Methods for treating hypercholesterolemia

[0001] This application claims the benefit of U.S. Provisional Application No. 61/059,169, filed Jun. 5, 2008, and U.S. Provisional Application No. 61/058,707, filed Jun. 4, 2008, which are herein incorporated by reference in their entirety. This application is a continuation-in-part of U.S. application Ser. No. 12/516,457, filed Jan. 20, 2010, which is a National Stage application of International PCT Application No. PCT/US07/024369, now WO 2008/066776, filed Nov. 27, 2007, which claims the benefit of U.S. Provisional Application No. 60/988,074, filed Nov. 14, 2007, U.S. Provisional Application No. 60/986,286, filed Nov. 7, 2007, U.S. Provisional Application No. 60/912,892, filed Apr. 19, 2007, and U.S. Provisional Application No. 60/867,395, filed Nov. 27, 2006, which all are herein incorporated by reference in their entirety.

[0002] Methods are provided for lowering LDL-C levels in an individual having elevated LDL-C levels. Such methods are further useful to treat hypercholesterolemia, and to reduce the risk of coronary heart disease.

[0003] The Sequence Listing attached below is herein incorporated by reference in its entirety.

[0004] Atherosclerosis is a complex, polygenic disease of arterial degeneration that can lead to coronary heart disease (CHD). In Western societies, complications arising from atherosclerosis are the most common causes of death. Several risk factors for CHD have been well-established, and include elevated low density lipoprotein-cholesterol (LDL-C), low levels of high density lipoprotein cholesterol (HDL-C), cigarette smoking, hypertension, age, and family history of early CHD. Given the abundant clinical data and epidemiological studies indicating that lowering LDL-C is beneficial for the prevention of adverse coronary events, the primary target of pharmacological intervention in the treatment and prevention of CHD is lowered LDL-C.

[0005] In individuals with autosomal dominant hypercholesterolemia (ADH), elevated LDL-C levels have been linked to mutations in the genes encoding LDL-receptor (LDL-R), apolipoprotein B (apoB), or proprotein convertase subtilisin/kexin type 9 (PCSK9) (Abifadel et al., Nat. Genet., 2003, 34:154-156). The PCSK9 gene (also known as FH3; NARC1; NARC-1; and HCHOLA3) was mapped to Chromosome 1 at location 1p32.3. PCSK9 was identified as a third locus associated with ADH when gain-of-function mutations in PCSK9 were found to be linked to elevated LDL-C levels. ApoB-100 participates in the intracellular assembly and secretion of triglyceride-rich lipoproteins and is a ligand for the LDL-R. PCSK9 is proposed to reduce LDL-R expression levels in the liver. Reduced LDL-R expression results in reduced hepatic uptake of circulating apoB-100-containing lipoproteins, which in turn leads to elevated cholesterol.

[0006] Familial hypercholesterolemia (FH) is caused by hundreds of different mutations in the LDL-R, and is phenotypically characterized by elevated plasma LDL-C levels and deposits of LDL-C in tendons, skin and arteries, leading to premature cardiovascular disease. Homozygous and heterozygous mutations in the LDL-R are associated with FH. Likewise, heterozygous and homozygous mutations in the ligand-binding domain of PCSK9 are associated with the FH phenotype. Mutations in this gene have been associated with a third form of autosomal dominant FH.

[0007] In mice genetically deficient in PCSK9, a marked increase in LDL-R expression and increased plasma LDL clearance rate were observed. Conversely, overexpression in mice of wild-type PCSK9, or certain PCSK9 mutants, promotes decreased LDL-R expression and elevated LDL-C levels.

[0008] Provided herein are methods for the treatment of hypercholesterolemia and/or atherosclerosis, as well as methods for the reduction of elevated LDL-C levels and CHD risk. Methods for treating hypercholesterolemia comprise administering to an individual an antisense compound targeted to a PCSK9 nucleic acid. In such methods, an antisense compound targeted to a PCSK9 nucleic acid may be targeted to sequences as set forth in GENBANK® Accession No. NM_—174936.2, nucleotides 25475000 to 25504000 of GENBANK® Accession No. NT_—032977.8, or GENBANK® Accession No. AK124635.1.

[0009] Methods for treating and/or preventing atherosclerosis comprise administering to an individual an antisense compound targeted to a PCSK9 nucleic acid. Methods for reducing LDL-C levels comprise administering to an individual an antisense compound targeted to a PCSK9 nucleic acid. Methods for reducing CHD risk comprise administering to an individual an antisense compound targeted to a PCSK9 nucleic acid. Such methods may comprise the administration of a therapeutically effective amount of an antisense compound targeted to a PCSK9 nucleic acid.

