Rhodopsin gene conserved regions in a viral vector enhance expression

[0001] This application is a continuation application under 35 U.S.C. §120 of a currently pending U.S. application Ser. No. 12/595,080 filed on Mar. 3, 2012 which is a 371 National Phase Entry Application of International Application No. PCT/GB2008/001310 filed Apr. 14, 2008, which designated the U.S., and which claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional No. 60/923,067 filed Apr. 12, 2007, the contents of which are incorporated herein by reference.

[0002] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 3, 2010, is named 20100303_ReplacementSequenceListing_TextFile_—740789_—066400_US.txt and is 127,586 bytes in size.

[0003] The invention relates to mutation independent suppression and replacement of disease-causing mutant genes.

[0004] Many mutation-based diseases are more genetically diverse than can be predicted from clinical presentation. Some mutation-based diseases are Mendelian and involve the inheritance of a single mutant gene, others are polygenic or multifactorial and involve multiple genetic insults. In the case of some Mendelian disorders, many different mutations within the same gene can give rise to, or can predispose an individual to, a disease. Similarly, for some multifactorial disorders, many different mutations within one or more genes can predispose an individual to a disease or can act in an additive manner with other genetic and environmental influences to give rise to a disease. This mutational heterogeneity underlying the molecular etiologies of many diseases represents a significant barrier to the development of therapies for such diseases. Moreover, genetic strategies for suppressing and replacing a mutant protein face many challenges with regard to the effectiveness of the machinery used to deliver and regulate the expression of the suppressor and replacement nucleic acids in vivo. Therefore, a need exists for effective mutation-independent therapeutics that achieve effective suppression and replacement.

[0005] The invention relates to gene suppression and replacement. In particular, the invention relates to enhanced expression of suppression agents for suppressing gene expression in a cell and in vivo and of replacement nucleic acids that are not inhibited and/or are partially inhibited by the suppression agent. Expression vectors used to express the suppression agent(s) and replacement nucleic acids comprise regulatory elements to optimize expression of the suppression agent(s) and or replacement nucleic acids.

[0006] The invention embodies use of replacement genes using sequences to enhance expression of replacement genes from viral and or non-viral vectors. In a further aspect the invection relates to enhanced expression of suppression agent(s) and or replacement genes from viral or and non-viral vectors. In a further embodiment the invention relates to enhanced expression of suppression agent(s) and or replacement genes and or genes encoding neurotrophic factors from viral and or non-viral vectors.

[0007] In one aspect the invention relates to use of conserved sequences from retinal genes to enhance expression of suppression agent(s) and or replacement genes and or genes encoding neurotrophic factors. The use of such conserved sequences has been found to result in surprisingly efficient expression. In a particular aspect the invention relates to use of conserved sequences from retinal genes to enhance expression of suppression agent(s) and or replacement genes and or genes encoding neurotrophic factors from adeno associated virus (AAV) vectors. In another aspect the invention provides vectors for expression of suppression agent(s) and or replacement gene(s) and or genes encoding neurotrophic factors using regulatory sequences from retinal gene(s) and or non-retinal gene(s) and or ubiquitously expressing genes to enhance expression from vectors.

[0008] In one aspect, the invention provides vectors for expressing a suppression agent for a disease causing gene and/or a replacement nucleic acid that is not recognized or is partially recognized by the suppression agent.

[0009] In an embodiment, the vector comprises an enhancer sequence, such as, for example, a sequence of SEQ ID NOs: 402-413 or functional variants or equivalents thereof. In another embodiment, the vector comprises at least one regulatory element selected from the group consisting of a promoter, a stuffer, an insulator, a silencer, an intron sequence, a post translational regulatory element, a polyadenylation site, and a transcription factor binding site.

[0010] In another embodiment, the vector comprises at least one of conserved regions A through I from the rhodopsin gene, as represented by SEQ ID NOs: 92-99, or functional variant or equivalent thereof. In another embodiment, the vector comprises at least one transcription factor binding site sequence selected from the group consisting of SEQ ID NOs: 100-401, or functional variant or equivalent thereof.

[0011] The suppression agent may be a nucleic acid, protein, amino acid(s), antibody, aptamer, or any such agent that can bind to and inhibit a DNA, RNA, or protein. In an embodiment, the suppression agent is a siRNA selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35-67, 75, 77, 79, 81, 83, 85, and 414-421 or functional variant or equivalent thereof.

[0012] The replacement nucleic acid is not recognized or is recognized partially by the suppression effector, because its sequence has been altered such that it cannot bind or binds less efficiently to the suppression agent but still encodes a normal or enhanced gene product. In an embodiment, the replacement nucleic acid is a siRNA selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 68, 76, 78, 80, 82, 84, and 86, or functional variant or equivalent thereof.

[0013] In an embodiment, the invention provides vectors, such as viral vectors, that comprise a suppression agent and/or a replacement nucleic acid. For example, the vector comprises at least one suppression agent nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35-67, 75, 77, 79, 81, 83, and 85, or functional variant or equivalent thereof, and at least one replacement nucleic acid nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 68, 76, 78, 80, 82, 84, and 86, or functional variant or equivalent thereof.

[0014] In another aspect, the invention provides therapeutic compositions comprising at least one vector comprising at least one suppression agent nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35-67, 75, 77, 79, 81, 83, 85 and 414-421 or functional variant or equivalent thereof, and at least one replacement nucleic acid nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 68, 76, 78, 80, 82, 84, and 86, or functional variant or equivalent thereof. In an embodiment, the vector of the therapeutic composition further comprises a regulatory element selected from the group consisting of an enhancer, a promoter, a stuffer, an insulator, a silencer, an antirepressor, an intron sequence, a post translational regulatory element, a polyadenylation signal (e.g. minimal poly A), a conserved region A through I, and a transcription factor binding site.

[0015] In another aspect the invention provides suppression and replacement in conjunction with provision of a gene encoding a neurotrophic/neuroprotective factor(s).

[0016] In another aspect, the invention provides cells comprising the nucleic acids and vectors of the invention.

[0017] In another aspect, the invention provides transgenic animals comprising the nucleic acids and vectors of the invention.

[0018] In yet another aspect, the invention provides methods of suppressing the expression of a mutant gene and replacing expression of the mutant gene with a replacement nucleic acid, the method comprising administering to a mammal a therapeutic composition of the invention.

[0019] In yet another aspect, the invention provides methods of suppressing the expression of a mutant gene and replacing expression of the mutant gene with a replacement nucleic acid in conjunction with a gene encoding a neurotrophic/neuropeotective factor(s), the method comprising administering to a mammal a therapeutic composition of the invention.

[0041] The instant invention utilises efficient gene suppression in conjunction with gene replacement to overcome the challenge of mutational heterogeneity. The suppression agent does not necessarily target a mutation (although it can encompass the site of a mutation), but is rather mutation independent. Suppression can involve one or both alleles of an endogenous gene. In conjunction with suppression, a replacement gene is provided that has been modified such that the replacement gene is refractory or partially refractory to suppression. The invention uses the degeneracy of the genetic code to modify the replacement gene. Alteration of “wobble” bases makes it possible for replacement nucleic acids to escape suppression at least in part, but does not change the protein product expressed from the replacement nucleic acids. Alternatively, replacement genes are modified in such a way that they encode altered amino acids but still encode a functional or partially functional protein that does not lead to pathology (e.g., because the amino acid changes are silent mutations or polymorphisms). Replacement has been demonstrated using rhodop sin nucleic acids, however, other genes or combinations of genes can be made and used in the practice of the invention. In particular, the invention relates to modulating and optimizing the expression levels of the suppression agents and/or replacement nucleic acids using one or more of the untranslated regions (UTRs) of a gene, intronic sequences, the degeneracy of the genetic code and/or polymorphisms to alter the sequence of replacement nucleic acids such that they are refractory or partially refractory to suppression.

[0042] In one aspect, the invention provides methods for preparing and using a suppression agent and replacement nucleic acid. The suppression agent binds to a coding region of a mature RNA or DNA encoding a mutant allele and inhibits expression of the mutant allele. The replacement nucleic acid encodes a wild-type or non-disease causing allele and comprises at least one degenerate/wobble nucleotide that is altered so that the replacement nucleic acid is not suppressed, or is only partially suppressed, by the suppression of one or both alleles of a gene.

[0043] The invention provides for replacement genes using sequences to enhance expression of replacement genes from viral and or non-viral vectors. In particular the invention relates to enhanced expression of suppression agent(s) and or replacement genes from viral or and non-viral vectors. The invention relates to use of conserved sequences from retinal genes to enhance expression of suppression agent(s) and or replacement genes. In a particular aspect the invention relates to use of conserved sequences from retinal genes to enhance expression of suppression agent(s) and or replacement genes from adeno associated virus (AAV) vectors. In another aspect the invention provides vectors for expression of suppression agent(s) and or replacement gene(s) using regulatory sequences from retinal gene(s) and or non-retinal gene(s) and or ubiquitously expressing genes such as those provided in the Tables below to enhance expression from viral and non-viral vectors.

[0044] In another aspect, the invention provides a composition comprising a suppression agent that binds to the coding region of a mature and/or immature RNA or DNA encoding a mutant allele to inhibit expression of the mutant allele and a replacement nucleic acid that encodes a wild-type or non-disease causing allele and comprises at least one degenerate/wobble nucleotide that is altered so that the replacement nucleic acid is not suppressed, or is only partially suppressed, by the suppression agent.

[0045] In yet another aspect, the invention provides a kit comprising a suppression agent that suppresses the expression of a mature and or immature RNA or DNA encoding a mutant allele and a replacement nucleic acid that encodes a wild-type or non-disease causing allele that is not suppressed, or is only partially suppressed, by the suppression agent and differs from the mutant allele in at least one degenerate/wobble nucleotide.

[0046] Suppression is achieved using a wide variety of molecular tools, such as, for example, RNA interference (RNAi) including non-coding RNAs such as small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNAs (miRNA), or other nucleotide-based molecules. In an embodiment, siRNAs in the order of 14-27 nucleotides in length are used for gene suppression. ShRNAs can be used to express functional siRNAs intracellularly and to achieve suppression in vitro and in vivo. Other suppression molecules include, for example, sense and antisense nucleic acids (single or double stranded), ribozymes, peptides, DNAzymes, peptide nucleic acids (PNAs), triple helix forming oligonucleotides, antibodies, and aptamers and modified form(s) thereof directed to sequences in gene(s), RNA transcripts, or proteins.

[0047] In an embodiment, the invention relates to vector(s) for supplying an endogenously generated suppression agent, such as, for example, a dsRNA in the form of a short hairpin (shRNA) which can be processed intracellularly into siRNA. dsRNA may be locally or systemically delivered. Expression vectors are used to generate functional siRNAs in cells and in animals typically using polymerase III promoters to drive expression, although polymerase II promoters are also used. For example, miRNA structures can be used to express double stranded RNAs from polymerase II promoters to enable tissue specific expression of double stranded RNA or polymerase II promoters can be juxtaposed to shRNA sequences to be expressed.

[0048] Suppression agents may be modified to alter the potency of the suppression agent, the target affinity of the suppression agent, the safety profile of the suppression agent and/or the stability of the suppression agent, for example, to render them resistant or partially resistant to intracellular degradation. Modifications, such as phosphorothioates, for example, can be made to oligonucleotides to increase resistance to nuclease degradation, binding affinity and/or uptake. In addition, hydrophobization and bioconjugation enhances siRNA delivery and targeting (De Paula et al., RNA. 13(4):431-56, 2007) and siRNAs with ribo-difluorotoluyl nucleotides maintain gene silencing activity (Xia et al., ASC Chem. Biol. 1(3):176-83, (2006). siRNAs with amide-linked oligoribonucleosides have been generated which are more resistant to 51 nuclease degradation (Iwase R et al. 2006 Nucleic Acids Symp Ser 50: 175-176). In addition, modification of siRNA at the 2′-sugar position and phosphodiester linkage confers improved serum stability without loss of efficacy (Choung et al., Biochem. Biophys. Res. Commun. 342(3):919-26, 2006). In one study, 2′-deoxy-2′-fluoro-beta-D-arabinonuclecic acid (FANA)-containing antisense oligonucleotides compared favourably to phosphorothioate oligonucleotides, 2′-0-methyl-RNA/DNA chimeric oligonucleotides and siRNAs in terms of suppression potency and resistance to degradation (Ferrari N et al. 2006 Ann N Y Acad Sci 1082: 91-102.)

[0049] Antisense and ribozyme suppression strategies have led to the reversal of a tumor phenotype by reducing expression of a gene product or by cleaving a mutant transcript at the site of the mutation (Carter and Lemoine Br. J. Cancer. 67(5):869-76, 1993; Lange et al., Leukemia. 6(11):1786-94, 1993; Valera et al., J. Biol. Chem. 269(46):28543-6, 1994; Dosaka-Akita et al., Am. J. Clin. Pathol. 102(5):660-4, 1994; Feng et al., Cancer Res. 55(10):2024-8, 1995; Quattrone et al., Cancer Res. 55(1):90-5, 1995; Lewin et al., Nat Med. 4(8):967-71, 1998). For example, neoplastic reversion was obtained using a ribozyme targeted to an H-ras mutation in bladder carcinoma cells (Feng et al., Cancer Res. 55(10):2024-8, 1995). Ribozymes have also been proposed as a means of both inhibiting gene expression of a mutant gene and of correcting the mutant by targeted trans-splicing (Sullenger and Cech Nature 371(6498):619-22, 1994; Jones et al., Nat. Med. 2(6):643-8, 1996). Ribozyme activity may be augmented by the use of, for example, non-specific nucleic acid binding proteins or facilitator oligonucleotides (Herschlag et al., Embo J. 13(12):2913-24, 1994; Jankowsky and Schwenzer Nucleic Acids Res. 24(3):423-9, 1996). Multitarget ribozymes (connected or shotgun) have been suggested as a means of improving efficiency of ribozymes for gene suppression (Ohkawa et al., Nucleic Acids Symp Ser. (29):121-2, 1993).

[0050] Triple helix approaches have also been investigated for sequence-specific gene suppression. Triplex forming oligonucleotides have been found in some cases to bind in a sequence-specific manner (Postel et al., Proc. Natl. Acad. Sci. U.S.A. 88(18):8227-31, 1991; Duval-Valentin et al., Proc. Natl. Acad. Sci. U.S.A. 89(2):504-8, 1992; Hardenbol and Van Dyke Proc. Natl. Acad. Sci. U.S.A. 93(7):2811-6, 1996; Porumb et al., Cancer Res. 56(3):515-22, 1996). Similarly, peptide nucleic acids have been shown to inhibit gene expression (Hanvey et al., Antisense Res. Dev. 1(4):307-17, 1991; Knudsen and Nielson Nucleic Acids Res. 24(3):494-500, 1996; Taylor et al., Arch. Surg. 132(11):1177-83, 1997). Minor groove binding polyamides can bind in a sequence-specific manner to DNA targets and hence may represent useful small molecules for future suppression at the DNA level (Trauger et al., Chem. Biol. 3(5):369-77, 1996). In addition, suppression has been obtained by interference at the protein level using dominant negative mutant peptides and antibodies (Herskowitz Nature 329(6136):219-22, 1987; Rimsky et al., Nature 341(6241):453-6, 1989; Wright et al., Proc. Natl. Acad. Sci. U.S.A. 86(9):3199-203, 1989). In some cases suppression strategies have lead to a reduction in RNA levels without a concomitant reduction in proteins, whereas in others, reductions in RNA have been mirrored by reductions in protein.

[0051] The diverse array of suppression strategies that can be employed includes the use of DNA and/or RNA aptamers that can be selected to target, for example, a protein of interest such as rhodopsin. In the case of age related macular degeneration (AMD), anti-VEGF aptamers have been generated and have been shown to provide clinical benefit in some AMD patients (Ulrich H, et al. Comb. Chem. High Throughput Screen 9: 619-632, 2006). Suppression and replacement using aptamers for suppression in conjunction with a modified replacement gene and encoded protein that is refractory or partially refractory to aptamer-based suppression could be used in the invention.

[0052] Recent evidence suggests that control of gene expression occurs endogenously in part by the activity of small non-coding RNAs, one broad category of which is termed microRNAs (miRNAs). miRNAs are expressed from polymerase II promoters, but can also be expressed from polymerase III promoters. miRNAs are processed intracellularly from larger transcripts to form small molecules approximately 20 nucleotides in length. miRNA structures can be used to express small double stranded RNAs and thus can be used to express the double stranded RNAs of the current invention.

[0053] Suppression targeted to coding sequence holds the advantage that such sequences are present in both precursor and mature RNAs, thereby enabling suppressor effectors to target all forms of RNA. A combined approach using a number of suppression effectors directed to multiple targets on an RNA or to multiple RNAs may also be used in the invention. As with suppression, multiple replacement nucleic acids can be used in the invention. For some disorders, it may be necessary to block expression of a disease allele completely to prevent disease symptoms whereas for others low levels of mutant protein may be tolerated. The invention can thus provide partial or complete suppression.

[0054] In one embodiment of the invention, suppressors are targeted to genes that are involved in the regulation of other genes. Suppression of these genes therefore may lead to up- or down-regulation of other genes.

[0055] In another embodiment, the invention relates to suppression of the expression of mutated genes that give rise to a dominant or deleterious effect or disease. A suppression effector may target either the disease allele or the normal allele. In another embodiment, the suppression effector targets both the disease allele and the normal allele.

[0056] In an embodiment of the invention, a replacement nucleic acid is provided that is altered at one or more degenerate or wobble bases from the endogenous wild type gene but that encodes the identical amino acids as the wild type or a non-disease causing gene. In another embodiment, the replacement nucleic acid encodes a beneficial replacement nucleic acid (e.g., a more active or stable product than that encoded by the wild-type gene). The replacement nucleic acid provides expression of a normal or non-disease causing protein product when required to ameliorate pathology associated with reduced levels of wild type protein. The same replacement nucleic acid can be used in conjunction with the suppression of many different disease mutations within a given gene. In addition, multiple replacement nucleic acids can be used in the invention.

[0057] Although the instant application provides numerous exemplary suppression agents and replacement nucleic acid sequences, these are only examples and other such sequences can be determined as described herein for the same targets or for any desired target. “Functional variant” includes any variant nucleic acid or other suppression agent that may have one or more nucleic acid substitutions but that does not have a materially different function than, or that can still hybridize under stringent hybridization conditions (0.2×SCC, 0.1% SDS) to, or that shares at least 70% identity, for example 80%, such as at least 90% or at least 95% sequence identity with the nucleic acid indicated.

[0058] In another embodiment of the invention, suppression effectors are targeted to the untranslated regions (either 5′UTR or 3′UTR) of at least one allele of a gene. In another embodiment of the invention replacement nucleic acids are provided that have been altered at the suppression site, such that replacement nucleic acids provide functional or partially functional protein and escape or partially escape from suppression by suppressors.

[0059] In another embodiment of the invention, suppression effectors are targeted to intronic sequences. In another embodiment, replacement nucleic acids are provided which have been altered at one or more nucleotides of the targeted site of the intron so that transcripts from the replacement nucleic acids escape or partially escape suppression by suppressors. In another embodiment the whole targeted intron may not be present in replacement nucleic acids.

[0060] In another embodiment of the invention, suppression effectors are targeted to polymorphic sites and at least one allele of the gene is suppressed or partially suppressed. In another embodiment, replacement nucleic acids are provided for the alternative polymorphic variant such that replacement nucleic acids encode functional or partially functional protein and escape or partially escape from suppression by suppressors.

[0061] In another embodiment of the invention the suppression agent and/or replacement nucleic acid is expressed from one or more promoter sequences. The invention provides promoter sequences that have been demonstrated to promote ubiquitous expression of nucleotides and/or promoters that have been demonstrated to exert tissue specific, temporal, inducible, and/or quantitative control of gene expression. The invention also provides enhancer sequences (Table 1) and/or post-translational regulatory elements and/or other regulatory elements and/or epigenetic elements that provide optimized expression of suppression agents and/or replacement nucleic acids.

[0062]

TABLE 1
Exemplary Enhancer Elements
Enhancer Element	Reference
Chicken ovalbumin upstream promoter	Eguchi et al., Biochimie
transcription factor II	89(3): 278-88, 2007
Mouse dystrophin muscle promoter/enhancer	Anderson et al., Mol.
	Ther. 14(5): 724-34, 2006
Tobacco eIF4A-10 promoter elements	Tian et al., J. Plant Physiol.
	162(12): 1355-66, 2005
Immunoglobulin (Ig) enhancer element	Frezza et al., Ann. Rheum.
HS1,2A	Dis. Mar. 28, 2007
Col9a1 enhancer element	Genzer and Bridgewater
	Nucleic Acids Res.
	35(4): 1178-86, 2007
Gata2 intronic enhancer	Khandekar et al.,
	Development Mar. 29,
	2007
TH promoter enhancer	Gao et al., Brain Res.
	1130(1): 1-16, 2007
CMV enhancer	InvivoGen cat# pdrive-cag
	05A13-SV
Woodchuck hepatitis virus	Donello et al., J. Virol.
posttranscriptional regulatory element	72(6): 5085-92, 1998
Woodchuck hepatitis virus	Schambach et al., Gene
posttranscriptional regulatory element	Ther. 13(7): 641-5, 2006
IRBP	Ying et al., Curr. Eye Res.
	17(8): 777-82, 1998
CMV enhancer and chicken β-actin promoter	InvivoGen cat# pdrive-cag
	05A13-SV
CMV enhancer and chicken β-actin promoter	InvivoGen cat# pdrive-cag
and 5′UTR	05A13-SV
CpG-island	Antoniou et al., Genomics
	82: 269-279, 2003

[0063] In a particular embodiment, sequences that influence chromatin structure, such as but not exclusive to insulator, antirepressor, cis-acting modulators of nucleosome positioning and/or silencer elements, sometimes termed epigenetic elements, are used to modulate expression of suppression agents and/or replacement nucleic acids. Exemplary epigenetic elements such as insulator and antirepressor sequences are provided in Table 2. It is clear that chromatin structures influence gene expression, for example, chromatin structures influence the ability of the transcriptional machinery to access promoter and/or enhancer elements amongst other sequence motifs. The inclusion of sequences which influence chromatin structures in viral and/or non-viral vectors and/or administered in conjunction with suppression and/or replacement nucleic acids can be used to optimize expression of either or both suppressors and replacement nucleic acids. In addition, chemical entities which influence chromatin structures can be used to optimize expression such as histone deacetylase (HDAC) inhibitors and/or DNA methyl transferase inhibitors and/or histone methyl transferase inhibitors. Such entities can be supplied in the form of DNA and/or RNA and/or protein amongst other forms. Similarly attracting enzymes and/or supplying enzymes (in the form of DNA and/or RNA and or protein) involved in chromatin remodelling such as but not exclusive to histone acetyl transferases to nucleic acids to be expressed and their associated regulatory regions can be used to optimize expression of suppression and/or replacement nucleic acids.

[0064]

TABLE 2
Exemplary Epigenetic Elements
Epigenetic elements	Reference
Mcp Insulators	Kyrchanova et al., Mol. Cell Biol.
	27(8): 3035-43, 2007
CpG-island region of the HNRPA2B1	Williams et al., BMC Biotechnol.
locus	5: 17, 2005
Chicken b-globin 5′hypersensitive	Kwaks and Otte 2006 Trends in
site 4 (cHS4)	Biotechnology 24: 137-142
Ubiquitous chromatin opening	Kwaks and Otte 2006 Trends in
elements (UCOEs)	Biotechnology 24: 137-142
Matrix associated regions (MARs)	Kwaks and Otte 2006 Trends in
	Biotechnology 24: 137-142
Stabilising and antirepressor	Kwaks and Otte 2006 Trends in
elements (STAR)	Biotechnology 24: 137-142
Human growth hormone gene	Trujillo MA et al. 2006 Mol
silencer	Endocrinol 20: 2559
S/MAR	Liebich et al., Nucleic Acids
	Res. 30: 3433-42, 2002

[0065] In another embodiment, expression of a suppression agent and/or replacement nucleic acid is optimized to enable efficient suppression in conjunction with sufficient replacement. In an additional embodiment, suppression and/or replacement nucleic acids are provided with agents that aid vector transfection, transduction, and/or expression of suppression and replacement nucleic acids.

[0066] The invention circumvents the need for a specific therapy for every disease-causing mutation within a given gene. Notably, the invention has the advantage that the same suppression agents can be used to suppress many mutations in a gene. This is particularly relevant when any one of a large number of mutations within a single gene can cause disease pathology. The compositions and methods of the invention allow greater flexibility in choice of target sequence for suppression of expression of a disease allele. Furthermore, the compositions and methods of the invention allow greater flexibility in terms of controlling expression of the suppression and/or replacement of a given gene and or allele of a gene.

[0067] Suppression and replacement can be undertaken in conjunction with each other or separately. Suppression and replacement utilizing the degeneracy of the genetic code may be undertaken in test tubes, in cells, in animals, or in plants and may be used for experimental research (e.g., for the study of development or gene expression) or for therapeutic purposes. Suppression and replacement may be used in conjunction with agents to promote cell transfection or cell transduction such as, for example, lipids and polymers. Suppression and replacement may be provided to consumers in a kit.

[0068] The suppression and replacement agents of the invention can be delivered to a target cell and or tissue and or animal and or plant using ‘naked’ reagents such as DNA, RNA, peptides or other reagents. Alternatively viral and or non-viral vectors can be used with or without ‘naked’ reagents.

[0069] In an embodiment, suppression and/or replacement construct(s) can be delivered to a cell using an AAV2/5 recombinant virus, however, other viral and non-viral vectors, such as other AAV serotypes, adenovirus, herpes virus, SV40, HIV, SIV and other lentiviral vectors, RSV and non-viral vectors including naked DNA, plasmid vectors, peptide-guided gene delivery, terplex gene delivery systems, calcium phosphate nanoparticles, magnetic nanoparticles, colloidal microgels and/or the integrase system from bacteriophage phiC31 may be utilised in the invention, for example. Suppression and replacement components may be found on separate vectors or may be incorporated into the same vector. Viral vectors useful in the invention include, but are not limited to, those listed in Table 3. Non-viral vectors useful in the invention include, but are not limited to, those listed in Table 4. Cationic lipid-based non-viral vectors can include glycerol-based (e.g. DOTMA, DOTAP, DMRIE, DOSPA), non-glycerol-based (e.g. DOGS, DOTIM) and/or cholesterol-based cationic lipids (e.g. BGTC, CTAP; Karmali PP and Chaudhuri A 2006 Med Res Rev). Viral and non-viral vector delivery may be accompanied by other molecules such as cationic lipids and/or polymers and/or detergents and/or agents to alter pH, such as, for example, polyethelene glycol (PEG), to enhance cellular uptake of vectors and/or to enhance expression from vectors and/or to evade the immune system. For example, polycationic molecules have been generated to facilitate gene delivery including but not exclusive to cationic lipids, poly-amino acids, cationic block co-polymers, cyclodextrins amongst others. Pegylation of vectors with polyethelene glycol (PEG) can shield vectors from, for example, the extracellular environment. Vectors may be used in conjunction with agents to avoid or minimise cellular immune responses such as PEG or as a Polyplex with Poly(L-Lysine) Vector delivery may be undertaken using physical methodologies such as electroporation, nucleofection and/or ionotophoresis, either alone or in combination with molecules to enhance delivery. Vectors may be used in conjunction with agents to promote expression of suppression and/or replacement components incorporated into vectors, for example, using histone deacetylase inhibitors (HDAC) and/or DNA methyl transferase inhibitors and/or histone methyl transferase inhibitors to modulate chromatin structures thereby aiding expression. HDAC inhibitors include but are not exclusive to short chain fatty acids such as valproic acid and sodium butyrate, ketones, benzamides, cyclic and non-cyclic hydroxamates such as suberoyl anilide hydroxamic acids (SAHA), trichostatin A (TSA), cyclic peptides or tetrapeptides amongst others (Liu T et al. 2006 Cancer Treatment Reviews 32: 157-165). DNA methyl transferase inhibitors including, for example, 5-AC, decitabine and zebularine can be used to modulate chromatin structures. In addition, histone methyl transferase inhibitors can influence chromatin states, for example, BIX-01294 (diazepin-quinazolin-amine derivative). In addition, to the chemical entities referred to above, nucleic acids-based inhibitors can be used to suppress expression of proteins and/or non-coding RNAs involved in chromatin remodelling. In one embodiment of the invention vectors are optimized to specifically transduce target cell type(s) or target tissue type(s). Viral and/or non-viral vectors may be modified to target specific cell types and/or to prevent targeting of some cell types. For example, the inclusion of the capsid from AAV serotype 5 in an AAV2/5 hybrid virus facilitates transduction of photoreceptor cells. Similarly, for example, peptides may be included in viral vectors to facilitate targeting. Synthetic non-viral vectors can be modified to include ligands to facilitate targeting of vectors to specific cell and/or tissue types, for example, folate can be conjugated to liposomes to target tumour cells which over express the folate receptor (Hattori Y et al. 2005; Curr Drug Deliv 3: 243-52). In another embodiment of the invention, suppression and replacement vectors are designed to optimize the generation and/or production of vector, for example, to optimize viral titre and/or to optimize the number or type of nucleotides incorporated into vector(s). For example, vector genomes may be modified such that large transgenes may be incorporated into vectors, for example, ‘gutless’ adenovirus vectors have an increased capacity in terms of size than previous generations of adenovirus vectors. Components of vectors can be modified to optimize generation and production of vectors, for example, genes involved in replication of AAV can be modified to optimize replication and/or self complementary AAV vectors can be used to optimize rates of transgene expression. In an additional embodiment, vectors are designed to optimize suppression in conjunction with replacement, to enable optimal expression of all components of a therapeutic. For example, to optimize expression of both elements of suppression and replacement from a given vector, additional sequences can be included in the vector. For example, inclusion of nucleotides to separate the ITRs of AAV and the shRNA sequences of an RNAi-based suppression agent can result in optimisation of expression of the suppression component. Nucleotides encoding suppressors and/or replacement nucleic acids can be juxtaposed or separated from each other and/or can be in the same orientation or opposing orientations. In addition, the suppressor(s) can be 5′ and/or 3′ to the replacement nucleic acids. Nucleotides encoding suppressors and/or replacement nucleic acids can be juxtaposed to nucleotides comprising vector(s) or can be separated from nucleotides comprising vector(s). Nucleotides encoding suppressors and/or replacement nucleic acids may be cloned within the backbone of the plasmid used to generate AAV and or may be cloned between the AAV ITRs and not within the plasmid backbone of the plasmid, and/or may be cloned in a combination of these positions. Additional sequences, such as, for example, stuffer sequences can be included in vectors to optimize vector design. In addition, multiple suppressors and/or replacement nucleic acids may be used in one vector.

