: 57	ctctctggggcatctatga	5139	5158	1	6
SEQ ID NO: 58	ctctcaagaccacagaggt	12976	12995	1	6
SEQ ID NO: 59	tctgaaagacaacgtgccc	12317	12336	1	6
SEQ ID NO: 60	tgttgctaaggttcagggt	5675	5694	1	6
SEQ ID NO: 61	cacaaattagtttcaccat	8941	8960	1	6
SEQ ID NO: 62	attccagcttccccacatc	8330	8349	1	6
SEQ ID NO: 63	ttttttggaaatgccattg	8643	8662	1	6
SEQ ID NO: 64	tcatgtgatgggtctctac	4371	4390	1	6
SEQ ID NO: 65	agtcaagaaggacttaagc	5304	5323	1	6
SEQ ID NO: 66	gacttcagagaaatacaaa	11400	11419	1	6
SEQ ID NO: 67	tgacttcagagaaatacaa	11399	11418	1	6
SEQ ID NO: 68	agcgagaatcaccctgcca	8219	8238	1	6
SEQ ID NO: 69	aaaggagatgtcaagggtt	10599	10618	1	6
SEQ ID NO: 70	tcatttgaaagaataaatg	7026	7045	1	6
SEQ ID NO: 71	taagaaccttactgacttt	7784	7803	1	6
SEQ ID NO: 72	aaggacttcaggaatggga	12004	12023	1	6
SEQ ID NO: 73	aaattaaaaagtcttgatg	6732	6751	1	6
SEQ ID NO: 74	taaaccaaaacttggttta	9019	9038	1	6
SEQ ID NO: 75	ttcaagacttaaaaaaata	8007	8026	1	6
SEQ ID NO: 76	ataaagaaattaaagtcat	7380	7399	1	6
SEQ ID NO: 77	atatattgtcagtgcctct	13382	13401	1	6
SEQ ID NO: 78	tctaaattcagttcttgtc	11327	11346	1	6
SEQ ID NO: 79	acaaagtcagtgccctgct	6007	6026	1	6
SEQ ID NO: 80	cctttgtgtacaccaaaaa	11230	11249	1	6
SEQ ID NO: 81	gcctttgtgtacaccaaaa	11229	11248	1	6
SEQ ID NO: 82	ttcagaaatactgttttcc	12824	12843	1	6
SEQ ID NO: 83	ctgggacctaccaagagtg	12523	12542	1	6
SEQ ID NO: 84	cacatttcaaggaattgtg	10063	10082	1	6
SEQ ID NO: 85	ggaactgttgactcaggaa	12569	12588	1	6
SEQ ID NO: 86	agagccaggtcgagctttc	11044	11063	1	6
SEQ ID NO: 87	acagctgaaagagatgaaa	13055	13074	1	6
SEQ ID NO: 88	ctgcacgctttgaggtaga	11761	11780	1	6
SEQ ID NO: 89	actgcacgctttgaggtag	11760	11779	1	6
SEQ ID NO: 90	ttggccaggaagtggccct	10957	10976	1	6
SEQ ID NO: 91	ctttttcaccaacggagaa	10838	10857	1	6
SEQ ID NO: 92	ataaactgcaagatttttc	13600	13619	1	6
SEQ ID NO: 93	agaaaatcaggatctgagt	14027	14046	1	6
SEQ ID NO: 94	gattaccaccagcagttta	13578	13597	1	6
SEQ ID NO: 95	cttcgtgaagaatattttg	13260	13279	1	6
SEQ ID NO: 96	aacacttacttgaattcca	10662	10681	3	5
SEQ ID NO: 97	cttcagagaaatacaaatc	11402	11421	3	5
SEQ ID NO: 98	acttcagagaaatacaaat	11401	11420	3	5
SEQ ID NO: 99	caaagaagtcaagattgat	4553	4572	2	5
SEQ ID NO: 100	ctggaaagttaaaacaaca	6955	6974	2	5
SEQ ID NO: 101	accaaagctggcaccaggg	13961	13980	2	5
SEQ ID NO: 102	aaagccattcagtctctca	12963	12982	2	5
SEQ ID NO: 103	acttttctaaacttgaaat	9055	9074	2	5
SEQ ID NO: 104	ttccggggaaacctgggat	12721	12740	2	5
SEQ ID NO: 105	agatggtacgttagcctct	11921	11940	2	5
SEQ ID NO: 106	tgggaactacaatttcatt	7012	7031	2	5
SEQ ID NO: 107	aatcttcaatttattcttc	13815	13834	2	5
SEQ ID NO: 108	cttcaggttccatcgtgca	11376	11395	2	5
SEQ ID NO: 109	caaaacctactgtctcttc	10459	10478	2	5
SEQ ID NO: 110	ccatatgaaagtcaagcat	12656	12675	2	5
SEQ ID NO: 111	cagattctcagatgaggga	8912	8931	2	5
SEQ ID NO: 112	acttttctaaacttgaaat	9055	9074	2	5
SEQ ID NO: 113	attatgttgaaacagtcct	11830	11849	2	5
SEQ ID NO: 114	attgttgctcatctccttt	10194	10213	2	5
SEQ ID NO: 115	ttctgattaccaccagcag	13574	13593	2	5
SEQ ID NO: 116	ttttaaaagaaatcttcaa	13805	13824	2	5
SEQ ID NO: 117	caaaacctactgtctcttc	10459	10478	2	5
SEQ ID NO: 118	aaatgtcagctcttgttca	10894	10913	2	5
SEQ ID NO: 119	gcaagtcagcccagttcct	10920	10939	2	5
SEQ ID NO: 120	cagccattgacatgagcac	5740	5759	1	5
SEQ ID NO: 121	cagctccacagactccgcc	3062	3081	1	5
SEQ ID NO: 122	agcagaaggtgcgaagcag	3224	3243	1	5
SEQ ID NO: 123	cctggattccacatgcagc	11846	11865	1	5
SEQ ID NO: 124	tttttcttcactacatctt	2584	2603	1	5
SEQ ID NO: 125	ccagacttccacatcccag	3915	3934	1	5
SEQ ID NO: 126	agcatgcctagtttctcca	9945	9964	1	5
SEQ ID NO: 127	cagcatgcctagtttctcc	9944	9963	1	5
SEQ ID NO: 128	tcttccatcacttgaccca	2042	2061	1	5
SEQ ID NO: 129	gcacaccttgacattgcag	11079	11098	1	5
SEQ ID NO: 130	gcaggctgaactggtggca	2717	2736	1	5
SEQ ID NO: 131	tgcaggctgaactggtggc	2716	2735	1	5
SEQ ID NO: 132	cctccacctctgatctgca	4744	4763	1	5
SEQ ID NO: 133	aacccctacatgaagcttg	13755	13774	1	5
SEQ ID NO: 134	tcaggaagcttctcaagag	13211	13230	1	5
SEQ ID NO: 135	ggtcttgagttaaatgctg	4977	4996	1	5
SEQ ID NO: 136	ctggacgctaagaggaagc	855	874	1	5
SEQ ID NO: 137	tggcatggcattatgatga	3604	3623	1	5
SEQ ID NO: 138	ctcaaccttaatgattttc	8286	8305	1	5
SEQ ID NO: 139	gcaagctatacagtattct	8377	8396	1	5
SEQ ID NO: 140	ctgcaggggatcccccaga	2526	2545	1	5
SEQ ID NO: 141	tggaagtgtcagtggcaaa	10372	10391	1	5
SEQ ID NO: 142	agaataaatgacgttcttg	7035	7054	1	5
SEQ ID NO: 143	cgtctacactatcatgtga	4360	4379	1	5
SEQ ID NO: 144	tccttgacatgttgataaa	7366	7385	1	5
SEQ ID NO: 145	ttccagaaagcagccagtg	12498	12517	1	5
SEQ ID NO: 146	cttcatacacattaatcct	9988	10007	1	5
SEQ ID NO: 147	gaagtagtactgcatcttg	6835	6854	1	5
SEQ ID NO: 148	ttcaattcttcaatgctgt	10500	10519	1	5
SEQ ID NO: 149	agatttgaggattccatca	7976	7995	1	5
SEQ ID NO: 150	caaggagaaactgactgct	6524	6543	1	5
SEQ ID NO: 151	tgtagtctcctggtgctgg	5094	5113	1	5
SEQ ID NO: 152	ctggagcttagtaatggag	8709	8728	1	5
SEQ ID NO: 153	tcagatgagggaacacatg	8919	8938	1	5
SEQ ID NO: 154	gccaccctggaactctctc	10869	10888	1	5
SEQ ID NO: 155	tgatcccacctctcattga	2965	2984	1	5
SEQ ID NO: 156	ctcagggatctgaaggtgg	8187	8206	1	5
SEQ ID NO: 157	tcttgagttaaatgctgac	4979	4998	1	5
SEQ ID NO: 158	aggtatattcgaaagtcca	12799	12818	1	5
SEQ ID NO: 159	caggtatattcgaaagtcc	12798	12817	1	5
SEQ ID NO: 160	tttcacatgccaaggagaa	6514	6533	1	5
SEQ ID NO: 161	ttgaagtgtagtctcctgg	5088	5107	1	5
SEQ ID NO: 162	catttctgattggtggact	7757	7773	1	5
SEQ ID NO: 163	aaagtgccacttttactca	6183	6202	1	5
SEQ ID NO: 164	acaaagtcagtgccctgct	6007	6026	1	5
SEQ ID NO: 165	aagtccataatggttcaga	12811	12830	1	5
SEQ ID NO: 166	ttctgaatatattgtcagt	13376	13395	1	5
SEQ ID NO: 167	ggctcaccctgagagaagt	12391	12410	1	5
SEQ ID NO: 168	aggaagatatgaagatgga	4712	4731	1	5
SEQ ID NO: 169	caaatttgtggagggtagt	10319	10338	1	5
SEQ ID NO: 170	tcagtataagtacaaccaa	9392	9411	1	5
SEQ ID NO: 171	tcccgattcacgcttccaa	11577	11596	1	5
SEQ ID NO: 172	cttcagaaagctaccttcc	7929	7948	1	5
SEQ ID NO: 173	tgaccttctctaagcaaaa	4876	4895	1	5
SEQ ID NO: 174	agcttggttttgccagtct	2458	2477	1	5
SEQ ID NO: 175	atctcgtgtctaggaaaag	5968	5987	1	5
SEQ ID NO: 176	ctcaaggataacgtgtttg	12609	12628	1	5
SEQ ID NO: 177	ttatcttattaattatatc	13079	13098	1	5
SEQ ID NO: 178	caacacttacttgaattcc	10661	10680	1	5
SEQ ID NO: 179	gacttcagagaaatacaaa	11400	11419	1	5
SEQ ID NO: 180	tccaatttccctgtggatc	3681	3700	1	5
SEQ ID NO: 181	tgaccacacaaacagtctg	5363	5382	1	5
SEQ ID NO: 182	tgaagtccggattcattct	11015	11034	1	5
SEQ ID NO: 183	cagctcaaccgtacagttc	11861	11880	1	5
SEQ ID NO: 184	tgacttcagtgcagaatat	11966	11985	1	5
SEQ ID NO: 185	tggccccgtttaccatgac	5809	5828	1	5
SEQ ID NO: 186	aggaggctttaagttcagc	7600	7619	1	5
SEQ ID NO: 187	gtctcttcctccatggaat	10470	10489	1	5
SEQ ID NO: 188	actgactgcacgctttgag	11756	11775	1	5
SEQ ID NO: 189	ctgagagaagtgtcttcaa	12399	12418	1	5
SEQ ID NO: 190	ggacggtactgtcccaggt	12784	12803	1	5
SEQ ID NO: 191	ggaaggcagagtttactgg	9148	9167	1	5
SEQ ID NO: 192	atttcctaaagctggatgt	11167	11186	1	5
SEQ ID NO: 193	ataaactgcaagatttttc	13600	13619	1	5
SEQ ID NO: 194	gaaacaatgcattagattt	9475	9764	1	5
SEQ ID NO: 195	tctcccgtgtataatgcca	11781	11800	1	5
SEQ ID NO: 196	tcaaaacctactgtctctt	10458	10477	1	5
SEQ ID NO: 197	gggaactacaatttcattt	7013	7032	1	5
SEQ ID NO: 198	caggctgattacgagtcat	4917	4936	1	5
SEQ ID NO: 199	ccacgaaaaatatggaagt	10360	10379	1	5
SEQ ID NO: 200	ccatcagttcagataaact	7989	8008	1	5
SEQ ID NO: 201	attgacctgtccattcaaa	13671	13690	1	5
SEQ ID NO: 202	ctggaattgtcattccttc	11728	11747	1	5
SEQ ID NO: 203	ttttaacaaaagtggaagt	6821	6840	1	5
SEQ ID NO: 204	catcactgccaaaggagag	8486	8505	1	5
SEQ ID NO: 205	tgactcactcattgatttt	12680	12699	1	5
SEQ ID NO: 206	ccacaaacaatgaagggaa	9256	9275	1	5
SEQ ID NO: 207	atgggaaaaaacaggcttg	9566	9585	1	5
SEQ ID NO: 208	atgggaagtataagaactt	4834	4853	1	5
SEQ ID NO: 209	agaaaaacaaacacaggca	9643	9662	1	5
SEQ ID NO: 210	tgaagtgtagtctcctggt	5089	5108	1	5
SEQ ID NO: 211	ctgaaatacaatgctctgg	5511	5530	1	5
SEQ ID NO: 212	agacacctgattttatagt	7948	7967	1	5
SEQ ID NO: 213	tgaaggctgactctgtggt	4282	4301	1	5
SEQ ID NO: 214	ctgagcaacaaatttgtgg	10311	10330	1	5
SEQ ID NO: 215	tttctctcatgattacaaa	5933	5952	1	5
SEQ ID NO: 216	tcaaggataacgtgtttga	12610	12629	1	5
SEQ ID NO: 217	gtcagatattgttgctcat	10187	10206	1	5
SEQ ID NO: 218	cattcattgaagatgttaa	7342	7361	1	5
SEQ ID NO: 219	ggcaaatttgaaggacttc	11994	12013	1	5
SEQ ID NO: 220	tggcaaatttgaaggactt	11993	12012	1	5
SEQ ID NO: 221	cagaatccagatacaagaa	6884	6903	1	5
SEQ ID NO: 222	tgggtctttccagagccag	11033	11052	1	5
SEQ ID NO: 223	tgagaagccccaagaattt	6248	6267	1	5
SEQ ID NO: 224	tcaaattcctggatacact	9848	9867	1	5
SEQ ID NO: 225	cctgaccttcacataccag	8310	8329	1	5
SEQ ID NO: 226	aagtaaaagaaaattttgc	10744	10763	1	5
SEQ ID NO: 227	tgaagtaaaagaaaatttt	10742	10761	1	5
SEQ ID NO: 228	ttaaggacttccattctga	13363	13382	1	5
SEQ ID NO: 229	agcccatcaatatcattga	6205	6224	1	5
SEQ ID NO: 230	tgtttcaactgcctttgtg	11219	11238	1	5
SEQ ID NO: 231	tgttttcctatttccaaga	12835	12854	1	5
SEQ ID NO: 232	agttattttgctaaacttg	14043	14062	1	5
SEQ ID NO: 233	ctgtttttagaggaaacca	7512	7531	1	5
SEQ ID NO: 234	tgtatagcaaattcctgtt	5890	5909	1	5
SEQ ID NO: 235	gttccttccatgatttccc	10933	10952	1	5
SEQ ID NO: 236	gagacagcatcttcgtgtt	11204	11223	1	5
SEQ ID NO: 237	ctgctaagaaccttactga	7780	7799	1	5
SEQ ID NO: 238	cctttcaagcactgactgc	11746	11765	1	5
SEQ ID NO: 239	ccaggttttccacaccaga	8038	8057	1	5
SEQ ID NO: 240	ctttttcaccaacggagaa	10838	10857	1	5
SEQ ID NO: 241	caggaggctttaagttcag	7599	7618	1	5
SEQ ID NO: 242	attccttcctttacaattg	8082	8101	1	5
SEQ ID NO: 243	gtcagcccagttccttcca	10924	10943	1	5
SEQ ID NO: 244	tgggctaaacgtatgaaag	7827	7846	1	5
SEQ ID NO: 245	atcttcataagttcaatga	13174	13193	1	5
SEQ ID NO: 246	gagtgaaatgctgtttttt	8630	8649	1	5
SEQ ID NO: 247	taatgattttcaagttcct	8294	8313	1	5
SEQ ID NO: 248	acgttagcctctaagacta	11928	11947	1	5
SEQ ID NO: 249	cttttacaattcattttcc	13014	13033	1	5
SEQ ID NO: 250	gctttctcttccacatttc	10052	10071	1	5
SEQ ID NO: 251	attgatgttagagtgcttt	6984	7003	1	5
SEQ ID NO: 252	tattgatgttagagtgctt	6983	7002	1	5
SEQ ID NO: 253	tcaaccttaatgattttca	8287	8306	1	5
SEQ ID NO: 254	caaagccatcactgatgat	1660	1679	1	5
SEQ ID NO: 255	tgaaatcattgaaaaatta	6719	6738	1	5
SEQ ID NO: 256	tgaagtagctgagaaaatc	7094	7113	1	5
SEQ ID NO: 257	aaacattcctttaacaatt	9488	9507	1	5
SEQ ID NO: 258	tattgaaaatattgatttt	6806	6825	1	5
SEQ ID NO: 259	atcatatccgtgtaatttt	6757	6776	1	5
SEQ ID NO: 260	ttaatcttcataagttcaa	13171	13190	1	5
SEQ ID NO: 261	agcttggttttgccagtct	2458	2477	1	5
SEQ ID NO: 262	attccttcctttacaattg	8082	8101	1	5
SEQ ID NO: 263	aattgttgaaagaaaacct	13147	13166	1	5
SEQ ID NO: 264	ggacaaggcccagaatctg	12545	12564	1	5
SEQ ID NO: 265	aaagaaaacctatgcctta	13155	13174	1	5
SEQ ID NO: 266	atttcttaaacattccttt	9481	9500	1	5
SEQ ID NO: 267	taaagccattcagtctctc	12962	12981	1	5
SEQ ID NO: 268	gacatgttgataaagaaat	7371	7390	1	5
SEQ ID NO: 269	atgtatcaaatggacattc	7677	7696	1	5
SEQ ID NO: 270	atctggaaccttgaagtaa	10731	10750	1	5
SEQ ID NO: 271	cttttcacattagatgcaa	8412	8431	1	5
SEQ ID NO: 272	ttgaaggacttcaggaatg	12001	12020	1	5
SEQ ID NO: 273	ggactcaaggataacgtgt	12606	12625	1	5
SEQ ID NO: 274	atcttcaatgattatatcc	13116	13135	1	5
SEQ ID NO: 275	tttatgattatgtcaacaa	12352	12371	1	5
SEQ ID NO: 276	actggacttctctagtcag	8801	8820	1	5
SEQ ID NO: 277	gaaaaatgaagtccggatt	11009	11028	1	5
SEQ ID NO: 278	tttgcaagttaaagaaaat	14015	14034	1	5
SEQ ID NO: 279	atttgatttaagtgtaaaa	9614	9633	1	5
SEQ ID NO: 280	tgcaagttaaagaaaatca	14017	14036	1	5
SEQ ID NO: 281	cattggtaggagacagcat	11195	11214	1	5
SEQ ID NO: 282	gcattggtaggagacagca	11194	11213	1	5
SEQ ID NO: 283	agctagagggcctcttttt	10825	10844	1	5
SEQ ID NO: 284	tccactcacatcctccagt	1281	1300	1	5
SEQ ID NO: 285	catgaacccctacatgaag	13751	13770	1	5
SEQ ID NO: 286	atttgaaagttcgttttcc	9274	9293	1	5
SEQ ID NO: 287	gagaacattatggaggccc	9432	9451	1	5
SEQ ID NO: 288	gtgtcttcaaagctgagaa	12408	12427	1	5
SEQ ID NO: 289	attccagcttccccacatc	8330	8349	1	5
SEQ ID NO: 290	cttatgggatttcctaaag	11159	11178	1	5
SEQ ID NO: 291	cttcatctgtcattgatgc	10219	10238	1	5
SEQ ID NO: 292	attccctgaagttgatgtg	11480	11499	1	5
SEQ ID NO: 293	tccatcacaaatcctttgg	9663	9682	1	5
SEQ ID NO: 294	ttccatcacaaatcctttg	9662	9681	1	5
SEQ ID NO: 295	tctcaagagttacagcaga	13221	13240	1	5
SEQ ID NO: 296	ttctcaagagttacagcag	13220	13239	1	5
SEQ ID NO: 297	tgggcctgccccagattct	8901	8920	1	5
SEQ ID NO: 298	gaaatcttcaatttattct	13813	13832	1	5
SEQ ID NO: 299	tttttgcaagttaaagaaa	14013	14032	1	5
SEQ ID NO: 300	aaagaaaatcaggatctga	14025	14044	1	5
SEQ ID NO: 301	atctatgccatctcttctg	5625	5644	1	5
SEQ ID NO: 302	atggagtctttattgtgta	14081	14100	1	5
SEQ ID NO: 303	tgagaactacgagctgact	4799	4818	1	5
SEQ ID NO: 304	actggtggcaaaaccctcc	2726	2745	1	5
SEQ ID NO: 305	attgaagtacctacttttg	8358	8377	1	5
SEQ ID NO: 306	aattgaagtacctactttt	8357	8376	1	5
SEQ ID NO: 307	cattatggcccttcgtgaa	13250	13269	1	5
SEQ ID NO: 308	tcaaaagaagcccaagagg	12939	12958	1	5
SEQ ID NO: 309	caagcatctgattgactca	12668	12687	1	5
SEQ ID NO: 310	cttgaacacaaagtcagtg	6000	6019	1	5
SEQ ID NO: 311	caaaaacattttcaacttc	5279	5298	1	5
SEQ ID NO: 312	ccatttacagatcttcagg	11364	11393	1	5
SEQ ID NO: 313	ctggattccacatgcagct	11847	11866	1	5
SEQ ID NO: 314	atcatatccgtgtaatttt	6757	6776	1	5
SEQ ID NO: 315	aaagctgagaagaaatctg	12416	12435	1	5
SEQ ID NO: 316	caaagctgagaagaaatct	12415	12434	1	5
SEQ ID NO: 317	atccaagatgagatcaaca	13095	13114	1	5
SEQ ID NO: 318	caactctctgattactatg	13623	13642	1	5
SEQ ID NO: 319	cttcaatttattcttcttt	13818	13837	1	5
SEQ ID NO: 320	cttcaaagacttaaaaaat	8006	8025	1	5
SEQ ID NO: 321	tctcttcctccatggaatt	10471	10490	1	5
SEQ ID NO: 322	tcttcataagttcaatgaa	13175	13194	1	5
SEQ ID NO: 323	tcttcataagttcaatgaa	12924	12943	1	5
SEQ ID NO: 324	actcaatggtgaaattcag	7457	7476	1	5
SEQ ID NO: 325	ttcagaagctaagcaatgt	7231	7250	1	5
SEQ ID NO: 326	ttttggcaagctatacagt	8372	8391	1	5
SEQ ID NO: 327	ttgcaagcaagtctttcct	3005	3024	1	5
SEQ ID NO: 328	ttctctctatgggaaaaaa	9558	9577	1	5
SEQ ID NO: 329	tgaaatgctgttttttgga	8633	8652	3	4
SEQ ID NO: 330	agaatctgtaccaggaact	12556	12575	3	4
SEQ ID NO: 331	acttcagagaaatacaaat	11401	11420	3	4
SEQ ID NO: 332	tgaaaccaatgacaaaatc	7421	7440	3	4
SEQ ID NO: 333	cagctgagcagacaggcac	6031	6050	2	4
SEQ ID NO: 334	cttcataagttcaatgaat	13176	13195	2	4
SEQ ID NO: 335	acttcccaactctcaagtc	13407	13426	2	4
SEQ ID NO: 336	ctgggcagctgtatagcaa	5881	5900	2	4
SEQ ID NO: 337	taagtatgatttcaattct	10490	10509	2	4
SEQ ID NO: 338	agttcaatgaatttattca	13183	13202	2	4
SEQ ID NO: 339	cagcccagccatttgagat	9229	9248	2	4
SEQ ID NO: 340	tcagcccagccatttgaga	9228	9247	2	4
SEQ ID NO: 341	gatgggaaagccgccctca	5208	5227	2	4
SEQ ID NO: 342	ttgaaagcagaacctctgg	5907	5926	2	4
SEQ ID NO: 343	ctttctcgggaatattcag	10623	10642	2	4
SEQ ID NO: 344	tgacaggcattttgaaaaa	9722	9741	2	4
SEQ ID NO: 345	ttgacaggcattttgaaaa	9721	9740	2	4
SEQ ID NO: 346	aattccaattttgagaatg	10406	10425	2	4
SEQ ID NO: 347	aaatgtcagctcttgttca	10894	10913	2	4
SEQ ID NO: 348	tgccagtttgaaaaacaaa	11807	11826	2	4
SEQ ID NO: 349	ttgacatgttgataaagaa	7369	7388	2	4
SEQ ID NO: 350	tgaagtagaccaacaaatc	7154	7173	2	4
SEQ ID NO: 351	cttcaggttccatcgtgca	11376	11395	2	4
SEQ ID NO: 352	atgttgataaagaaattaa	7374	7393	2	4
SEQ ID NO: 353	attcaaactgcctatattg	13868	13887	2	4
SEQ ID NO: 354	caagagcacacggtcttca	10679	10698	2	4
SEQ ID NO: 355	aaattccctgaagttgatg	11478	11497	2	4
SEQ ID NO: 356	aagtaagtgctaggttcaa	9373	9392	2	4
SEQ ID NO: 357	ttctgcacagaaatattca	13438	13457	2	4
SEQ ID NO: 358	gcttgctaacctctctgaa	12304	12323	2	4
SEQ ID NO: 359	gggacctaccaagagtgga	12525	12544	2	4
SEQ ID NO: 360	agttcaatgaatttattca	13183	13202	2	4
SEQ ID NO: 361	gattactatgaaaaattta	13632	13651	2	4
SEQ ID NO: 362	ttttaaaagaaatcttcaa	13805	13824	2	4
SEQ ID NO: 363	cgaggccaggccgcagccc	76	95	1	4
SEQ ID NO: 364	ccgaggccaggccgcagcc	75	94	1	4
SEQ ID NO: 365	aaccgtgcctgaatctcag	11549	11568	1	4
SEQ ID NO: 366	tcagctgacctcatcgaga	2160	2179	1	4
SEQ ID NO: 367	gctctgcagcttcatcctg	368	387	1	4
SEQ ID NO: 368	cagcacagaccatttcagc	4244	4263	1	4
SEQ ID NO: 369	ctggatgtaaccaccagca	11178	11197	1	4
SEQ ID NO: 370	catcctgaagaccagccag	380	399	1	4
SEQ ID NO: 371	actcaccctggacattcag	3383	3402	1	4