[0070]

TABLE 3
Exemplary Viral Vectors
Delivery Method	Serotype	Reference
AAV	All serotypes,	Lebkowski et al., Mol.
	including but	Cell Biol. 8(10): 3988-
	not limited to	96, 1988
	1, 2, 3, 4, 5, 6,	Flannery et al., Proc.
	7, 8, 9, 10, 11,	Natl. Acad. Sci. U.S.A.
	12,	94(13): 6916-21, 1997
Lentivirus (for example	VSV-G	Pang et al., Mol. Vis.
but not exclusively	Rabies-G	12: 756-67, 2006
Feline—FIV,	Further	Takahashi Methods Mol.
Equine—EIAV,	serotypes**	Biol. 246: 439-49, 2004
Bovine—BIV and		Balaggan et al., J. Gene
Simian—SIV).		Med. 8(3): 275-85, 2006
Adenovirus	Various	Bennett et al., Nat. Med.
		2(6): 649-54, 1996
Simian papovirius SV40	Various	Kimchi-Sarfaty et al., Hum.
		Gene Ther. 13(2): 299-310,
		2002
Semliki Forest Virus	Various	DiCiommo et al., Invest.
		Ophthalmol. Vis. Sco.
		45(9): 3320-9, 2004
Sendai Virus	Various	Ikeda et al., Exp. Eye Res.
		75(1): 39-48, 2002

• • [0071] The list provided is not exhaustive; other viral vectors and derivatives, natural or synthesized could be used in the invention.

[0072]

TABLE 4
Exemplary Non-Viral Vectors or Delivery Methods
Delivery Method	Reference
Cationic	Sakurai et al., Gene Ther. 8(9): 677-86,
liposomes	2001
HVJ liposomes	Hangai et al., Arch. Ophthalmol.
	116(3): 342-8, 1998
Polyethylenimine	Liao and Yau Biotechniques 42(3): 285-
	6, 2007
DNA nanoparticles	Farjo et al., PloS ONE 1: e38, 2006
Dendrimers	Marano et al., Gene Ther. 12(21):
	1544-50, 2005
Bacterial	Brown and Giaccia Cancer Res.
	58(7): 1408-16, 1998
Macrophages	Griffiths et al., Gene Ther. 7(3): 255-62,
	2000
Stem cells	Hall et al., Exp. Hematol. 34(4): 433-42,
	2006
Retinal transplant	Ng et al., Chem. Immunol. Allergy 92:
	300-16, 2007
Marrow/Mesenchymal	Kicic et al., J. Neurosci. 23(21): 7742-9,
stromal cells	2003
	Chng et al., J. Gene Med. 9(1): 22-32, 2007
Implant (e.g.,	Montezuma et al., Invest. Ophthalmol. Vis.
Poly(imide)uncoated	Sci. 47(8): 3514-22, 2006
or coated)
Electroporation	Featherstone A. Biotechnol. Lab. 11(8): 16,
	1993
Targeting peptides	Trompeter et al., J. Immunol Methods.
(for example but	274(1-2): 245-56, 2003
not exclusively Tat)
Lipid mediated (e.g.,	Nagahara et al., Nat. Med. 4(12): 1449-52,
DOPE, PEG)	1998
	Zeng et al., J. Virol. 81(5): 2401-17, 2007
	Caplen et al., Gene Ther. 2(9): 603-13,
	1995Manconi et al., Int. J. Pharm. 234(1-
	2): 237-48, 2006
	Amrite et al., Invest. Ophthalmol. Vis.
	Sci. 47(3): 1149-60, 2006
	Chalberg et al., Invest. Ophthalmol. Vis.
	Sci. 46(6): 2140-6, 2005

[0073] The list provided is not exhaustive. Other non-viral vectors and derivatives, natural or synthesized and other delivery methods could be used with the invention.

[0074] In an embodiment, the replacement nucleic acid encodes mammalian rhodopsin, collagen 1A1, collagen 1A2, collagen 7A1, or peripherin. In another embodiment, the replacement nucleic acid encodes a protein that has been mutated to cause an autosomal or X-linked dominant retinitis pigmentosa, such as those listed in Table 5. Suppression agents and replacement nucleic acids may be generated for one or more of these genes, for example.

[0075]

TABLE 5
Genes known to be involved in retinitis pigmentosa (Table
adapted from RETNET) (http://www.sph.uth.tmc.edu/Retnet/)
Symbols;
OMIM Numbers	Location	Diseases; Protein	References
LCA9	1p36	recessive Leber congenital	Keen et al., Hum. Mol.
		amaurosis	Genet. 3: 367-368
			(1994)
NPHP4,	1p36.31	recessive Senior-Loken	Mollet et al., Nat.
SLSN4;		syndrome; recessive	Genet. 32: 300-305
		nephronophthisis, juvenile;	(2002); Otto et al., Am.
		protein: nephronophthisis 4	J. Hum. Genet.
		protein	71: 1161-1167 (2002);
			Schuermann et al., Am.
			J. Hum. Genet.
			70: 1240-1246 (2002)
RP32;	1p34.3-p13.3	recessive RP, severe	Zhang et al., Hum.
			Genet. 118: 356-365
			(2005)
RPE65,	1p31.2	recessive Leber congenital	Acland Nat. Genet.
LCA2, RP20;		amaurosis; recessive RP; protein:	28: 92-95 (2001)
		retinal pigment epithelium-
		specific 65 kD protein
ABCA4,	1p22.1	recessive Stargardt disease,	Lewis et al., Am. J.
ABCR, RP19,		juvenile and late onset; recessive	Hum. Genet. 64: 422-
STGD1;		MD; recessive RP; recessive	1434 (1999)
		fundus flavimaculatus; recessive
		cone-rod dystrophy; protein:
		ATP-binding cassette transporter -
		retinal
COL11A1,	1p21.1	dominant Stickler syndrome, type	Annunen et al., Am. J.
STL2;		II; dominant Marshall syndrome;	Hum. Genet. 65: 974-
		protein: collagen, type XI, alpha 1	983 (1999)
GNAT2,	1p13.3	recessive achromatopsia; protein:	Aligianis et al., J. Med.
ACHM4;		guanine nucleotide binding	Genet. 39: 656-660
		protein (G protein) cone-specifc	(2002)
		transducin alpha subunit
PRPF3,	1q21.2	dominant RP; protein: human	Chakarova et al., Hum.
HPRP3,		homolog of yeast pre-mRNA	Mol. Genet. 11: 87-92
PRP3, RP18;		splicing factor 3	(2002)
SEMA4A,	1q22	dominant RP; dominant cone-rod	Abid et al., J. Med.
SEMAB;		dystrophy; protein: semaphorin	Genet. 43: 378-381
		4A	(2005)
CORD8;	1q23.1-q23.3	recessive cone-rod dystrophy	Ismail et al., J. Hum.
			Genet. 51: 827-831
			(2006)
AXPC1	1q31-q32	recessive ataxia, posterior column	Higgins et al., Neurol.
		with RP	52: 146-150 (1999)
ARMD1,	1q31.1	dominant MD, age-related;	Schultz et al., Hum.
FIBL6,		protein: hemicentin 1 (fibulin 6)	Mol. Genet. 12: 3315-
FBLN6;			3123 (2003)
CFH, HF1;	1q31.3	age-related macular degeneration,	Edwards et al., Science
		complex etiology; protein:	308: 421-424 (2005)
		complement factor H
CRB1, RP12;	1q31.3	recessive RP with para-arteriolar	Jacobson et al., Hum.
		preservation of the RPE (PPRPE);	Mol. Genet. 9: 1073-
		recessive RP; recessive Leber	1078 (2003)
		congenital amaurosis; dominant
		pigmented paravenous
		chorioretinal atrophy; protein:
		crumbs homolog 1
RD3,	1q32.3	recessive Leber congenital	Friedman et al., Am. J.
C1ORF36;		amaurosis; protein: RD3 protein	Hum. Genet. 79: 1059-
			1070 (2006)
USH2A;	1q41	recessive Usher syndrome, type	Seyedahmadi et al.,
		2a; recessive RP; protein: usherin	Exp. Eye. Res. 79: 167-
			173 (2004)
RP28;	2p16-p11	recessive RP	Kumar et al., Mol. Vis.
			10: 399-402 (2004)
EFEMP1,	2p16.1	dominant radial, macular drusen;	Kermani et al., Hum.
DHRD,		dominant Doyne honeycomb	Genet. 104: 77-82
MTLV,		retinal degeneration (Malattia	(1999)
FBLN3;		Leventinese); protein: EGF-
		containing fibrillin-like
		extracellular matrix protein 1
		(fibulin 3)
ALMS1,	2p13.1	recessive Alström syndrome;	Hearn et al., Nat.
ALSS		protein: ALMS1 protein	Genet. 31: 79-83 (2002)
RP33	2cen-q12.1	dominant RP	Zhao et al., Hum.
			Genet. 119: 617-623
			(2006)
LOC619531	2q11	recessive cone-rod dystrophy and	Michaelides et al., J.
		amelogenesis imperfecta	Med. Genet. 41: 468-
			473 (2004)
CNGA3,	2q11.2	recessive achromatopsia; protein:	Nishiguchi et al., Hum.
ACHM2,		cone photoreceptor cGMP-gated	Mutat. 25: 248-258
CNCG3,		cation channel alpha subunit	(2005)
RMCH2
MERTK	2q13	recessive RP; protein: c-mer	Vollrath et al., Proc.
		protooncogene receptor tyrosine	Natl. Acad. Sci. USA
		kinase	98: 12584-12589 (2001)
NPHP1,	2q13	recessive Senior-Loken	Hildebrandt et al., Nat.
JBTS4,		syndrome; recessive	Genet. 17: 149-153
SLSN1		nephronophthisis, juvenile;	(1997)
		recessive Joubert syndrome;
		protein: nephronophthisis 3
		protein
BBS5	2q31.1	recessive Bardet-Biedl syndrome;	Li et al., Cell. 117: 541-
		protein: flagellar apparatus-basal	552 (2004)
		body protein DKFZp7621194
CERKL,	2q31.3	recessive RP; protein: ceramide	Tuson et al., Am. J.
RP26		kinase-like protein	Hum. Genet. 74: 128-
			138 (2004)
SVD	2q36	dominant vitreoretinal	Jiao et al., Invest.
		degeneration, snowflake	Ophthalmol. Vis. Sci.
			45: 4498-503 (2004)
SAG	2q37.1	recessive Oguchi disease;	Nakazawa et al., Arch.
		recessive RP; protein: arrestin (s-	Ophthalmol. 116: 498-
		antigen)	501 (1998)
USH2B,	3p24.2-p23	recessive Usher syndrome, type 2;	Hmani et al., Eur. J.
DFNB6		recessive sensorineural deafness	Hum. Genet. 7: 363-367
		without RP	(1999)
CRV,	3p21.3-p21.1	dominant hereditary vascular	Ophoff et al., Am. J.
HERNS, HVR		retinopathy with Raynaud	Hum. Genet. 69: 447-
		phenomenon and migraine	453 (2001)
GNAT1	3p21.31	dominant CSNB, Nougaret type;	Dryja et al., Nat. Genet.
		protein: rod transducin alpha	13: 358-360 (1996)
		subunit
ATXN7,	3p14.1	dominant spinocerebellar ataxia	Aleman et al., Exp.
ADCA2,		w/MD or retinal degeneration;	Eye. Res. 74: 737-745
OPCA3,		protein: ataxin 7	(2002)
SCA7
ARL6, BBS3	3q11.2	recessive Bardet-Biedl syndrome;	Fan et al., Nat. Genet.
		protein: ADP-ribosylation factor-	36: 989-993 (2004)
		like 6
IQCB1,	3q13.33	recessive Senior-Loken	Otto et al., Nat. Genet.
NPHP5,		syndrome; protein: IQ motif	37: 282-288 (2005)
SLSN5		containing B1 protein
NPHP3,	3q22.1	recessive Senior-Loken	Olbrich et al., Nat.
SLSN3		syndrome; recessive	Genet. 34: 455-459
		nephronophthisis, adolescent;	(2003)
		protein: nephronophthisis 3
		protein
RHO, RP4	3q22.1	dominant RP; dominant CSNB;	Dryja et al., Nat. Genet.
		recessive RP; protein: rhodopsin	4: 280-283 (1993)
RP5	same as RHO	not distinct from RHO/RP4	Farrar et al., Hum. Mol.
			Genet. 1: 769-771
			(1992)
USH3A,	3q25.1	recessive Usher syndrome, type 3;	Joensuu et al., Am. J.
USH3		protein: clarin-1	Hum. Genet. 69: 673-
			684 (2001)
OPA1	3q29	dominant optic atrophy, Kjer	Aung et al., Hum.
		type; dominant optic atrophy with	Genet. 110: 52-56
		sensorineural hearing loss;	(2002)
		protein: OPA1 protein
STGD4	4p	dominant Stargardt-like macular	Kniazeva et al., Am. J.
		dystrophy	Hum. Genet. 64: 1394-
			1399 (1999)
MCDR2	4p16.3-p15.2	dominant MD, bull's-eye	Michaelides et al.,
			Invest. Ophthalmol.
			Vis. Sci. 44: 1657-1662
			(2003)
PDE6B,	4p16.3	recessive RP; dominant CSNB;	Pearce-Kelling et al.,
CSNB3		protein: rod cGMP	Mol. Vis. 7: 42-47
		phosphodiesterase beta subunit	(2001)
WFS1,	4p16.1	recessive Wolfram syndrome;	Hum. Mol. Genet.
DFNA38		dominant low frequency	10: 2501-2508 (2001)
		sensorineural hearing loss;
		protein: wolframin
PROML1	4p15.32	recessive retinal degeneration;	Maw et al., Hum. Mol.
		protein: prominin (mouse)-like 1	Genet. 9: 27-34 (2000)
CNGA1,	4p12	recessive RP; protein: rod cGMP-	Dryja et al., Proc. Natl.
CNCG,		gated channel alpha subunit	Acad. Sci. USA
CNCG1			192: 10177-10181 (1995)
WFS2	4q22-q24	recessive Wolfram syndrome;	El-Shanti et al., Am. J.
		dominant	Hum. Genet. 66: 1229-
			1236 (2000)
MTP, ABL	4q23	recessive abetalipoproteinemia;	Narcisi et al., Am. J.
		protein: microsomal triglyceride	Hum. Genet. 57: 1298-
		transfer protein	1310 (1995)
BBS7,	4q27	recessive Bardet Biedl syndrome;	Badano et al., Am. J.
BBS2L1		protein: BBS7 protein	Hum. Genet. 72: 650-
			658 (2003)
BBS12,	4q27	recessive Bardet-Biedl syndrome;	Stoetzel et al., Am. J.
FLJ35630		protein: BBS12 protein	Hum. Genet. 80: 1-11
			(2007)
RP29	4q32-q34	recessive RP	Hameed et al., Invest.
			Ophthalmol. Vis. Sci.
			42: 1436-1438 (2001)
LRAT	4q32.1	recessive RP, severe early-onset;	Thompson et al., Nat.
		recessive Leber congenital	Genet. 128: 123-124
		amaurosis; protein: lecithin	(2001)
		retinol acyltransferase
CYP4V2,	4q35.2	recessive Bietti crystalline	Li et al., Am. J. Hum.
BCD		corneoretinal dystrophy; protein:	Genet. 74: 817-826
		cytochrome P450 4V2	(2004)
MCDR3	5p15.33-p13.1	dominant MD	Michaelides et al.,
			Invest. Ophthalmol.
			Vis. Sci. 44: 2178-2183
			(2003)
CSPG2,	5q14.3	dominant Wagner disease and	Kloeckener-Gruissem
WGN1,		erosive vitreoretinopathy; protein:	et al., Mol. Vis. 12: 350-
ERVR		chondroitin sulfate proteoglycan 2	355 (2006)
		(versican)
MASS1,	5q14.3	recessive Usher syndrome, type 2;	Weston et al., Am. J.
USH2C,		dominant/recessive febrile	Hum. Genet. 74: 357-
VLGR1		convulsions; protein: monogenic	366 (2004)
		audiogenic seizure susceptibility
		1 homolog
BSMD	5q21.2-q33.2	dominant MD, butterfly-shaped	den Hollander et al., J.
			Med. Genet. 41: 699-
			702 (2004)
PDE6A	5q33.1	recessive RP; protein: cGMP	Dryja et al., Invest.
		phosphodiesterase alpha subunit	Ophthalmol. Vis. Sci.
			40: 1859-1865 (1999).
GRM6	5q35.3	recessive CSNB; protein:	Dryja et al., Proc. Natl.
		metabotropic glutamate receptor 6	Acad. Sci. USA
			102: 4884-4889 (2005)
C2	6p21.32	age-related macular degeneration,	Gold et al., Nat. Genet.
		complex etiology; protein:	38: 458-462(2006)
		complement component 2
CFB, BF,	6p21.32	age-related macular degeneration,	Gold et al., Nat. Genet.
BFD		complex etiology; protein:	38: 458-462 (2006)
		complement factor B, properdin
TULP1, RP14	6p21.31	recessive RP; recessive Leber	Banerjee et al., Nat.
		congenital amaurosis; protein:	Genet. 18: 177-179
		tubby-like protein 1	(1998)
RDS, RP7	6p21.2	dominant RP; dominant MD;	Hum. Mutat. 10: 301-
		digenic RP with ROM1;	309 (1997)
		dominant adult vitelliform MD;
		protein: peripherin 2
GUCA1A,	6p21.1	dominant cone dystrophy;	Payne et al., Am. J.
COD3,		dominant cone-rod dystrophy;	Hum. Genet. 61: A290
GCAP1		protein: guanylate cyclase	(1997)
		activating protein 1A
GUCA1B,	6p21.1	dominant RP; dominant MD;	Sato et al., Graefes
GCAP2		protein: guanylate cyclase	Arch. Clin. Exp.
		activating protein 1B	Ophthalmol. 243: 235-
			242 (2004)
BCMAD	6p12.3-q16	dominant MD, benign concentric	van Lith-Verhoeven et
		annular	al., Invest. Ophthalmol.
			Vis. Sci. 45: 30-35
			(2004)
RP25	6cen-q15	recessive RP	Abd El-Aziz et al.,
			Ann. Hum. Genet.
			(2006)
LCA5	6q11-q16	recessive Leber congenital	Dharmaraj et al., Am. J.
		amaurosis	Hum. Genet. 66: 319-
			326 (2000)
COL9A1	6q13	recessive Stickler syndrome;	Van Camp et al., Am.
		dominant multiple epiphyseal	J. Hum. Genet. 79: 449-
		dysplasia (MED); protein:	457 (2006)
		collagen, type IX, alpha-1
RIMS1,	6q13	dominant cone-rod dystrophy;	Kelsell et al., Am. J.
CORD7,		protein: regulating synaptic	Hum. Genet. 63: 274-
RIM1		membrane exocytosis protein 1or	279 (1998)
		rab3A-interacting molecule
MCDR1,	6q14-q16.2	dominant MD, North Carolina	Small et al., Mol. Vis.
PBCRA		type; dominant progressive	5: 38 (1999)
		bifocal chorioretinal atrophy
ELOVL4,	6q14.1	dominant MD, Stargardt-like;	Edwards et al., Invest.
STGD3		protein: elongation of very long	Ophthalmol. Vis. Sci.
		fatty acids protein	42: 2652-2663 (2001)
AHI1, JBTS3	6q23.3	recessive Joubert syndrome;	Parisi et al., J. Med.
		protein: Abelson helper	Genet. 43: 334-339
		integration site 1	(2006)
PEX7,	6q23.3	recessive Refsum disease, adult	van den Brink 0et al.,
PTS2R,		form; protein: peroxisome	Am. J. Hum. Genet.
RCDP1,		biogenesis factor 7	72: 471-477 (2003)
RCD1	6q25-q26	dominant retinal-cone dystrophy	OMIM 07
		1
MDDC,	7p21-p15	dominant MD, cystoid	Inglehearn et al., Am. J.
CYMD			Hum. Genet. 55: 581-
			582 (1994)
PTHB1,	7p14.3	recessive Bardet Biedl syndrome;	Nishimura et al., Am. J.
BBS9, PHTB1		protein: parathyroid hormone-	Hum. Genet. 77: 1021-
		responsive B1 protein	1033 (2005)
RP9, PAP1,	7p14.3	dominant RP; protein: RP9	Sullivan et al., Invest.
PIM1K		protein or PIM1-kinase associated	Ophthalmol. Vis. Sci.
		protein 1	47: 3052-3064 (2006)
PEX1, IRD	7q21.2	recessive Refsum disease,	Portsteffen et al., Nat.
		infantile form; protein:	Genet. 17: 449-452
		peroxisome biogenesis factor 1	(1997)
IMPDH1,	7q32.1	dominant RP; dominant Leber	Mortimer et al.,
RP10		congenital amaurosis; protein:	Biochem. J. 390: 41-47
		inosine monophosphate	(2005)
		dehydrogenase 1
OPN1SW,	7q32.1	dominant tritanopia; protein: blue	Fitzgibbon et al., Hum.
BCP, CBT		cone opsin	Genet. 93: 79-80 (1994)
CORD9	8p11	recessive cone-rod dystrophy	Danciger et al., Invest.
			Ophthalmol. Vis. Sci.
			42: 2458-2465 (2001)
RP1	8q12.1	dominant RP; recessive RP;	Bowne et al., Hum.
		protein: RP1 protein	Mol. Genet. 11: 2121-
			2128 (1999)
TTPA	8q12.3	recessive RP and/or recessive or	Yokota et al., New
		dominant ataxia; protein: alpha-	Eng. J. Med. 335: 1770-
		tocopherol-transfer protein	1771 (1996)
ROA1	8q21-q22	recessive optic atrophy	Barbet et al., Eur. J.
			Hum. Genet. 11: 966-
			971 (2003)
PXMP3,	8q21.13	recessive Refsum disease,	Gartner et al., Nat.
PAF1, PEX2,		infantile form; protein:	Genet. 1: 16-23 (1992)
PMP35		peroxisomal membrane protein 3
CNGB3,	8q21.3	recessive achromatopsia	Kohl et al., Eur. J.
ACHM3		Pingelapese; recessive,	Hum. Genet. 13: 302-
		progressive cone dystrophy;	308 (2005)
		protein: cone cyclic nucleotide-
		gated cation channel beta 3
		subunit
VMD1	not 8q24	dominant MD, atypical	Sohocki et al., Am. J.
		vitelliform	Hum. Genet. 61: 239-
			241 (1997)
RP31	9p22-p13	dominant RP	Papaioannou et al.,
			Hum. Mut. 118: 501-
			503 (2005)
KCNV2	9q24.2	recessive cone dystrophy with	Wu et al., Am. J. Hum.
		supernormal rod	Genet. 79: 574-579
		electroretinogram; protein:	(2006)
		potasium channel subfamily V
		member 2
INVS, NPHP2	9q31.1	recessive Senior-Loken	O'Toole et al., Nephrol.
		syndrome; recessive	Dial. Transplant.
		nephronophthisis; protein:	21: 1989-1991 (2006)
		inverson
DFNB31	9q32	recessive Usher syndrome, type 2;	Ebermann et al., Hum.
		recessive deafness without RP;	Genet. (2006)
		protein: whirlin
TLR4	9q33.1	age-related macular degeneration,	Zareparsi et al., Hum.
		complex etiology; protein: toll-	Mol. Genet. 14: 1449-
		like receptor 4	1455 (2005)
TRIM32,	9q33.1	recessive Bardet-Biedl syndrome;	Chiang et al., Proc.
BBS11, HT2A		recessive limb-girdle muscular	Natl. Acad. Sci. USA
		dystrophy; protein: tripartite	103: 6287-6292 (2006)
		motif-containing protein 32
RP21, RP8	not 9q34-qter	dominant RP with sensorineural	Mansergh et al., Am. J.
		deafness	Hum. Genet. 64: 971-
			985 (1999)
JBTS1,	9q34	recessive Joubert syndrome	Saar et al., Am. J. Hum.
CORS1			Genet. 65: 1666-1671
			(1999)
PHYH,	10p13	recessive Refsum disease, adult	Jansen et al., Nat.
PAHX, RDPA		form; protein: phytanoyl-CoA	Genet. 17: 190-193
		hydroxylase	(1997)
RNANC	10q21	recessive nonsyndromal	Ghiasvand et al., Am. J.
		congenital retinal nonattachmen	Med. Genet. 90: 165-
			168 (2000)
PCDH15,	10q21.1	recessive Usher syndrome, type	Ahmed et al., Hum.
DFNB23,		1f; recessive deafness without	Mol. Genet. 12: 3215-
USH1F		RP; protein: protocadherin 15	3223 (2003)
CDH23,	10q22.1	recessive Usher syndrome, type	Astuto et al., Am. J.
DFNB12,		1d; recessive deafness without	Hum. Genet. 71: 262-
USH1D		RP; protein: cadherin-like gene	275 (2002)
		23
RGR	10q23.1	recessive RP; dominant choroidal	Morimura et al., Nat.
		sclerosis; protein: RPE-retinal G	Genet. 23: 393-394
		protein-coupled receptor	(1999)
RBP4	10q23.33	recessive RPE degeneration;	Seeliger et al., Invest.
		protein: retinol-binding protein 4	Ophthalmol. Vis. Sci.
			40: 3-11 (1999)
PAX2, ONCR	10q24.31	dominant renal-coloboma	Favor et al., Proc. Natl.
		syndrome; protein: paired	Acad. Sci. USA
		homeotic gene 2 protein	93: 13870-13875 (1996)
HTRA1,	10q26.13	age-related macular degeneration,	DeWan et al., Science
PRSS11		complex etiology; protein: HtrA	314: 989-992 (2006)
		serine peptidase 1
LOC387715	10q26.13	age-related macular degeneration,	Jakobsdottir et al., Am.
		complex etiology; protein:	J. Hum. Genet. 77: 389-
		hypothetical protein with Entrez	407 (2005)
		ID 387715
OAT	10q26.13	recessive gyrate atrophy; protein:	D Valle, O Simell. In
		ornithine aminotransferase	‘The Metabolic and
			Molecular Bases of
			Inherited Disease’, 8th
			Ed. C R Schriver, et al.
			eds., McGraw-Hill.
			(2000)
TEAD1, AA,	11p15.3	dominant atrophia areata; protein:	Fossdal et al., Hum.
TCF13, TEF1		TEA domain family member 1	Mol. Genet. 13: 975-
		[Entrez]	981 (2004)
USH1C,	11p15.1	recessive Usher syndrome,	Ahmed et al., Hum.
DFNB18		Acadian; recessive deafness	Genet. 110: 527-531
		without RP; protein: harmonin	(2002)
EVR3	11p13-p12	dominant familial exudative	Downey et al., Am. J.
		vitreoretinopathy	Hum. Genet. 68: 778-
			781 (2001)
CORS2,	11p12-q13.3	recessive Joubert syndrome	Valente et al., Ann.
JBTS2			Neurol. 57: 513-519
			(2005)
ROM1	11q12.3	dominant RP; digenic RP with	Dryja et al., Invest.
		RDS; protein: retinal outer	Ophthalmol. Vis. Sci.
		segment membrane protein 1	18: 1972-1982 (1997)
VMD2	11q12.3	dominant MD, Best type;	Weber et al., Am. J.
		dominant	Hum. Genet. 55: 1182-
		vitreoretinochoroidopathy;	1187 (1994a)
		protein: bestrophin
BBS1	11q13	recessive Bardet-Biedl syndrome;	Mykytyn et al., Nat.
		protein: BBS1 protein	Genet. 31: 435-438
			(2002)
VRNI	11q13	dominant neovascular	Stone et al., Hum. Mol.
		inflammatory vitreoretinopathy	Genet. 1: 685-689
			(1992)
CABP4	11q13.1	recessive CSNB; protein: calcium	Zeitz et al., Am. J.
		binding protein 4	Hum. Genet. 79: 657-
			667 (2006)
LRP5, EVR4,	11q13.2	dominant familial exudative	Jiao et al., Am. J. Hum.
HBM, OPPG		vitreoretinopathy; dominant high	Genet. 75: 878-884
		bone mass trait; recessive	(2004)
		osteoporosis-pseudoglioma
		syndrome; recessive FEVR;
		protein: low density lipoprotein
		receptor-related protein 5
MYO7A,	11q13.5	recessive Usher syndrome, type 1 ;	Gibbs et al., Natl.
DFNB2,		recessive congenital deafness	Acad. Sci. USA
USH1B		without RP; recessive atypical	100: 6481-6486 (2003)
		Usher syndrome (USH3-like);
		protein: myosin VIIA
FZD4, EVR1,	11q14.2	dominant familial exudative	Müller et al., Genomics
FEVR		vitreoretinopathy; protein:	20: 317-319(1994)
		frizzled-4 Wnt receptor homolog
C1QTNF5,	11q23.3	dominant MD, late onset;	Ayyagari et al., Invest.
CTRP5		dominant MD with lens zonules;	Ophthalmol. Vis. Sci.
		protein: C1q and tumor necrosis-	46: 3363-3371 (2005)
		related protein 5 collagen
COL2A1,	12q13.11	dominant Stickler syndrome, type	Snead et al., J. Med.
AOM, STL1		I; dominant Wagner syndrome;	Genet. 36: 353-659
		dominant epiphyseal dysplasia;	(1999)
		protein: collagen, type II, alpha 1
RDH5, RDH1	12q13.2	recessive fundus albipunctatus;	Cideciyan et al., Vis.
		recessive cone dystrophy, late	Neurosci. 17: 667-678
		onset; protein: 11-cis retinol	(2000)
		dehydrogenase 5
BBS10,	12q21.2	recessive Bardet-Biedl syndrome;	Stoetzel et al., Nat.
FLJ23560		protein: BBS10 (C12orf58)	Genet. 38: 521-524
		chaperonin	(2006)
CEP290,	12q21.32	recessive Senior-Loken	Chang et al., Hum.
JBTS5,		syndrome; recessive Joubert	Mol. Genet. 15: 1847-
NPHP6,		syndrome; recessive Leber	1857 (2006)
SLSN6		congenital amaurosis; protein:
		centrosomal protein 290 kDa
RB1	13q14.2	dominant germline or somatic	Lohmann et al., Am. J.
		retinoblastoma; benign retinoma;	Hum. Genet. 58: 940-
		pinealoma; osteogenic sarcoma;	949 (1996)
		protein: retinoblastoma protein 1
GRK1,	13q34	recessive CSNB, Oguchi type;	Cideciyan et al., Proc.
RHOK, RK		protein: rhodopsin kinase	Natl. Acad. Sci. USA
			95: 328-333 (1998)
STGD2	not 13q34	dominant MD, Stargardt type	Zhang et al., Nat.
			Genet. 27: 89-93 (2001)
ACHM1,	14	recessive rod monochromacy or	Pentao et al., Am. J.
RMCH		achromatopsia	Hum. Genet. 50: 690-
			699 (1992)
RP16	not 14	recessive RP	Bruford et al., Am. J.
			Hum. Genet. 55: A181
			(1994)
MCDR4	14q	dominant MD, North Carolina-	Francis et al., Br. J.
		like with progressive	Ophthalmol. 87: 893-
		sensorineural hearing loss	898 (2003)
NRL, RP27	14q11.2	dominant RP; recessive RP;	Mears et al., Nat.
		protein: neural retina lucine	Genet. 29: 447-452
		zipper	(2001)
RPGRIP1,	14q11.2	recessive Leber congenital	Mellersh et al.,
LCA6		amaurosis; protein: RPGR-	Genomics 88: 293-301
		interacting protein 1	(2006)
LCA3	14q24	recessive Leber congenital	Stockton et al., Hum.
		amaurosis	Genet. 103: 328-333
			(1998)
RDH12	14q24.1	recessive Leber congenital	Janecke et al., Nat.
		amaurosis with severe childhood	Genet. 36: 850-854
		retinal dystrophy; protein: retinol	(2004)
		dehydrogenase 12
USH1A,	not 14q32	recessive Usher syndrome,	Gerber et al., Am. J.
USH1		French	Hum. Genet. 78: 357-
			359 (2006)
TTC8, BBS8	14q32.11	recessive Bardet-Biedl syndrome;	Ansley et al., Nat.
		protein: tetratricopeptide repeat	425: 628-633 (2003)
		domain 8
FBLN5	14q32.12	familial MD, age-related; protein:	Arch. Ophthalmol.
		fibulin 5	112: 765-772 (1994)
NR2E3,	15q23	recessive enhanced S-cone	Sharon et al., Arch.
ESCS, PNR		syndrome; recessive RP in	Ophthalmol. 121: 1316-
		Portuguese Crypto Jews;	1323 (2003)
		Goldmann-Favre syndrome;
		protein: nuclear receptor
		subfamily 2 group E3
MRST	15q24	recessive retardation, spasticity	Mitchell et al., Am. J.
		and retinal degeneration	Hum. Genet. 62: 1070-
			1076 (1998)
BBS4	15q24.1	recessive Bardet-Biedl syndrome;	Katsanis et al., Nat.
		protein: BBS4 protein	Genet. 26: 67-70 (2000)
RLBP1,	15q26.1	recessive RP; recessive Bothnia	Burstedt et al., Invest.
CRALBP		dystrophy; recessive retinitis	Ophthalmol. Vis. Sci.
		punctata albescens; recessive	40: 995-1000 (1999)
		Newfoundland rod-cone
		dystrophy; protein: retinaldehyde-
		binding protein 1
ABCC6,	16p13.11	recessive pseudoxanthoma	Bergen et al., Nat.
ARA, MRP6,		elasticum; dominant	Genet. 25: 228-231
PXE		pseudoxanthoma elasticum;	(2000)
		protein: ATP-binding casette,
		subfamily C, member 6
RP22	16p12.3-p12.1	recessive RP	Finckh et al., Genomics
			48: 341-345 (1998)
CLN3, JNCL	16p11.2	recessive Batten disease (ceroid-	Kremmidiotis et al.,
		lipofuscinosis, neuronal 3),	Hum. Mol. Genet.
		juvenile; protein: Batten disease	8: 523-531 (1999)
		protein
BBS2	16q12.2	recessive Bardet-Biedl syndrome;	Beales et al., Am. J.
		protein: BBS2 protein	Hum. Genet. 68: 606-
			616 (2001)
CNGB1,	16q13	recessive RP; protein: rod cGMP-	Bareil et al., Hum.
CNCG2,		gated channel beta subunit	Genet. 108: 328-334
CNCG3L,			(2001)
GAR1, GARP
CDH3,	16q22.1	recessive MD, juvenile with	Indelman et al., J.
CDHP, PCAD		hypotrichosis; protein: cadherin 3,	Invest. Dermatol.
		type 1, placental	119: 1210-1213 (2002)
FHASD	16q23.2-q24.2	recessive foveal hypoplasia and	Pal et al., J. Med.
		anterior segment dysgenesis	Genet. 41: 772-777
			(2004)
CACD	17p13	dominant central areolar	Hughes et al., J. Med.
		choroidal dystrophy	Genet. 35: 770-772
			(1998)
PRPF8,	17p13.3	dominant RP; protein: human	Kojis et al., Am. J.
PRPC8, RP13		homolog of yeast pre-mRNA	Hum. Genet. 58: 347-
		splicing factor C8	355 (1996)
AIPL1, LCA4	17p13.2	recessive Leber congenital	Hanein et al., Hum.
		amaurosis; dominant cone-rod	Mutat. 23: 306-317
		dystrophy; protein:	(2004)
		arylhydrocarbon-interacting
		receptor protein-like 1
GUCY2D,	17p13.1	recessive Leber congenital	Hanein et al., Hum.
CORD6,		amaurosis; dominant cone-rod	Mutat. 23: 306-317
LCA1,		dystrophy; protein: retinal-	(2004)
RETGC,		specific guanylate cyclase
RETGC1
CORD5,	same as	dominant cone-rod dystrophy,	Udar et al., Hum. Mut.
RCD2	GUCY2D	progressive; recessive cone-rod	21: 170-171 (2003)
		dystrophy
CORD4	17q	cone-rod dystrophy	Klystra et al.,
UNC119,	17q11.2	dominant cone-rod dystrophy;	Kobayashi et al.,
HRG4		protein: human homolog of C.	Invest. Ophthalmol.
		elegans unc119 protein	Vis. Sci. 41: 3268-3277
			(2000)
CA4, RP17	17q23.2	dominant RP; protein: carbonic	Rebello et al., Proc.
		anhydrase IV	Natl. Acad. Sci. USA
			101: 6617-6622 (2004)
USH1G,	17q24-q25	recessive Usher syndrome;	Kikkawa et al., Hum.
SANS		protein: human homolog of	Mol. Genet. 12: 453-
		mouse scaffold protein containing	461 (2003)
		ankyrin repeats and SAM domain
RGS9	17q24.1	recessive delayed cone	Nishiguchi et al.,
		adaptation; protein: regulator of	Nature 427: 75-78
		G-protein signalling 9	(2004)
PRCD	17q25.1	recessive RP; protein: progressive	Zangerl et al.,
		rod-cone degneration protein	Genomics (2006)
FSCN2, RP30	17q25.3	dominant RP; dominant MD;	Wada et al., Arch.
		protein: retinal fascin homolog 2,	Ophthalmol. 121: 1613-
		actin bundling protein	1620 (2003)
OPA4	18q12.2-q12.3	dominant optic atrophy, Kjer type	Kerrison et al., Arch.
			Ophthalmol. 117: 805-
			810 (1999)
CORD1	18q21.1-q21.3	cone-rod dystrophy; de Grouchy	Manhant et al., Am. J.
		syndrome	Hum. Genet. 57: A96
			(1995)
R9AP	19q13.12	recessive delayed cone	Nishiguchi et al.,
		adaptation; protein: regulator of	Nature 427: 75-78
		G-protein signalling 9-binding	(2004)
		protein
MCDR5	19q13.31-q13.32	dominant macular dystrophy	Yang et al., Science
			314: 992-993 (2006)
CRX, CORD2	19q13.32	dominant cone-rod dystrophy;	Hanein et al., Hum.
		recessive, dominant and de novo	Mutat. 23: 306-317
		Leber congenital amaurosis;	(2004)
		dominant RP; protein: cone-rod
		otx-like photoreceptor homeobox
		transcription factor
OPA3, MGA3	19q13.32	recessive optic atrophy with	Anikster et al., Am. J.
		ataxia and 3-methylglutaconic	Hum. Genet. 69: 1218-
		aciduria; protein: OPA3 protein	1224 (2001)
PRPF31,	19q13.42	dominant RP; protein: human	Sullivan et al., Invest.
PRP31, RP11		homolog of yeast pre-mRNA	Ophthalmol. Vis. Sci.
		splicing factor 31	47: 4579-4588 (2006)
JAG1, AGS	20p12.2	dominant Alagille syndrome;	Li et al., Nat. Genet.
		protein: Jagged protein 1	16: 243-251 (1997)
MKKS, BBS6	20p12.2	recessive Bardet-Biedl syndrome;	Beales et al., Am. J.
		protein: McKusick-Kaufman	Hum. Genet. 68: 606-
		syndrome protein	616 (2001)
PANK2,	20p13	recessive HARP	Hartig et al., Ann.
HARP, PKAN		(hypoprebetalipoproteinemia,	Neurol. 59: 248-256
		acanthocytosis, RP, and palladial	(2006)
		degeneration); recessive
		Hallervorden-Spatz syndrome;
		protein: pantothenate kinase 2
USH1E	21q21	recessive Usher syndrome, type 1	Chäib et al., Hum. Mol.
			Genet. 6: 27-31 (1997)
OPA5	22q12.1-q12.3	dominant optic atrophy	Rozet et al., Invest.
			Ophthalmol. Vis. Sci.
			46: E-Abstract 2292
			(2005)
TIMP3, SFD	22q12.3	dominant Sorsby's fundus	Felbor et al., Am. J.
		dystrophy; protein: tissue	Hum. Genet. 60: 57-62
		inhibitor of metalloproteinases-3	(1997)
RP23	Xp22	X-linked RP	Hardcastle et al.,
			Invest. Ophthalmol.
			Vis. Sci. 41: 2080-2086
			(2000)
RS1, XLRS1	Xp22.13	retinoschisis; protein:	Grayson et al., Hum.
		retinoschisin	Mol. Genet. 9: 1873-
			1879 (2000)
(- - -)	Xp21-q21	RP with mental retardation	Aldred et al., Am. J.
			Hum. Genet. 55: 916-
			922 (1994)
RP6	Xp21.3-p21.2	X-linked RP	Breuer et al., Invest.
			Ophthalmol. Vis. Sci.
			41: S191 (2000)
DMD	Xp21.2-p21.1	Oregon eye disease (probably);	D'Souza et al., Hum.
		protein: dystrophin	Mol. Genet. 5: 837-842
			(1995)
AIED, OA2	Xp11.4-q21	Åland island eye disease	Wutz et al., Eur. J.
			Hum. Genet. 10: 449-
			456 (2002)
COD4	Xp11.4-q13.1	X-linked progressive cone-rod	Jalkanen et al., J. Med.
		dystrophy	Genet. 40: 418-423
			(2003)
OPA2	Xp11.4-p11.2	X-linked optic atrophy	Assink et al., Am. J.
			Hum. Genet. 61: 934-
			939 (1997)
NYX, CSNB1	Xp11.4	X-linked CSNB; protein:	Bech-Hansen et al.,
		nyctalopin	Nat. Genet. 26: 319-323
			(2000)
CSNB4	same as NYX	X-linked CSNB	Pusch et al., Nat.
			Genet. 26: 324-327
			(2000)
RPGR, RP3	Xp11.4	X-linked RP, recessive; X-linked	Bader et al., Invest.
		RP, dominant; X-linked CSNB;	Ophthalmol. Vis. Sci.
		X-linked cone dystrophy 1; X-	44: 1458-1463(2003)
		linked atrophic MD, recessive;
		protein: retinitis pigmentosa
		GTPase regulator
COD1	same as RPGR	X-linked cone dystrophy 1	Demirci et al., Am. J.
			Hum. Genet. 70: 1049-
			1053 (2002)
RP15	same as RPGR	X-linked RP, dominant	Mears et al., Am. J.
			Hum. Genet. 67: 1000-
			1003 (2000)
PRD	Xp11.3-p11.23	retinal dysplasia, primary	Ravia et al., Hum. Mol.
			Genet. 8: 1295-1297
			(1993)
NDP, EVR2	Xp11.3	Norrie disease; familial exudative	Black et al., Hum. Mol.
		vitreoretinopathy; Coats disease;	Genet. 11: 2021-2035
		protein: Norrie disease protein	(1999)
CACNA1F,	Xp11.23	X-linked CSNB, incomplete;	Nakamura et al., Arch.
CSNB2,		ÅIED-like disease; severe CSNB;	Ophthalmol. 121: 1028-
CSNBX2		protein: L-type voltage-gated	1033 (2003)
		calcium channel alpha-1 subunit
RP2	Xp11.23	X-linked RP; protein: novel	Hardcastle et al., Am. J.
		XRP2 protein similar to human	Hum. Genet. 64: 1210-
		cofactor C	1215 (1999)
PGK1	Xq21.1	RP with myopathy; protein:	Tonin et al., Neurol.
		phosphoglycerate kinase	43: 387-391 (1993)
CHM	Xq21.2	choroideremia; protein:	van den Hurk et al.,
		geranylgeranyl transferase Rab	Hum. Mutat. 9: 110-117
		escort protein 1	(1997)
TIMM8A,	Xq22.1	optic atrophy with deafness-	Koehler et al., Proc.
DDP, DDP2,		dystonia syndrome; protein: inner	Natl. Acad. Sci USA
DFN1		mitochondrial membrane	96: 2141-1246 (1999)
		translocase 8 homolog A
RP24	Xq26-q27	X-linked RP	Gieser et al., Am. J.
			Hum. Genet. 63: 1439-
			1447 (1998)
COD2,	Xq27	X-linked progressive cone	Bergen et al.,
XLPCD		dystrophy, 2
RP34	Xq28-qter	X-linked RP	Melamud et al., J. Med.
			Genet. 43: e27 (2006)
OPN1LW,	Xq28	deuteranopia and rare macular	Ayyagari et al., Mol.
GCP, CBD		dystrophy in blue cone	Vis. 58: 98-101 (1999)
		monochromacy with loss of locus
		control element; protein: green
		cone opsin
OPN1MW,	Xq28	protanopia and rare macular	Ayyagari et al., Mol.
RCP, CBP		dystrophy in blue cone	Vis. 58: 98-101 (1999)
		monochromacy with loss of locus
		control element; protein: red cone
		opsin
KSS	mitochondrion	Kearns-Sayre syndrome including	al., Science 283: 1482-
		retinal pigmentary degeneration;	1488 (1999)
		protein: several mitochondrial
		proteins
LHON,	mitochondrion	Leber hereditary optic	Brown et al., Am. J.
MTND1,		neuropathy; protein: complex I,	Hum. Genet. 60: 381-
MTND4,		III or IV proteins	387 (1997)
MTND6
MTTL1,	mitochondrion	macular pattern dystrophy with	Bonte et al., Retina
DMDF,		type II diabetes and deafness;	17: 216-221 (1997)
TRNL1		protein: leucine tRNA 1
		(UUA/G), nt 3230-3304
MTATP6,	mitochondrion	RP with developmental and	White et al., J. Inherit.
ATP6, NARP		neurological abnormalities; Leigh	Metab. Dis. 22: 899-914
		syndrome; LHON; protein:	(1999)
		complex V ATPase 6 subunit, nt
		8527-9207
MTTH,	mitochondrion	pigmentary retinopathy and	Crimi et al., Neurology
TRNH		sensorineural hearing loss;	60: 1200-1203 (2003)
		protein: histidine tRNA, nt
		12138-12206
MTTS2,	mitochondrion	RP with progressive sensorineural	Mansergh et al., Am. J.
TRNS2		hearing loss; protein: serine tRNA	Hum. Genet. 64: 971-
		2 (AGU/C), nt 12207-12265	985 (1999)

[0076] In an embodiment of the invention, suppression agents are siRNAs or shRNAs targeting human rhodopsin. Exemplary siRNAs and replacement rhodopsin sequences are provided in Table 6A.

[0077]

TABLE 6A
Exemplary siRNA Sequences Targeting Human Rhodopsin and
Replacement Rhodopsin Sequences
	SEQ		SEQ
siRNA Target Site	ID NO	Replacement Site	ID NO
1. TACGTCACCGTCCAGCACAAG	1	TATGTGACGGTGCAACATAA	2
2. CTCAACTACATCCTGCTCAAC	3	CTGAATTATATTTTATTGAAT	4
3. CAGCTCGTCTTCACCGTCAAG	5	CAATTGGTGTTTACGGTGAAA	6
4. ATCTATATCATGATGAACAAG	7	ATTTACATTATGATGAATAAA	8
5. GCCTACATGTTTCTGCTGATC	9	GCTTATATGTTCTTATTAATT	10
6. TACATGTTTCTGCTGATCGTG	11	TATATGTTCTTATTAATTGTC	12
7. CTGCGCACGCCTCTCAACTAC	13	TTACGGACCCCCTTGAATTAT	14
8. CGCACGCCTCTCAACTACATC	15	CGGACCCCCTTGAATTATATT	16
9. CTCAAGCCGGAGGTCAACAAC	17	TTGAAACCCGAAGTGAATAAT	18
10. CAGCTCGTCTTCACCGTCA	19	CAATTGGTGTTTACGGTGA	20
11. TACGCCAGCGTGGCATTCTAC	21	TATGCTTCTGTCGCCTTTTAC	22
12. CCAGCGTTCTTTGCCAAGA	23	CCCGCCTTTTTCGCTAAAA	24
13. GTCATCTATATCATGATGAAC	25	GTGATTTACATTATGATGAAT	26
14. AACTGCATGCTCACCACCATC	27	AATTGTATGTTGACGACGATT	28
15. ACCATCTGCTGCGGCAAGA	29	ACGATTTGTTGTGGGAAAA	30
16. GACGATGAGGCCTCTGCTA	31	GAGGACGAAGCTAGCGCCA	32
17. CACCTCTCTGCATGGATACT	33	CACGAGCTTACACGGGTATT	34
siRNAs Targeting 5′ UTR
18. AGCTCAGGCCTTCGCAGCA	35
19. CAGGCCTTCGCAGCATTCT	36
siRNAs Targeting 3′ UTR
20. TCACTTTCTTCTCCTATAA	37
21. TAGTTAATGTTGTGAATAA	38
22. GCTCCTATGTTGGTATTAA	39
23. AGTCACATAGGCTCCTTAA	40
24. GATTCTTGCTTTCTGGAAA	41
25. ACAGTAGGTGCTTAATAAA	42
26. GAACATATCTATCCTCTCA	43
27. CTGTACAGATTCTAGTTAA	44
28. TGTGAATAACATCAATTAA	45
29. CAATTAATGTAACTAGTTA	46
30. TGATTATCACCTCCTGATA	47
31. GCAGTCATCAGACCTGAAA	48
32. TGTCATCCTTACTCGAAGA	49
33. GAATTAAGCTGCCTCAGTA	50
34. GCCAGAAGCTCTAGCTTTA	51
35. AGCTCTGCCTGGAGACTAA	52
siRNAs Targeting an Intron
36. GATCTTATTTGGAGCAATA	53
37. TGGCTGTGATCCAGGAATA	54
38. GATGCATTCTTCTGCTAAA	55
39. GCAATATGCGCTTGTCTAA	56
40. TTGTCTAATTTCACAGCAA	57
41.TGTTTGTTGCATTCAATAA	58
42. CCAGAGCGCTAAGCAAATA	59
43. GTCTTGCATTTAACAGGAA	60
44. GGCTGTGATCCAGGAATAT	61
45. TGCAGGAGGAGACGCTAGA	62
46. CTTTCACTGTTAGGAATGT	63
47. TTTGGTTGATTAACTATAT	64
48. TTAACTATATGGCCACTCT	65
49. AGATGTTCGAATTCCATCA	66
siRNAs Targeting a Polymorphism
50. TCTTCACCGTCAAGGAGGTAT	67	TGTTTACGGTGAAAGAAGTAC	68

[0078] siRNA sequences 1-17 target the human rhodopsin coding sequence. siRNA sequences 18 and 19 target the human rhodopsin 5′UTR. siRNA sequences 20-35 target the human rhodopsin 3′UTR. siRNA sequences 36-49 target human rhodopsin intronic sequence. The sequence of the sense strand of the siRNA is given. Notably, siRNAs may also target a combination of these. For example, an siRNA target site may be in the 5′UTR and exon 1. Or an siRNA target site may be in the coding region and an intron. Or an siRNA target site may be in an exon and the 3′UTR. siRNA sequence 50 is an example of an siRNA that has a target site that spans Exon 3/intron 3 of the human rhodopsin gene. The site contains a known polymorphism in intron 3. If this site was used as an siRNA target, the replacement gene would have the wildtype base at the polymorphic site but degeneracy of the genetic code could be used to change other bases at the replacement site. The siRNA(s) may comprise all or part of the sequence provided. The sequences of replacement human rhodopsin nucleic acids over the target for siRNA-mediated suppression are provided for siRNA sequences 1-17. Replacement nucleic acids include at least one altered nucleotide(s) at degenerate position(s) over the siRNA target site (highlighted in bold print). Thus, replacement sequences here provide one of multiple replacement options. Some replacement constructs contain nucleotide changes in the coding sequence. These replacement constructs while altered in nucleotide sequence encode the same amino acids as the wild type rhodopsin protein. Other replacement constructs are altered at either silent or non-silent polymorphic sites. These replacement constructs encode wild type protein, with wild type function. For siRNAs targeting the UTRs or intronic sequence, no replacement constructs have been suggested because the number of base changes within the site is not limited to degenerate positions (as is the case for sequence coding for amino acids).

• • [0079] It is notable that suppression of a given gene such as rhodopsin may be evaluated in a variety of animal species. The siRNA sequences provided in Table 6B represent examples of RNAi sequences that are homologous between porcine and human rhodopsin. In some transgenic animal models the presence of the human transgene enables direct evaluation of sequences that target the human gene in that animal model. In other instances suppressor sequences may be chosen to maximise the homology between the human gene (for example, rhodopsin) and the endogenous gene in the animal under evaluation.

[0080]

TABLE 6B
Exemplary siRNA Sequences Targeting Homologous
Sequences Between Human and Porcine Rhodopsin
		SEQ		Suppression
		ID	Position in	levels in
siRNA	Sequence	NO:	NM_000539.2	HeLa Cells
P1	ACCTCTCTGCATG	414	384-403	69%
	GATAGT-TT
P2	CATGTTCGTGGTC	415	713-732	81%
	CACTTC-TT

• • [0081] siRNA can be expressed in miR vectors using polymerase II promoters. For this purpose pcDNA6.2-GW/EmGFP-miR from Invitrogen is used where the cloned miR-155 gene is recombined in order to express the choice of siRNA. The antisense strand of the siRNA is kept intact followed by a modified terminal loop and the sense strand, which is modified by introducing a deletion of 2 central nucleotides in order to form an internal loop. See Catalogue no K4936-00, Block-IT, POLII, miR RNAi expression vector kits catalogue, Invitrogen, page 7 for figure showing the native miR-155 sequence and the converted sequence of siRNA-lacZ in the form of miR-lacZ.
    Exemplary miRNA Sequences Targeting Human Rhodopsin:

[0082]

CC miRNA oligos:

Top strand:

(SEQ ID NO: 416)

5′ - TGCTGCTTCTTGTGCTGGACGGTGACGTTTTGGCCACTGACTG

ACGTCACCGTAGCACAAGAAG - 3′

Bottom strand:

(SEQ ID NO: 417)

5′ - CCTGCTTCTTGTGCTACGGTGACGTCAGTCAGTGGCCAAAACG

TCACCGTCCAGCACAAGAAGC - 3′

Q1 miRNA oligos:

Top strand:

(SEQ ID NO: 418)

5′ - TGCTGGTAGTAGTCGATTCCACACGAGTTTTGGCCACTGACTG

ACTCGTGTGGTCGACTACTAC - 3′

Bottom strand:

(SEQ ID NO: 419)

5′ - CCTGGTAGTAGTCGACCACACGAGTCAGTCAGTGGCCAAAACT

CGTGTGGAATCGACTACTACC - 3′

BB miRNA oligos:

Top strand:

(SEQ ID NO: 420)

5′ - TGCTGGTAGAGCGTGAGGAAGTTGATGTTTTGGCCACTGACTG

ACATCAACTTTCACGCTCTAC - 3′

Bottom strand:

(SEQ ID NO: 421)

5′ - CCTGGTAGAGCGTGAAAGTTGATGTCAGTCAGTGGCCAAAACA

TCAACTTCCTCACGCTCTACC - 3′

[0083] In an embodiment of the invention, suppression agents and replacement genes are expressed in photoreceptor cells to alleviate disease pathology. In a further embodiment, replacement nucleic acids encode a gene which when mutated may cause retinal degeneration other than retinitis pigmentosa, for example, Stargarts Syndrome, glaucoma, cod-rod dystrophy, corneal dystrophy or Age-related Macular Degeneration (AMD) (Table 5).

[0084] In another aspect, the invention provides cells expressing a suppression effector such as a dsRNA, either transiently or stably, for experimental or therapeutic use. In an embodiment, the cells express an siRNA that targets rhodopsin. In another embodiment, the cells express a replacement nucleic acid expressing rhodopsin that is not targeted by the siRNA. In another embodiment, the cells comprise a vector encoding at least one or more siRNAs. In another embodiment, the cells comprise a vector encoding a replacement nucleic acid. In an additional embodiment, the cells comprise one or more vectors encoding siRNA(s) and replacement nucleic acid(s).