SEQ ID NO: 372	tcccggagccaaggctgga	2675	2694	1	4
SEQ ID NO: 373	ggaaccctctccctcacct	4728	4747	1	4
SEQ ID NO: 374	ctgggaggcatgatgctca	9163	9182	1	4
SEQ ID NO: 375	ttcaaatataatcggcaga	3261	3280	1	4
SEQ ID NO: 376	tcttccgttctgtaatggc	5794	5813	1	4
SEQ ID NO: 377	tgcaagaatattttgagag	6340	6359	1	4
SEQ ID NO: 378	ccagtttccggggaaacct	12716	12735	1	4
SEQ ID NO: 379	tctttttgggaagcaagga	2219	2238	1	4
SEQ ID NO: 380	aatggtcaagttcctgatg	2277	2296	1	4
SEQ ID NO: 381	gtctctgaactcagaagga	13988	14007	1	4
SEQ ID NO: 382	aacaaataaatggagtctt	14072	14091	1	4
SEQ ID NO: 383	ccagagccaggtcgagctt	11042	11061	1	4
SEQ ID NO: 384	tccatgtcccatttacaga	11356	11375	1	4
SEQ ID NO: 385	ttgattttaacaaaagtgg	6817	6836	1	4
SEQ ID NO: 386	ttgcaagcaagtctttcct	3005	3024	1	4
SEQ ID NO: 387	atttccataccccgtttgc	3480	3499	1	4
SEQ ID NO: 388	tattcttcttttccaattg	13826	13845	1	4
SEQ ID NO: 389	gtggcttcccatattgcca	1887	1906	1	4
SEQ ID NO: 390	cacattacatttggtctct	2930	2949	1	4
SEQ ID NO: 391	tgggaaagccgccctcaca	5210	5229	1	4
SEQ ID NO: 392	atgggaaagccgccctcac	5209	5228	1	4
SEQ ID NO: 393	gatgctgaacagtgagctg	8144	8163	1	4
SEQ ID NO: 394	tcataacagtactgtgagc	10337	10356	1	4
SEQ ID NO: 395	ctgagtgggtttatcaagg	12445	12464	1	4
SEQ ID NO: 396	gctgagtgggtttatcaag	12444	12463	1	4
SEQ ID NO: 397	ttgcaatgagctcatggct	3805	3824	1	4
SEQ ID NO: 398	gttgcaatgagctcatggc	3804	3823	1	4
SEQ ID NO: 399	gtaggaataaatggagaag	9453	9472	1	4
SEQ ID NO: 400	ttattgctgaatccaaaag	13648	13667	1	4
SEQ ID NO: 401	aaagccatcactgatgatc	1661	1680	1	4
SEQ ID NO: 402	caaagccatcactgatgat	1660	1679	1	4
SEQ ID NO: 403	tcacaaatcctttggctgt	9667	9686	1	4
SEQ ID NO: 404	caaaatagaagggaatctt	2069	2088	1	4
SEQ ID NO: 405	ctggtaactactttaaaca	5487	5506	1	4
SEQ ID NO: 406	gttcaatgaatttattcaa	13184	13203	1	4
SEQ ID NO: 407	atggcattttttgcaagtt	14006	14025	1	4
SEQ ID NO: 408	agattgatgggcagttcag	4564	4583	1	4
SEQ ID NO: 409	ctcaaagaatgactttttt	2570	2589	1	4
SEQ ID NO: 410	tctccagataaaaaactca	12201	12220	1	4
SEQ ID NO: 411	ctcagatcaaagttaattg	12265	12284	1	4
SEQ ID NO: 412	gagggtagtcataacagta	10329	10348	1	4
SEQ ID NO: 413	actgttgactcaggaaggc	12572	12591	1	4
SEQ ID NO: 414	tggccacatagcatggact	8858	8877	1	4
SEQ ID NO: 415	cagctgacctcatcgagat	2161	2180	1	4
SEQ ID NO: 416	tcagctgacctcatcgaga	2160	2179	1	4
SEQ ID NO: 417	acctgcaccaaagctggca	13955	13974	1	4
SEQ ID NO: 418	caccaaaaaccccaatggc	11240	11259	1	4
SEQ ID NO: 419	accagatgctgaacagtga	6140	8159	1	4
SEQ ID NO: 420	accacttacagctagaggg	10816	10835	1	4
SEQ ID NO: 421	gggcgacctaagttgtgac	3431	3450	1	4
SEQ ID NO: 422	tggctggtaacctaaaagg	5578	5597	1	4
SEQ ID NO: 423	ggtcctttatgattatgtc	12347	12366	1	4
SEQ ID NO: 424	ttcccaaaagcagtcagca	9930	9949	1	4
SEQ ID NO: 425	ttcaggtccatgcaagtca	10909	10928	1	4
SEQ ID NO: 426	tcttgaacacaaagtcagt	5999	6018	1	4
SEQ ID NO: 427	aaatgaaagtaaagatcat	8110	8129	1	4
SEQ ID NO: 428	gagtaaaccaaaacttggt	9016	9035	1	4
SEQ ID NO: 429	gagttactgaaaaagctgc	13719	13738	1	4
SEQ ID NO: 430	attggatatccaagatctg	1925	1944	1	4
SEQ ID NO: 431	ccatgacctccagctcctg	2477	2496	1	4
SEQ ID NO: 432	ctgaaatacaatgctctgg	5511	5530	1	4
SEQ ID NO: 433	gaaaaacttggaaacaacc	4431	4450	1	4
SEQ ID NO: 434	agaatccagatacaagaaa	6885	6904	1	4
SEQ ID NO: 435	cagcatgcctagtttctcc	9944	9963	1	4
SEQ ID NO: 436	tcaatatcaaaagcccagc	12037	12056	1	4
SEQ ID NO: 437	atatctggaaccttgaagt	10729	10748	1	4
SEQ ID NO: 438	ctgaactcagaaggatggc	13992	14011	1	4
SEQ ID NO: 439	cttccattctgaatatatt	13370	13389	1	4
SEQ ID NO: 440	gataaaagattactttgag	7265	7284	1	4
SEQ ID NO: 441	tcttcaatttattcttctt	13817	13836	1	4
SEQ ID NO: 442	atcttcaatttattcttct	13816	13835	1	4
SEQ ID NO: 443	ttcacataccagaattcca	8317	8336	1	4
SEQ ID NO: 444	tttttaaccagtcagatat	10177	10196	1	4
SEQ ID NO: 445	ctttttaaccagtcagata	10176	10195	1	4
SEQ ID NO: 446	ctaaattcccatggtcttg	4965	4984	1	4
SEQ ID NO: 447	actaaattcccatggtctt	4964	4983	1	4
SEQ ID NO: 448	tctttctcgggaatattca	10622	10641	1	4
SEQ ID NO: 449	gaagcacatatgaactgga	13937	13956	1	4
SEQ ID NO: 450	ttcctttaacaattcctga	9493	9512	1	4
SEQ ID NO: 451	gctgacatagggaatggaa	8433	8452	1	4
SEQ ID NO: 452	tattctatccaagattggg	7812	7831	1	4
SEQ ID NO: 453	ttctatccaagattgggct	7814	7833	1	4
SEQ ID NO: 454	tttgaaaaacaaagcagat	11813	11832	1	4
SEQ ID NO: 455	attttttgcaagttaaaga	14011	14030	1	4
SEQ ID NO: 456	gcatggcattatgatgaag	3606	3625	1	4
SEQ ID NO: 457	ttcagggtgtggagtttag	5686	5705	1	4
SEQ ID NO: 458	tttcttaaacattccttta	9482	9501	1	4
SEQ ID NO: 459	ttccctccattaagttctc	11701	11720	1	4
SEQ ID NO: 460	ttccaatgaccaagaaaag	11060	11079	1	4
SEQ ID NO: 461	gcttactggacgaactctg	6134	6153	1	4
SEQ ID NO: 462	caaattcctggatacactg	9849	9868	1	4
SEQ ID NO: 463	acaagaatacgtctacact	4351	4370	1	4
SEQ ID NO: 464	acctcggaacaatcctcag	3325	3344	1	4
SEQ ID NO: 465	gacctgcgcaacgagatca	8823	8842	1	4
SEQ ID NO: 466	ccatgatctacatttgttt	6788	6807	1	4
SEQ ID NO: 467	caaaaacattttcaacttc	5279	5298	1	4
SEQ ID NO: 468	ctttaagttcagcatcttt	7606	7625	1	4
SEQ ID NO: 469	cagatttgaggattccatc	7975	7994	1	4
SEQ ID NO: 470	caatcacaagtcgattccc	9075	9094	1	4
SEQ ID NO: 471	gaagtgtcagtggcaaaaa	10374	10393	1	4
SEQ ID NO: 472	catggcattatgatgaaga	3607	3626	1	4
SEQ ID NO: 473	cattatggaggcccatgta	9437	9456	1	4
SEQ ID NO: 474	caaaatcaactttaatgaa	6599	6618	1	4
SEQ ID NO: 475	tcaacacaatcttcaatga	13108	13127	1	4
SEQ ID NO: 476	ctccccaggacctttcaaa	9834	9853	1	4
SEQ ID NO: 477	gctccccaggacctttcaa	8333	9852	1	4
SEQ ID NO: 478	agctccccaggacctttca	9832	9851	1	4
SEQ ID NO: 479	ctgtttctgagtcccagtg	9336	9355	1	4
SEQ ID NO: 480	actgtttctgagtcccagt	9335	9354	1	4
SEQ ID NO: 481	gatgatgccaaaatcaact	6591	6610	1	4
SEQ ID NO: 482	agatgatgccaaaatcaac	6590	6609	1	4
SEQ ID NO: 483	actcagaaggatggcattt	13996	14015	1	4
SEQ ID NO: 484	ttggttacaggaggcttta	7592	7611	1	4
SEQ ID NO: 485	ttttcttttcagcccagcc	9220	9239	1	4
SEQ ID NO: 486	attttcaagcaaatgcaca	8630	8549	1	4
SEQ ID NO: 487	atgcgtctaccttacacaa	9513	9532	1	4
SEQ ID NO: 488	ggaagctgaagtttatcat	2869	2888	1	4
SEQ ID NO: 489	cagagctatcactgggaag	5227	5246	1	4
SEQ ID NO: 490	gagcttactggacgaactc	6132	6151	1	4
SEQ ID NO: 491	cagcctccccagccgtagg	12112	12131	1	4
SEQ ID NO: 492	aaactgttaatttacagct	5455	5474	1	4
SEQ ID NO: 493	agtttccggggaaacctgg	12718	12737	1	4
SEQ ID NO: 494	tacagtattctgaaaatcc	8385	8404	1	4
SEQ ID NO: 495	aatgagctcatggcttcag	3809	3828	1	4
SEQ ID NO: 496	attttgagaggaatcgaca	6349	6368	1	4
SEQ ID NO: 497	aacacatgaatcacaaatt	8930	8949	1	4
SEQ ID NO: 498	tcaaaacgagcttcaggaa	13199	13218	1	4
SEQ ID NO: 499	aacttgtacaactggtccg	4203	4222	1	4
SEQ ID NO: 500	tcatcaattggttacagga	7585	7604	1	4
SEQ ID NO: 501	tctgcagaacaatgctgag	12431	12450	1	4
SEQ ID NO: 502	aattgactttgtagaaatg	8096	8115	1	4
SEQ ID NO: 503	ccatgcaagtcagcccagt	10916	10935	1	4
SEQ ID NO: 504	aaactgcctatattgataa	13872	13891	1	4
SEQ ID NO: 505	ctggacttctcttcaaaac	5400	5419	1	4
SEQ ID NO: 506	tctgggtgtcgacagcaaa	5264	5283	1	4
SEQ ID NO: 507	ttctgggtgtcgacagcaa	5263	5282	1	4
SEQ ID NO: 508	agtcaagattgatgggcag	4559	4578	1	4
SEQ ID NO: 509	ggaggctttaagttcagca	7601	7620	1	4
SEQ ID NO: 510	ctgtatagcaaattcctgt	5889	5908	1	4
SEQ ID NO: 511	catggacttcttctggaaa	8869	8888	1	4
SEQ ID NO: 512	tgcccagcaagcaagttgg	9353	9372	1	4
SEQ ID NO: 513	catccttaacaccttccac	8063	8082	1	4
SEQ ID NO: 514	ttcactgttcctgaaatca	7863	7882	1	4
SEQ ID NO: 515	caaaaacattttcaacttc	5279	5298	1	4
SEQ ID NO: 516	attcataatcccaactctc	8270	8289	1	4
SEQ ID NO: 517	tgcctttgtgtacaccaaa	11228	11247	1	4
SEQ ID NO: 518	ggtaacctaaaaggagcct	5583	5602	1	4
SEQ ID NO: 519	attgaagtacctacttttg	8358	8377	1	4
SEQ ID NO: 520	aattgaagtacctactttt	8357	8376	1	4
SEQ ID NO: 521	aaatccaatctcctctttt	8398	8417	1	4
SEQ ID NO: 522	gtctgtgggattccatctg	4082	4101	1	4
SEQ ID NO: 523	tcataagttcaatgaattt	13178	13197	1	4
SEQ ID NO: 524	ccattgaccagatgctgaa	8134	8153	1	4
SEQ ID NO: 525	tggaaatgggcctgcccca	8895	8914	1	4
SEQ ID NO: 526	aatcacaactcctccactg	9533	9552	1	4
SEQ ID NO: 527	caaaactaccacacatttc	13686	13705	1	4
SEQ ID NO: 528	tttgagaggaatcgacaaa	6351	6370	1	4
SEQ ID NO: 529	catcaattggttacaggag	7586	7605	1	4