[0085] In another aspect, the invention provides transgenic animals and their experimental or therapeutic use. In an embodiment, the transgenic animal is a model for Retinitis Pigmentosa, for example, an animal with a mutation observed in humans such as the Pro23His and or Pro347ser mutations. In another embodiment, the transgenic animal expresses a dsRNA that targets human rhodopsin. In another embodiment, the transgenic animal expresses a replacement nucleic acid transgene that has been altered at one or more wobble position(s) such that it escapes suppression.

[0086] Suppression agents and replacement nucleic acids of the invention can be administered to cells, tissues, plants and/or animals, either separately or together. In yet another aspect administration of suppression agent and/or replacement nucleic acid may be systemic or local. In yet another aspect, administration of suppression agent and replacement nucleic acid may be used in conjunction with chemical and/or physical agents to aid administration. In another aspect, the invention provides methods for suppressing rhodopsin expression in an animal by intraocular (e.g., subretinal or intravitreal) injection of a suppression agent into the animal. In another aspect intraocular administration (e.g., subretinal injection, intravitreal) is used to administer a suppression agent and/or replacement nucleic acid to an animal. In another embodiment, ionthophoresis or electroporation is used to administer suppression agents and/or replacement nucleic acids. In another embodiment, suppression agents and/or replacement nucleic acids are administered using nanotechnology (Kawasaki and Player Nanomedicine 1(2):101-9, 2005; Silva Surg. Neurol. 67(2):113-6, 2007; Andrieu-Solar et al., Mol. Vis. 12:1334-47, 2006) or bacteria (Daudel et al., Expert Rev. Vaccines 6(1):97-110, 2007).

[0087] Suppression agents and replacement nucleic acids may be optimally combined with conserved regions A-I and/or transcription factor binding sites identified within conserved regions A-I and/or with enhancer elements and/or other regulatory elements (see Tables 1 and 2 above and Tables 9-12 below).

[0088] In one aspect of the invention, there is provided a vector for expression of a suppression agent for a disease causing gene and/or a replacement nucleic acid that is not recognized by the suppression agent, wherein the vector comprises at least one of the conserved regions selected from: conserved region B from the rhodopsin gene represented by SEQ ID NO: 93, or a variant or equivalent thereof; conserved region C from the rhodopsin gene represented by SEQ ID NO: 94, or a variant or equivalent thereof; conserved region F and G from the rhodopsin gene represented by SEQ ID NO: 97 or a variant or equivalent thereof; and conserved region A from the rhodopsin gene represented by SEQ ID NO: 92, or a variant or equivalent thereof. In a particular embodiment, the vector comprises at least one of the conserved regions selected from: conserved region B from the rhodopsin gene represented by SEQ ID NO: 93, conserved region C from the rhodopsin gene represented by SEQ ID NO: 94, conserved region F and G from the rhodopsin gene represented by SEQ ID NO: 97; and conserved region A from the rhodopsin gene represented by SEQ ID NO: 92.

[0089] In one embodiment of the invention the use of suppression and replacement constructs in combination with one or more factors to facilitate cell survival, cell viability and/or cell functioning is contemplated. In relation to neurons, a range of neurotrophic and/or neuroprotective factors may be used inter alia brain derived neurotrophic factor (BDNF), glial derived neurotrophic factor (GDNF), neurturin, ciliary derived neurotrophic factor (CNTF), nerve growth factor (NGF), fibroblast growth factors (FGF), insulin-like growth factors (IGF), pigment epithelium-derived factor (PEDG), hepatocyte growth factor (HGF), thyrotrophin releasing hormone (TRH) and rod derived cone viability factor (RDCVF) amongst others. There is substantial evidence in the literature that such factors may increase cell viability and/or cell survival for a range of cell types. For example, these factors have been shown to provide beneficial effects to a wide range of neuronal cell types including, for example, photoreceptors, when delivered either in protein or DNA forms (Buch et al., Mol. Ther., 2006; 14(5):700-709). The use of GDNF to augment gene-based therapies for recessive disease has been demonstrated in mice (Buch et al., Mol. Ther., 2006; 14(5):700-709). Genes encoding neurotrophic/neuroprotective factors may be expressed from general promoters such as the CBA promoter (Buch et al., Mol. Ther., 2006; 14(5):700-709) or from tissue specific promoters. Sequences to optimise expression of neurotrophic/neuroprotective factors such as those sequences identified in Tables 1, 2, 9-13 may be included in constructs.

• • [0090] Sequences of a number of exemplary neurotrophic factors are provided in FIG. 17. DNA encoding one or more neurotrophic and/or neuroprotective factors may be utilised in conjunction with suppression and replacement. FIG. 18 provides examples of constructs incorporating suppression and replacement sequences together with sequence encoding a factor promoting cell viability and/or cell functioning such as GDNF, neurturin, CNTF and/or RDCVF amongst others as described above. Well established art known methods involving DNA restriction digestion, DNA ligation into plasmids, bacterial transformation, characterization of transfected bacterial colonies, plasmid purification and DNA sequencing may be used to clone suppression and replacement and neuroprotection/neurotrophic sequences into DNA-based vectors. Examples of the design of such constructs are provided in FIG. 18
  • [0091] Constructs incorporating suppression and replacement and neurotrophic/neuroprotective factor(s) may be delivered using viral and/or non-viral vectors using art known methods (Andrieu-Soler et al., Mil. Vis., 2006; 12:1334-47). Naked DNA, lipids, polymers, nanoparticles, electrotransfer amongst other methods have been used to achieve gene/nucleotide delivery in cells and animals. For example, lentiviral vectors and/or adenoassociated viral (AAV) vectors may be used to deliver constructs incorporating the 3 components defined above (suppression, replacement and neurotrophism/neuroprotection). 3-component constructs in some instances may require vectors that have significant capacity in terms of size of DNA inserts. Many viral and non-viral vectors have been characterised that can facilitate large DNA fragments including inter alia lentiviral vectors and some of adenoassociated viral serotypes. For example, AAV serotype 2 capsid 5 vectors (AAV2/5) have been shown to accommodate 8-9 kilobases of DNA (Alberto Aurrichio; British Society of Gene Therapy, 2008). One or more components (suppression, replacement, neurotrophism/neuroprotection) may, for example, in the case of AAV be cloned between the AAV ITRS and or one or more components may be cloned into the backbone of the plasmid used to generate AAV. FIG. 18 provides key elements of the construct design (B). Utilisation of backbone plasmid sequences to carry components of a multi-component construct can be used to optimise the population of AAV vectors generated using that plasmid. Moreover, in relation to eye disease, it is notable that there is significant evidence that AAV2/5 transduces photoreceptor efficiently. Generation of AAV vectors carrying suppression and replacement sequences in conjunction with sequences encoding neurotrophic and/or neuroprotective agents is contemplated. While AAV may be of value as a vector to deliver 3-component constructs for some target tissues, a range of additional viral and non-viral vectors are available for this purpose, such as those described above, and vectors that are well know in the art.

[0092] While utilisation of a single vector to deliver 3-component constructs involving suppression and replacement and a neurotrophic/neuroprotective sequence to a cell, a tissue and or an animal is contemplated, the use of multiple vectors in combination to deliver all 3 components is also contemplated. The multivalent approach involving suppression, replacement and neuroprotection may involve the use of 1 or more vectors for delivery. In addition, the 3 components may be delivered using a combination of a vector or vectors incorporating DNA sequences together with RNA and or dsRNA and or protein. In the current invention, delivery of protein, of RNA encoding protein and/or of DNA encoding protein or a combination thereof to achieve delivery of all 3 components, suppression, replacement and neuroprotection, is contemplated.

[0093] In another embodiment of the invention the size of the backbone of the AAV plasmid vector is either increased or decreased so as to increase expression from the virus. For example, it has been described in the art that increasing the AAV virus backbone in size such that it is larger than the insert cloned within ITSI and ITS2 favours AAV packaging of the insert over packaging of the backbone, thereby increasing expression of DNA cloned within the ITR regions (Bennet et al., Reversal of visual defects in animal models of LCA within weeks of treatment with an optimized AAV. Molecular Therapy Vol. 15, supplement 1, s286).

[0094] In a further embodiment of the invention the size of the backbone is increased with a gene which is therapeutically beneficial driven by a promoter. In this embodiment a portion of packaged AAV consists of the backbone and hence a portion of AAV particles will express the gene encoded within the backbone. In one embodiment the therapeutically beneficial gene cloned in the backbone is a neurotrophic factor such GDNF, Neurturin or others.

[0095] While the invention can be used for dominant and or polygenic disorders, it may also be practised for recessive disorders. For example, the art describes that when treating the recessive disorder phenylketonuria (PKU) with replacement genes, endogenous protein expressed from mutant genes interfered with protein from replacement genes (Described in a thesis submitted to the University of Florida in partial fulfillment of the requirements for the degree of Doctor of Philosophy, by Catherine Elisabeth Charron, August 2005 and entitled “Gene therapy for phenylketonuria: dominant-negative interference in a recessive disease”). Thus, suppression and replacement constructs may be targeted to recessive disorders which like PKU require suppression and replacement.

[0096] Suppression and replacement technology provides a strategy that may be applicable to a wide range of genetic disorders including disorders characterized by either a recessive, dominant, polygenic, multifactorial or a dominant negative pathology. In a further embodiment of the invention conserved regions identified in the promoter region of mammalian rhodopsin genes and/or enhancer elements and/or other regulatory elements and/or epigenetic elements such as listed in Table 5 may be combined with suppressors targeting genes with mutations other than rhodopsin and providing replacement genes other than rhodopsin. Osteogenesis imperfecta, epidermolysis bullosa, autosomal dominant early onset Alzheimer's disease, autosomal dominant polycystic kidney disease, Rett syndrome, familial platelet disorder, dominant negative diabetes insipidus, autosomal dominant Stargardt like macular dystrophy, nemaline myopathy, familial pulmonary arterial hypertension, APC and p53 related cancers and several other disorders (OMIM) may potentially benefit from a suppression and replacement therapeutic approach. Triplet repeat disorders, 14 of which have been characterised to date, including Huntington's disease, spinocerebellar ataxia and myotonic dystrophy may benefit from a suppression and replacement approach. For each disorder, promoters of the endogenous gene or constitutive promoters or promotes from other genes, or inducible promoters may be used to express the suppression agent or replacement nucleic acid.

[0097] In another embodiment of the invention, promoter and/or enhancer elements and/or other regulatory elements and/or epigenetic elements may be combined with other promoters than rhodopsin in combination with suppression and/or replacement elements. For example, but not exclusively, promoter and enhancer elements can be combined with the COL1A1 and or COL1A2 and or COL7A1 and or Keratin 5 and or Keratin 14 and or peripherin and/or IMPDH1 promoters and/or genes. Depending upon the tissue in which the suppression agent and/or replacement nucleic acid is administered or active in vivo, tissue specific regulatory elements are used to enhance expression of the suppression agent and/or replacement nucleic acid.

[0098] The suppressors and/or replacement nucleic acids of the invention can be targeted to suppress and replace a gene where mutations in the gene can give rise, predispose or work in combination with other genetic factors and/or environmental factors to cause disease pathology. For example, in the case of dominant retinopathies the rhodopsin geen may be suppressed and replaced. For example, siRNAs targeting RHO- (NM_—000539.2) can be designed and provided commercially. Likewise control siRNAs, for example, targeting EGFP (U57608) and or other reporter genes and or other non-targeting siRNAs can be designed and sythesised. siRNAs are chosen to target sequences which differed by at least one and preferable many more nucleotides from any known gene in mouse and human databases (http://www.ncbi.nlm.nih.gov/blast, BLASTN2.2.6, Altschul et al., Nuc Acids Res. 25: (17:3389-402, 1997). siRNAs can be cloned downstream of, for example, polymerase III promoters such as the H1 or U6 promoters to generate short hairpin RNAs (shRNAs; Brummelkamp et al., Science 296: (5567:505-3, 2001). Alternatively, polymerase II promoters which drive expression in many or all cell or tissue types including the CMV promoter, ubquitin promoter and or the β-actin promoter, for example, may be used to express shRNAs Likewise tissue specific promoters such as the rhodopsin promoter, peripherin promoter and or enolase promoter amongst others may be used to express shRNAs. shRNA sequences can be cloned into vectors with a reporter gene to facilitate monitoring expression from vectors, for example, shRNAs can be cloned in pEGFP-1 amongst other plasmids (BD Biosciences, Clontech, Palo Alto, Calif.). Suppressors can be delivered to cells, tissues and or animals with or without replacement nucleic acids.

[0099] Replacement nucleic acids with nucleotide sequence changes over the target site for siRNA-mediated suppression, for example, at degenerative nucleotides can be generated by primer directed mutagenesis and cloned into vectors such as pcDNA3.1- (Invitrogen). Replacement nucleic acids may also be modified at the UTRs and or at polymorphic sites within the target gene. Ubiquitous promoters such as the CMV promoter and or the ubiquitin promoter and or the β-actin promoter amongst others can be used to drive expression of replacement nucleic acids. Alternatively, tissue specific promoters such as the rhodopsin promoter, peripherin promoter, Col1A1 promoter, Col1A2 promoter, Col1A7 promoter, Keratin promoters and/or the enolase promoter amongst others and/or inducible promoters such as a tetracycline responsive promoter can be used to drive expression of replacement nucleic acids. Replacement human rhodopsin nucleic acids which have been altered in nucleotide sequence at degenerate positions over siRNA target sites for example, replacement nucleic acids for siRNA sequences 1-17 are provided in Table 5. Replacement nucleic acids can be delivered to cells, tissues and or animals with or without suppressor agents.

Suppression and Replacement in Cells and Tissues

[0100] Promoter driven replacement nucleic acids such as rhodopsin nucleic acids and siRNAs and/or shRNAs targeting rhodopsin can be co-transfected into cells, for example, HeLa and or Cos-7 cells amongst other cell types using art known methods. For example, 24 hours post-transfection of suppressor agents and/or replacement nucleic acids, RNA and cytoplasmic protein can be isolated from cells using well established methodologies. Additionally, suppression and replacement can be evaluated in tissues. In the case of retinal genes, for example, organotypic retinal explant cultures from mouse or rat, for example, can be prepared and maintained using art known methods and suppressor agents and or replacement nucleic acids can be delivered to organotypic cultures. For example, electroporation can be used to deliver siRNA and/or shRNA constructs and/or shRNA constructs and replacement nucleic acids to retinal explants as described in Palfi et al., Hum. Mutat. 27(3):260-8, 2006. Subsequent to electroporation of retinal explants, retinas can be treated with trypsin to expedite dissociation of cells. Retinal cell sub-populations within the dissociated cell population which have a particular feature, for example, that express a reporter gene such as EGFP can be identified. One method of identification that can be invoked is FACS (Palfi et al., Hum. Mutat. 27(3):260-8, 2006). Levels of suppression and replacement of a target gene can be evaluated in FACS isolated cell populations. For example, suppression and/or suppression and replacement can be evaluated in electroporated EGFP positive cells from retinal explants.

Evaluation of Suppression and Replacement Using RNA Assays

[0101] Suppression and replacement can be evaluated in cells, tissues and/or animals using RNA assays including real time RT-PCR, northern blotting, RNA in situ hybridisation and or RNAse protection assays. RNA expression levels of suppressors and/or of endogenous genes and or replacement nucleic acids can be assessed by real time RT-PCR using, for example, a 7300 Real Time PCR System (Applied Biosystems, Foster City, Calif., USA) and using, for example, a QuantiTect SYBR Green RT-PCR kit (Qiagen Ltd). RT-PCR assays are undertaken using levels of expression of housekeeping controls such as β-actin or GAPDH, for example, for comparative purposes. Levels of RNA expression can be evaluated using sets of primers targeting the nucleic acids of interest including suppressors, target genes and/or replacements, for example, the following primers can be used for the evaluation of levels of expression of human rhodopsin, β-actin and GAPDH.

[0102]

TABLE 7
PCR Primers for measuring rhodopsin,
β-actin, and GAPDH
		SEQ
		ID
Primer	Sequence	NO
RHO forward	5′ CTTTCCTGATCTGCTGGGTG 3′	69
primer
RHO reverse	5′ GGCAAAGAACGCTGGGATG 3′	70
primer
β-actin forward	5′ TCACCCACACTGTGCCCATCTACGA 3′	71
primer
β-actin reverse	5′ CAGCGGAACCGCTCATTGCCAATGG 3′	72
primer
GAPDH forward	5′-CAGCCTCAAGATCATCAGCA-3′	73
primer:
GAPDH reverse	5′-CATGAGTCCTTCCACGATAC-3′	74
primer:

[0103] Expression of replacement constructs and/or shRNAs may be confirmed, for example, by Northern blotting. RNA may also be detected by in situ hybridisations using single stranded RNA probes that have been labelled with, for example, DIG. To evaluate levels of expression of suppression agents and/or replacement nucleic acids and/or endogenous target genes, RNase protections assays can be performed using art known methods, such as that described in the Ambion mirVana™ Probe and Marker kit manual (catalogue number 1554) and the Ambion RPAIII™ Ribonuclease protection assay kit manual (catalogue number 1414). For example, RNA probes approximately 15-25 nucleotides in length specific for transcripts from, for example, an endogenous target gene and/or a suppressor and/or a replacement nucleic acid can be synthesized. For example, RNA probes targeting mouse rhodopsin and/or human rhodopsin and/or suppression agents targeting rhodopsin and/or rhodopsin replacement nucleic acids can be synthesized using companies such as Sigma-Proligo or Ambion. RNA probes and size standards can be labelled to aid visualization after separation of samples on denaturing polyacrylamide gels. For example, RNA probes and Decade™ size marker (Ambion Inc) can be 5′ end-labelled with P³²-γATP (GE Healthcare) using the mirVana™ probe and marker kit according to the manufacturer's protocol (Ambion Inc.). RNase protection assays can be performed using art known methods, for example, using the RPA III™ Ribonuclease Protection Assay Kit and the manufacturer's protocol (Ambion Inc.). Expression of suppressors and/or replacement nucleic acids and/or endogenous genes can be undertaken and determined in cells, in tissues and or in animals using, for example, the assays and associated methodologies provided above.

Evaluation of Suppression and Replacement Using Protein Assays

[0104] Suppression and replacement can be evaluated in cells, tissues and/or animals using protein assays including ELISA, western blotting and immunocytochemistry assays. ELISAs can be undertaken to evaluate levels of suppression by assessing levels of expression of a target endogenous gene and/or can be used to evaluate levels expression of replacement nucleic acids—such proteins assays are well know in the art and methods are provided in, for example, Palfi et al., Hum. Mutat. 27(3):260-8, 2006. For example, in the case of retinal genes such as the rhodopsin gene, ELISA is undertaken using a rhodopsin primary antibody which is typically used in a diluted form, for example, using a 1/10-1/10000 dilution (but possibly outside of this range) of an antibody for the target protein. In addition, Western Blotting may be undertaken to determine relative quantities of a specific protein, for example rhodopsin. Briefly, protein samples are separated using SDS-PAGE and transferred to a membrane. The membrane is incubated with generic protein (for example milk proteins) to bind to “sticky” places on the membrane. A primary antibody is added to a solution which is able to bind to its specific protein and a secondary antibody-enzyme conjugate, which recognizes the primary antibody is added to find locations where the primary antibody bound.

[0105] In addition to the protein assays referred to above, assays using antibodies in conjunction with microscopy can be used to evaluate protein levels. For example, in the case of rhodopsin immunocytochemistry (for example, using a 1/10-1:1000 dilution of a primary rhodopsin antibody) and fluorescent microscopy can be carried out as has been documented in Kiang et al., 2005 Mol. Ther. 12(3):555-61, 2005. Immunocytochemistry can be undertaken on cells and/or tissues. In the case of the retina, various modes of sectioning can be implemented to evaluate retinal sections. For example, frozen sections, agar embedded sections and/or resin embedded sections can be used. To obtain thin sections, for example of the retina, epon embedding and semi-thin sectioning can be performed using art known methods such as those provided in McNally et al., Hum. Mol. Genet. 11(9):1005-16, 2002. Immunocytochemistry may be used to evaluate suppression of a target gene and or expression of replacement nucleic acids. Additionally, histological analyses can be used to evaluate the histological effect(s) associated with the administration of suppressors and or replacement nucleic acids. In animal models of retinal degenerations such as the rho−/−, rds, rhodopsin Pro23H is, rhodopsin Pro2347Ser mice and others there is a degeneration of the photoreceptor cell layer over time. Histological analyses can be used to evaluate if this degeneration has been modulated subsequent to administration of suppression agents and/or replacement nucleic acids.

Delivery of Suppression and Replacement

[0106] Both non-viral and/or viral vectors can be used in the invention to deliver the suppression agents and/or replacement nucleic acids. For example, in the case of retina, recombinant adenoassociated virus (AAV) and more specifically AAV2/5 has previously been found to elicit efficient transduction of photoreceptor cells. Other AAV serotypes may also be used to deliver to retina, for example, AAV2/2 elicits efficient delivery to the retinal pigment epithelium (RPE) as does AAV4. AAV vectors can be generated using protocols with and without helper virus. For example, a helper virus free protocol using a triple transfection approach is well documented (Xiao et al., J. Virol. 72(3):2224-32, 1998). Expression cassettes carrying suppression and/or replacement elements can be cloned into plasmids such as pAAV-MCS provided by Stratagene Inc. Suppressors and/or replacement nucleic acids are cloned between the inverted terminal repeats of AAV2 and transfected into 293 cells (Stratagene; ATACC cat no CRL-1573) with two other plasmids, hence the term triple transfection. For example, the pRep2/Cap5 plasmid (Hildinger et al., J. Virol. 75(13):6199-203, 2001) together with the pHelper plasmid (Stratagene), at, for example, a ratio of 1:1:2, can be used to generate AAV2/5 vectors. Virus can be generated using a variety of art known procedures including the method outlined below. For example, to generate virus fifty 150 mm plates of confluent HEK293 cells were transfected (50 μg DNA/plate) with polyethyleminine (Reed et al., J. Virol. Methods 138(1-2):85-98, 2006). 48 hrs post-transfection crude viral lysates were cleared (Auricchio et al., 2001) and purified by CsCl₂ gradient centrifugation (Zolotukhin et al., Gene Ther. 6(6):973-85, 1999). The AAV containing fraction was dialyzed against PBS. Genomic titres, viral particles (vp/ml), were determined by quantitative real-time PCR using art known methods (Rohr et al., J. Virol. Methods 106(1):81-8, 2002). AAVs can be generated that contain, for example, either targeting shRNAs or control shRNAs and/or replacement nucleic acids such as rhodopsin and/or reporter nucleic acids such as EGFP and/or stuffer sequences and/or sequences aiding expression of suppression agents and/or replacement nucleic acids such as promoter and/or enhancer sequences and/or other regulatory sequences and/or epigenetic elements.

Administration of Suppression and Replacement Vectors

[0107] Animal models can be used to mirror human disorders. For example, animal models of human retinopathies or that express a human retinal gene have been generated, for example, rho−/− mice (Humphries et al., Nat. Genet. 15(2):216-9, 1997), NHR+/− mice (Olsson et al., Neuron 9(5):815-30, 1992), Pro23H is mice (Olsson et al., Neuron 9(5):815-30, 1992), Pro347Ser mice (Li et al., Proc. Natl. Acad. Sci. U.S.A. 95(20):11933-8, 1998) and RHO-M mice (see below). Mice typically are maintained under specific pathogen free (SPF) housing conditions and in a controlled light environment. The suppression agents and/or replacement nucleic acids of the invention can be administered to animals either locally and/or systemically. Local administration can include direct injection to the target tissues and/or in the proximity of the target tissue as has been described in detail in the art in, for example, Xia et al. (ACS Chem. Biol. 1(3):176-83, 2004) delivered AAV vectors with shRNAs to brain to treat spinocerebellar ataxia. In the case of the retina, subretinal injection can be used to administer suppression agents and/or replacement nucleic acids according to the following procedure. For example, mice can be anaesthetised by intraperitoneal injection of Domitor and Ketalar (10 and 50 μg/g of body weight respectively). The pupils are dilated with phenylephrine and under local analgesia (amethocaine) a small puncture is made in the sclera. A micro-needle attached to a 10 μl syringe (Hamilton Company Europe) is inserted through the puncture to the subretinal space and 1-3 μl of vector is administered. For example, in the case of AAV 1-3 μl of a 10^12-14 vp/ml AAV vector preparation in PBS is administered. A reverse anaesthetic (antisedan, 50 μg/g of body weight) can be applied by intraperitoneal injection post-delivery. Body temperature during the procedure is sustained using a homeothermic heating device. In addition newborn mice can be prepared for subretinal injection according to Matsuda and Cepko (Proc. Natl. Acad. Sci. U.S.A. 101(1):16-22, 2004).

Assay for Function

[0108] To evaluate if suppression and/or replacement modulates the function of a target tissue and/or cell type, one or more assays may be employed that are well described in the prior art. In the case of the retina, functional assays include but are not limited to electrophysiology, such as pattern electroretinogram (ERG), full field ERG, and visual evoked potentials. In addition, visual field assessments, color vision assessments, and pupilometry may be performed. For example, electroretinography can be used to evaluate the response of the retina to light. This can be performed using, for example, the following procedure or an adapted procedure. Animals can be dark-adapted overnight and prepared for ERG under dim red light. Pupils are dilated with 1% cyclopentalate and 2.5% phenylephrine. Animals are anesthetized with ketamine and xylazine (16 and 1.6 μg/10 g body weight respectively) injected intraperitoneally. Standardized flashes of light are presented to the animal, for example a mouse, in a Ganzfeld bowl. ERG responses are recorded simultaneously from both eyes by means of contact lens electrodes (Medical Workshop, Netherlands) using 1% amethocaine as topical anesthesia. Reference and ground electrodes are positioned subcutaneously, approximately one mm from the temporal canthus and anterior to the tail respectively. Responses are analysed using a RetiScan RetiPort electrophysiology unit (Roland Consulting Gmbh). The protocol is based on that approved by the International Clinical Standards Committee for human electroretinography. Rod-isolated responses are recorded using a dim white flash (−25 dB maximal intensity where maximal flash intensity was 3 candelas/m²/s) presented in the dark-adapted state. Maximal combined rod-cone responses to the maximal intensity flash are then recorded. Following a 10 minute light adaptation to a background illumination of 30 candelas/m², cone-isolated responses are recorded to the maximal intensity flash presented initially as a single flash and subsequently as 10 Hz flickers. A-waves are measured from the baseline to the trough and b-waves from the baseline (in the case of rod-isolated responses) or from the a-wave to the trough.

[0109] The agents of the invention are administered in effective amounts. An effective amount is a dosage of the agent sufficient to provide a medically desirable result. An effective amount means that amount necessary to delay the onset of, inhibit the progression of or halt altogether the onset or progression of the particular condition or disease being treated. An effective amount may be an amount that reduces one or more signs or symptions of the disease. When administered to a subject, effective amounts will depend, of course, on the particular condition being treated; the severity of the condition; individual patient parameters including age, physical condition, size and weight, concurrent treatment, frequency of treatment, and the mode of administration. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.

[0110] Actual dosage levels of active ingredients in the pharmaceutical compositions of the invention can be varied to obtain an amount of the agent(s) that is effective to achieve the desired therapeutic response for a particular patient, compositions, and mode of administration. The selected dosage level depends upon the activity of the particular agent, the route of administration, the severity of the condition being treated, the condition, and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the agent(s) at levels lower than required to achieve the desired therapeutic effort and to gradually increase the dosage until the desired effect is achieved.

[0111] The agents and pharmaceutical compositions of the invention can be administered to a subject by any suitable route. For example, the compositions can be administered orally, including sublingually, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically and transdermally (as by powders, ointments, or drops), bucally, or nasally. The term “parenteral” administration as used herein refers to modes of administration other than through the gastrointestinal tract, which include intravenous, intramuscular, intraperitoneal, intrasternal, intramammary, intraocular, retrobulbar, intrapulmonary, intrathecal, subcutaneous and intraarticular injection and infusion. Surgical implantation also is contemplated, including, for example, embedding a composition of the invention in the body such as, for example, in the brain, in the abdominal cavity, under the splenic capsule, brain, or in the cornea.

[0112] Agents of the present invention also can be administered in the form of liposomes. As is known in the art, liposomes generally are derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any nontoxic, physiologically acceptable, and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a compound of the present invention, stabilizers, preservatives, excipients, and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33, et seq.

[0113] Dosage forms for topical administration of an agent of this invention include powders, sprays, ointments, and inhalants as described herein. The agent is mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives, buffers, or propellants which may be required. Ophthalmic formulations, eye ointments, powders, and solutions also are contemplated as being within the scope of this invention.

[0114] Pharmaceutical compositions of the invention for parenteral injection comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water ethanol, polyols (such as, glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils (such, as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0115] These compositions also can contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It also may be desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.

[0116] In some cases, in order to prolong the effect of the agent, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This result can be accomplished by the use of a liquid suspension of crystalline or amorphous materials with poor water solubility. The rate of absorption of the agent then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug from is accomplished by dissolving or suspending the agent in an oil vehicle.

[0117] Injectable depot forms are made by forming microencapsule matrices of the agent in biodegradable polymers such a polylactide-polyglycolide. Depending upon the ratio of agent to polymer and the nature of the particular polymer employed, the rate of agent release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations also are prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.

[0118] The injectable formulations can be sterilized, for example, by filtration through a bacterial- or viral-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.

[0119] The invention provides methods for oral administration of a pharmaceutical composition of the invention. Oral solid dosage forms are described generally in Remington's Pharmaceutical Sciences, 18th Ed., 1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89. Solid dosage forms for oral administration include capsules, tablets, pills, powders, troches or lozenges, cachets, pellets, and granules. Also, liposomal or proteinoid encapsulation can be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Pat. No. 4,925,673). Liposomal encapsulation may include liposomes that are derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556). In general, the formulation includes an agent of the invention and inert ingredients which protect against degradation in the stomach and which permit release of the biologically active material in the intestine.

[0120] In such solid dosage forms, the agent is mixed with, or chemically modified to include, a least one inert, pharmaceutically acceptable excipient or carrier. The excipient or carrier preferably permits (a) inhibition of proteolysis, and (b) uptake into the blood stream from the stomach or intestine. In a most preferred embodiment, the excipient or carrier increases uptake of the agent, overall stability of the agent and/or circulation time of the agent in the body. Excipients and carriers include, for example, sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, cellulose, modified dextrans, mannitol, and silicic acid, as well as inorganic salts such as calcium triphosphate, magnesium carbonate and sodium chloride, and commercially available diluents such as FAST-FLO®, EMDEX®, STA-RX 1500®, EMCOMPRESS® and AVICEL®, (b) binders such as, for example, methylcellulose ethylcellulose, hydroxypropyhnethyl cellulose, carboxymethylcellulose, gums (e.g., alginates, acacia), gelatin, polyvinylpyrrolidone, and sucrose, (c) humectants, such as glycerol, (d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate, starch including the commercial disintegrant based on starch, EXPLOTAB®, sodium starch glycolate, AMBERLITE®, sodium carboxymethylcellulose, ultramylopectin, gelatin, orange peel, carboxymethyl cellulose, natural sponge, bentonite, insoluble cationic exchange resins, and powdered gums such as agar, karaya or tragacanth; (e) solution retarding agents such a paraffm, (f) absorption accelerators, such as quaternary ammonium compounds and fatty acids including oleic acid, linoleic acid, and linolenic acid (g) wetting agents, such as, for example, cetyl alcohol and glycerol monosterate, anionic detergent surfactants including sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate, cationic detergents, such as benzalkonium chloride or benzethonium chloride, nonionic detergents including lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65, and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose; (h) absorbents, such as kaolin and bentonite clay, (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils, waxes, CARBOWAX® 4000, CARBOWAX® 6000, magnesium lauryl sulfate, and mixtures thereof; (j) glidants that improve the flow properties of the drug during formulation and aid rearrangement during compression that include starch, talc, pyrogenic silica, and hydrated silicoaluminate. In the case of capsules, tablets, and pills, the dosage form also can comprise buffering agents.

[0121] Solid compositions of a similar type also can be employed as fillers in soft and hard-filled gelatin capsules, using such excipients as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

[0122] The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They optionally can contain opacifying agents and also can be of a composition that they release the active ingredients(s) only, or preferentially, in a part of the intestinal tract, optionally, in a delayed manner. Exemplary materials include polymers having pH sensitive solubility, such as the materials available as EUDRAGIT® Examples of embedding compositions which can be used include polymeric substances and waxes.

[0123] The active compounds also can be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0124] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms can contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol ethyl carbonate ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydroflirfuryl alcohol, polyethylene glycols, fatty acid esters of sorbitan, and mixtures thereof.

[0125] Besides inert diluents, the oral compositions also can include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, coloring, flavoring, and perfuming agents. Oral compositions can be formulated and further contain an edible product, such as a beverage.

[0126] Suspensions, in addition to the agent(s), can contain suspending agents such as, for example ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, and mixtures thereof.

[0127] Also contemplated herein is pulmonary delivery of the agent(s) of the invention. The agent(s) is delivered to the lungs of a mammal while inhaling, thereby promoting the traversal of the lung epithelial lining to the blood stream. See, Adjei et al., Pharmaceutical Research 7:565-569 (1990); Adjei et al., International Journal of Pharmaceutics 63:135-144 (1990) (leuprolide acetate); Braquet et al., Journal of Cardiovascular Pharmacology 13 (suppl.5): s.143-146 (1989) (endothelin-1); Hubbard et al., Annals of Internal Medicine 3:206-212 (1989) (α1-antitrypsin); Smith et al., J. Clin. Invest. 84:1145-1146 (1989) (α1-proteinase); Oswein et al., “Aerosolization of Proteins,” Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colo., March, 1990 (recombinant human growth hormone); Debs et al., The Journal of Immunology 140:3482-3488 (1988) (interferon-γ and tumor necrosis factor α) and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor).

[0128] Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including, but not limited to, nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.

[0129] Some specific examples of commercially available devices suitable for the practice of the invention are the ULTRAVENT® nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the ACORN II® nebulizer, manufactured by Marquest Medical Products, Englewood, Colo.; the VENTOL® metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, N.C.; and the SPINHALER® powder inhaler, manufactured by Fisons Corp., Bedford, Mass.

• • [0130] All such devices require the use of formulations suitable for the dispensing of a agent(s) of the invention. Typically, each formulation is specific to the type of device employed and can involve the use of an appropriate propellant material, in addition to diluents, adjuvants, and/or carriers useful in therapy.

[0131] The composition is prepared in particulate form, preferably with an average particle size of less than 10 □m, and most preferably 0.5 to 5 □m, for most effective delivery to the distal lung.

• • [0132] Carriers include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for use in formulations may include lipids, such as DPPC, DOPE, DSPC and DOPC, natural or synthetic surfactants, polyethylene glycol (even apart from its use in derivatizing the inhibitor itself), dextrans, such as cyclodextran, bile salts, and other related enhancers, cellulose and cellulose derivatives, and amino acids.
  • [0133] Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.

[0134] Formulations suitable for use with a nebulizer, either jet or ultrasonic, typically comprise a compound of the invention dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein per mL of solution. The formulation also can include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure). The nebulizer formulation also can contain a surfactant to reduce or prevent surface-induced aggregation of the inhibitor composition caused by atomization of the solution in forming the aerosol.

[0135] Formulations for use with a metered-dose inhaler device generally comprise a finely divided powder containing the agent suspended in a propellant with the aid of a surfactant. The propellant can be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid also can be useful as a surfactant.

[0136] Formulations for dispensing from a powder inhaler device comprise a finely divided dry powder containing the agent and also can include a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol, in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation.

[0137] Nasal delivery of the agent(s) and composition of the invention also is contemplated. Nasal delivery allows the passage of the agent or composition to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran. Delivery via transport across other mucous membranes also is contemplated.

[0138] Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the agent(s) of the invention with suitable nonirritating excipients or carriers, such as cocoa butter, polyethylene glycol, or suppository wax, which are solid at room temperature, but liquid at body temperature, and therefore melt in the rectum or vaginal cavity and release the active compound.

[0139] In order to facilitate delivery of the agent(s) across cell and/or nuclear membranes, compositions of relatively high hybrophobicity are preferred. The agent(s) can be modified in a manner which increases hydrophobicity, or the agent(s) can be encapsulated in hydrophobic carriers or solutions which result in increased hydrophobicity.

[0140] Practice of the invention will be still more fully understood from the following examples, which are presented herein for illustration only and should not be construed as limiting the invention in any way.

• • [0141] siRNAs targeting human rhodopsin were synthesized and evaluated for RNAi-mediated suppression (listed in Table 8). Suppression and replacement constructs with suppressors targeting the human rhodopsin mRNA sequence and replacement rhodopsin genes that escape suppression by the suppressor due to subtle changes in the sequence were subsequently designed. These changes, while enabling replacement nucleic acids to escape suppression at least in part, did not change the protein product expressed from the replacement genes. Short hairpin RNAs (shRNAs) were used to demonstrate suppression in vivo (FIG. 1). The sequence of the sense and antisense strands of the shRNAs is the same as the sequence used for the siRNAs. An intervening loop is included between the sense and antisense strands in the same manner as Brummelkamp et al., Science 296(5567):550-3, 2001. Notably, the number of nucleotides and the make up of the nucleotides in the intervening loop can vary. The construct(s) were delivered using an AAV2/5 recombinant virus. Non-targeting siRNA can be used as controls, for example, a non-targeting siRNA directed towards an EGFP reporter gene can be used—for example.
  • [0142] siRNAs were designed according to the method of Elbashir et al., Nature 411(6836):494-8, 2001, or by using the HiPerformance siRNA design algorithm (Qiagen Ltd. Crawley, UK). siRNA target sequences differed by at least 4 nucleotides from any non-rhodopsin sequences in mouse and human databases (http://www.ncbi.nlm.nih.gov/blast, BLAST2.2.6 (Altschul et al., Nucleic Acids Res. 25(17):3389-402, 1997). siBB, siQ1 and a non-targeting siRNA siNT (5′ UUCUCCGAACGUGUCACGU 3′; SEQ ID NO:75) or EGFP (U57608), siEGFP (nt 256-277) were initially cloned downstream of the H1 promoter using BglII/BamH1 and Hind III restriction sites to generate shRNAs and subsequently in pEGFP-1 (BD Biosciences, Clontech, Palo Alto, Calif.) using EcoRI and Hind III sites generating shBB-EGFP, shQ1-EGFP and shNT-EGFP (FIG. 1A). The EGFP gene enabled viral transduction to be monitored. Six siRNAs sequences targeted the coding region of human rhodopsin. Replacement nucleic acids were cloned into pCDNA3.1-plasmid (Invitrogen, Karlsruhe, Germany). The CMV promoter was replaced with either the human ubiquitin C promoter (pUB6/V5-His, Invitrogen) or a 1.7 kb fragment of the mouse rho promoter (rhoP). Sequence alterations were introduced into replacement nucleic acids using primer directed PCR-based mutagenesis using art known methods. Replacement nucleic acids with sequence alterations over the target sites for siB, siBB, siC, siCC, siQ1 and siQ2 were termed rB, rBB, rC, rCC, rQ1, and rQ2. Altered nucleotides in the replacement rhodopsin sequences are at wobble positions (highlighted in bold print). These replacement genes were designed to avoid suppression by the siRNAs yet encode wild type protein. Table 8 provides one replacement example for each siRNA target site; however, in each case there are several alternative possible replacement sequences because some amino acids have as many as six codons and others have four or three codons.

[0143]

TABLE 8
siRNA Sequence and Replacement Rhodopsin Sequence
		SEQ	Replacement	SEQ
		ID	rhodopsin	ID	Position in
siRNA	Sequence	NO	sequence	NO	NM_000539.2
siB	TCAACTTCCTCA	75	ATAAATTTTTTGACC	76	256-277
	CGCTCTA		CTGTAT
siBB	TCACCGTCCAGC	77	CTGTATGTGACGGTG	78	254-274
	ACAAGAA		CAGCAC
siC	CGTGTGGAATCG	79	AGCTGCGGTATAGAT	80	270-292
	ACTACTA		TATTA
siCC	CGCTCAAGCCGG	81	ACCTTGAAACCCGAA	82	274-294
	AGGTCAA		GTGAA
siQ1	TCAACTTCCTCA	83	CTGTATGTGACGGTG	84	650-670
	CGCTCTACGT		CAGCAC
siQ2	CTCTACGTCACC	85	CTGTATGTGACGGTG	86	671-694
	GTCCAGCACAA		CAGCAC

Suppression of RHO in HeLa Cells

• • [0144] RNAi-mediated suppression of RHO was initially evaluated in HeLa cells. siRNAs targeting RHO were co-transfected with a CMV promoter-driven wild type RHO. Transfections were carried out three times in quadruplicate using lipofectamine 2000 to aid transfections (Gibco-BRL). Real time RT-PCRs, performed on RNAs extracted from transfected cells 24 hours post-transfection, demonstrated up to 87% suppression (p<0.01, FIG. 2A) (see Table 7 for primer sequences). siRNAs siBB, siCC and siQ1 were selected for further analysis. Similar levels of rhodopsin protein suppression were quantified by ELISA (up to 88%, p<0.01, FIG. 2A) and demonstrated by immunocytochemistry 24 hours post-transfection (FIG. 2B). Subsequently, replacement RHO constructs, rBB, rCC and rQ1, were generated incorporating nucleotide changes at degenerate positions over the target sites for siRNAs, siBB, siQ1 and siCC as described above and shown in Table 8. Transfections were performed three times in quadruplicate in HeLa cells according to art known methods as described above. Results indicated that replacement RHO constructs were not suppressed by corresponding siRNAs, for example, rBB by siBB (FIG. 3). However, significant levels of suppression were obtained with other non-corresponding siRNAs, for example siQ1 suppressed rBB and rCC (FIG. 3).
    Long Term Suppression of RHO in Retinal Explants

[0145] To provide long term RHO suppression, siBB and siQ1 were cloned as shRNAs into an EGFP expressing vector (shBB-EGFP and shQ1-EGFP, FIG. 1A). Plasmids were electroporated into retinal explants from newborn NHR+/− rho−/− mice using the methods described in Matsuda and Cepko (2004) Proc. Natl. Acad. Sci. USA 101: 16-22. NHR+/− mice express a wild type human RHO gene and display a wild type phenotype. Cells from retinal explants (n=6) were dissociated two weeks post-electroporation and EGFP-positive cells isolated by FACS (FIG. 4A). Real time RT-PCR was performed on RNA extracted from EGFP-positive FACS-isolated cells using the primers described in Table 8 and results obtained in explants mirrored those found in HeLa cells. Results indicated that RHO suppression of greater than 85% was achieved (p<0.001, FIG. 4B).

Long Term Suppression Using AAV Vectors

[0146] Long-term expression of therapies will be required for a progressive retinopathy such as adRP. To achieve long-term suppression in vivo, shBB-EGFP and the non-targeting shNT-EGFP were engineered into AAV vectors (AAV-shBB-EGFP and AAV-shNT-EGFP) (FIG. 1A). Recombinant AAV2/5 viruses were generated using a helper virus free system. Expression cassettes were cloned into pAAV-MCS (Stratagene, La Jolla, Calif., USA), between the inverted terminal repeats of AAV2, and transfected into HEK-293 cells (ATCC no. CRL-1573) with pRep2/Cap5 and pHelper (Stratagene), at a ratio of 1:1:2. Fifty 150 mm plates of confluent cells were transfected (50 μg DNA/plate) with polyethyleminine. Forty eight hours post-transfection crude viral lysates were cleared and purified by CsCl₂ gradient centrifugation. AAV-containing fractions were dialysed against PBS. Genomic titres, i.e., viral particles (vp/ml), were determined by quantitative real time PCR. AAVs generated contained the shBB-EGFP and shNT-EGFP constructs (AAV-shBB-EGFP and AAV-shNT-EGFP, FIG. 1A).

[0147] The EGFP gene enabled viral transduction to be monitored. Three μl of AAV-shBB-EGFP (2×1012 vp/ml) or AAV-shNT-EGFP (3×1012 vp/ml) were subretinally injected into adult NHR+/− rho−/− mice. Two weeks post-injection two animals were sacrificed and expression of the 21 nucleotide shBB shown in two retinas using RNase protection (FIG. 5A). Retinas were dissociated and EGFP-positive cells collected by FACS. RNAi-mediated suppression of RHO, as evaluated by real time RT-PCR (see Table 8 for primer sequences) two weeks post-injection (n=6), was approximately 90% (p<0.001) in AAV-shBB-EGFP-transduced photoreceptor cells (FIG. 5B). Four retinas were dissociated and significant suppression of rhodopsin protein expression was demonstrated in vivo in EGFP-positive transduced cells by immunocytochemistry (FIG. 5C).

Suppression in Transgenic Animals

[0148] A transgenic mouse expressing a sequence-modified RHO gene was generated (RHO-M). RHO-M+/− rho−/− were evaluated at two months of age for rescue of the retinal pathology present in rho−/− mice by histology (FIG. 6A-C) and ERG (FIG. 6D). Rhodopsin immunolabeling in rod outer segments and the thickness of outer nuclear layers were similar in wild type rho+/+(FIG. 6A), NHR+/− rho−/− (FIG. 6B) and RHO-M+/− rho−/− (FIG. 6C) mice. Additionally, ERG responses were similar in wild type rho+/+, rho+/−, NHR+/− rho−/− and RHO-M+/− rho−/− mice. ERG b-waves of rod-isolated responses of 500-700 μV were observed in mice of all genotypes (FIG. 6D). The amplitudes and timings of the combined rod and cone responses to the maximal intensity flash presented in the dark-adapted state, as well as the light adapted cone-isolated responses both to single flash and 10 Hz flickers, were equivalent in all the genotypes examined (data not shown). These results validate the use of the degeneracy of the genetic code to engineer codon-modified human RHO genes which can provide functional human rhodopsin protein.

AAV-Delivered Suppression and Replacement of Human RHO In Vivo

[0149] Having established shBB and shQ1 as potent suppressors and rBB and rQ1 as being refractory to their corresponding suppressors, shBB-rBB and shQ1-rQ1 were cloned into AAV vectors using the triple plasmid system detailed above and viruses containing both elements of the therapeutics were generated (AAV-shBB-rBB (also termed AAV-BB8) and AAV-shQ1-rQ1 (also termed AAV-Q1)) using the method detailed above. Three μl of AAV-shBB-rBB was subretinally injected into adult wild type rho+/+ mice (n=12) and replacement RHO mRNA expression confirmed by RT-PCR and RNase protection using RNA extracted 10 days post-injection (data not shown). To demonstrate that AAV-delivered rBB is translated into protein, 2 μl of a 1:1 mix of AAV-shBB-rBB and AAV-CMV-EGFP was subretinally injected into 10 day old rho−/− mice (n=6). Two weeks post-injection rhodopsin and EGFP protein expression were determined using fluorescent microscopy. Marked rhodopsin expression, overlapping with EGFP, was observed in transduced areas (FIG. 7).

[0150] Subsequently, 1 μl of AAV-shBB-rBB or AAV-shQ1-rQ1 was subretinally injected into newborn Pro23His+/− rho+/− mice (n=10) that present with a retinal degeneration resulting in complete loss of photoreceptors by two weeks of age. In all animals one eye was injected with therapeutic virus (either AAV-shBB-rBB or AAV-shQ1-rQ1) and the other with a control virus (AAV-EGFP). The early onset and rapid nature of the retinopathy in young Pro23H is pups precluded use of ERG as a readout for benefit. However, at ten days of age retinal histology was evaluated in semi-thin resin embedded sections cut at approximately 50 μm intervals throughout the central meridian of the eye (n=10). From each section approximately 40 measurements of ONL thickness were taken. Since only a part of the retina is transduced by a single subretinal injection of AAV (particularly in newborn pups), to identify the transduced area ONL measurements were ordered by thickness and the 15% highest and lowest values grouped for analysis. Lowest values represent thinnest ONL readings, most likely corresponding to peripheral areas of the retina and thus not in close proximity to injection sites. Highest values represent thickest ONL readings, most likely corresponding to central areas of the retina and thus in closer proximity to injection sites. Significant differences in ONL thickness between AAV-shBB-rBB- and AAV-EGFP-treated eyes were observed. The ONL of treated eyes was found to be approximately 33% (p<0.001) thicker than control injected counterparts for the highest value groupings (FIG. 8A-C). In the lowest value groupings a difference of approximately 10% was observed (FIG. 8A). These data provide evidence at the histological level that AAV2/5-delivered RNAi in conjunction with provision of a codon-modified replacement gene can beneficially modulate the retinopathy in Pro23His+/− rho−/− mice.

• • [0151] RNAi-mediated suppression was evaluated in retinal tissue after sub-retinal injection of AAV vectors expressing either a suppressor targeting rhodopsin (AAV-shBB-EGFP, AAV-shCC-EGFP and AAV-shQ1-EGFP) or a non-targeting control (AAV-shNT-EGFP). Mice expressing a human rhodopsin replacement gene (referred to as RHO-M mice and detailed in the section on suppression in transgenic animals) were subretinally injected with AAV vectors (AAV2/5), containing shRNA sequences for BB, CC and Q1 and an EGFP reporter gene (AAV-shBB-EGFP, AAV-shCC-EGFP and AAV-shQ1-EGFP). The presence of the EGFP reporter gene enabled isolation of the population of retinal cells that are EGFP positive and therefore have received AAV using FACS to isolate these cell populations. AAV-delivered RNAi-mediated suppression with each suppressor (BB, CC and Q1) was evaluated using real-time RT-PCR in cell populations characterised by FACS and was compared to suppression obtained using AAV with non-targeting control shRNA sequences (AAV-shNT-EGFP). Significant rhodopsin suppression was obtained with BB and Q1 suppressors, however, significantly lower levels of suppression were obtained with the CC suppressor (FIG. 6E). The replacement gene in RHO-M mice was partially protected from suppression due to the presence of two nucleotide mismatches between the CC suppressor sequence and the target site for suppression in the human rhodopsin replacement gene. The replacement gene is partially protected from siRNA CC-based suppression by the introduction of two nucleotide changes at degenerate sites in the replacement gene. FIG. 6F illustrates depression of the ERG response in RHO-M eyes that have received AAV-shBB-EGFP (panel 1) or AAV-shQ1-EGFP (panel 2) when compared to eyes subretinally injected with AAV-shNT-EGFP. The top tracing in each panel represents the right eye which received the targeting AAV-shRNA vector and the bottom tracing in each panel represents the left eye which received the control non-targeting AAV-shNT vector. In contrast no reduction/depression of the ERG was observed in RHO-M mice subretinally injected with AAV-shCC-EGFP (panel 3) vector; this is likely due to the reduced levels of rhodopsin suppression observed with AAV-shCC-EGFP (see FIG. 6E above).

• • [0152] Expression of suppression and/or replacement vectors was optimized by including in the vectors sequences that enhanced and/or modulate expression levels at the RNA and/or protein level. A list of exemplary sequence elements is provided in Table 1, however, the enhancing and/or modulating elements of the invention are not exclusive to this list. For example, one or more of a promoter, a stuffer, an insulator, a silencer, a chromatin remodelling sequence, an intron sequence, a poly adenylation signal, a post translational regulatory element, and a transcription factor binding site can be included in suppression and/or replacement constructs to modulate expression of suppression and/or replacement components relating to the invention. Such elements and derivatives thereof can be used to modulate levels of expression, tissue specificity, timing of expression, and/or induction of expression. Table 9 provides some exemplary sequences that can be used to modulate expression of suppression and/or replacement constructs relating to the invention. The sequences provided are within conserved regions as evaluated by comparison of sequences from multiple species. At any one position a nucleotide may not be conserved between all species—the sequences represent regions where overall there is a high degree of conservation. Such conserved sequences from any species such as human, mouse, rat, bacteria, virus and/or indeed a hybrid sequence from more than one species could be used in the invention.

[0153]

TABLE 9
Exemplary Enhancer Sequences
CMV enhancer element amplified from pCDNA3.1 Invitrogen nt
308-734 http://www.invitrogen.com/ (SEQ ID NO: 87)
CCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCG
CCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCA
TTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGT
GTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTG
GCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTA
TTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGA
TAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGT
TTG TTTTGGCACC AAAATCAACG GGAC
pAAV.BB11 The WPR element from pSin11 CMV GFPpre mut FL
(Gene Therapy (7): 641-5 (2006)) (SEQ ID NO: 88)
GAGCAT CTTACCGCCATTTATTCCCA TATTTGTTCT GTTTTTCTTG ATTTGGGTAT
ACATTTAAATGTTAATAAAA CAAAATGGTG GGGCAATCAT TTACATTTTT
AGGGATATGTAATTACTAGT TCAGGTGTAT TGCCACAAGA CAAACATGTT
AAGAAACTTTCCCGTTATTT ACGCTCTGTT CCTGTTAATC AACCTCTGGA
TTACAAAATTTGTGAAAGAT TGACTGATAT TCTTAACTAT GTTGCTCCTT
TTACGCTGTGTGGATATGCT GCTTTATAGC CTCTGTATCT AGCTATTGCT
TCCCGTACGGCTTTCGTTTT CTCCTCCTTG TATAAATCCT GGTTGCTGTC
TCTTTTAGAGGAGTTGTGGC CCGTTGTCCG TCAACGTGGC GTGGTGTGCT
CTGTGTTTGCTGACGCAACC CCCACTGGCT GGGGCATTGC CACCACCTGT
CAACTCCTTTCTGGGACTTT CGCTTTCCCC CTCCCGATCG CCACGGCAGA
ACTCATCGCCGCCTGCCTTG CCCGCTGCTG GACAGGGGCT AGGTTGCTGG
GCACTGATAATTCCGTGGTG TTGTC
pAAV.BB13 The WPR element from pBSK11 (Donello JE, et al. J.
Virol. 1998 72(6): 5085-92.) (SEQ ID NO: 89)
AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTT
GCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTC
CCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGG
AGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAA
CCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTT
CCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGAC
AGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTC
CTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGC
TACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTC
TGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGC
CGCCTCCCC
(Wild type woodchuck hepatitis B virus genome sequence ACCESSION J04514)

[0154] In addition to adding enhancing and/or modulating elements to suppression and/or replacement vectors, the rhodopsin promoter was studied in detail. A comparison of rhodopsin genes present in different mammals resulted in identification of 9 highly conserved regions in the rhodopsin gene (conserved regions A though I, Sequence 1, Table 10). Regions A, B, C and D are in the rhodopsin promoter region, conserved region E is in intron 2 of the gene and conserved regions F, G, H and I are in the 3′ region.

• • [0155] The following sequence (Sequence 1; Table 10) shows the conserved regions within the mouse promoter human intronic and exonic and 3′ sequence. Notably, conserved sequences in the mouse promoter are nearly the same in the human rhodopsin promoter and it is contemplated that the human or other mammalian rhodopsin promoters and/or derivatives and/or hybrids thereof may be used in suppression and replacement constructs. Additionally, it is contemplated that other promoters could be combined with some or all of conserved regions A though I and used in suppression and/or replacement constructs, for example, other retinal promoter sequences may be used.