SEQ ID NO: 530	agtccttcatgtccctaga	10025	10044	1	4
SEQ ID NO: 531	ggcattttgaaaaaaacag	9727	9746	1	4
SEQ ID NO: 532	aactctcaaaccctaagat	8548	8567	1	4
SEQ ID NO: 533	ggacattcctctagcgaga	8207	8226	1	4
SEQ ID NO: 534	aatgaatacagccaggact	6078	6097	1	4
SEQ ID NO: 535	actttgtagaaatgaaagt	8101	8120	1	4
SEQ ID NO: 536	ttgaaggacttcaggaatg	12001	12020	1	4
SEQ ID NO: 537	gagtaaaccaaaacttggt	9016	9035	1	4
SEQ ID NO: 538	cctttaacaattcctgaaa	9495	9514	1	4
SEQ ID NO: 539	tcattctgggtctttccag	11027	11046	1	4
SEQ ID NO: 540	tagaattacagaaaatgat	6557	6576	1	4
SEQ ID NO: 541	cgtaggcaccgtgggcatg	12125	12144	1	4
SEQ ID NO: 542	ttgatgatgctgtcaagaa	7300	7319	1	4
SEQ ID NO: 543	cagaattccagcttcccca	8326	8345	1	4
SEQ ID NO: 544	ttgaggctattgatgttag	6976	6995	1	4
SEQ ID NO: 545	attgaggctattgatgtta	6975	6994	1	4
SEQ ID NO: 546	cattgaggctattgatgtt	6974	6993	1	4
SEQ ID NO: 547	tctccatctgcgctaccag	12065	12084	1	4
SEQ ID NO: 548	tcatctcctttcttcatct	1202	10221	1	4
SEQ ID NO: 549	cagatatatatctcaggga	8176	8195	1	4
SEQ ID NO: 550	tcaggctcttcagaaagct	7922	7941	1	4
SEQ ID NO: 551	tgtcaagataaacaatcag	8732	8751	1	4
SEQ ID NO: 552	gaagtagtactgcatcttg	6835	6854	1	4
SEQ ID NO: 553	ctgagtcccagtgcccagc	9342	9361	1	4
SEQ ID NO: 554	cagcaagtacctgagaacg	8603	8622	1	4
SEQ ID NO: 555	actcagatcaaagttaatt	12264	12283	1	4
SEQ ID NO: 556	agcacagtacgaaaaacca	10801	10820	1	4
SEQ ID NO: 557	tgtccctagaaatctcaag	10034	10053	1	4
SEQ ID NO: 558	atgtccctagaaatctcaa	10033	10052	1	4
SEQ ID NO: 559	gatggaaccctctccctca	4725	4744	1	4
SEQ ID NO: 560	aggaaactcagatcaaagt	12259	12278	1	4
SEQ ID NO: 561	agcagccagtggcaccact	12506	12525	1	4
SEQ ID NO: 562	tcagccaggtttatagcac	7726	7745	1	4
SEQ ID NO: 563	aatttctgattaccaccag	13571	13590	1	4
SEQ ID NO: 564	tgttttttggaaatgccat	8641	8660	1	4
SEQ ID NO: 565	ctttgacaggcattttgaa	9719	9738	1	4
SEQ ID NO: 566	tcgatgcacatacaaatgg	5830	5849	1	4
SEQ ID NO: 567	ttgatgttagagtgctttt	6985	7004	1	4
SEQ ID NO: 568	tgtcctacaacaagttaag	7247	7266	1	4
SEQ ID NO: 569	atgtcctacaacaagttaa	7246	7265	1	4
SEQ ID NO: 570	agcatctttggctcacatg	7616	7635	1	4
SEQ ID NO: 571	atttatcaaaagaagccca	12934	12953	1	4
SEQ ID NO: 572	ttttggcaagctatacagt	8372	8391	1	4
SEQ ID NO: 573	tcaattgggagagacaagt	6496	6515	1	4
SEQ ID NO: 574	tgctttgtgagtttatcag	9685	9704	1	4
SEQ ID NO: 575	cagtcatgtagaaaaactt	4421	4440	1	4
SEQ ID NO: 576	tgtactggaaaacgtacag	6380	6399	1	4
SEQ ID NO: 577	aatattgatcaatttgtaa	6417	6436	1	4
SEQ ID NO: 578	gtttgaaaaacaaagcaga	11812	11831	1	4
SEQ ID NO: 579	tttcatttgaaagaataaa	7024	7043	1	4
SEQ ID NO: 580	atttcaagcaagaacttaa	10426	10445	1	4
SEQ ID NO: 581	attggcgtggagcttactg	6123	6142	1	4
SEQ ID NO: 582	ttttgctggagaagccaca	10757	10776	1	4
SEQ ID NO: 583	ctttgcactatgttcataa	12756	12775	1	4
SEQ ID NO: 584	aaacacctaagagtaaacc	9006	9025	1	4
SEQ ID NO: 585	aaatgctgacatagggaat	8429	8448	1	4
SEQ ID NO: 586	gaaatattatgaacttgaa	13304	13323	1	4
SEQ ID NO: 587	tttcctaaagctggatgta	11168	11187	1	4
SEQ ID NO: 588	caggtccatgcaagtcagc	10911	10930	1	4
SEQ ID NO: 589	ccagcttccccacatctca	8333	8352	1	4
SEQ ID NO: 590	tcttcgtgtttcaactgcc	11213	11232	1	4
SEQ ID NO: 591	caagtaagtgctaggttca	9372	9391	1	4
SEQ ID NO: 592	ccaacacttacttgaattc	10660	10679	1	4
SEQ ID NO: 593	tcagaaagctaccttccag	7931	7950	1	4
SEQ ID NO: 594	atggacttcttctggaaaa	8870	8889	1	4
SEQ ID NO: 595	ctcatctcctttcttcatc	10201	10220	1	4
SEQ ID NO: 596	cttttctaaacttgaaatt	9056	9075	1	4
SEQ ID NO: 597	ttatgaacttgaagaaaag	13310	13329	1	4
SEQ ID NO: 598	ttttcacattagatgcaaa	8413	8432	1	4
SEQ ID NO: 599	aaaattgatgatatctgga	10719	10738	1	4
SEQ ID NO: 600	aaaagggtcatggaaatgg	8885	8904	1	4
SEQ ID NO: 601	ctcaattttgattttcaag	8520	8539	1	4
SEQ ID NO: 602	attccctccattaagttct	11700	11719	1	4
SEQ ID NO: 603	tttcaagcaagaacttaat	10427	10446	1	4
SEQ ID NO: 604	atcagttcagataaacttc	7991	8010	1	4
SEQ ID NO: 605	aattccctgaagttgatgt	11479	11498	1	4
SEQ ID NO: 606	gctttctcttccacatttc	10052	10071	1	4
SEQ ID NO: 607	cccatttacagatcttcag	11363	11382	1	4
SEQ ID NO: 608	tcccatttacagatcttca	11362	11381	1	4
SEQ ID NO: 609	tttgaggattccatcagtt	7979	7998	1	4
SEQ ID NO: 610	tctggctccctcaactttt	9042	9061	1	4
SEQ ID NO: 611	gtttattgaaaatattgat	6803	6822	1	4
SEQ ID NO: 612	aatattattgatgaaatca	6708	6727	1	4
SEQ ID NO: 613	gcaagaacttaatggaaat	10433	10452	1	4
SEQ ID NO: 614	catcacactgaataccaat	10151	10170	1	4
SEQ ID NO: 615	caagagcttatgggatttc	11153	11172	1	4
SEQ ID NO: 616	attactttgagaaattagt	7273	7292	1	4
SEQ ID NO: 617	acttgacttcagagaaata	11396	11415	1	4
SEQ ID NO: 618	gtcttcagtgaagctgcag	10691	10710	1	4
SEQ ID NO: 619	agcctcacctcttactttt	10563	10582	1	4
SEQ ID NO: 620	aagtagctgagaaaatcaa	7096	7115	1	4
SEQ ID NO: 621	ttttcacattagatgcaaa	8413	8432	1	4
SEQ ID NO: 622	ggtggactcttgctgctaa	7768	7787	1	4
SEQ ID NO: 623	ttctcaattttgattttca	8518	8537	1	4
SEQ ID NO: 624	ttagccacagctctgtgtc	10293	10312	1	4
SEQ ID NO: 625	tcaagaagcttaatgaatt	7312	7331	1	4
SEQ ID NO: 626	aaaacgagcttcaggaagc	13201	13220	1	4
SEQ ID NO: 627	atgtcctacaacaagttaa	7246	7265	1	4
SEQ ID NO: 628	aatcctttgacaggcattt	9715	9734	1	4
SEQ ID NO: 629	tgaaattcaatcacaagtc	9068	9087	1	4
SEQ ID NO: 630	gttctcaattttgattttc	8517	8536	1	4
SEQ ID NO: 631	ttcaggaactattgctagt	10637	10656	1	4
SEQ ID NO: 632	atgatttccctgaccttgg	10942	10961	1	4
SEQ ID NO: 633	ttgaagtaaaagaaaattt	10741	10760	1	4
SEQ ID NO: 634	caaatctggatttcttaaa	9472	9491	1	4
SEQ ID NO: 635	caagggttcactgttcctg	7857	7876	1	4
SEQ ID NO: 636	gattctcagatgagggaac	8914	8933	1	4
SEQ ID NO: 637	cttgaacacaaagtcagtg	6000	6019	1	4
SEQ ID NO: 638	tcttgaacacaaagtcagt	5999	6018	1	4
SEQ ID NO: 639	actgttgactcaggaaggc	12572	12591	1	4
SEQ ID NO: 640	ggaagcttctcaagagtta	13214	13233	1	4
SEQ ID NO: 641	aaatttctctgctggaaac	9410	9429	1	4
SEQ ID NO: 642	atctgcagaacaatgctga	12430	12449	1	4
SEQ ID NO: 643	ggcagcttctggcttgcta	12293	12312	1	4
SEQ ID NO: 644	aactgttgactcaggaagg	12871	12590	1	4
SEQ ID NO: 645	agctgccagtccttcatgt	10018	10037	1	4
SEQ ID NO: 646	cattaatcctgccatcatt	9997	10016	1	4
SEQ ID NO: 647	ccatttgagatcacggcat	9237	9256	1	4
SEQ ID NO: 648	ttcgttttccattaaggtt	9283	9302	1	4
SEQ ID NO: 649	ggaagtggccctgaatgct	10964	10983	1	4
SEQ ID NO: 650	agggaaagagaagattgca	13493	13512	1	4
SEQ ID NO: 651	gagaacttactatcatcct	13780	13799	1	4
SEQ ID NO: 652	tcaatgaatttattcaaaa	13186	13205	1	4
SEQ ID NO: 653	tcttttcagcccagccatt	9223	9242	1	4
SEQ ID NO: 654	gctgactttaaaatctgac	4811	4830	1	4
SEQ ID NO: 655	gggatttcctaaagctgga	11164	11183	1	4
SEQ ID NO: 656	ccagtttccagggactcaa	12595	12614	1	4
SEQ ID NO: 657	aagtcgattcccagcatgt	9082	9101	1	4
SEQ ID NO: 658	tcagatggaaaaatgaagt	11002	11021	1	4
SEQ ID NO: 659	gaaagtccataatggttca	12809	12828	1	4
SEQ ID NO: 660	ggaagaagaggcagcttct	12284	12303	1	4
SEQ ID NO: 661	atctaaatgcagtagccaa	11626	11645	1	4
SEQ ID NO: 662	attgataaaaccatacagt	13883	13902	1	4
SEQ ID NO: 663	tattgataaaaccatacag	13882	13901	1	4
SEQ ID NO: 664	gggaatctgatgaggaaac	12247	12266	1	4
SEQ ID NO: 665	ttgagttgcccaccatcat	11659	11678	1	4
SEQ ID NO: 666	caagatcgcagactttgag	11645	11664	1	4
SEQ ID NO: 667	aacagaaacaatgcattag	9741	9760	1	4
SEQ ID NO: 668	ccaagaaaaggcacacctt	11069	11088	1	4
SEQ ID NO: 669	ccctaacagatttgaggat	7969	7988	1	4
SEQ ID NO: 670	aaacaaacacaggcattcc	9647	9666	1	4
SEQ ID NO: 671	gaaatactgttttcctatt	12828	12847	1	4
SEQ ID NO: 672	ataaactgcaagatttttc	13600	13619	1	4
SEQ ID NO: 673	gctttccaatgaccaagaa	11057	11076	1	4
SEQ ID NO: 674	ctgtgctttgtgagtttat	9682	9701	1	4
SEQ ID NO: 675	gaatttgaaagttcgtttt	9272	9291	1	4
SEQ ID NO: 676	aggaagtggccctgaatgc	10963	10982	1	4
SEQ ID NO: 677	tgttgaaagatttatcaaa	12925	12944	1	4
SEQ ID NO: 678	gacaagaaaaaggggattg	10271	10290	1	4
SEQ ID NO: 679	ctgagaacttcatcatttg	11430	11449	1	4
SEQ ID NO: 680	ctggacttctctagtcagg	8802	8821	1	4
SEQ ID NO: 681	tgaatctggctccctcaac	9038	9057	1	4
SEQ ID NO: 682	agaatccagatacaagaaa	6885	6904	1	4
SEQ ID NO: 683	cagaatccagatacaagaa	6884	6903	1	4
SEQ ID NO: 684	ggacagtgaaatattatga	13297	13316	1	4
SEQ ID NO: 685	ctggatgtaaccaccagca	11178	11197	1	4
SEQ ID NO: 686	atgaagcttgctccaggag	13764	13783	1	4