[0156]

TABLE 10
Conserved Regions of Rhodopsin
Sequence 1: Mouse rhodopsin promoter sequence (upper case) ending at the Xho I
site (highlighted in bold print), followed by the human rhodopsin 5′UTR, human
rhodopsin exons and introns and human rhodopsin 3′ region sequence (lower case).
Conserved regions A - I are highlighted in bold print. (SEQ ID NO: 90).
GTTCCAGGGC CCAGGGGCTT CCAGCCATGA GGGCACCTAG ACTTGTAATC
CCTAGAGTCC TCCTGATGCC ACTGCCCAGG GACAGACAGC ACACAGCACC
CCTCCCCCAC TCTCTTAACA GGCAGAAGCA GGGAGATGGA GGCATGCTGA
AGATGTCCAT GTGAGGCTGG TGGTAGCATG CCCACTGCTG GGATGAAGAG
ATGGGGGCAA AGTGAGTGGC AGAGGCCAGG CCAGGTCCAG GCCCTTCCAG
GCTTCCTCTG CCACTGTGGA GATGAAAGAG GGAGCCAGGC AAGGTCCAGG
CCCTCCCCAC CCCCTCTGCC TCTATGGAGA TGAAGGGGGA ATGAAGAAGG
GAGCCAGACA GTTGTGCCAA CACAACTCCT CCGTCGAGTG TCTAATTGCT
TATGATCATG CATGCTCTCT CTCCCACTAA ACATTTATTA ATGTGTTAGG cons reg A
ATTTCCATTA GCGCGTGCCT TGAACTGAAA TCATTTGCAT ATGGCTGGGA
AAAAGTGGGG TGAGGGAGGA AACAGTGCCA GCTCCCCAAC AGGCGTCAAT
CACAGTGACA GATCAGATGG TTTCTGGCTG GAGGCAGGGG GGCTGTCTGA
GATGGCGGCA TGCATCCTTT CAGTGCATAT CACAGAAATT CAGGTGACTC
CTGCTGGGAG CCAAGACCCT GAGGCTGAGC CTGGCCACAG CTCCAATAGC
TGCTGGATAT CATCATGTCT GGGCTGAGCA GCCTCTAGAG GTACCCTTTT
ACAGATAGTA AAACTGAGGC TCAGTGACTG CTGAGCCAAA GTTGGACCCA
CCCACACTCA TTTGCAGACT GCCGTGGGCC ATGTTCTGAT CTCTTCCCTA
CCTGGACTCA GCCCAGCACA CTCGGCACAC AAGGCCCTTC TTCAGCTTGA
ATACAGCGTC CTCAGCTATA GCCAGCATCT ATGAATGGAG CTCAGTGACC
CTGACTGGAG GAAGTTAGGA CAGGGATTTT TTCTGGAGTT TTGGCAGGAA
GAGGCCAGGG TCAGGTGACT GCTGGAGCAC ACAGCTTGGT AAGACTAGTC
AGGACCTGCG TCCTGAGGCT ACATGTCATA TCCACAGTAA GGAAGTGGAA
GATGGGAGAT GACTGGCTGG GCCACAACCA GTGAGTGGAA TGTCCTTGTG
CATCTTTGTT TCCTAACCTT CCCCTCTGTA GCTGCTGAAA CACACACACA
CCCCATGCTC TGTTATGCCT CTTCCCTGGC CTGGGATTTC CATGGCTGAG
GTGATGGGGC ACTGAGGCAC CGCCAGGAAA GGCTGTAACC CATCTGCTCC
CCCATCCTTC ACCAGACTTC AAGCACCTAC CTAGAGCACA GGTGCAATTT
TGTACCCTCC CTGTCTGGGA CCCACAGTGG TTCCTCAATG CCGGCCAACC
AGACTCATAG GCCTGCCCAC AAGGCCCTTG GGGCTATCTG TCTGAGGCCT
GCAGGTGCCC TCCTGGCCAC CTAGGCTCCT GTGAGACTTA GACTTCCATA
GATTCTTCCT GAAAGACTAC TGAGGGCAGG AGCCCCCAAG CCTCAGGGTT
AGCTTTCCTC AGCCCTGCCT CTTTGCTAGC TCCGTTTCCA CATTGAAGGC
AGGGCTGAGC AGGGCAGGCG CAGCGAGGAG CTAACTGCTG CTTCTCTCTC
GTTCATTTGT CTGCTGCCCT GAGACGCCAC AGCACCTAAT AAGAGCATGT
TATGTGTAGC AAACATTAGG CCTGTAAGGA AGGAAAGGAG TGACGTCCCT
TGACGTCCTC AGCTAGGCTG TGGTGACACA AGCAAGAGGA CTAAGCCACA
GGTGAGGAGA AAGGGGGGGG GGGGTCTGCT GACCCAGCAA CACTCTTTCC cons reg B
TTCTGAGGCT TAAGAGCTAT TAGCGTAGGT GACTCAGTCC CTAATCCTCC
ATTCAATGCC CTGTGACTGC CCCTGCTTCT GAAGGGCCAA CATGGCTACA
GCTAGCTCCA GAGACAGCTT TTCAGGGCCC CAGCATCCAA GCATCTCACA
GTTCTCCACT GACCACACTC CTGTGCAGCA CTGGGCTTTT CAATGCCCCT
GACTTGAAGA GAACTCAAAC TGCAGGTCAA CTAGACTCTG CAAACTTCAC
CTGTGCTGGG GGTTCCTAGC CTGTGGGGAC AGTGTATCTT GAATACCTGC
TGCTATGGAC CAAGAGCTGA ACACACAGAC AAACAGGCTC AGCTGGCCGG
CATTCTGGAA CCACAAATGA GTGTGGATGA GCAGGAGGGC AACAAAATGG
TCTGGGTGTT GTCAACACAG TCAGTAAACA ATGCACGCAG TGGGGCTGGG
CCCTGATGTG GAGCTAGGTG GGGTTGGCTC TCCTTGGAAA CCTGAAGGGA
GAAGGAGAGG GAGCGAGATG ATGAGGTTTA TCAGCCTGCA GAGGCAGGGG
GTCAGGAAGG AGTGCCACTG TACTGACCCA GGACCTCTGT GGGACATCAA
GCCATGCCAA GGAGCCATGG AGCCTCGATT GCACTGGCAG GGACAGGTTG
TGATGCCCCA GAGTCCCCAG ACCCAGCAAA CAGAGGCCCA GAGTGGGAAG
TGGAGCTTTC CAGGGTATCG GGGTGACTCA GAGACACAGG GTAGAATCTG
CCTTGGGTGC TCACTGCCCT ATCTGAGTCC ACATGGCTCA GTCCCCAGGC
CCTGTTCTCT AGTGACTGTT GCTTTGATGA GGTAGAGACA GGCAGCCCTC
TTCTAAGAAC TATGTTTTGA TGGGGGACTC AGAGTTGGGG TGGGGTGGCA
ATGAAATTCT GTAGACTGTG TGGTTATAAC CCTGGCTGTT ACTAGCTAGT
TCTGTGACCT TGGTGACCCA CTTCAGACTC TAGGCCTCAG CCTCTGTAAG
TGCAGATACA CAGCGCCAAT CAGCCGATGA CTTCTAACAA TACTCTTAAC
TCACACAGAG CTTGTCTCAC TGAGCCAACA CCCTGTACCC TCAGCTCAGT
GACGGCTTTC AACCTGTGGG GCTGCCTCTG TTACCCAAGT GAGAGAGGGC
CAGTGCTCCC AGAGGTGACC TTGTTTGCCC ATTCTCTCCC TGGGTCAGCC
AGTGTTTATC TGTTGTATAC CCAGTCCACC CTGCAGGCTC ACATCAGAGC
CTAGGAGATG GCTAGTGTCC CCGCGGAGAC CACGATGAAG CTTCCCAGCT HindIII
GTCTCAAGCA CAAGCTGGCT GCAGAGGCTG CTGAGGCACT GCTAGCTGGG start 1.734 kb
GATGGGGGCA GGGTAGATCT GGGGCTGACC ACCAGGGTCA GAATCAGAAC
CTCCACCTTG ACCTCATTAA CGCTGGTCTT AATCACCAAG CCAAGCTCCT cons reg C
TAAACTGCTA GTGGCCAACT CCCAGGCCCT GACACACATA CCTGCCCTGT
GTTCCCAAAC AAGACACCTG CATGGAAGGA AGGGGGTTGC TTTTCTAAGC
AAACATCTAG GAATCCCGGG TGCAGTGTGA GGAGACTAGG CGAGGGAGTA
CTTTAAGGGC CTCAAGGCTC AGAGAGGAAT ACTTCTTCCC TGGTTAGCCT
CGTGCCTAGG CTCCAGGGTC TTTGTCCTGC CTGGATACCT ATGTGGCAAG
GGGCATAGCA TTTCCCCCAC CATCAGCTCT TAGCTCAACC TTATCTTCTC
GGAAAGACTG CGCAGTGTAA CAACACAGCA GAGACTTTTC TTTTGTCCCC
TGTCTACCCC TGTAACTGCT ACTCAGAAGC ATCTTTCTCA CAGGGTACTG
GCTTCTTGCA TCCAGAGTTT TTTGTCTCCC TCGGGCCCCC AGAATCAAAT
TCTTCCTCTG GGACTCAGTG GATGTTTCAC ACACGTATCG GCCTGACAGT
CATCCTGGAG CATCCTACAC AGGGGCCATC ACAGCTGCAT GTCAGAAATG
CTGGCCTCAC ATCCTCAGAC ACCAGGCCTA GTGCTGGTCT TCCTCAGACT
GGCGTCCCCA GCAGGCCAGT AGGATCATCT TTTAGCCTAC AGAGTTCTGA
AGCCTCAGAG CCCCAGGTCC CTGGTCATCT TCTCTGCCCC TGAGATTTTT
CCAAGTTGTA TGCCTTCTAG GTAAGGCAAA ACTTCTTACG CCCCTCCTCG
TGGCCTCCAG GCCCCACATG CTCACCTGAA TAACCTGGCA GCCTGCTCCC
TCATGCAGGG ACCACGTCCT GCTGCACCCA GCAGGCCATC CCGTCTCCAT
AGCCCATGGT CATCCCTCCC TGGACAGGAA TGTGTCTCCT CCCCGGGCTG
AGTCTTGCTC AAGCTAGAAG CACTCCGAAC AGGGTTATGG GCGCCTCCTC
CATCTCCCAA GTGGCTGGCT TATGAATGTT TAATGTACAT GTGAGTGAAC
AAATTCCAAT TGAACGCAAC AAATAGTTAT CGAGCCGCTG AGCCGGGGGG
CGGGGGGTGT GAGACTGGAG GCGATGGACG GAGCTGACGG CACACACAGC
TCAGATCTGT CAAGTGAGCC ATTGTCAGGG CTTGGGGACT GGATAAGTCA
GGGGGTCTCC TGGGAAGAGA TGGGATAGGT GAGTTCAGGA GGAGACATTG
TCAACTGGAG CCATGTGGAG AAGTGAATTT AGGGCCCAAA GGTTCCAGTC
GCAGCCTGAG GCCACCAGAC TGACATGGGG AGGAATTCCC AGAGGACTCT
GGGGCAGACA AGATGAGACA CCCTTTCCTT TCTTTACCTA AGGGCCTCCA
CCCGATGTCA CCTTGGCCCC TCTGCAAGCC AATTAGGCCC CGGTGGCAGC
AGTGGGATTA GCGTTAGTAT GATATCTCGC GGATGCTGAA TCAGCCTCTG
GCTTAGGGAG AGAAGGTCAC TTTATAAGGG TCTGGGGGGG GTCAGTGCCT
GGAGTTGCGC TGTGGGAGCC GTCAGTGGCT GAGCTCGCCA AGCAGCCTTG
GTCTCTGTCT ACGAAGAGCC CGTGGGGCAG CCTCGAG XhoI
ggatcctgag tacctctcct ccctgacctc aggcttcctc ctagtgtcac cttggcccct conserved region D
cttagaagcc aattaggccc tcagtttctg cagcggggat taatatgatt atgaacaccc
ccaatctccc agatgctgat tcagccagga gcttaggagg gggaggtcac tttataaggg
tctggggggg tcagaaccca gagtcatcca gctggagccc tgagtggctg agctcaggcc
ttcgcagcat tcttgggtgg gagcagccac gggtcagcca caagggccac agccatgaat
ggcacagaag gccctaactt ctacgtgccc ttctccaatg cgacgggtgt ggtacgcagc
cccttcgagt acccacagta ctacctggct gagccatggc agttctccat gctggccgcc
tacatgtttc tgctgatcgt gctgggcttc cccatcaact tcctcacgct ctacgtcacc
gtccagcaca agaagctgcg cacgcctctc aactacatcc tgctcaacct agccgtggct
gacctcttca tggtcctagg tggcttcacc agcaccctct acacctctct gcatggatac
ttcgtcttcg ggcccacagg atgcaatttg gagggcttct ttgccaccct gggcggtatg
agccgggtgt gggtggggtg tgcaggagcc cgggagcatg gaggggtctg ggagagtccc
gggcttggcg gtggtggctg agaggccttc tcccttctcc tgtcctgtca atgttatcca
aagccctcat atattcagtc aacaaacacc attcatggtg atagccgggc tgctgtttgt
gcagggctgg cactgaacac tgccttgatc ttatttggag caatatgcgc ttgtctaatt
tcacagcaag aaaactgagc tgaggctcaa aggccaagtc aagcccctgc tggggcgtca
cacagggacg ggtgcagagt tgagttggaa gcccgcatct atctcgggcc atgtttgcag
caccaagcct ctgtttccct tggagcagct gtgctgagtc agacccaggc tgggcactga
gggagagctg ggcaagccag accectectc tctgggggcc caagctcagg gtgggaagtg
gattttccat tctccagtca ttgggtcttc cctgtgctgg gcaatgggct cggtcccctc
tggcatcctc tgcctcccct ctcagcccct gtcctcaggt gcccctccag cctccctgcc
gcgttccaag tctcctggtg ttgagaaccg caagcagccg ctctgaagca gttccttttt
gctttagaat aatgtcttgc atttaacagg aaaacagatg gggtgctgca gggataacag
atcccactta acagagagga aaactgaggc agggagaggg gaagagactc atttagggat
gtggccaggc agcaacaaga gcctaggtct cctggctgtg atccaggaat atctctgctg
agatgcagga ggagacgcta gaagcagcca ttgcaaagct gggtgacggg gagagcttac
cgccagccac aagcgtctct ctgccagcct tgccctgtct cccccatgtc caggctgctg
cctcggtccc attctcaggg aatctctggc cattgttggg tgtttgttgc attcaataat
cacagatcac tcagttctgg ccagaaggtg ggtgtgccac ttacgggtgg ttgttctctg
cagggtcagt cccagtttac aaatattgtc cctttcactg ttaggaatgt cccagtttgg
ttgattaact atatggccac tctccctatg aaacttcatg gggtggtgag caggacagat
gttcgaattc catcatttcc ttcttcttcc tctgggcaaa acattgcaca ttgcttcatg
gctcctagga gaggccccca catgtccggg ttatttcatt tcccgagaag ggagagggag
gaaggactgc caattctggg tttccaccac ctctgcattc cttcccaaca aggaactctg
ccccacatta ggatgcattc ttctgctaaa cacacacaca cacacacaca cacacaacac
acacacacac acacacacac acacacacac aaaactccct accgggttcc cagttcaatc
ctgaccccct gatctgattc gtgtccctta tgggcccaga gcgctaagca aataacttcc
cccattccct ggaatttctt tgcccagctc tcctcagcgt gtggtccctc tgccccttcc
ccctcctccc agcaccaagc tctctccttc cccaaggcct cctcaaatcc ctctcccact
cctggttgcc ttcctagcta ccctctccct gtctaggggg gagtgcaccc tccttaggca
gtggggtctg tgctgaccgc ctgctgactg ccttgcaggt gaaattgccc tgtggtcctt
ggtggtcctg gccatcgagc ggtacgtggt ggtgtgtaag cccatgagca acttccgctt
cggggagaac catgccatca tgggcgttgc cttcacctgg gtcatggcgc tggcctgcgc
cgcaccccca ctcgccggct ggtccaggta atggcactga gcagaaggga agaagctccg
ggggctcttt gtagggtcct ccagtcagga ctcaaaccca gtagtgtctg gttccaggca
ctgaccttgt atgtctcctg gcccaaatgc ccactcaggg taggggtgta gggcagaaga
agaaacagac tctaatgttg ctacaagggc tggtcccatc tcctgagccc catgtcaaac conserved region E
agaatccaag acatcccaac ccttcacctt ggctgtgccc ctaatcctca actaagctag
gcgcaaattc caatcctctt tggtctagta ccccgggggc agccccctct aaccttgggc
ctcagcagca ggggaggcca caccttccta gtgcaggtgg ccatattgtg gccccttgga
actgggtccc actcagcctc taggcgattg tctcctaatg gggctgagat gagactcagt
ggggacagtg gtttggacaa taggactggt gactctggtc cccagaggcc tcatgtccct
ctgtctccag aaaattccca ctctcacttc cctttcctcc tcagtcttgc tagggtccat
ttctacccct tgctgaattt gagcccaccc cctggacttt ttccccatct tctccaatct
ggcctagttc tatcctctgg aagcagagcc gctggacgct ctgggtttcc tgaggcccgt
ccactgtcac caatatcagg aaccattgcc acgtcctaat gacgtgcgct ggaagcctct
agtttccaga agctgcacaa agatccctta gatactctgt gtgtccatct ttggcctgga
aaatactctc accctggggc taggaagacc tcggtttgta caaacttcct caaatgcaga
gcctgagggc tctccccacc tcctcaccaa ccctctgcgt ggcatagccc tagcctcagc
gggcagtgga tgctggggct gggcatgcag ggagaggctg ggtggtgtca tctggtaacg
cagccaccaa acaatgaagc gacactgatt ccacaaggtg catctgcatc cccatctgat
ccattccatc ctgtcaccca gccatgcaga cgtttatgat ccccttttcc agggagggaa
tgtgaagccc cagaaagggc cagcgctcgg cagccacctt ggctgttccc aagtccctca
caggcagggt ctccctacct gcctgtcctc aggtacatcc ccgagggcct gcagtgctcg
tgtggaatcg actactacac gctcaagccg gaggtcaaca acgagtcttt tgtcatctac
atgttcgtgg tccacttcac catccccatg attatcatct ttttctgcta tgggcagctc
gtcttcaccg tcaaggaggt acgggccggg gggtgggcgg cctcacggct ctgagggtcc
agcccccagc atgcatctgc ggctcctgct ccctggagga gccatggtct ggacccgggt
cccgtgtcct gcaggccgct gcccagcagc aggagtcagc caccacacag aaggcagaga
aggaggtcac ccgcatggtc atcatcatgg tcatcgcttt cctgatctgc tgggtgccct
acgccagcgt ggcattctac atcttcaccc accagggctc caacttcggt cccatcttca
tgaccatccc agcgttcttt gccaagagcg ccgccatcta caaccctgtc atctatatca
tgatgaacaa gcaggtgcct actgcgggtg ggagggcccc agtgccccag gccacaggcg
ctgcctgcca aggacaagct actcccaggg caggggaggg gctccatcag ggttactggc
agcagtcttg ggtcagcagt cccaatgggg agtgtgtgag aaatgcagat tcctggcccc
actcagaact gctgaatctc agggtgggcc caggaacctg catttccagc aagccctcca
caggtggctc agatgctcac tcaggtggga gaagctccag tcagctagtt ctggaagccc
aatgtcaaag tcagaaggac ccaagtcggg aatgggatgg gccagtctcc ataaagctga
ataaggagct aaaaagtctt attctgaggg gtaaaggggt aaagggttcc tcggagaggt
acctccgagg ggtaaacagt tgggtaaaca gtctctgaag tcagctctgc cattttctag
ctgtatggcc ctgggcaagt caatttcctt ctctgtgctt tggtttcctc atccatagaa
aggtagaaag ggcaaaacac caaactcttg gattacaaga gataatttac agaacaccct
tggcacacag agggcaccat gaaatgtcac gggtgacaca gcccccttgt gctcagtccc
tggcatctct aggggtgagg agcgtctgcc tagcaggttc ccaccaggaa gctggatttg
agtggatggg gcgctggaat cgtgaggggc agaagcaggc aaagggtcgg ggcgaacctc
actaacgtgc cagttccaag cacactgtgg gcagccctgg ccctgactca agcctcttgc
cttccagttc cggaactgca tgctcaccac catctgctgc ggcaagaacc cactgggtga
cgatgaggcc tctgctaccg tgtccaagac ggagacgagc caggtggccc cggcctaaga
cctgcctagg actctgtggc cgactatagg cgtctcccat cccctacacc ttcccccagc
cacagccatc ccaccaggag cagcgcctgt gcagaatgaa cgaagtcaca taggctcctt conserved region F
aatttttttt ttttttttaa gaaataatta atgaggctcc tcactcacct gggacagcct
gagaagggac atccaccaag acctactgat ctggagtccc acgttcccca aggccagcgg
gatgtgtgcc cctcctcctc ccaactcatc tttcaggaac acgaggattc ttgctttctg
gaaaagtgtc ccagcttagg gataagtgtc tagcacagaa tggggcacac agtaggtgct conserved region G
taataaatgc tggatggatg caggaaggaa tggaggaatg aatgggaagg gagaacatat
ctatcctctc agaccctcgc agcagcagca actcatactt ggctaatgat atggagcagt
tgtttttccc tccctgggcc tcactttctt ctcctataaa atggaaatcc cagatccctg
gtcctgccga cacgcagcta ctgagaagac caaaagaggt gtgtgtgtgt ctatgtgtgt
gtttcagcac tttgtaaata gcaagaagct gtacagattc tagttaatgt tgtgaataac
atcaattaat gtaactagtt aattactatg attatcacct cctgatagtg aacattttga
gattgggcat tcagatgatg gggtttcacc caaccttggg gcaggttttt aaaaattagc
taggcatcaa ggccagacca gggctggggg ttgggctgta ggcagggaca gtcacaggaa
tgcaggatgc agtcatcaga cctgaaaaaa caacactggg ggagggggac ggtgaaggcc
aagttcccaa tgagggtgag attgggcctg gggtctcacc cctagtgtgg ggccccaggt
cccgtgcctc cccttcccaa tgtggcctat ggagagacag gcctttctct cagcctctgg
aagccacctg ctcttttgct ctagcacctg ggtcccagca tctagagcat ggagcctcta
gaagccatgc tcacccgccc acatttaatt aacagctgag tccctgatgt catccttact conserved region H
cgaagagctt agaaacaaag agtgggaaat tccactgggc ctaccttcct tggggatgtt
catgggcccc agtttccagt ttcccttgcc agacaagccc atcttcagca gttgctagtc
cattctccat tctggagaat ctgctccaaa aagctggcca catctctgag gtgtcagaat
taagctgcct cagtaactgc tcccccttct ccatataagc aaagccagaa gctctagctt
tacccagctc tgcctggaga ctaaggcaaa ttgggccatt aaaagctcag ctcctatgtt
ggtattaacg gtggtgggtt ttgttgcttt cacactctat ccacaggata gattgaaact conserved region I
gccagcttcc acctgatccc tgaccctggg atggctggat tgagcaatga gcagagccaa
gcagcacaga gtcccctggg gctagaggtg gaggaggcag tcctgggaat gggaaaaacc
ccaactttgg ggtcatagag gcacaggtaa cccataaaac tgcaaacaag ctt

• • [0157] Conserved regions A through I and some sequence flanking the regions (5′ and 3′, were combined (Table 11, SEQ ID NO: 92 through SEQ ID NO: 99, Sequence 2). This sequence was analyzed using MatInspector Release Professional 7.4.1 to identify other regions that may be involved in transcriptional and/or translational control of rhodopsin gene expression. (A small portion of the Matinspector results are presented in Table 12). This table illustrates some sequences within conserved regions A through I that are thought to be involved in the transcription and/or translation and/or stability of rhodopsin. Some of these sequences, such as the CRX binding element in conserved region D and the TATA box in region G are known in the art. Others, such as the CRX binding region in E, are not. The complete set of results from MatInspector are presented in Table 13. 302 putative transcription binding sites and/or regulatory sequences were identified and some are highlighted in bold. On the basis of the conserved nature of regions A though I and the important transcription factor binding sites thought to be located within these regions, the constructs in FIG. 9 were generated. Construct BB16 contains conserved regions A, B, C, D, F and G. In addition an artificial CRX-NRL element (below) was inserted between conserved regions A and B. The components of the artificial CRX-NRL enhancer element include the CRX motif from conserved region D, the CRX motif from conserved region E and NRL binding sites are underlined.

[0158]

(SEQ ID NO: 91)

TTTCTGCAGCGGGGATTAATATGATTATGAACACCCCCAATCTCCCAGAT

GCTGATTCAGCCAGGAGGTACC

[0159] All these constructs contain transcription binding sites identified within conserved regions A though I.

• • [0160] Sequence 2: Conserved regions A through I in the rhodopsin gene are highlighted in bold below. The nucleotides of these sequences and a small section of 5′ and 3′ sequence surrounding conserved regions have been numbered 1-1600. This sequence was analysed with MatInspector and the nucleotide numbering system of sequence 2 (1-1600) relates to the nucleotide numbering system in Table 13.

[0161]

TABLE 11
(Conserved regions are in bold)
Conserved region A 1-210 (SEQ ID NO: 92)
CACAACTCCT CCGTCGAGTG TCTAATTGCT TATGATCATG
CATGCTCTCT CTCCCACTAA ACATTTATTA ATGTGTTAGG
ATTTCCATTA GCGCGTGCCT TGAACTGAAA TCATTTGCAT
ATGGCTGGGA AAAAGTGGGG TGAGGGAGGA AACAGTGCCA
GCTCCCCAAC AGGCGTCAAT CACAGTGACA GATCAGATGG
TTTCTGGCTG 210
Conserved region B 210-310 (SEQ ID NO: 93)
AAGGGGGGGG GGGGTCTGCT GACCCAGCAA CACTCTTTCC
TTCTGAGGCT TAAGAGCTAT TAGCGTAGGT GACTCAGTCC
CTAATCCTCC ATTCAATGCC 310
Conserved region C 310-410 (SEQ ID NO: 94)
GGGGCTGACC ACCAGGGTCA GAATCAGAAC CTCCACCTTG
ACCTCATTAA CGCTGGTCTT AATCACCAAG CCAAGCTCCT
TAAACTGCTA GTGGCCAACT 410
Conserved region D 410-690 (SEQ ID NO: 95)
aggcttcctc ctagtgtcac cttggcccct cttagaagcc aattaggccc tcagtttctg cagcggggat
taatatgatt atgaacaccc ccaatctccc agatgctgat tcagccagga gcttaggagg gggaggtcac
tttataaggg tctggggggg tcagaaccca gagtcatcca gctggagccc tgagtggctg agctcaggcc
ttcgcagcat tcttgggtgg gagcagccac gggtcagcca caagggccac agccatgaat ggcacagaag
690
Conserved region E 690-850 (SEQ ID NO: 96)
tcctgagccc catgtcaaac agaatccaag acatcccaac ccttcacctt ggctgtgccc ctaatcctca
actaagctag gcgcaaattc caatcctctt tggtctagta ccccgggggc agccccctct aaccttgggc
ctcagcagca ggggaggcca 850
Conserved regions F and G 850-1220 (SEQ ID NO: 97)
cccctacacc ttcccccagc cacagccatc ccaccaggag cagcgcctgt gcagaatgaa cgaagtcaca
taggctcctt aatttttttt ttttttttaa gaaataatta atgaggctcc tcactcacct gggacagcct
gagaagggac atccaccaag acctactgat ctggagtccc acgttcccca aggccagcgg gatgtgtgcc
cctcctcctc ccaactcatc tttcaggaac acgaggattc ttgctttctg gaaaagtgtc ccagcttagg
gataagtgtc tagcacagaa tggggcacac agtaggtgct taataaatgc tggatggatg caggaaggaa
tggaggaatg aatgggaagg 1220
Conserved region H 1220 1230-1316 1330 (SEQ ID NO: 98)
tctagagcat ggagcctcta gaagccatgc tcacccgccc acatttaatt aacagctgag tccctgatgt
catccttact cgaagagctt agaaacaaag agtgggaaat 1330
Conserved region I 1330 1342-1425 1600 (SEQ ID NO: 99)
gctctagctt tacccagctc tgcctggaga ctaaggcaaa ttgggccatt aaaagctcag ctcctatgtt
ggtattaacg gtggtgggtt ttgttgcttt cacactctat ccacaggata gattgaaact gccagcttcc
acctgatccc tgaccctggg atggctggat tgagcaatga gcagagccaa gcagcacaga gtcccctggg
gctagaggtg gaggaggcag tcctgggaat gggaaaaacc ccaactttgg ggtcatagag 1600

[0162]

TABLE 12
Conserved sequence motifs in Rhodopsin
	Conserved
	region	Position	Name
	B	288-304	CRX
	C	366-382	CRX
	D	470-486	CRX
	E	784-764	CRX
	G	1172-1177	TATA box
	D	500-520	Neuron-restrictive silencer factor
	E	794-814	Neuron-restrictive silencer factor
	E	831-851	Neuron-restrictive silencer factor

[0163]