SEQ ID NO: 687	ctgcgctaccagaaagaca	12072	12091	1	4
SEQ ID NO: 688	tttgagttgcccaccatca	11658	11677	1	4
SEQ ID NO: 689	gttgaccacaagcttagct	10539	10558	1	4
SEQ ID NO: 690	caaagctggcaccagggct	13963	13982	1	4
SEQ ID NO: 691	gcttcaggaagcttctcaa	13208	13227	1	4
SEQ ID NO: 692	aggaaggccaagccagttt	12583	12602	1	4
SEQ ID NO: 693	atgattatgtcaacaagta	12355	12374	1	4
SEQ ID NO: 694	ctgacatcttaggcactga	4993	5012	1	4
SEQ ID NO: 695	gaactcagaaggatggcat	13994	14013	1	4
SEQ ID NO: 696	ttctcaattttgattttca	8518	8537	1	4
SEQ ID NO: 697	ttttctaaatggaacttct	12165	12184	1	4
SEQ ID NO: 698	ggatctaaatgcagtagcc	11624	11643	1	4
SEQ ID NO: 699	atttcttaaacattccttt	9481	9500	1	4
SEQ ID NO: 700	gaatctggctccctcaact	9039	9058	1	4
SEQ ID NO: 701	tcattctgggtctttccag	11027	11046	1	4
SEQ ID NO: 702	atagcatggacttcttctg	8865	8884	1	4
SEQ ID NO: 703	cttctggcttgctaacctc	12298	12317	1	4
SEQ ID NO: 704	cacggagttactgaaaaag	13715	13734	1	4
SEQ ID NO: 705	aaggcatctccacctcagc	12094	12113	1	4
SEQ ID NO: 706	tccaagatgagatcaacac	13096	13115	1	4
SEQ ID NO: 707	gttgagaagccccaagaat	6246	6265	1	4
SEQ ID NO: 708	aaattctcttttcttttca	9212	9231	1	4
SEQ ID NO: 709	tgaaagtcaagcatctgat	12661	12680	1	4
SEQ ID NO: 710	catccttaacaccttccac	8063	8082	1	4
SEQ ID NO: 711	tgtaccataagccatattt	10080	10099	1	4
SEQ ID NO: 712	ttgatgttagagtgctttt	6985	7004	1	4
SEQ ID NO: 713	tctgcacagaaatattcag	13439	13458	1	4
SEQ ID NO: 714	taaatggagtctttattgt	14078	14097	1	4
SEQ ID NO: 715	ataaatggagtctttattg	14077	14096	1	4
SEQ ID NO: 716	atattgtcagtgcctctaa	13384	13403	1	4
SEQ ID NO: 717	attactatgaaaaaattat	13633	13652	1	4
SEQ ID NO: 718	gttattttgctaaacttgg	14044	14063	1	4
SEQ ID NO: 719	tcatcctctaattttttaa	13792	13811	1	4
SEQ ID NO: 720	tttcatttgaaagaataaa	7024	7043	1	4
SEQ ID NO: 721	gaataccaatgctgaactt	10160	10179	1	4
SEQ ID NO: 722	ctgagagaagtgtcttcaa	12399	12418	1	4
SEQ ID NO: 723	atatctggaaccttgaagt	10729	10748	1	4
SEQ ID NO: 724	attccctgaagttgatgtg	11480	11499	1	4
SEQ ID NO: 725	atttttattcctgccatgg	10095	10114	1	4
SEQ ID NO: 726	aaaattcaaactgcctata	13865	13884	1	4
SEQ ID NO: 727	atttgtaagaaaatacaga	6428	6447	1	4
SEQ ID NO: 728	cagcatgcctagtttctcc	9944	9963	1	4
SEQ ID NO: 729	tctcctttcttcatcttca	10205	10224	1	4
SEQ ID NO: 730	ttggtagagcaagggttca	7848	7867	1	4
SEQ ID NO: 731	cctcctacagtggtggcaa	4222	4241	1	4
SEQ ID NO: 732	tgaaaacgacaaagcaatc	9595	9614	3	3
SEQ ID NO: 733	acttttctaaacttgaaat	9055	9074	3	3
SEQ ID NO: 734	tcagcccagccatttgaga	9228	9247	3	3
SEQ ID NO: 735	gctggatgtaaccaccagc	11177	11196	2	3
SEQ ID NO: 736	catcagaaccattgaccag	8126	8145	2	3
SEQ ID NO: 737	actcaatggtgaaattcag	7457	7476	2	3
SEQ ID NO: 738	cctcacttcctttggactg	8969	8988	2	3
SEQ ID NO: 739	tgcaaacttgacttcagag	11391	11410	2	3
SEQ ID NO: 740	atgacgttcttgagcatgt	7042	7061	2	3
SEQ ID NO: 741	actggacttctctagtcag	8801	8820	2	3
SEQ ID NO: 742	atgcctacgttccatgtcc	11346	11365	2	3
SEQ ID NO: 743	gagaagtgtcttcaaagct	12403	12422	2	3
SEQ ID NO: 744	gagatcaacacaatcttca	13104	13123	2	3
SEQ ID NO: 745	ctgaattactgcacctcag	3027	3046	2	3
SEQ ID NO: 746	ttggtagagcaagggttca	7848	7867	2	3
SEQ ID NO: 747	tggcactgtttggagaagg	9130	9149	2	3
SEQ ID NO: 748	gcaagtcagcccagttcct	10920	10939	2	3
SEQ ID NO: 749	atgaaaccaatgacaaaat	7420	7439	2	3
SEQ ID NO: 750	tgaaatacaatgctctgga	5512	5531	2	3
SEQ ID NO: 751	gaaataccaagtcaaaacc	10447	10466	2	3
SEQ ID NO: 752	tgaaaaagctgcaatcaac	13726	13745	2	3
SEQ ID NO: 753	aactgcttctccaaatgga	3544	3563	2	3
SEQ ID NO: 754	agaattcataatcccaact	8267	8286	2	3
SEQ ID NO: 755	caaaacctactgtctcttc	10459	10478	2	3
SEQ ID NO: 756	cttcacataccagaattcc	8316	8335	2	3
SEQ ID NO: 757	aacaaacacaggcattcca	9648	9667	2	3
SEQ ID NO: 758	cttcatgtccctagaaatc	10029	10048	2	3
SEQ ID NO: 759	cttcagcctgctttctgga	4943	4962	2	3
SEQ ID NO: 760	cattagagctgccagtcct	10012	10031	2	3
SEQ ID NO: 761	tgaagatgacgacttttct	12152	12171	2	3
SEQ ID NO: 762	tgccagtttgaaaaacaaa	11807	11826	2	3
SEQ ID NO: 763	aatgtcagctcttgttcag	10895	10914	2	3
SEQ ID NO: 764	tcatttgccctcaacctac	11442	11461	2	3
SEQ ID NO: 765	gggaactgttgaaagattt	12919	12938	2	3
SEQ ID NO: 766	caggagaacttactatcat	13777	13796	2	3
SEQ ID NO: 767	aactcctccactgaaagat	9539	9558	2	3
SEQ ID NO: 768	acttccgtttaccagaaat	8239	8258	2	3
SEQ ID NO: 769	cagagctttctgccagtgc	13510	13529	2	3
SEQ ID NO: 770	aaattcaaactgcctatat	13866	13885	2	3
SEQ ID NO: 771	aaatacttccacaaattga	8772	8791	2	3
SEQ ID NO: 772	attgaacatccccaaactg	8786	8805	2	3
SEQ ID NO: 773	tttcaactgcctttgtgta	11221	11240	2	3
SEQ ID NO: 774	ttattgctgaatccaaaag	13648	13667	2	3
SEQ ID NO: 775	ttttcaagcaaatgcacaa	8531	8550	2	3
SEQ ID NO: 776	aaaagaaaattttgctgga	10748	10767	2	3
SEQ ID NO: 777	tgaagtagaccaacaaatc	7154	7173	2	3
SEQ ID NO: 778	aaactaaatgatctaaatt	11316	11335	2	3
SEQ ID NO: 779	gcaatttctgcacagaaat	13433	13452	2	3
SEQ ID NO: 780	aaagccattcagtctctca	12963	12982	2	3
SEQ ID NO: 781	aattccatatgaaagtcaa	12652	12671	1	3
SEQ ID NO: 782	agaatattttgatccaagt	13268	13287	2	3
SEQ ID NO: 783	aaatctggatttcttaaac	9473	9492	2	3
SEQ ID NO: 784	aaataaatggagtctttat	14075	14094	2	3
SEQ ID NO: 785	ctcagttaactgtgtcccg	11563	11582	1	3
SEQ ID NO: 786	agcatctgattgactcact	12670	12689	1	3
SEQ ID NO: 787	gctgattgaggtgtccagc	1217	1236	1	3
SEQ ID NO: 788	ctggatcacagagtccctc	3744	3763	1	3
SEQ ID NO: 789	ggccctgatccccgagccc	1355	1374	1	3
SEQ ID NO: 790	ctcccggagccaaggctgg	2674	2693	1	3
SEQ ID NO: 791	gctgttttgaagactctcc	1080	1099	1	3
SEQ ID NO: 792	tttcaagttcctgaccttc	8301	8320	1	3
SEQ ID NO: 793	aatcttattggggattttg	7077	7096	1	3
SEQ ID NO: 794	cttccacatttcaaggaat	10059	10078	1	3
SEQ ID NO: 795	ccagcaagtacctgagaac	8602	8621	1	3
SEQ ID NO: 796	acttgaagaaaagatagtc	13316	13335	1	3
SEQ ID NO: 797	cagtgaagctgcagggcac	10696	10715	1	3
SEQ ID NO: 798	ctcacctccacctctgatc	4740	4759	1	3
SEQ ID NO: 799	tccactcacatcctccagt	1281	1300	1	3
SEQ ID NO: 800	gatgtggtcacctacctgg	1335	1354	1	3
SEQ ID NO: 801	tggtgctggagaatgagct	5104	5123	1	3
SEQ ID NO: 802	gctggagtaaaactggaag	2688	2707	1	3
SEQ ID NO: 803	ttcaagatgactgcactgg	1531	1550	1	3
SEQ ID NO: 804	cctcacagagctatcactg	5222	5241	1	3
SEQ ID NO: 805	gcccactggtcgcctgcca	3525	3544	1	3
SEQ ID NO: 806	tttgagccaacattggaag	2199	2218	1	3
SEQ ID NO: 807	ctttgacaggcattttgaa	9719	9738	1	3
SEQ ID NO: 808	cttgaaattcaatcacaag	9066	9085	1	3
SEQ ID NO: 809	ttctgctgccttatcagca	5639	5658	1	3
SEQ ID NO: 810	ctggtcagtttgcaagcaa	2996	3015	1	3
SEQ ID NO: 811	cctggtcagtttgcaagca	2995	3014	1	3
SEQ ID NO: 812	ctcacatcctccagtggct	1285	1304	1	3
SEQ ID NO: 813	ggaactaccacaaaaagct	7481	7500	1	3
SEQ ID NO: 814	gaaatcttcaatttattct	13813	13832	1	3
SEQ ID NO: 815	aggacaccaaaataacctt	7564	7583	1	3
SEQ ID NO: 816	taagaactttgccacttct	4844	4863	1	3
SEQ ID NO: 817	ccctaacagatttgaggat	7969	4988	1	3
SEQ ID NO: 818	cccctaacagatttgagga	7968	7987	1	3
SEQ ID NO: 819	tgcctgcctttgaagtcag	7900	7919	1	3
SEQ ID NO: 820	tgataaaaaccaagatgtt	6290	6309	1	3
SEQ ID NO: 821	atgataaaaaccaagatgt	6289	6308	1	3
SEQ ID NO: 822	accaccagtttgtagatga	7405	7424	1	3
SEQ ID NO: 823	tcttccacatttcaaggaa	10058	10077	1	3
SEQ ID NO: 824	acaccttccacattccttc	8071	8090	1	3
SEQ ID NO: 825	acactaaatacttccacaa	8767	8786	1	3
SEQ ID NO: 826	gtggaggcaacacattaca	2920	2939	1	3
SEQ ID NO: 827	aaagaaacagcatttgttt	4532	4551	1	3
SEQ ID NO: 828	tcttacttttccattgagt	10572	10591	1	3
SEQ ID NO: 829	cctccagctcctgggaaag	2483	2502	1	3
SEQ ID NO: 830	ttcctaaagctggatgtaa	11169	11188	1	3
SEQ ID NO: 831	ccacaagtcatcatctcgt	5956	5975	1	3
SEQ ID NO: 832	gccagaagtgagatcctcg	3507	3526	1	3
SEQ ID NO: 833	tgccagtctccatgacctc	2468	2487	1	3
SEQ ID NO: 834	gttgctcttaaggacttcc	13356	13375	1	3
SEQ ID NO: 835	tgttgatgaggagtccttc	1801	1820	1	3
SEQ ID NO: 836	gcaagtctttcctggcctg	3011	3030	1	3
SEQ ID NO: 837	agcaagtctttcctggcct	3010	3029	1	3
SEQ ID NO: 838	atgaaagtcaagcatctga	12660	12679	1	3
SEQ ID NO: 839	gctgactttaaaatctgac	4811	4830	1	3
SEQ ID NO: 840	atgcactgtttctgagtcc	9331	9350	1	3
SEQ ID NO: 841	ggaatatcttagcatcctt	13457	13476	1	3
SEQ ID NO: 842	aggaatatcttagcatcct	13456	13475	1	3
SEQ ID NO: 843	aaggctgactctgtggttg	4284	4303	1	3
SEQ ID NO: 844	aaagcaggccgaagctgtt	1067	1086	1	3