TABLE 13
Putative Rhodopsin Transcription Regulatory Factors
							Sequence
							(red: ci-
							value >60
							capitals:	SEQ
	Further				Core	Matrix	core	ID
Family/matrix	Information	Opt.	Position	Str.	sim.	sim.	sequence)	NO
V$PDX1/1SL1.01	Pancreatic and	0.82	14-34	(+)	1.000	0.860	tcgagtgtcTAA	100
	intestinal lim-						Ttgcttatg
	homeodomain
	factor
V$HOMF/MSX.01	Homeodomain	0.97	18-30	(+)	1.000	0.995	gtgtcTAATtgc	101
	proteins MSX-1						t
	and MSX-2
V$HOXF/GSH2.01	Homeodomain	0.95	19-35	(+)	1.000	0.975	tgtcTAATtgctt	102
	transcription						atga
	factor Gsh-2
V$GABF/GAGA.01	GAGA-Box	0.78	33-57	(−)	1.000	0.825	gtgggAGAGag	103
							agcatgcatgatca
V$FKHD/FREAC2.0	Fork head related	0.84	52-68	(+)	1.000	0.884	tcccacTAAAc	104
1	activator-2						atttat
	(FOXF2)
V$HOXF/HOXC13.	Homeodomain	0.91	58-74	(−)	1.000	0.914	acattaaTAAAt	105
01	transcription						gttta
	factor HOXC13
V$NKXH/HMX2.02	Hmx2/Nkx5-2	0.82	58-72	(−)	0.750	0.835	attaatAAATgtt	106
	homeodomain						ta
	transcription
	factor
V$SATB/SATB1.01	Special AT-rich	0.94	58-72	(−)	1.000	0.956	attAATAaatgtt	107
	sequence-binding						ta
	protein 1,
	predominantly
	expressed in
	thymocytes,
	binds to matrix
	attachment
	regions (MARs)
V$BRNF/BRN3.02	Brn-3, POU-IV	0.89	59-77	(−)	1.000	0.892	aacacatTAATa	108
	protein class						aatgttt
V$PDX1/PDX1.01	Pdx1	0.74	59-79	(−)	1.000	0.744	ctaacacatTAA	109
	(IDX1/IPF1)						Taaatgttt
	pancreatic and
	intestinal
	homeodomain TF
V$PIT1/PIT1.01	Pit1, GHF-1	0.84	61-73	(+)	1.000	0.857	acatTTATtaatg	110
	pituitary specific
	pou domain
	transcription
	factor
V$BRNF/BRN3.02	Brn-3, POU-IV	0.89	62-80	(+)	1.000	0.893	catttatTAATgt	111
	protein class						gttagg
V$LHXF/LMX1B.0	LIM-	0.91	62-76	(−)	1.000	0.946	acacatTAATaa	112
1	homeodomain						atg
	transcription
	factor
V$HOXH/MEIS1B_	Meis1b and	0.78	64-78	(−)	0.750	0.823	TAACacattaat	113
HOXA9.01	Hoxa9 form						aaa
	heterodimeric
	binding
	complexes on
	target DNA
V$HOXF/HOX1-	Hox-1.3,	0.82	65-81	(+)	1.000	0.826	ttatTAATgtgtt	114
3.01	vertebrate						agga
	homeobox
	protein
V$OCT1/OCT1.04	Octamer-binding	0.80	77-91	(−)	0.846	0.866	ctAATGgaaatc	115
	factor 1						cta
V$HOXF/PHOX2.01	Phox2a (ARIX)	0.87	78-94	(−)	1.000	0.969	gcgcTAATgga	116
	and Phox2b						aatcct
V$AHRR/AHRARN	Aryl hydrocarbon	0.92	83-107	(+)	1.000	0.932	ttccattagcgCG	117
T.01	receptor/Arnt						TGccttgaactg
	heterodimers
V$MOKF/MOK2.02	Ribonucleopro-	0.98	85-105	(+)	1.000	0.988	ccattagcgcgtg	118
	tein associated						CCTTgaac
	zinc finger
	protein MOK-2
	(human)
V$EBOX/MYCMA	MYC-MAX	0.91	87-101	(−)	1.000	0.918	aaggcaCGCGc	119
X.03	binding sites						taat
V$HESF/HELT.01	Hey-like bHLH-	0.91	87-101	(−)	1.000	0.947	aaggCACGcgc	120
	transcriptional						taat
	repressor
V$HOMF/EN1.01	Homeobox	0.77	97-109	(+)	0.782	0.776	gccTTGAactg	121
	protein engrailed						aa
	(en-1)
V$OCT1/OCT1.02	Octamer-binding	0.85	109-123	(−)	1.000	0.992	catATGCaaatg	122
	factor 1						att
V$OCTP/OCT1P.01	Octamer-binding	0.86	113-125	(−)	1.000	0.910	gccATATgcaa	123
	factor 1, POU-						at
	specific domain
V$AIRE/AIRE.01	Autoimmune	0.86	119-145	(+)	0.916	0.862	atatggctgggaaa	124
	regulator						aagTGGGgtga
							gg
V$RBPF/RBPJK.02	Mammalian	0.94	122-136	(+)	1.000	0.941	tggcTGGGaaa	125
	transcriptional						aagt
	repressor RBP-
	Jkappa/CBF1
V$RXRF/VDR_RX	Bipartite binding	0.75	123-147	(+)	0.812	0.760	ggctgggaaaaag	126
R.06	site of						tgGGGTgaggg
	VDR/RXR						a
	heterodimers: 4
	spacer
	nucleotides
	between the two
	directly repeated
	motifs
V$NKXH/HMX3.01	H6	0.89	127-141	(+)	1.000	0.910	gggaaaAAGTg	127
	homeodomain						gggt
	HMX3/Nkx5.1
	transcription
	factor
V$CIZF/NMP4.01	NMP4 (nuclear	0.97	128-138	(+)	1.000	0.998	ggAAAAagtgg	128
	matrix protein 4)/
	CIZ (Cas-
	interacting zinc
	finger protein)
V$EBOX/SREBP.01	Sterol regulatory	0.90	132-146	(−)	1.000	0.960	cccTCACccca	129
	element binding						cttt
	protein 1 and 2
V$RXRF/VDR_RX	VDR/RXR	0.86	134-158	(+)	1.000	0.878	agtggggtgagg	130
R.02	Vitamin D						GAGGaaacagt
	receptor RXR						gc
	heterodimer site
V$ETSF/PU1.01	Pu.1 (Pu120) Ets-	0.89	141-157	(+)	1.000	0.895	tgagggaGGAA	131
	like transcription						acagtg
	factor identified
	in lymphoid B-
	cells
V$NFAT/NFAT.01	Nuclear factor of	0.95	145-155	(+)	1.000	0.989	ggaGGAAaca	132
	activated T-cells						g
V$AREB/AREB6.04	AREB6 (Atp1a1	0.98	146-158	(−)	1.000	0.991	gcactGTTTcct	133
	regulatory						c
	element binding
	factor 6)
V$COMP/COMP1.0	COMP1,	0.77	163-185	(−)	1.000	0.811	ctgtgATTGacg	134
1	cooperates with						cctgttgggga
	myogenic
	proteins in
	multicomponent
	complex
V$PAX6/PAX6.01	Pax-6 paired	0.77	163-181	(−)	0.808	0.781	gaTTGAcgcct	135
	domain binding						gttgggga
	site
V$MYBL/CMYB.01	c-Myb, important	0.90	165-177	(+)	1.000	0.945	ccCAACaggcg	136
	in hematopoesis,						tc
	cellular
	equivalent to
	avian
	myoblastosis
	virus oncogene
	v-myb
V$CREB/CREB.02	cAMP-	0.89	167-187	(−)	1.000	0.902	cactgtgatTGA	137
	responsive						Cgcctgttg
	element binding
	protein
V$WHZF/WHN.01	Winged helix	0.95	169-179	(−)	1.000	0.955	ttgACGCctgt	138
	protein, involved
	in hair
	keratinization
	and thymus
	epithelium
	differentiation
V$HOXC/PBX1.01	Homeo domain	0.78	170-186	(−)	1.000	0.840	actgtGATTgac	139
	factor Pbx-1						gcctg
V$PBXC/PBX1_ME	Binding site for a	0.77	170-186	(−)	1.000	0.875	actgTGATtgac	140
IS1.02	Pbx1/Meis1						gcctg
	heterodimer
V$AP1R/TCF11MA	TCF11/MafG	0.81	177-201	(+)	1.000	0.838	caatcacagTGA	141
FG.01	heterodimers,						Cagatcagatggt
	binding to
	subclass of AP1
	sites
V$TALE/MEIS1.01	Binding site for	0.95	183-193	(−)	1.000	0.971	atcTGTCactg	142
	monomeric
	Meis1
	homeodomain
	protein
V$HOXH/MEIS1A_	Meis1a and	0.77	186-200	(+)	1.000	0.770	TGACagatcag	143
HOXA9.01	Hoxa9 form						atgg
	heterodimeric
	binding
	complexes on
	target DNA
V$GATA/GATA3.0	GATA-binding	0.91	187-199	(+)	1.000	0.950	gacAGATcaga	144
2	factor 3						tg
V$AP4R/TAL1BET	Tal-1beta/E47	0.87	189-205	(+)	1.000	0.955	cagatCAGAtg	145
AE47.01	heterodimer						gtttct
V$NEUR/NEUROG.	Neurogenin 1	0.92	191-203	(−)	1.000	0.925	aaaCCATctgat	146
01	and 3 (ngn1/3)						c
	binding sites
V$ZBPF/ZBP89.01	Zinc finger	0.93	205-227	(−)	1.000	0.966	agacccccccCC	147
	transcription						CCcttcagcca
	factor ZBP-89
V$ZBPF/ZNF219.01	Kruppel-like zinc	0.91	207-229	(−)	1.000	0.997	gcagaccCCCC	148
	finger protein						ccccccttcagc
	219
V$INSM/INSM1.01	Zinc finger	0.90	209-	(+)	1.000	0.914	tgaagGGGGgg	149
	protein		221nc				gg
	insulinoma-
	associated 1 (IA-
	1) functions as a
	transcriptional
	repressor
V$EKLF/KKLF.01	Kidney-enriched	0.91	210-	(+)	1.000	0.934	gaagggGGGG	150
	kruppel-like		226nc				gggggtc
	factor, KLF15
V$EGRF/WT1.01	Wilms Tumor	0.92	211-	(+)	0.837	0.945	aagggGGGGg	151
	Suppressor		227nc				ggggtct
V$SP1F/GC.01	GC box elements	0.88	211-	(+)	0.819	0.897	aagggGGGGg	152
			225nc				ggggt
V$EKLF/KKLF.01	Kidney-enriched	0.91	212-	(+)	1.000	0.949	agggggGGGG	153
	kruppel-like		228nc				gggtctg
	factor, KLF15
V$SP1F/GC.01	GC box elements	0.88	213-	(+)	0.819	0.908	gggggGGGGg	154
			227nc				ggtct
V$EGRF/WT1.01	Wilms Tumor	0.92	214-	(+)	0.837	0.932	gggggGGGGg	155
	Suppressor		230nc				gtctgct
V$GLIF/ZIC2.01	Zinc finger	0.89	214-228	(−)	1.000	0.967	cagacccCCCC	156
	transcription						cccc
	factor, Zic family
	member 2 (odd-
	paired homolog,
	Drosophila)
V$MAZF/MAZR.01	MYC-associated	0.88	215-	(+)	1.000	0.972	ggggggGGGG	157
	zinc finger		227nc				tct
	protein related
	transcription
	factor
V$AP1R/BACH2.01	Bach2 bound	0.89	221-245	(−)	0.813	0.897	gagtgttgcTGG	158
	TRE						Gtcagcagacccc
V$AP1R/VMAF.01	v-Maf	0.82	221-245	(+)	1.000	0.957	ggggtctgcTGA	159
							Cccagcaacactc
V$XBBF/MIF1.01	MIBP-1/RFX1	0.76	225-243	(−)	0.800	0.778	gtgttgctggGTC	160
	complex						Agcaga
V$XBBF/RFX1.01	X-box binding	0.89	227-245	(+)	1.000	0.907	tgctgacccaGC	161
	protein RFX1						AAcactc
V$NFAT/NFAT.01	Nuclear factor of	0.95	243-253	(−)	1.000	0.971	gaaGGAAaga	162
	activated T-cells						g
V$NKXH/HMX2.01	Hmx2/Nkx5-2	0.83	253-267	(−)	1.000	0.911	gctCTTAagcct	163
	homeodomain						cag
	transcription
	factor
V$PAX8/PAX8.01	PAX 2/5/8	0.88	254-266	(−)	0.800	0.901	ctcTTAAgcctc	164
	binding site						a
V$NKXH/HMX2.01	Hmx2/Nkx5-2	0.83	256-270	(+)	1.000	0.931	aggCTTAagag	165
	homeodomain						ctat
	transcription
	factor
V$HOXF/PHOX2.01	Phox2a (ARIX)	0.87	260-276	(−)	1.000	0.898	acgcTAATagc	166
	and Phox2b						tcttaa
V$CLOX/CDPCR3.	Cut-like	0.73	266-284	(−)	0.880	0.770	agtcacctacgcta	167
01	homeodomain						ATAGc
	protein
V$EGRF/NGFIC.01	Nerve growth	0.80	269-285	(+)	1.000	0.855	attaGCGTaggt	168
	factor-induced						gactc
	protein C
V$AP1R/BACH2.01	Bach2 bound	0.89	271-295	(−)	1.000	0.957	attagggacTGA	169
	TRE						Gtcacctacgcta
V$CREB/TAXCRE	Tax/CREB	0.71	274-294	(+)	1.000	0.744	cgtaggTGACtc	170
B.02	complex						agtccctaa
V$AP1F/AP1.01	Activator protein	0.94	278-288	(+)	0.904	0.967	ggtgACTCagt	171
	1
V$AP1F/AP1.03	Activator protein	0.94	278-288	(−)	1.000	0.976	acTGAGtcacc	172
	1
V$HOXF/CRX.01	Cone-rod B	0.94	288-304	(+)	1.000	0.972	tcccTAATcctc	173
	homeobox-						cattc
	containing
	transcription
	factor/otx-like
	homeobox gene
V$SORY/HBP1.01	HMG box-	0.86	298-310	(−)	1.000	0.905	ggcattgAATG	174
	containing						ga
	protein 1
V$IRFF/IRF7.01	Interferon	0.86	329-347	(+)	0.936	0.865	caGAATcagaa	175
	regulatory factor						cctccacc
	7 (IRF-7)
V$RORA/RORA1.0	RAR-related	0.93	342-360	(−)	1.000	0.953	ttaatgaGGTCa	176
1	orphan receptor						aggtgga
	alpha1
V$CSEN/DREAM.0	Downstream	0.95	344-354	(−)	1.000	0.960	agGTCAaggtg	177
1	regulatory
	element-
	antagonist
	modulator,
	Ca2+-binding
	protein of the
	neuronal
	calcium sensors
	family that
	binds DRE
	(downstream
	regulatory
	element) sites as
	a tetramer
V$E4FF/E4F.01	GLI-Krueppel-	0.82	345-357	(−)	0.789	0.824	atgAGGTcaag	178
	related						gt
	transcription
	factor, regulator
	of adenovirus E4
	promoter
V$HOXF/BARX2.0	Barx2,	0.95	347-363	(−)	1.000	0.980	gcgtTAATgag	179
1	homeobox						gtcaag
	transcription
	factor that
	preferentially
	binds to paired
	TAAT motifs
V$MYBL/VMYB.04	v-Myb, AMV v-	0.85	356-368	(+)	1.000	0.881	attAACGctggt	180
	myb						c
V$HOXF/CRX.01	Cone-rod (C)	0.94	366-382	(+)	1.000	0.962	gtctTAATcacc	181
	homeobox-						aagcc
	containing
	transcription
	factor/otx-like
	homeobox gene
V$RCAT/CLTR_CA	Mammalian C-	0.71	375-399	(+)	1.000	0.718	aCCAAgccaag	182
AT.01	type LTR						ctccttaaactgct
	CCAAT box
V$ETSF/ETS1.01	c-Ets-1 binding	0.92	409-425	(−)	1.000	0.921	actaggaGGAA	183
	site						gcctag
V$SF1F/FTF.01	Alpha (1)-	0.94	426-438	(−)	1.000	0.940	gggcCAAGgtg	184
	fetoprotein						ac
	transcription
	factor (FTF),
	liver receptor
	homologue-1
	(LRH-1)
V$BCL6/BCL6.02	POZ/zinc finger	0.77	436-452	(+)	1.000	0.785	cccctctTAGAa	185
	protein,						gccaa
	transcriptional
	repressor,
	translocations
	observed in
	diffuse large cell
	lymphoma
V$HOXF/GSH2.01	Homeodomain	0.95	443-459	(−)	1.000	1.000	ggccTAATtgg	186
	transcription						cttcta
	factor Gsh-2
V$CAAT/CAAT.01	Cellular and viral	0.90	445-459	(+)	1.000	0.949	gaagCCAAtta	187
	CCAAT box						ggcc
V$NKXH/NKX25.0	Homeo domain	0.88	446-460	(−)	1.000	0.938	gggccTAATtg	188
2	factor Nkx-						gctt
	2.5/Csx, tinman
	homolog low
	affinity sites
V$HOMF/S8.01	Binding site for	0.97	448-460	(−)	1.000	0.999	gggccTAATtg	189
	S8 type						gc
	homeodomains
V$HOXF/CRX.01	Cone-rod (D)	0.94	470-486	(−)	1.000	0.985	atatTAATcccc	190
	homeobox-						gctgc
	containing
	transcription
	factor/otx-like
	homeobox gene
V$MZF1/MZF1.01	Myeloid zinc	0.99	473-481	(+)	1.000	0.991	gcGGGGatt	191
	finger protein
	MZF1
V$OCTB/TST1.01	POU-factor Tst-	0.90	475-487	(+)	1.000	0.947	ggggATTAatat	192
	1/Oct-6						g
V$CART/CART1.01	Cart-1 (cartilage	0.86	477-493	(−)	1.000	0.926	caTAATcatatt	193
	homeoprotein 1)						aatcc
V$CART/CART1.01	Cart-1 (cartilage	0.86	479-495	(+)	1.000	0.914	atTAATatgatta	194
	homeoprotein 1)						tgaa
V$SATB/SATB1.01	Special AT-rich	0.94	479-493	(+)	1.000	0.957	attAATAtgatta	195
	sequence-binding						tg
	protein 1,
	predominantly
	expressed in
	thymocytes,
	binds to matrix
	attachment
	regions (MARs)
V$PDX1/PDX1.01	Pdx1	0.74	480-500	(+)	0.826	0.775	ttaatatgaTTAT	196
	(IDX1/IPF1)						gaacaccc
	pancreatic and
	intestinal
	homeodomain TF
V$GLIF/ZIC2.01	Zinc finger	0.89	491-505	(+)	1.000	0.932	atgaacaCCCCc	197
	transcription						aat
	factor, Zic family
	member 2 (odd-
	paired homolog,
	Drosophila)
V$CAAT/ACAAT.0	Avian C-type	0.83	497-511	(+)	1.000	0.905	acccCCAAtctc	198
1	LTR CCAAT						cca
	box
V$RREB/RREB1.01	Ras-responsive	0.80	499-513	(+)	1.000	0.841	cCCCAatctccc	199
	element binding						aga
	protein 1
V$NRSF/NRSF.01	Neuron-	0.69	500-520	(−)	1.000	0.696	atcAGCAtctgg	200
	restrictive						gagattggg
	silencer factor
V$IKRS/LYF1.01	LyF-1 (Ikaros 1),	0.98	502-514	(−)	1.000	1.000	atcTGGGagatt	201
	enriched in B and						g
	T lymphocytes
V$AP4R/TAL1 ALP	Tal-1 alpha/E47	0.87	505-521	(+)	1.000	0.905	tctccCAGAtgc	202
HAE47.01	heterodimer						tgatt
V$RP58/RP58.01	Zinc finger	0.84	507-519	(−)	1.000	0.865	tcagCATCtggg	203
	protein RP58						a
	(ZNF238),
	associated
	preferentially
	with
	heterochromatin
V$AP1R/NFE2.01	NF-E2 p45	0.85	508-532	(+)	1.000	0.904	cccagatgCTG	204
							Attcagccaggag
							c
V$AP1R/NFL2.01	NF-E2 p45	0.85	508-532	(−)	1.000	0.882	gctcctggCTGA	205
							atcagcatctggg
V$BEL1/BEL1.01	Bel-1 similar	0.81	510-532	(−)	1.000	0.818	gctcctggctgaaT	206
	region (defined						CAGcatctg
	in Lentivirus
	LTRs)
V$NRLF/NRL.01	Neural retinal	0.85	511-529	(−)	1.000	0.991	cctggCTGAatc	207
	basic leucine						agcatct
	zipper factor
	(bZIP)
V$AP1F/AP1.03	Activator protein	0.94	515-525	(+)	0.885	0.970	gcTGATtcagc	208
	1
V$AP1F/AP1.03	Activator protein	0.94	515-525	(−)	0.857	0.963	gcTGAAtcagc	209
	1
V$HOXF/PTX1.01	Pituitary	0.94	523-539	(−)	1.000	0.944	ctcCTAAgctcc	210
	Homeobox 1						tggct
	(Ptx1, Pitx-1)
V$ZBPF/ZNF219.01	Kruppel-like zinc	0.91	528-550	(−)	1.000	0.926	gtgacctCCCCc	211
	finger protein						tcctaagctcc
	219
V$RXRF/VDR_RX	VDR/RXR	0.85	531-555	(+)	1.000	0.889	gcttaggaggggG	212
R.01	Vitamin D						AGGtcactttat
	receptor RXR
	heterodimer site
V$ZBPF/ZBP89.01	Zinc finger	0.93	531-553	(−)	1.000	0.958	aaagtgacctCC	213
	transcription						CCctcctaagc
	factor ZBP-89
V$EKLF/KKLF.01	Kidney-enriched	0.91	534-550	(+)	1.000	0.913	taggagGGGGa	214
	kruppel-like						ggtcac
	factor, KLF15
V$GLIF/ZIC2.01	Zinc finger	0.89	536-550	(−)	1.000	0.945	gtgacctCCCCc	215
	transcription						tcc
	factor, Zic family
	member 2 (odd-
	paired homolog,
	Drosophila)
V$RORA/TR2.01	Nuclear hormone	0.92	538-556	(+)	1.000	0.950	agggggaGGTC	216
	receptor TR2,						actttata
	half site
V$TBPF/TATA.01	Cellular and viral	0.90	543-559	(−)	1.000	0.915	ccttaTAAAgtg	217
	TATA box						acctc
	elements
V$SRFF/SRF.01	Serum response	0.66	545-563	(−)	1.000	0.722	agaccctTATAa	218
	factor						agtgacc
V$SRFF/SRF.01	Serum response	0.66	546-564	(+)	1.000	0.712	gtcacttTATAa	219
	factor						gggtctg
V$TBPF/LTATA.01	Lentivirus LTR	0.82	550-566	(+)	1.000	0.829	cttTATAagggt	220
	TATA box						ctggg
V$MOKF/MOK2.01	Ribonucleopro-	0.74	552-572	(−)	1.000	0.772	gacccccccagac	221
	tein associated						CCTTataa
	zinc finger
	protein MOK-2
	(mouse)
V$ZBPF/ZNF219.01	Kruppel-like zinc	0.91	553-575	(−)	1.000	0.948	tctgaccCCCCc	222
	finger protein						agacccttata
	219
V$GLIF/ZIC2.01	Zinc finger	0.89	560-574	(−)	1.000	0.967	ctgacccCCCCa	223
	transcription						gac
	factor, Zic family
	member 2 (odd-
	paired homolog,
	Drosophila)
V$MAZF/MAZR.01	MYC-associated	0.88	561-573	(+)	1.000	0.919	tctgggGGGGtc	224
	zinc finger						a
	protein related
	transcription
	factor
V$ZNFP/SZF1.01	SZF1,	0.82	579-603	(−)	0.801	0.829	tcaGGGCtccag	225
	hematopoietic						ctggatgactctg
	progenitor-
	restricted KRAB-
	zinc finger
	protein
V$AP4R/AP4.01	Activator protein	0.85	584-	(+)	1.000	0.916	tcatcCAGCtgg	226
	4		600UTR				agccc
V$EBOX/ATF6.01	Member of b-zip	0.93	596-610	(−)	1.000	0.970	cagCCACtcag	227
	family, induced						ggct
	by ER
	damage/stress,
	binds to the
	ERSE in
	association with
	NF-Y
V$CAAT/CAAT.01	Cellular and viral	0.90	597-611	(−)	0.826	0.937	tcagCCACtcag	228
	CCAAT box						ggc
V$HEAT/HSF1.01	Heat shock factor	0.84	621-645	(−)	1.000	0.857	tgctcccacccaA	229
	1						GAAtgctgcgaa
V$OAZF/ROAZ.01	Rat C2H2 Zn	0.73	625-641	(+)	0.750	0.779	caGCATtcttgg	230
	finger protein						gtggg
	involved in
	olfactory
	neuronal
	differentiation
V$OAZF/ROAZ.01	Rat C2H2 Zn	0.73	626-	(−)	0.750	0.744	tcCCACccaag	231
	finger protein		642nc				aatgct
	involved in
	olfactory
	neuronal
	differentiation
V$EGRF/EGR2.01	Egr-2/Krox-20	0.79	631-	(+)	0.766	0.828	tcttGGGTggga	232
	early growth		647UTR				gcagc
	response gene
	product
V$EGRF/WT1.01	Wilms Tumor	0.92	633-649	(+)	1.000	0.930	ttgggTGGGag	233
	Suppressor						cagcca
V$RBPF/RBPJK.01	Mammalian	0.84	634-	(+)	1.000	0.847	tgggTGGGagc	234
	transcriptional		648UTR				agcc
	repressor RBP-
	Jkappa/CBF1
V$OAZF/ROAZ.01	Rat C2H2 Zn	0.73	641-657	(+)	0.750	0.818	gaGCAGccacg	235
	finger protein						ggtcag
	involved in
	olfactory
	neuronal
	differentiation
V$EBOX/USF.03	Upstream	0.89	643-657	(−)	1.000	0.904	ctgaccCGTGg	236
	stimulating factor						ctgc
V$EBOX/MYCMA	MYC-MAX	0.91	644-658	(+)	0.842	0.919	cagccaCGGGt	237
X.03	binding sites						cagc
V$PAX5/PAX5.03	PAXS paired	0.80	659-	(+)	0.894	0.833	cacaagggCCA	238
	domain protein		687gen				Cagccatgaatgg
							cacag
V$CLOX/CDPCR3.	Cut-like	0.73	665-	(+)	1.000	0.735	ggccacagccatg	239
01	homeodomain		683gen				aATGGc
	protein
V$PAX5/PAX5.03	PAX5 paired	0.80	674-	(+)	1.000	0.800	catgaatgGCAC	240
	domain protein		702gen				agaagtcctgagcc
							cca
V$ZNFP/ZBRK1.01	Transcription	0.77	680-704	(−)	0.813	0.847	catggggcTCA	241
	factor with 8						Ggacttctgtgcca
	central zinc
	fingers and an N-
	terminal KRAB
	domain
V$PBXC/PBX1_ME	Binding site for a	0.77	699-715	(−)	0.750	0.860	attcTGTTtgaca	242
IS1.02	Pbx1/Meis1						tggg
	heterodimer
V$TALE/TGIF.01	TG-interacting	1.00	700-	(+)	1.000	1.000	ccatGTCAaac	243
	factor belonging		710nc
	to TALE class of
	homeodomain
	factors
V$SNAP/PSE.02	Proximal	0.73	745-763	(+)	1.000	0.734	gtgccCCTAatc	244
	sequence element						ctcaact
	(PSE) of RNA
	polymerase III-
	transcribed genes
V$HOXF/CRX.01	Cone-rod (E)	0.94	748-764	(+)	1.000	0.965	ccccTAATcctc	245
	homeobox-						aacta
	containing
	transcription
	factor/otx-like
	homeobox gene
V$FAST/FAST1.01	FAST-1 SMAD	0.81	749-763	(−)	0.983	0.829	agttgagGATTa	246
	interacting						ggg
	protein
V$NR2F/HPF1.01	HepG2-specific	0.78	767-787	(+)	0.750	0.801	ctaggcgcAAA	247
	P450 2C factor-1						Ttccaatcct
V$SORY/HMGIY.0	HMGI(Y) high-	0.92	770-782	(−)	1.000	0.938	tggAATTtgcgc	248
1	mobility-group						c
	protein I (Y),
	architectural
	transcription
	factor organizing
	the framework of
	a nuclear protein-
	DNA
	transcriptional
	complex
V$HMTB/MTBF.01	Muscle-specific	0.90	774-782	(−)	1.000	0.953	tggaATTTg	249
	Mt binding site
V$LEFF/LEF1.01	TCF/LEF-1,	0.86	783-799	(−)	1.000	0.889	actagacCAAA	250
	involved in the						gaggat
	Wnt signal
	transduction
	pathway
V$NRSF/NRSE.01	Neural-	0.67	794-	(+)	0.782	0.762	tctagtacccCGG	251
	restrictive-		814nc				Gggcagcc
	silencer-element
V$ZBPF/ZF9.01	Core promoter-	0.87	803-	(+)	0.769	0.878	ccgggggCAG	252