SEQ ID NO: 845	atccatgatctacatttgt	6786	6805	1	3
SEQ ID NO: 846	catcactttacaagccttc	1238	1257	1	3
SEQ ID NO: 847	gtctcttcgttctatgcta	4584	4603	1	3
SEQ ID NO: 848	agtctcttcgttctatgct	4583	4602	1	3
SEQ ID NO: 849	aagtgtagtctcctggtgc	5091	5110	1	3
SEQ ID NO: 850	tttgaggattccatcagtt	7979	7998	1	3
SEQ ID NO: 851	gtacctacttttggcaagc	8364	8383	1	3
SEQ ID NO: 852	cttatgggatttcctaaag	11159	11178	1	3
SEQ ID NO: 853	gagggtagtcataacagta	10329	10346	1	3
SEQ ID NO: 854	tggaagtgtcagtggcaaa	10372	10391	1	3
SEQ ID NO: 855	gatggatatgaccttctct	4868	4887	1	3
SEQ ID NO: 856	gagaacatactgggcagct	5872	5891	1	3
SEQ ID NO: 857	gagaaaatcaatgccttca	7104	7123	1	3
SEQ ID NO: 858	agagccaggtcgagctttc	11044	11063	1	3
SEQ ID NO: 859	tctgatgaggaaactcaga	12252	12271	1	3
SEQ ID NO: 860	aacctcccattttttgaga	6318	6337	1	3
SEQ ID NO: 861	ctgatccccgagccctcag	1359	1376	1	3
SEQ ID NO: 862	gagaaaatcaatgccttca	7104	7123	1	3
SEQ ID NO: 863	aagaggcagcttctggctt	12289	12308	1	3
SEQ ID NO: 864	atcaaaagaagcccaagag	12938	12957	1	3
SEQ ID NO: 865	tcaaagttaattgggaaga	12271	12290	1	3
SEQ ID NO: 866	ctcaattttgattttcaag	8520	8539	1	3
SEQ ID NO: 867	gatggaaccctctccctca	4725	4744	1	3
SEQ ID NO: 868	ctgacatcttaggcactga	4993	5012	1	3
SEQ ID NO: 869	ttcagaagctaagcaatgt	7231	7250	1	3
SEQ ID NO: 870	tctctgaaagacaacgtgc	12315	12334	1	3
SEQ ID NO: 871	agataacattaaacagctg	13043	13062	1	3
SEQ ID NO: 872	ggctcaacacagacatcgc	5710	5729	1	3
SEQ ID NO: 873	tcccagaaaacctcttctt	3928	3947	1	3
SEQ ID NO: 874	caatggagagtccaacctg	4652	4671	1	3
SEQ ID NO: 875	gcaagggttcactgttcct	7856	7875	1	3
SEQ ID NO: 876	aattgggaagaagaggcag	12279	12298	1	3
SEQ ID NO: 877	gaatattttgagaggaatc	6345	6364	1	3
SEQ ID NO: 878	atgaagtagaccaacaaat	7153	7172	1	3
SEQ ID NO: 879	atttgttagaaaatacaga	6428	6447	1	3
SEQ ID NO: 880	agtttctccatcctaggtt	9954	9973	1	3
SEQ ID NO: 881	ttctgaaaatccaatctcc	8392	8411	1	3
SEQ ID NO: 882	aagatcgcagactttgagt	11646	11665	1	3
SEQ ID NO: 883	tgaactcagaagaattgga	1912	1931	1	3
SEQ ID NO: 884	cagtcatgtagaaaaactt	4421	4440	1	3
SEQ ID NO: 885	ctggaactctctccatggc	10875	10894	1	3
SEQ ID NO: 886	tctgaactcagaaggatgg	13991	14010	1	3
SEQ ID NO: 887	ggatttcctaaagctggat	11165	11184	1	3
SEQ ID NO: 888	cctgaaatacaatgctctg	5510	5529	1	3
SEQ ID NO: 889	agaaacagcatttgtttgt	4534	4553	1	3
SEQ ID NO: 890	agaagctaagcaatgtcct	7234	7253	1	3
SEQ ID NO: 891	tgaaggctgactctgtggt	4282	4301	1	3
SEQ ID NO: 892	atcaggaagggctcaaaga	2559	2578	1	3
SEQ ID NO: 893	tcattactcctgggctgaa	11299	11318	1	3
SEQ ID NO: 894	tgaatctggctccctccac	9038	9057	1	3
SEQ ID NO: 895	ttaatcgagaggtatgaag	7140	7159	1	3
SEQ ID NO: 896	gttaatcgagaggtatgaa	7139	7158	1	3
SEQ ID NO: 897	aattgcattagatgatgcc	6581	6600	1	3
SEQ ID NO: 898	tggagtttgtgacaaatat	2752	2771	1	3
SEQ ID NO: 899	agaaacagcatttgtttgt	4534	4553	1	3
SEQ ID NO: 900	caaatgacatgatgggctc	5326	5345	1	3
SEQ ID NO: 901	aagcatctgattgactcac	12669	12688	1	3
SEQ ID NO: 902	tgggcctgccccagattct	8901	8920	1	3
SEQ ID NO: 903	caataagatcaatagcaaa	8990	9009	1	3
SEQ ID NO: 904	ttggctcacatgaaggcca	7623	7642	1	3
SEQ ID NO: 905	gatatacactagggaggaa	12737	12756	1	3
SEQ ID NO: 906	agatcaaagttaattggga	12268	12287	1	3
SEQ ID NO: 907	gagtcccagtgcccagcaa	9344	9363	1	3
SEQ ID NO: 908	tcagtataagtacaaccaa	9392	9411	1	3
SEQ ID NO: 909	aacttccaactgtcatgga	1978	1997	1	3
SEQ ID NO: 910	ctttgaagtcagtcttcag	7907	7926	1	3
SEQ ID NO: 911	agaatctcaacttccaact	1970	1989	1	3
SEQ ID NO: 912	acaggggtcctttatgatt	12342	12361	1	3
SEQ ID NO: 913	atttgaaagaataaatgac	7028	7047	1	3
SEQ ID NO: 914	aacacattgaggctattga	6970	6989	1	3
SEQ ID NO: 915	gaaaaaggggattgaagtt	1276	10295	1	3
SEQ ID NO: 916	ataagcaaactgttaattt	5449	5468	1	3
SEQ ID NO: 917	aaatgcactgctgcgttct	4892	4911	1	3
SEQ ID NO: 918	caaaaacattttcaacttc	5279	5298	1	3
SEQ ID NO: 919	aaggaagaaagaaaaatca	3453	3472	1	3
SEQ ID NO: 920	gtcagcccagttccttcca	10924	10943	1	3
SEQ ID NO: 921	tgaggaaactcagatcaaa	12257	12276	1	3
SEQ ID NO: 922	aaagcattggtagagcaag	7842	7861	1	3
SEQ ID NO: 923	tcaagtctgtgggattcca	4078	4097	1	3
SEQ ID NO: 924	ccaagaggtatttaaagcc	12950	12969	1	3
SEQ ID NO: 925	agctttctgccactgctca	13513	13532	1	3
SEQ ID NO: 926	gagctttctgccactgctc	13512	13531	1	3
SEQ ID NO: 927	aaaagaaacagcatttgtt	4531	4550	1	3
SEQ ID NO: 928	ttccggcacgtgggttcca	3777	3796	1	3
SEQ ID NO: 929	ccttactgactttgcagag	7790	7809	1	3
SEQ ID NO: 930	aatcattgaaaaattaaaa	6722	6741	1	3
SEQ ID NO: 931	ttgatgaaatcattgaaaa	6715	6734	1	3
SEQ ID NO: 932	tcattgctcccggagccaa	2668	2687	1	3
SEQ ID NO: 933	tgttgcttttgtaaagtat	6272	6291	1	3
SEQ ID NO: 934	catttcagccttcgggctc	4254	4273	1	3
SEQ ID NO: 935	gcatgcctagtttctccat	9946	9965	1	3
SEQ ID NO: 936	agcacagtacgaaaaacca	10801	10820	1	3
SEQ ID NO: 937	ctgaaagagatgaaattta	13059	13078	1	3
SEQ ID NO: 938	aacagatttgaggattcca	7973	7992	1	3
SEQ ID NO: 939	atcacaactcctccactga	9534	9553	1	3
SEQ ID NO: 940	aatcacaactcctccactg	9533	9552	1	3
SEQ ID NO: 941	taatcacaactcctccact	9532	9551	1	3
SEQ ID NO: 942	ggatactaagtaccaaatc	6866	6885	1	3
SEQ ID NO: 943	cttccgtttaccagaaatc	8240	8259	1	3
SEQ ID NO: 944	acttccgtttaccagaaat	8239	8258	1	3
SEQ ID NO: 945	ttccaatttccctgtggat	3680	3699	1	3
SEQ ID NO: 946	cttccaatttccctgtgga	3679	3698	1	3
SEQ ID NO: 947	agattaatccgctggctct	8563	8582	1	3
SEQ ID NO: 948	aagattaatccgctggctc	8562	8581	1	3
SEQ ID NO: 949	accactgggacctaccaag	12519	12538	1	3
SEQ ID NO: 950	actggtggcaaaaccctcc	2726	2745	1	3
SEQ ID NO: 951	tggagaagccacactccaa	10763	10782	1	3
SEQ ID NO: 952	ctggtcgcctgccaaactg	3530	3549	1	3
SEQ ID NO: 953	ggagtcattgctcccggag	2664	2683	1	3
SEQ ID NO: 954	tcagaaagctaccttccag	7931	7950	1	3
SEQ ID NO: 955	agccagaagtgagatcctc	3506	3525	1	3
SEQ ID NO: 956	gaaggcatctgggagtctt	3827	3846	1	3
SEQ ID NO: 957	gatgcttacaacactaaag	6099	6118	1	3
SEQ ID NO: 958	ggcacttccaaaattgatg	10710	10729	1	3
SEQ ID NO: 959	aaagttaattgggaagaag	12273	12292	1	3
SEQ ID NO: 960	tgggctggcttcagccatt	5729	5748	1	3
SEQ ID NO: 961	cagtctgaacattgcaggc	5375	5394	1	3
SEQ ID NO: 962	cagtgcaacgaccaacttg	5072	5091	1	3
SEQ ID NO: 963	tactccaacgccagctcca	3051	3070	1	3
SEQ ID NO: 964	ctgccatctcgagagttcc	4098	4117	1	3
SEQ ID NO: 965	tgcctttgtgtacaccaaa	11228	11247	1	3
SEQ ID NO: 966	gatgggtctctacgccaca	4377	4396	1	3
SEQ ID NO: 967	cccaaggccacaggggtcc	12333	12352	1	3
SEQ ID NO: 968	gtgttctagacctctccac	4171	4190	1	3
SEQ ID NO: 969	ccagaatctgtaccaggaa	12554	12573	1	3
SEQ ID NO: 970	tgagaactacgagctgact	4799	4818	1	3
SEQ ID NO: 971	catgaaggccaaattccga	7631	7650	1	3
SEQ ID NO: 972	cttccagacacctgatttt	7943	7962	1	3
SEQ ID NO: 973	gaatttacaattgttgctt	6261	6280	1	3
SEQ ID NO: 974	gagcttcaggaagcttctc	13206	13225	1	3
SEQ ID NO: 975	accagtcagatattgttgc	10183	10202	1	3
SEQ ID NO: 976	tagaatatgaactaaatgt	11881	11900	1	3
SEQ ID NO: 977	gtagctgagaaaatcaatg	7098	7117	1	3
SEQ ID NO: 978	ggtggataccctgaagttt	3197	3216	1	3
SEQ ID NO: 979	aggaaaagcgcacctcaat	12023	12042	1	3
SEQ ID NO: 980	ccagcttccccacatctca	8333	8352	1	3
SEQ ID NO: 981	gtaagaaaatacagagcag	6432	6451	1	3
SEQ ID NO: 982	aggacagagccttggtgga	3184	3203	1	3
SEQ ID NO: 983	atctgatgaggaaactcag	12251	12270	1	3
SEQ ID NO: 984	ggcctctctggggcatcta	5136	5155	1	3
SEQ ID NO: 985	ctctcaaaaaaaagtatag	6541	6560	1	3
SEQ ID NO: 986	cttcaggaagcttctcaag	13209	13228	1	3
SEQ ID NO: 987	ccttacacaataatcacaa	9522	9541	1	3
SEQ ID NO: 988	gcacctagctggaaagtta	6947	6966	1	3
SEQ ID NO: 989	tctgtgggattccatctgc	4083	4102	1	3
SEQ ID NO: 990	gtctgtgggattccatctg	4082	4101	1	3
SEQ ID NO: 991	tcaccaacggagaacatac	10843	10862	1	3
SEQ ID NO: 992	ttcaccaacggagaacata	10842	10861	1	3
SEQ ID NO: 993	aatctcaagctttctcttc	10044	10063	1	3
SEQ ID NO: 994	tgtacaactggtccgcctc	4207	4226	1	3
SEQ ID NO: 995	ctgttaggacaccagccct	4054	4073	1	3
SEQ ID NO: 996	tgaaattcaatcacaagtc	9068	9087	1	3
SEQ ID NO: 997	tcttttcttttcagcccag	9216	9237	1	3
SEQ ID NO: 998	caactgcagacatatatga	6627	6646	1	3
SEQ ID NO: 999	tcactccattaacctccca	6308	6327	1	3
SEQ ID NO: 1000	cttcttttccaattgaact	13830	13849	1	3
SEQ ID NO: 1001	tcttcatcttcatctgtca	10212	10231	1	3
SEQ ID NO: 1002	ttcttcatcttcatctgtc	10211	10230	1	3
SEQ ID NO: 1003	cttgtcatgcctacgttcc	11340	11359	1	3
SEQ ID NO: 2007	gaccttgcaagaatatttt	6335	6354	1	3
SEQ ID NO: 2008	tgttaacaaattccttgac	7355	7374	1	3
SEQ ID NO: 2009	ttcaagttcctgaccttca	8302	8321	1	3
SEQ ID NO: 2010	gaatctggctccctcaact	9039	9058	1	3
SEQ ID NO: 2011	ggaaataccaagtcaaaac	10446	10465	1	3
SEQ ID NO: 2012	aaggaaaagcgcacctcaa	12022	12041	1	3
SEQ ID NO: 2013	cagttgaccacaagcttag	10537	10556	1	3
SEQ ID NO: 2014	tccttaacaccttccacat	8065	8084	1	3
SEQ ID NO: 2015	gacttctctagtcaggctg	8805	8824	1	3
SEQ ID NO: 2016	agacatcgctgggctggct	5720	5739	1	3
SEQ ID NO: 2017	ctctcaaatgacatgatgg	5322	5341	1	3
SEQ ID NO: 2018	gttgagaagccccaagaat	6246	6265	1	3
SEQ ID NO: 2019	gagatcaagacactgttga	8835	8854	1	3
SEQ ID NO: 2020	tggaaccctctccctcacc	4727	4746	1	3
SEQ ID NO: 2021	gctggtaacctaaaaggag	5580	5599	1	3
SEQ ID NO: 2022	gttgcccaccatcatcgtg	11663	11682	1	3
SEQ ID NO: 2023	agttgcccaccatcatcgt	11662	11682	1	3
SEQ ID NO: 2024	tgttagttgctcttaagga	13351	13370	1	3
SEQ ID NO: 2025	cagcaagtacctgagaacg	8603	8622	1	3
SEQ ID NO: 2026	aacctatgccttaatcttc	13161	13180	1	3
SEQ ID NO: 2027	ggaaagttaaaacaacaca	6957	6976	1	3
SEQ ID NO: 2028	cacaccttgacattgcagg	11080	11099	1	3
SEQ ID NO: 2029	gcacaccttgacattgcag	11079	11098	1	3
SEQ ID NO: 2030	atccgctggctctgaagga	8569	8588	1	3
SEQ ID NO: 2031	acgtccgtgtgccttcata	9976	9995	1	3
SEQ ID NO: 2032	gacgtccgtgtgccttcat	9975	9994	1	3
SEQ ID NO: 2033	tgattatctgaattcattc	6479	6498	1	3
SEQ ID NO: 2034	tgatttacatgatttgaaa	6677	6696	1	3
SEQ ID NO: 2035	tgaagtagctgagaaaatc	7094	7113	1	3
SEQ ID NO: 2036	tttgaaaaattctcttttc	9208	9225	1	3
SEQ ID NO: 2037	taaattcattactcctggg	11294	11313	1	3
SEQ ID NO: 2038	tattcaaaactgagttgag	12223	12242	1	3
SEQ ID NO: 2039	atattcaaaactgagttga	12222	12241	1	3
SEQ ID NO: 2040	gaatttgaaagttcgtttt	9272	9291	1	3
SEQ ID NO: 2041	gacagcatcttcgtgtttc	11206	11225	1	3
SEQ ID NO: 2042	acttaaaaaatataaaaat	8014	8033	1	3
SEQ ID NO: 2043	aactctcaagtcaagttga	13414	13433	1	3
SEQ ID NO: 2044	agaagatggcaaatttgaa	11987	12006	1	3
SEQ ID NO: 2045	atagcatggacttcttctg	8865	8884	1	3
SEQ ID NO: 2046	ccatttgagatcacggcat	9237	9256	1	3
SEQ ID NO: 2047	caagttggcaagtaagtgc	9364	9383	1	3
SEQ ID NO: 2048	taaagtgccacttttactc	6182	6201	1	3
SEQ ID NO: 2049	ttaacagggaagatagact	9300	9319	1	3
SEQ ID NO: 2050	ttggcaagtaagtgctagg	9368	9387	1	3
SEQ ID NO: 2051	gggaagaagaggcagcttc	12283	12302	1	3
SEQ ID NO: 2052	gatgaggaaactcagatca	12255	12274	1	3
SEQ ID NO: 2053	tgatgaggaaactcagatc	12254	12273	1	3
SEQ ID NO: 2054	tctcgtgtctaggaaaagc	5969	5988	1	3
SEQ ID NO: 2055	cagcttaagagacacatac	6912	6931	1	3
SEQ ID NO: 2056	tcattttccaactaataga	13024	13043	1	3
SEQ ID NO: 2057	gatacaagaaaaactgcag	6893	6912	1	3
SEQ ID NO: 2058	ggctcatatgctgaaatga	5340	5359	1	3
SEQ ID NO: 2059	ggaaggacaaggcccagaa	12541	12560	1	3
SEQ ID NO: 2060	atttttattcctgccatgg	10095	10114	1	3
SEQ ID NO: 2061	attttttgcaagttaaaga	14011	14030	1	3
SEQ ID NO: 2062	atccatgatctacatttgt	6786	6805	1	3
SEQ ID NO: 2063	cttcagggaacacaatgca	5177	5196	1	3
SEQ ID NO: 2064	gagatgagagatgccgttg	6231	6250	1	3
SEQ ID NO: 2065	attctcttttcttttcagc	9214	9233	1	3
SEQ ID NO: 2066	cagatacaagaaaaactgc	6891	6910	1	3
SEQ ID NO: 2067	ttcattcaattgggagaga	6491	6510	1	3
SEQ ID NO: 2068	atttgtaagaaaatacaga	9428	6447	1	3
SEQ ID NO: 2069	ctgaagcattaaaactgtt	7498	7517	1	3
SEQ ID NO: 2070	ccagatgctgaacagtgag	8141	8160	1	3
SEQ ID NO: 2071	gcctacgttccatgtccca	11348	11367	1	3
SEQ ID NO: 2072	cttcagtgcagaatatgaa	11969	11988	1	3
SEQ ID NO: 2073	atgattatctgaattcatt	6478	697	1	3
SEQ ID NO: 2074	ttcagccattgacatgagc	5738	5757	1	3
SEQ ID NO: 2075	aaatagctattgctaatat	6694	6713	1	3
SEQ ID NO: 2076	taagaaccagaagatcaga	10988	11007	1	3
SEQ ID NO: 2077	tgatatcgacgtgaggttc	12482	12501	1	3
SEQ ID NO: 2078	gtcctggattccacatgca	11844	11863	1	3
SEQ ID NO: 2079	aaattccttgacatgttga	7362	7381	1	3
SEQ ID NO: 2080	acttaaaaaatataaaaat	8014	8033	1	3
SEQ ID NO: 2081	cttacttgaattccaagag	10665	16085	1	3
SEQ ID NO: 2082	gctgcatgtggctggtaac	5570	5589	1	3
SEQ ID NO: 2083	taaaagattactttgagaa	7267	7286	1	3
SEQ ID NO: 2084	ttggcaagtaagtgctagg	9368	9387	1	3
SEQ ID NO: 2085	atttacaattgttgctttt	6263	6282	1	3
SEQ ID NO: 2086	attacctatgatttctcct	10119	10138	1	3
SEQ ID NO: 2087	atgtcaaacactttgttat	7057	7076	1	3
SEQ ID NO: 2088	gatgaagatgacgactttt	12150	12169	1	3
SEQ ID NO: 2089	cacaagtcgattcccagca	9079	9098	1	3
SEQ ID NO: 2090	tgaggtgactcagagactc	7442	7461	1	3
SEQ ID NO: 2091	gtcagtgaagttctccagc	8588	8607	1	3
SEQ ID NO: 2092	cggagcatgggagtgaaat	8620	8639	1	3
SEQ ID NO: 2093	acggagcatgggagtgaaa	8619	8638	1	3
SEQ ID NO: 2094	aacggagcatgggagtgaa	8618	8637	1	3
SEQ ID NO: 2095	agaagtgtcttcaaagctg	12404	12423	1	3
SEQ ID NO: 2096	cattcaattgggagagaca	6493	6512	1	3
SEQ ID NO: 2097	ccattcagtctctcaagac	12967	12986	1	3
SEQ ID NO: 2098	cagataaaaaactcaccat	12205	12224	1	3
SEQ ID NO: 2099	gctgttttgaagactctcc	1080	1099	1	3
SEQ ID NO: 2100	cagaattcataatcccaac	8266	8285	1	3
SEQ ID NO: 2101	actgcaagatttttcagac	13604	13623	1	3
SEQ ID NO: 2102	caagaacctgttagttgct	13343	13362	1	3
SEQ ID NO: 2103	gcacatcaatattgatcaa	6410	6429	1	3
SEQ ID NO: 2104	tttcagatggcattgctgc	11602	11621	1	3
SEQ ID NO: 2105	aaatcccatccaggttttc	8029	8048	1	3
SEQ ID NO: 2106	tcctttggctgtgctttgt	9674	9693	1	3
SEQ ID NO: 2107	agtgaagttctccagcaag	8591	8610	1	3
SEQ ID NO: 2108	ccagaattcataatcccaa	8265	8284	1	3
SEQ ID NO: 2109	ggctattgatgttagagtg	6980	6999	1	3
SEQ ID NO: 2110	ggcatgatgctcatttaaa	9169	9188	1	3
SEQ ID NO: 2111	taaagccattcagtctctc	12962	12981	1	3
SEQ ID NO: 2112	tttaaccagtcagatattg	10179	10198	1	3
SEQ ID NO: 2113	tttattgctgaatccaaaa	13647	13666	1	3
SEQ ID NO: 2114	ttttgagaggaatcgacaa	6350	6369	1	3
SEQ ID NO: 2115	gggaaaaaacaggcttgaa	9568	9587	1	3
SEQ ID NO: 2116	ttctctctatgggaaaaaa	9558	9577	1	3
SEQ ID NO: 2117	caaaagaagcccaagaggt	12940	12959	1	3
SEQ ID NO: 2118	acaaagcagattatgttga	11821	11840	1	3
SEQ ID NO: 2119	ttttcagaccaactctctg	13614	13633	1	3
SEQ ID NO: 2120	ctgtctctggtcagccagg	7716	7735	1	3
SEQ ID NO: 2121	attacacttcctttcgagt	12861	12880	1	3
SEQ ID NO: 2122	tctcttcctccatggaatt	10471	10490	1	3
SEQ ID NO: 2123	cttggagtgccagtttgaa	11800	11819	1	3
SEQ ID NO: 2124	agagcttatgggatttcct	11155	11174	1	3
SEQ ID NO: 2125	ttttggcaagctatacagt	8372	8391	1	3
SEQ ID NO: 2126	ctttgtgagtttatcagtc	9687	9706	1	3
SEQ ID NO: 2127	attggatatccaagatctg	1925	1944	1	3
SEQ ID NO: 2128	ttaaaagaaatcttcaatt	13807	13826	1	3
SEQ ID NO: 2129	tcaatgattatatcccata	13120	13139	1	3
SEQ ID NO: 2130	agcacagaaaaaattcaaa	13856	13875	1	3
SEQ ID NO: 2131	tagcacagaaaaaattcaa	13855	13874	1	3
SEQ ID NO: 2132	aataaatggagtctttatt	14076	14095	1	3
SEQ ID NO: 2133	caataccagaattcataat	8260	8279	1	3
SEQ ID NO: 2134	tagtgattacacttccttt	12856	12875	1	3
SEQ ID NO: 2135	atagcaacactaaatactt	8761	8780	1	3
SEQ ID NO: 2136	atatccaagatgagatcaa	13093	13112	1	3
SEQ ID NO: 2137	attgagattccctccatta	11694	11713	1	3
SEQ ID NO: 2138	tggagtgccagtttgaaaa	11802	11821	1	3
SEQ ID NO: 2139	atttcctaaagctggatgt	11167	11186	1	3
SEQ ID NO: 2140	cactgttccagttgtcaat	9863	9882	1	3
SEQ ID NO: 2141	cttcaaagacttaaaaaat	8006	8025	1	3
SEQ ID NO: 2142	tgtaccataagccatattt	10080	10099	1	3
SEQ ID NO: 2143	ttttctaaacttgaaattc	9057	9076	1	3
SEQ ID NO: 2144	ttcttaaacattcctttaa	9483	9502	1	3
SEQ ID NO: 2145	ttccaatttccctgtggat	3680	3699	1	3
SEQ ID NO: 2146	aaagtgccacttttactca	6183	6202	1	3
SEQ ID NO: 2147	caaatgacatgatgggctc	5326	5345	1	3
SEQ ID NO: 2148	gattatatcccatatgttt	13125	13144	1	3
SEQ ID NO: 2149	gaaggaaaagcgcacctca	12021	12040	1	3
SEQ ID NO: 2150	tttgtggagggtagtcata	10323	10342	1	3
SEQ ID NO: 2151	tggatgaagatgacgactt	12148	12167	1	3
SEQ ID NO: 2152	gaataccaatgctgaactt	10160	10179	1	3
SEQ ID NO: 2153	atctaaattcagttcttgt	11326	11345	1	3
SEQ ID NO: 2154	caaaatagaagggaatctt	2069	2088	1	3
SEQ ID NO: 5255	ctaaacttgaaattcaatc	9061	9080	1	3
SEQ ID NO: 2156	aaatccgtgaggtgactca	7435	7454	1	3
SEQ ID NO: 2157	aaatccgtgaggtgactca	10411	10430	1	3
SEQ ID NO: 5158	caattttgagaatgaattt	9945	9964	1	3
SEQ ID NO: 2159	agcatgcctagtttctcca	7414	7433	1	3