	binding protein		825nc				Ccccctctaacct
	(CPBP) with 3
	Krueppel-type
	zinc fingers
V$HICF/HIC1.01	Hypermethylated	0.93	804-816	(−)	1.000	0.970	ggggcTGCCcc	253
	in cancer 1,						cg
	transcriptional
	repressor
	containing five
	Krüppel-like
	C2H2 zinc
	fingers, for
	optimal binding
	multiple binding
	sites are required.
V$NFKB/NFKAPP	NF-kappaB (p50)	0.83	806-818	(−)	0.750	0.865	aggGGGCtgcc	254
AB50.01							cc
V$STAF/ZNF76_14	ZNF143 is the	0.76	810-832	(+)	0.809	0.761	cagcCCCCtcta	255
3.01	human ortholog						accttgggcct
	of Xenopus Staf,
	ZNF76 is a DNA
	binding protein
	related to
	ZNF143 and Staf
V$SF1F/SF1.01	SF1	0.95	819-831	(−)	1.000	0.966	ggccCAAGgtt	256
	steroidogenic						ag
	factor 1
V$RXRF/VDR_RX	Bipartite binding	0.75	825-	(+)	0.812	0.787	ttgggcctcagcag	257
R.06	site of		849nc				cAGGGgaggc
	VDR/RXR						c
	heterodimers: 4
	spacer
	nucleotides
	between the two
	directly repeated
	motifs
V$MYOD/MYF5.01	Myf5 myogenic	0.90	831-	(+)	1.000	0.903	ctcagCAGCag	258
	bHLH protein		847nc				gggagg
V$NRSF/NRSF.01	Neuron-	0.69	831-	(+)	1.000	0.705	ctcAGCAgcag	259
	restrictive		851nc?				gggaggccac
	silencer factor
V$ZBPF/ZF9.01	Core promoter-	0.87	832-854	(−)	0.820	0.890	ggggtggCCTC	260
	binding protein						ccctgctgctga
	(CPBP) with 3
	Krueppel-type
	zinc fingers
V$ZBPF/ZF9.01	Core promoter-	0.87	841-	(+)	0.923	0.937	ggggaggCCA	261
	binding protein		863nc				Cccctacaccttc
	(CPBP) with 3
	Krueppel-type
	zinc fingers
V$PLAG/PLAG1.01	Pleomorphic	0.88	847-867	(−)	0.958	0.929	GGGGgaaggtg	262
	adenoma gene						taggggtggc
	(PLAG) 1, a
	developmentally
	regulated C2H2
	zinc finger
	protein
V$ZBPF/ZNF202.01	Transcriptional	0.73	859-881	(+)	1.000	0.776	ccttccCCCAgc	263
	repressor, binds						cacagccatcc
	to elements
	found
	predominantly in
	genes that
	participate in
	lipid metabolism
V$INSM/INSM1.01	Zinc finger	0.90	860-872	(−)	1.000	0.965	tggctGGGGga	264
	protein						ag
	insulinoma-
	associated 1 (IA-
	1) functions as a
	transcriptional
	repressor
V$MZF1/MZF1.02	Myeloid zinc	0.99	860-868	(−)	1.000	0.994	tgGGGGaag	265
	finger protein
	MZF1
V$HAML/AML3.01	Runt-related	0.84	863-877	(−)	1.000	0.845	ggctGTGGctg	266
	transcription						gggg
	factor 2/CBFA1
	(core-binding
	factor, runt
	domain, alpha
	subunit 1)
V$NRF1/NRF1.01	Nuclear	0.78	889-	(−)	0.750	0.828	tctGCACaggc	267
	respiratory factor		905nc				gctgct
	1 (NRF1), bZIP
	transcription
	factor that acts on
	nuclear genes
	encoding
	mitochondrial
	proteins
V$NRF1/NRF1.01	Nuclear	0.78	890-	(+)	1.000	0.801	gcaGCGCctgt	268
	respiratory factor		906nc				gcagaa
	1 (NRF1), bZIP
	transcription
	factor that acts on
	nuclear genes
	encoding
	mitochondrial
	proteins
V$SORY/HBP1.01	HMG box-	0.86	898-	(+)	1.000	0.862	tgtgcagAATG	269
	containing		910nc				aa
	protein 1
V$BRNF/BRN2.03	Brn-2, POU-III	0.92	923-941	(+)	1.000	0.932	ggctccttaATT	270
	protein class						Ttttttt
V$NKXH/NKX25.0	Homeo domain	0.88	925-939	(+)	1.000	0.956	ctcctTAATttttt	271
2	factor Nkx-						t
	2.5/Csx, tinman
	homolog low
	affinity sites
V$CDXF/CDX1.01	Intestine specific	0.94	939-957	(+)	1.000	0.948	tttttttTTTAaga	272
	homeodomain						aataa
	factor CDX-1
V$HOXF/HOXB9.0	Abd-B-like	0.88	940-956	(−)	1.000	0.888	tatttctTAAAaa	273
1	homeodomain						aaaa
	protein Hoxb-9
V$CEBP/CEBPB.01	CCAAT/enhancer	0.94	943-	(+)	1.000	0.942	ttttttaaGAAAt	274
	binding protein		957nc				aa
	beta
V$HNF1/HNF1.01	Hepatic nuclear	0.80	947-963	(−)	0.790	0.824	cATTAattatttct	275
	factor 1						taa
V$HOXF/BARX2.0	Barx2,	0.95	948-964	(−)	1.000	0.967	tcatTAATtatttc	276
1	homeobox						tta
	transcription
	factor that
	preferentially
	binds to paired
	TAAT motifs
V$BRNF/BRN3.01	Brn-3, POU-IV	0.78	949-967	(−)	1.000	0.990	gcctcattaATT	277
	protein class						Atttctt
V$BRNF/BRN4.01	POU domain	0.89	949-	(+)	1.000	0.894	aagaaataatTA	278
	transcription		967nc				ATgaggc
	factor brain 4
V$LHXF/LMX1B.0	LIM-	0.91	949-	(+)	1.000	0.962	aagaaaTAATta	279
1	homeodomain		963nc				atg
	transcription
	factor
V$HOMF/S8.01	Binding site for	0.97	950-	(+)	1.000	0.997	agaaaTAATtaa	280
	S8 type		962nc				t
	homeodomains
V$HOXF/GSH1.01	Homeobox	0.85	952-	(+)	1.000	0.863	aaataatTAATg	281
	transcription		968nc				aggct
	factor Gsh-1
V$LHXF/LMX1B.0	LIM-	0.91	952-966	(−)	1.000	0.946	cctcatTAATtat	282
1	homeodomain						tt
	transcription
	factor
V$RBIT/BRIGHT.0	Bright, B cell	0.92	952-	(+)	1.000	0.961	aaataATTAatg	283
1	regulator of IgH		964nc				a
	transcription
V$HOMF/S8.01	Binding site for	0.97	953-965	(−)	1.000	0.992	ctcatTAATtatt	284
	S8 type
	homeodomains
V$LHXF/LHX3.01	Homeodomain	0.81	953-	(+)	1.000	0.851	aataaTTAAtga	285
	binding site in		967nc				ggc
	LIM/Homeodo-
	main factor LHX3
V$SORY/HBP1.01	HMG box-	0.86	953-	(+)	1.000	0.876	aataattAATGa	286
	containing		965nc				g
	protein 1
V$HOXF/BARX2.0	Barx2,	0.95	955-	(+)	1.000	0.987	taatTAATgagg	287
1	homeobox		971nc				ctcct
	transcription
	factor that
	preferentially
	binds to paired
	TAAT motifs
V$RXRF/VDR_RX	VDR/RXR	0.86	960-984	(−)	1.000	0.871	tcccaggtgagtG	288
R.02	Vitamin D						AGGagcctcatt
	receptor RXR
	heterodimer site
V$AP1F/AP1.03	Activator protein	0.94	969-979	(−)	1.000	0.940	ggTGAGtgagg	289
	1
V$AREB/AREB6.01	AREB6 (Atp1a1	0.93	972-	(+)	1.000	0.933	cactcACCTgg	290
	regulatory		984nc				ga
	element binding
	factor 6)
V$PAX6/PAX6.02	PAX6 paired	0.89	973-991	(−)	1.000	0.893	caggctgtcCCA	291
	domain and						Ggtgagt
	homeodomain
	are required for
	binding to this
	site
V$AP2F/AP2.01	Activator protein	0.90	1033-	(−)	1.000	0.911	ctgGCCTtggg	292
	2		1047				gaac
V$EREF/ERR.01	Estrogen related	0.87	1033-	(+)	1.000	0.897	gttccccAAGG	293
	receptor		1051nc				ccagcggg
V$MZF1/MZF1.02	Myeloid zinc	0.99	1033-	(−)	1.000	0.994	ttGGGGaac	294
	finger protein		1041
	MZF1
V$SF1F/SF1.01	SF1	0.95	1035-	(+)	1.000	0.992	tcccCAAGgcc	295
	steroidogenic		1047nc				ag
	factor 1
V$TEAF/TEF.01	Thyrotrophic	0.88	1044-	(−)	0.968	0.894	ggcacaCATCc	296
	embryonic factor		1060				cgctgg
V$SP1F/TIEG.01	TGFbeta-	0.83	1046-	(+)	0.750	0.878	agcGGGAtgtgt	297
	inducible early		1060nc				gcc
	gene (TIEG)/
	Early growth
	response gene
	alpha
	(EGRalpha)
V$MAZF/MAZ.01	Myc associated	0.90	1056-	(−)	1.000	0.909	ggagGAGGgg	298
	zinc finger		1068				cac
	protein (MAZ)
V$RXRF/VDR_RX	Bipartite binding	0.74	1056-	(−)	0.823	0.750	gatgAGTTggg	299
R.03	site of		1080				aggaggaggggc
	VDR/RXR						ac
	heterodimers
	without a spacer
	between directly
	repeated motifs
V$EVI1/MEL1.02	MEL1	0.99	1071-	(−)	1.000	0.997	cctgaaaGATG	300
	(MDS1/EVI1-		1087				agttgg
	like gene 1)
	DNA-binding
	domain 2
V$HEAT/HSF1.01	Heat shock factor	0.84	1073-	(−)	0.857	0.849	tcctcgtgttccTG	301
	1		1097				AAagatgagtt
V$MYT1/MYT1L.0	Myelin	0.92	1073-	(−)	0.818	0.927	tgaaAGATgag	302
1	transcription		1085				tt
	factor 1-like,
	neuronal C2HC
	zinc finger
	factor 1
V$STAT/STAT1.01	Signal transducer	0.77	1075-	(−)	0.767	0.774	cgtgttcctGAA	303
	and activator of		1093				Agatgag
	transcription 1
V$STAT/STAT.01	Signal	0.87	1077-	(+)	1.000	0.911	catctttcaGGA	304
	transducers and		1095				Acacgag
	activators of
	transcription
V$EBOX/NMYC.01	N-Myc	0.92	1085-	(−)	1.000	0.923	aatcctCGTGttc	305
			1099				ct
V$HEAT/HSF2.02	Heat shock factor	0.95	1089-	(−)	1.000	0.967	ttccagaaagcaA	306
	2		1113				GAAtcctcgtgt
V$HEAT/HSF1.01	Heat shock factor	0.84	1097-	(−)	1.000	0.874	ggacacttttccA	307
	1		1121				GAAagcaagaat
V$STAT/STAT1.01	Signal transducer	0.77	1099-	(−)	0.767	0.798	acttttccaGAA	308
	and activator of		1117				Agcaaga
	transcription 1
V$STAT/STAT.01	Signal	0.87	1101-	(+)	1.000	0.895	ttgctttctGGAA	309
	transducers and		1119				aagtgt
	activators of
	transcription
V$BCL6/BCL6.02	POZ/zinc finger	0.77	1102-	(+)	0.800	0.808	tgctttcTGGAa	310
	protein,		1118				aagtg
	transcriptional
	repressor,
	translocations
	observed in
	diffuse large cell
	lymphoma
V$BNCF/BNC.01	Basonuclin,	0.85	1107-	(+)	1.000	0.852	tctggaaaagTG	311
	cooperates with		1125				TCccagc
	USF1 in rDNA
	Po1I
	transcription)
V$GATA/GATA2.0	GATA-binding	0.92	1127-	(+)	1.000	0.938	taggGATAagt	312
1	factor 2		1139				gt
V$NKXH/NKX32.0	Homeodomain	0.96	1128-	(+)	1.000	0.962	agggataAGTG	313
1	protein NKX3.2		1142				tcta
	(BAPX1,
	NKX3B,
	Bagpipe
	homolog)
V$PAX1/PAX1.01	Pax1 paired	0.62	1135-	(−)	0.750	0.696	cCATTctgtgct	314
	domain protein,		1153				agacact
	expressed in the
	developing
	vertebral column
	of mouse
	embryos
V$SORY/HBP1.01	HMG box-	0.86	1142-	(+)	1.000	0.860	agcacagAATG	315
	containing		1154nc				gg
	protein 1
V$NKXH/NKX25.0	Homeo domain	0.88	1166-	(+)	1.000	0.898	gtgctTAATaaa	316
2	factor Nkx-		1180				tgc
	2.5/Csx, tinman
	homolog low
	affinity sites
V$HOXF/HOXC13.	Homeodomain	0.91	1167-	(+)	1.000	0.944	tgcttaaTAAAt	317
01	transcription		1183				gctgg
	factor HOXC13
V$HOXC/HOX_PB	HOX/PBX	0.81	1178-	(+)	0.944	0.862	tgctGGATggat	318
X.01	binding sites		1194				gcagg
V$AIRE/AIRE.01	Autoimmune	0.86	1184-	(+)	1.000	0.877	atggatgcaggaa	319
	regulator		1210				ggaaTGGAgga
							atg
V$ETSF/ELF2.01	Ets-family	0.90	1186-	(+)	1.000	0.933	ggatgcaGGAA	320
	member ELF-2		1202				ggaatg
	(NERF1a)
V$GKLF/GKLF.01	Gut-enriched	0.86	1191-	(+)	0.779	0.864	caggaaggaAT	321
	Krueppel-like		1203				GG
	factor
V$SORY/HBP1.01	HMG box-	0.86	1192-	(+)	1.000	0.904	aggaaggAATG	322
	containing		1204				ga
	protein 1
V$TEAF/TEF1.01	TEF-1 related	0.84	1192-	(−)	1.000	0.859	ttcctcCATTcct	323
	muscle factor		1208				tcct
V$ETSF/PU1.01	Pu.1 (Pu120) Ets-	0.89	1198-	(+)	1.000	0.899	gaatggaGGAA	324
	like transcription		1214				tgaatg
	factor identified
	in lymphoid B-
	cells
V$SORY/HBP1.01	HMG box-	0.86	1200-	(+)	1.000	0.916	atggaggAATG	325
	containing		1212				aa
	protein 1
V$TEAF/TEF1.01	TEF-1 related	0.84	1200-	(−)	1.000	0.884	cccattCATTcct	326
	muscle factor		1216				ccat
V$SORY/HBP1.01	HMG box-	0.86	1204-	(+)	1.000	0.949	aggaatgAATG	327
	containing		1216				gg
	protein 1
V$IRFF/IRF7.01	Interferon	0.86	1208-	(+)	0.936	0.885	atGAATgggaa	328
	regulatory factor		1226nc				ggtctaga
	7 (IRF-7)
V$RBPF/RBPJK.02	Mammalian	0.94	1209-	(+)	1.000	0.942	tgaaTGGGaag	329
	transcriptional		1223nc				gtct
	repressor RBP-
	Jkappa/CBF1
V$IKRS/IK1.01	Ikaros 1,	0.92	1210-	(+)	1.000	0.925	gaatGGGAagg	330
	potential		1222nc				tc
	regulator of
	lymphocyte
	differentiation
V$RORA/NBRE.01	Monomers of the	0.89	1212-	(+)	1.000	0.947	atgggAAGGtct	331
	nur subfamily of		1230nc				agagcat
	nuclear receptors
	(nur77, nurr-1,
	nor-1)
V$ZFIA/ZID.01	Zinc finger with	0.85	1225-	(−)	1.000	0.916	agGCTCcatgct	332
	interaction		1237				c
	domain
V$AIRE/AIRE.01	Autoimmune	0.86	1238-	(−)	0.916	0.863	atgtgggcgggtg	333
	regulator		1264				agcaTGGCttct
							ag
V$EGRF/WT1.01	Wilms Tumor	0.92	1246-	(−)	0.953	0.930	gtgggCGGGtg	334
	Suppressor		1262				agcatg
V$SP1F/SP1.01	Stimulating	0.88	1250-	(−)	1.000	0.907	atgtGGGCgggt	335
	protein 1,		1264				gag
	ubiquitous zinc
	finger
	transcription
	factor
V$NKXH/HMX3.02	Hmx3/Nkx5-1	0.92	1258-	(−)	1.000	0.933	ttaaTTAAatgtg	336
	homeodomain		1272				gg
	transcription
	factor
V$CREB/E4BP4.01	E4BP4, bZIP	0.80	1259-	(+)	0.758	0.801	ccacatttaaTTA	337
	domain,		1279				Acagctga
	transcriptional
	repressor
V$BRNF/BRN3.02	Brn-3, POU-IV	0.89	1260-	(−)	1.000	0.940	cagctgtTAATt	338
	protein class		1278				aaatgtg
V$LHXF/LHX3.01	Homeodomain	0.81	1260-	(+)	1.000	0.944	cacatTTAAtta	339
	binding site in		1274				aca
	LIM/Homeo-
	domain factor LHX3
V$OCT1/OCT1.05	Octamer-binding	0.89	1260-	(+)	0.900	0.942	caCATTtaattaa	340
	factor 1		1274				ca
V$HOMF/S8.01	Binding site for	0.97	1261-	(+)	1.000	0.997	acattTAATtaa	341
	S8 type		1273				c
	homeodomains
V$HOXF/PHOX2.01	Phox2a (ARIX)	0.87	1262-	(+)	1.000	0.877	cattTAATtaac	342
	and Phox2b		1278				agctg
V$NKXH/NKX25.0	Homeo domain	0.88	1262-	(−)	1.000	0.898	gctgtTAATtaa	343
2	factor Nkx-		1276				atg
	2.5/Csx, tinman
	homolog low
	affinity sites
V$PBXC/PBX1_ME	Binding site for a	0.77	1262-	(+)	0.750	0.781	cattTAATtaac	344
IS1.02	Pbx1/Meis1		1278				agctg
	heterodimer
V$RBIT/BRIGHT.0	Bright, B cell	0.92	1262-	(−)	1.000	0.967	tgttaATTAaatg	345
1	regulator of IgH		1274
	transcription
V$FAST/FAST1.01	FAST-1 SMAD	0.81	1263-	(−)	0.850	0.845	agctgttAATTa	346
	interacting		1277				aat
	protein
V$LHXF/LHX3.01	Homeodomain	0.81	1263-	(−)	1.000	0.870	agctgTTAAtta	347
	binding site in		1277				aat
	LIM/Homeo-
	domain factor
	LHX3
V$RBIT/BRIGHT.0	Bright, B cell	0.92	1263-	(+)	1.000	0.941	atttaATTAaca	348
1	regulator of IgH		1275				g
	transcription
V$ZNFP/SZF1.01	SZF1,	0.82	1263-	(−)	0.875	0.866	tcaGGGActca	349
	hematopoietic		1287				gctgttaattaaat
	progenitor-
	restricted KRAB-
	zinc finger
	protein
V$ATBF/ATBF1.01	AT-binding	0.79	1264-	(−)	1.000	0.812	ctcagctgttAAT	350
	transcription		1280				Taaa
	factor 1
V$HOMF/S8.01	Binding site for	0.97	1264-	(−)	1.000	0.997	gctgtTAATtaa	351
	S8 type		1276				a
	homeodomains
V$HEN1/HEN1.02	HEN1	0.81	1265-	(−)	1.000	0.845	agggactcaGCT	352
			1285				Gttaattaa
V$NKXH/HMX3.02	Hmx3/Nkx5-1	0.92	1265-	(+)	1.000	0.927	ttaaTTAAcagc	353
	homeodomain		1279				tga
	transcription
	factor
V$AP4R/AP4.02	Activator protein	0.92	1267-	(−)	1.000	0.950	ggactcAGCTgt	354
	4		1283				taatt
V$AP1R/NFE2.01	NF-E2 p45	0.85	1268-	(+)	1.000	0.865	attaacagCTGA	355
			1292				gtccctgatgtca
V$BEL1/BEL1.01	Bel-1 similar	0.81	1270-	(−)	1.000	0.842	tgacatcagggac	356
	region (defined		1292				TCAGctgtta
	in Lentivirus
	LTRs)
V$CREB/CREBP1.0	cAMP-	0.85	1278-	(−)	1.000	0.851	taaggaTGACat	357
1	responsive		1298				cagggactc
	element binding
	protein 1
V$CREB/ATF2.01	Activating	0.87	1279-	(+)	0.814	0.871	agtcccTGATgt	358
	transcription		1299				catccttac
	factor 2
V$E4FF/E4F.01	GLI-Krueppel-	0.82	1284-	(+)	0.842	0.824	ctgATGTcatcc	359
	related		1296				t
	transcription
	factor, regulator
	of adenovirus E4
	promoter
V$HOXF/PTX1.01	Pituitary	0.94	1299-	(−)	1.000	0.949	tttCTAAgctctt	360
	Homeobox 1		1315				cgag
	(Ptx1, Pitx-1)
V$TBPF/ATATA.01	Avian C-type	0.78	1302-	(−)	1.000	0.781	ttgtttcTAAGct	361
	LTR TATA box		1318				cttc
V$XBBF/RFX1.01	X-box binding	0.89	1302-	(+)	0.881	0.890	gaagagcttaGA	362
	protein RFX1		1320				AAcaaag
V$LEFF/LEF1.01	TCF/LEF-1,	0.86	1309-	(+)	1.000	0.884	ttagaaaCAAA	363
	involved in the		1325nc				gagtgg
	Wnt signal
	transduction
	pathway
V$RBPF/RBPJK.02	Mammalian	0.94	1319-	(+)	1.000	0.977	agagTGGGaaa	364
	transcriptional		1333 nc				tgct
	repressor RBP-
	Jkappa/CBF1
V$CP2F/CP2.01	CP2	0.90	1331-	(−)	1.000	0.932	agCTGGgtaaa	365
			1349				gctagagc
V$SRFF/SRF.02	Serum response	0.84	1362-	(+)	0.888	0.842	taaggCAAAttg	366
	factor		1380				ggccatt
V$CART/XVENT2.	Xenopus	0.82	1366-	(+)	0.750	0.882	gcAAATtgggc	367
01	homeodomain		1382				cattaa
	factor Xvent-2;
	early BMP
	signaling
	response
V$CART/XVENT2.	Xenopus	0.82	1367-	(−)	1.000	0.835	ttTAATggccca	368
01	homeodomain		1383				atttg
	factor Xvent-2;
	early BMP
	signaling
	response
V$PDX1/1SL1.01	Pancreatic and	0.82	1370-	(−)	1.000	0.875	ctgagctttTAAT	369
	intestinal lim-		1390				ggcccaat
	homeodomain
	factor
V$NKXH/HMX3.02	Hmx3/Nkx5-1	0.92	1372-	(−)	1.000	0.946	gcttTTAAtggc	370
	homeodomain		1386				cca
	transcription
	factor
V$HOXF/HOXC13.	Homeodomain	0.91	1373-	(+)	1.000	0.932	gggccatTAAA	371
01	transcription		1389				agctca
	factor HOXC13
V$NKXH/HMX3.02	Hmx3/Nkx5-1	0.92	1375-	(+)	1.000	0.953	gccaTTAAaag	372
	homeodomain		1389				ctca
	transcription
	factor
V$MYBL/VMYB.05	v-Myb, variant of	0.90	1404-	(+)	1.000	0.990	attAACGgtggt	373
	AMV v-myb		1416				g
V$AHRR/AHRARN	Aryl hydrocarbon/	0.77	1423-	(−)	0.750	0.781	cctgtggataGA	374
T.02	Arnt		1447				GTgtgaaagcaa
	heterodimers,						c
	fixed core
V$EVI1/EVI1.06	Ecotropic viral	0.83	1440-	(+)	0.750	0.835	tccacaGGATa	375
	integration site 1		1456				gattga
	encoded factor,
	amino-terminal
	zinc finger
	domain
V$HOXC/HOX_PB	HOX/PBX	0.81	1442-	(+)	0.944	0.814	cacaGGATaga	376
X.01	binding sites		1458				ttgaaa
V$HOXC/PBX1.01	Homeo domain	0.78	1446-	(+)	1.000	0.809	ggataGATTga	377
	factor Pbx-1		1462				aactgc
V$IRFF/ISRE.01	Interferon-	0.81	1447-	(+)	1.000	0.829	gatagattGAAA	378
	stimulated		1465				ctgccag
	response element
V$HOXH/MEIS1B_	Meis1b and	0.78	1450-	(−)	0.750	0.781	TGGCagtttcaat	379
HOXA9.01	Hoxa9 form		1464				ct
	heterodimeric
	binding
	complexes on
	target DNA
V$NR2F/ARP1.01	Apolipoprotein	0.82	1469-	(−)	0.857	0.897	ccagggtcaggG	380
	AI regulatory		1489				ATCaggtgg
	protein 1, NR2F2
V$MEF3/MEF3.01	MEF3 binding	0.89	1474-	(−)	1.000	0.943	gggTCAGggat	381
	site, present in		1486				ca
	skeletal muscle-
	specific
	transcriptional
	enhancers
V$RORA/TR4.01	Nuclear hormone	0.84	1474-	(−)	1.000	0.841	atcccagGGTC	382
	receptor TR4		1492				agggatca
	homodimer
	binding site
V$CSEN/DREAM.0	Downstream	0.95	1476-	(−)	1.000	0.974	ggGTCAgggat	383
1	regulatory		1486
	element-
	antagonist
	modulator,
	Ca2+-binding
	protein of the
	neuronal
	calcium sensors
	family that
	binds DRE
	(downstream
	regulatory
	element) sites as
	a tetramer
V$CP2F/CP2.01	CP2	0.90	1493-	(+)	1.000	0.969	ggCTGGattga	384
			1511				gcaatgag
V$HOXC/PBX1.01	Homeo domain	0.78	1493-	(+)	1.000	0.811	ggctgGATTga	385
	factor Pbx-1		1509				gcaatg
V$CEBP/CEBPB.01	CCAAT/enhancer	0.94	1496-	(+)	1.000	0.984	tggattgaGCAA	386
	binding protein		1510				tga
	beta
V$CAAT/NFY.03	Nuclear factor Y	0.81	1513-	(+)	1.000	0.873	agagCCAAgca	387
	(Y-box binding		1527				gcac
	factor)
V$STAF/ZNF76_14	ZNF143 is the	0.76	1522-	(−)	1.000	0.765	tagcCCCAggg	388
3.01	human ortholog		1544				gactctgtgctg
	of Xenopus Staf,
	ZNF76 is a DNA
	binding protein
	related to
	ZNF143 and Staf
V$NOLF/OLF1.01	Olfactory	0.82	1526-	(+)	1.000	0.879	acagagTCCCct	389
	neuron-specific		1548				ggggctagagg
	factor
V$AP2F/AP2.02	Activator protein	0.92	1531-	(−)	0.905	0.941	ctaGCCCcagg	390
	2 alpha		1545				ggac
V$ZBPF/ZNF202.01	Transcriptional	0.73	1536-	(−)	0.761	0.739	gcctccTCCAcc	391
	repressor, binds		1558				tctagccccag
	to elements
	found
	predominantly in
	genes that
	participate in
	lipid metabolism
V$IKRS/IK1.01	Ikaros 1,	0.92	1561-	(+)	1.000	0.933	tcctGGGAatgg	392
	potential		1573				g
	regulator of
	lymphocyte
	differentiation
V$TEAF/TEF1.01	TEF-1 related	0.84	1561-	(−)	1.000	0.855	ttttccCATTccc	393
	muscle factor		1577				agga
V$IRFF/IRF7.01	Interferon	0.86	1565-	(+)	0.936	0.895	ggGAATggga	394
	regulatory factor		1583nc				aaaacccca
	7 (IRF-7)
V$LTUP/TAACC.01	Lentiviral TATA	0.71	1565-	(+)	1.000	0.721	gggaatgggaaa	395
	upstream element		1587nc				AACCccaactt
V$RBPF/RBPJK.02	Mammalian	0.94	1566-	(+)	1.000	0.947	ggaaTGGGaaa	396
	transcriptional		1580nc				aacc
	repressor RBP-
	Jkappa/CBF1
V$IKRS/IK1.01	Ikaros 1,	0.92	1567-	(+)	1.000	0.927	gaatGGGAaaa	397
	potential		1579nc				ac
	regulator of
	lymphocyte
	differentiation
V$NFKB/CREL.01	c-Rel	0.91	1571-	(−)	1.000	0.971	tggggtttTTCCc	398
			1583
V$CIZF/NMP4.01	NMP4 (nuclear	0.97	1572-	(+)	1.000	0.986	ggAAAAacccc	399
	matrix protein 4)/		1582nc
	CIZ (Cas-
	interacting zinc
	finger protein)
V$SRFF/SRF.02	Serum response	0.84	1576-	(−)	0.888	0.881	gacccCAAAgt	400
	factor		1594				tggggttt
V$MYT1/MYT1.02	MyT1 zinc	0.88	1578-	(−)	1.000	0.882	ccaAAGTtggg	401
	finger		1590				gt
	transcription
	factor involved
	in primary
	neurogenesis
Cartharius K, et al. (2005) Bioinformatics 21, 2933-42.