SEQ ID NO: 2160	ttgtagatgaaaccaatga	7413	7432	1	3
SEQ ID NO: 2161	tttgtagatgaaaccaatg	10487	10506	1	3
SEQ ID NO: 2162	atttaagtatgatttcaat	10486	10505	1	3
SEQ ID NO: 2163	aatttaagtatgatttcaa	10485	10504	1	3
SEQ ID NO: 2164	gaatttaagtatgatttca	11479	11498	1	3
SEQ ID NO: 2165	caaattgaacatccccaaa	8783	8802	1	3
SEQ ID NO: 2166	agtccccctaacagatttg	7964	7983	1	3
SEQ ID NO: 2167	gagtgaaatgctgtttttt	8630	8649	1	3
SEQ ID NO: 2168	ttgatgatatctggaacct	10723	10742	1	3
SEQ ID NO: 2169	tccaatctcctcttttcac	8401	8420	1	3
SEQ ID NO: 2170	tttcaagcaaatgcacaac	8532	8551	1	3
SEQ ID NO: 2171	tttcaagcaaatgcacaac	10165	10184	1	3
SEQ ID NO: 2172	ccaatgctgaactttttaa	10205	10224	1	3
SEQ ID NO: 2173	tctcctttcttcatcttca	11013	11032	1	3
SEQ ID NO: 2174	gttgagaagccccaagaat	6246	6265	1	3
SEQ ID NO: 2175	tggcaagtaagtgctaggt	9369	9388	1	3
SEQ ID NO: 2176	ctggacttctctagtcagg	8802	8821	1	3
SEQ ID NO: 2177	gctaaaggagcagttgacc	10527	10546	1	3
SEQ ID NO: 2178	aagtccggattcattctgg	11017	11036	1	3
SEQ ID NO: 2179	tgacctgtccattcaaaac	13673	13692	1	3
SEQ ID NO: 2180	agaaaaaggggattgaagt	10275	10294	1	3
SEQ ID NO: 2181	gcaagttaaagaaaatcag	14018	14037	1	3
SEQ ID NO: 2182	gctcatctcctttcttcat	10200	10219	1	3
SEQ ID NO: 2183	tgctcatctcctttcttca	10199	10218	1	3
SEQ ID NO: 2184	tttcaccatagaaggaccc	8951	8970	1	3
SEQ ID NO: 2185	gtccccctaacagatttga	7965	7984	1	3
SEQ ID NO: 2186	ggcaccagggctcggaagg	13970	13989	1	3
SEQ ID NO: 2187	gcttgaaggaattcttgaa	9580	9599	1	3
SEQ ID NO: 2188	tcttcataagttcaatgaa	13175	13194	1	3
SEQ ID NO: 2189	ttttaacaaaagtggaagt	6821	6840	1	3
SEQ ID NO: 2190	tggagaagcaaatctggat	9464	9483	1	3
SEQ ID NO: 2191	tgttgaagtgtctccattc	9881	9900	1	3
SEQ ID NO: 2192	aattccaattttgagaatg	10406	10425	1	3
SEQ ID NO: 2193	ctgttgaaagatttatcaa	12924	12943	1	3
SEQ ID NO: 2194	gaagttctcaattttgatt	8514	8533	1	3
SEQ ID NO: 2195	ttcttctggaaaagggtca	8876	8895	1	3
SEQ ID NO: 2196	agattctcagatgagggaa	8913	8932	1	3
SEQ ID NO: 2197	tcagatggcattgctgctt	11604	11623	1	3
SEQ ID NO: 2198	gagataaccgtgcctgaat	11544	11563	1	3
SEQ ID NO: 2199	attttgaaaaaaacagaaa	9730	9749	1	3
SEQ ID NO: 2200	ttccatcacaaatcctttg	9662	9681	1	3
SEQ ID NO: 2201	actttacttcccaactctc	13402	13421	1	3
SEQ ID NO: 2202	aactttacttcccaactct	13401	13420	1	3
SEQ ID NO: 2203	ttgattcccttttttgaga	11529	11548	1	3
SEQ ID NO: 2204	tgctgaatccaaaagattg	13652	13671	1	3
SEQ ID NO: 2205	ggtttatcaaggggccatt	12452	12471	1	3
SEQ ID NO: 2206	aggttccatcgtgcaaact	11380	11399	1	3
SEQ ID NO: 2207	tgctccaggagaacttact	13772	13791	1	3
SEQ ID NO: 2208	aactctcaagtcaagttga	13414	13433	1	3
SEQ ID NO: 2209	tccattctgaatatattgt	13372	13391	1	3
SEQ ID NO: 2210	attttctgaacttccccag	12694	12713	1	3
SEQ ID NO: 2211	atctgatgaggaaactcag	12251	12270	1	3
SEQ ID NO: 2212	ttcatgtccctagaaatct	10030	10049	1	3
SEQ ID NO: 2213	tcaaggataacgtgtttga	12610	12629	1	3
SEQ ID NO: 2214	ttgatgatgctgtcaagaa	7300	7319	1	3
SEQ ID NO: 2215	cgacgaagaaaataatttc	13558	13577	1	3
SEQ ID NO: 2216	ttccagaaagcagccagtg	12498	12517	1	3
SEQ ID NO: 2217	cttccccaaagagaccagt	2890	2909	1	3
SEQ ID NO: 2218	ctgattactatgaaaaatt	13630	13649	1	3
SEQ ID NO: 2219	atggaaaagggaaagagaa	13486	13505	1	3
SEQ ID NO: 2220	tggaagtgtcagtggcaaa	10372	10937	1	3
SEQ ID NO: 2221	aggacctttcaaattcctg	9840	9859	1	3
SEQ ID NO: 2222	aaatcaggatctgagttat	14030	14049	1	3
SEQ ID NO: 2223	tccattctgaatatattgt	13372	13391	1	3
SEQ ID NO: 2224	tgctggaattgtcattcct	11726	11745	1	3
SEQ ID NO: 2225	tagattctctgtacctgct	11711	11730	1	3
SEQ ID NO: 2226	ttcaaaacgagcttcagga	13198	13217	1	3
SEQ ID NO: 2227	catttgatttaagtgtaaa	9613	9632	1	3
SEQ ID NO: 2228	ggagacagcatcttcgtgt	11203	11222	1	3
SEQ ID NO: 2229	tcccagaaaacctcttctt	3928	3947	1	3
SEQ ID NO: 2230	ccttttacaattcattttc	13013	13032	1	3
SEQ ID NO: 2231	ttttgagaatgaatttcaa	10414	10433	1	3
SEQ ID NO: 2232	gttttggctgataaattca	11283	11302	1	3
SEQ ID NO: 2233	gtttgataagtacaaagct	9797	9816	1	3
SEQ ID NO: 2234	tgcctgagcagaccattga	11680	11699	1	3
SEQ ID NO: 2235	aactttgcactatgttcat	12754	12773	1	3
SEQ ID NO: 2236	aacacatgaatcacaaatt	8930	8949	1	3
SEQ ID NO: 2237	tcaaaacgagcttcaggaa	13199	13218	1	3
SEQ ID NO: 2238	atgggaagtataagaactt	4834	4853	1	3
SEQ ID NO: 2239	agaaaaggcacaccttgac	11072	11091	1	3
SEQ ID NO: 2240	gtaagaaaatacagagcag	6432	6451	1	3
SEQ ID NO: 2241	agttgaaggagactattca	7216	7235	1	3
SEQ ID NO: 2242	aaggaaacataaactaata	12881	12900	1	3
SEQ ID NO: 2243	agaagaaatctgcagaaca	12423	12442	1	3
SEQ ID NO: 2244	gcagtagactataagcaga	13920	13939	1	3
SEQ ID NO: 2245	ctcagggatctgaaggtgg	8187	8206	1	3
SEQ ID NO: 2246	atgaagtagaccaacaaat	7153	7172	1	3
SEQ ID NO: 2247	gccacactccaacgcatat	10770	10789	1	3
SEQ ID NO: 2248	ttttacaattcattttcca	13015	13034	1	3
SEQ ID NO: 2249	agacctagtgattacactt	12851	12870	1	3
SEQ ID NO: 2250	ccatgcaagtcagcccagt	10916	10935	1	3
SEQ ID NO: 2251	ttaatcgagaggtatgaag	7140	7159	1	3
SEQ ID NO: 2252	gagttgagggtccgggaat	12234	12253	1	3
SEQ ID NO: 2253	aagcgcacctcaatatcaa	12028	12047	1	3
SEQ ID NO: 2254	ggaactattgctagtgagg	10641	10660	1	3
SEQ ID NO: 2255	ttgggaagaagaggcagct	12281	12300	1	3
SEQ ID NO: 2256	gatatacactagggaggaa	12737	12756	1	3
SEQ ID NO: 2257	gcttggttttgccagtctc	2459	2476	1	3
SEQ ID NO: 2258	aagaggtatttaaagccat	12952	12971	1	3
SEQ ID NO: 2259	caagaggtatttaaagcca	12951	12970	1	3
SEQ ID NO: 2260	cttgggggaggaggaacaa	14058	14077	1	3
SEQ ID NO: 2261	acttgggggaggaggaaca	14057	14076	1	3
SEQ ID NO: 2262	ggaatctgatgaggaaact	12248	12267	1	3
SEQ ID NO: 2263	ctggatgtaaccaccagca	11178	11197	1	3
SEQ ID NO: 2264	tgatcaagaacctgttagt	13339	13358	1	3
SEQ ID NO: 2265	ctgatcaagaacctgttag	13338	13357	1	3
SEQ ID NO: 2266	ttttcagaccaactctctg	13614	13633	1	3
SEQ ID NO: 2267	gaatttgaaagttcgtttt	9272	9291	1	3
SEQ ID NO: 2268	gaaaacctatgccttaatc	13158	13177	1	3
SEQ ID NO: 2269	tcaaaacctactgtctctt	10458	10477	1	3
SEQ ID NO: 2270	aggacaccaaaataacctt	7564	7583	1	3
SEQ ID NO: 2271	tgtcaacaagtaccactgg	12362	12381	1	3
SEQ ID NO: 2272	gtttttaaattgttgaaag	13140	13159	1	3
SEQ ID NO: 2273	aaaagggtcatggaaatgg	8885	8904	1	3
SEQ ID NO: 2274	aagatagtcagtctgatca	13326	13345	1	3
SEQ ID NO: 2275	atgagatcaacacaatctt	13102	13121	1	3
SEQ ID NO: 2276	ttggtacgagttactcaaa	12633	12652	1	3
SEQ ID NO: 2277	ctcaattttgattttcaag	8520	8539	1	3
SEQ ID NO: 2278	cactcattgattttctgaa	12685	12704	1	3
SEQ ID NO: 2279	agcagattatgttgaaaca	11825	11844	1	3
SEQ ID NO: 2280	tcttttcagcccagccatt	9223	9242	1	3
SEQ ID NO: 2281	atctgatgaggaaactcag	12251	12270	1	3
SEQ ID NO: 2282	aatctgatgaggaaactca	12250	12269	1	3
SEQ ID NO: 2283	ttaatcttcataagttcaa	13171	13190	1	3
SEQ ID NO: 2284	tcaattgggagagacaagt	6496	6515	1	3
SEQ ID NO: 2285	ctgagaacttcatcatttg	11430	11449	1	3
SEQ ID NO: 2286	gatccaagtatagttggct	13278	13297	1	3
SEQ ID NO: 2287	tggacctgcaccaaagctg	1952	13971	1	3
SEQ ID NO: 2288	tctgatatacatcacggag	13703	13722	1	3
SEQ ID NO: 2289	ttgagttgcccaccatcat	11659	11678	1	3
SEQ ID NO: 2290	agcaagtctttcctggcct	3010	3029	1	3
SEQ ID NO: 2291	ttgggagagacaagtttca	6500	6519	1	3
SEQ ID NO: 2292	actttgcactatgttcata	12755	12774	1	3
SEQ ID NO: 2293	atcaacacaatcttcaatg	13107	13126	1	3
SEQ ID NO: 2294	cttggtacgagttactcaa	12632	12651	1	3
SEQ ID NO: 2295	agtgattacacttcctttc	12857	12876	1	3
SEQ ID NO: 2296	tttctgccactgctcagga	13516	13535	1	3
SEQ ID NO: 2297	tcttccgttctgtaatggc	5794	5813	1	3
SEQ ID NO: 2298	cctgcaccaaagctggcac	13956	13975	1	3
SEQ ID NO: 2299	aatttattcaaaacgagct	13192	13211	1	3
SEQ ID NO: 2300	ttaaaagaaatcttcaatt	13807	13826	1	3
SEQ ID NO: 2301	ttctctctatgggaaaaaa	9558	9577	1	3
SEQ ID NO: 2302	attgagattccctccatta	11694	11713	1	3
SEQ ID NO: 2303	tataagcagaagcacatat	13929	13948	1	3
SEQ ID NO: 2304	tcaaccttaatgattttca	8287	8306	1	3
SEQ ID NO: 2305	tattcttcttttccaattg	13826	13845	1	3
SEQ ID NO: 2306	gaaatcttcaatttattct	13813	13832	1	3
SEQ ID NO: 2307	atcagttcagataaacttc	7991	8010	1	3
SEQ ID NO: 2308	ttttgagaatgaatttcaa	10414	10433	1	3
SEQ ID NO: 2309	aaattccttgacatgttga	7362	7381	1	3
SEQ ID NO: 2310	gaagtgtcagtggcaaaaa	10374	10393	1	3
SEQ ID NO: 2311	gcaagggttcactgttcct	7856	7675	1	3
SEQ ID NO: 2312	ctccccaggacctttcaaa	9834	9853	1	3