[0010] Methods are provided for reducing LDL-C levels in an individual having elevated LDL-C levels, comprising administering to the individual a therapeutically effective amount of an antisense oligonucleotide targeted to a PCSK9 nucleic acid, thereby reducing LDL-C levels. Also provided are methods for reducing LDL-C levels in an individual comprising selecting an individual having elevated LDL-C levels, and administering to the individual a therapeutically effective amount of an antisense compound targeted to a PCSK9 nucleic acid, and additionally monitoring LDL-cholesterol levels. Further provided are methods for treating atherosclerosis in an individual, comprising selecting an individual diagnosed with atherosclerosis, administering to the individual a therapeutically effective amount of an antisense compound targeted to a PCSK9 nucleic acid, and monitoring atherosclerosis. Also provided are methods for reducing coronary heart disease risk, comprising selecting an individual having elevated LDL-C levels and one or more additional indicators of coronary heart disease, administering to the individual a therapeutically effective amount of an antisense compound targeted to a PCSK9 nucleic acid, and monitoring LDL-C levels. Additionally provided are methods for treating hypercholesterolemia, comprising administering to an individual diagnosed with hypercholesterolemia a therapeutically effective amount of an antisense oligonucleotide targeted to a PCSK9 nucleic acid, thereby reducing cholesterol levels.

[0011] Further provided is a use of an effective amount of one or more of the disclosed antisense compounds for treating atherosclerosis or hypercholesterolemia in an individual in need of such treatment. Also provided is a use of an effective amount of one or more of the disclosed antisense compounds for reducing LDL-C levels or reducing CHD risk in an individual in need of such treatment. Also provided is the use of one or more of the disclosed antisense compounds in the manufacture of a medicament for the treatment of atherosclerosis or hypercholesterolemia. Further provided is the use of one or more of the disclosed antisense compounds in the manufacture of a medicament for reducing LDL-C levels or for reducing CHD risk.

[0012] In any of the methods provided, a PCSK9 nucleic acid may be the sequence set forth in SEQ ID NO: 1. Thus, the antisense compound may be targeted to a PCSK9 nucleic acid as set forth in SEQ ID NO: 1.

[0013] Any of the methods provided herein may further comprise monitoring LDL-C levels.

[0014] An individual may be selected for administration of an antisense compound targeted to a PCSK9 nucleic acid when the individual exhibits an LDL-C level above 100 mg/dL, above 130 mg/dL, above 160 mg/dL, or above 190 mg/dL. Administration of an antisense compound targeted to a PCSK9 nucleic acid may result in an LDL-C level below 190 mg/dL, below 160 mg/dL, below 130 mg/dL, below 100 mg/dL, below 70 mg/dL, or below 50 mg/dL.

[0015] In any of the aforementioned methods, administration of the antisense compound may comprise parenteral administration. The parenteral administration may further comprise subcutaneous or intravenous administration.

[0016] In any of the methods provided herein, the antisense compound may have least 80%, at least 90%, or at least 95% complementarity to SEQ ID NO: 1. Alternatively, the antisense compound may have 100% complementarity to SEQ ID NO: 1.

[0017] The antisense compounds provided herein and employed in any of the described methods may be 8 to 80 subunits in length, 12 to 50 subunits in length, 12 to 30 subunits in length, 15 to 30 subunits in length, 18 to 24 subunits in length, 19 to 22 subunits in length, or 20 subunits in length. Further, the antisense compounds employed in any of the described methods may be antisense oligonucleotides 8 to 80 nucleotides in length, 12 to 50 nucleotides in length, 12 to 30 nucleotides in length 15 to 30 nucleotides in length, 18 to 24 nucleotides in length, 19 to 22 nucleotides in length, or 20 nucleotides in length.

[0018] In any of the methods provided, the antisense compound may be an antisense oligonucleotide. Moreover, the antisense oligonucleotide may be a gapmer antisense oligonucleotide. The gapmer antisense oligonucleotide may comprise a gap segment of ten 2′-deoxynucleotides positioned between wing segments of five 2′-MOE nucleotides. The gapmer antisense oligonucleotide may be a gap-widened antisense oligonucleotide, comprising a gap segment of fourteen 2′-deoxynucleotides positioned between wing segments of three 2′-MOE nucleotides.

[0019] In any of the methods provided, the antisense compounds may have at least one modified internucleoside linkage. Additionally, each internucleoside linkage may be a phosphorothioate internucleoside linkage. Each cytosine may be a 5-methyl cytosine.

[0020] Also provided herein are antisense compounds targeted to a PCSK9 nucleic acid. Further provided are antisense oligonucleotides targeted to a PCSK9 nucleic acid. The antisense compounds, including antisense oligonucleotides, may be targeted to PCSK9 nucleic acids, which include the sequences as set forth in GENBANK® Accession No. NM_—174936.2, nucleotides 25475000 to 25504000 of GENBANK® Accession No. NT_—032977.8, or GENBANK® Accession No. AK124635.1. The antisense compounds, including antisense oligonucleotides, may have at least 70%, at least 80%, at least 90%, or at least 95% complementarity to a PCSK9 nucleic acid. The antisense compounds, including antisense oligonucleotides, may have 99% complementarity to a PCSK9 nucleic acid. The antisense compounds, including antisense oligonucleotides, may have 100% complementarity to a PCSK9 nucleic acid. For any of the antisense compounds provided, including antisense oligonucleotides, the PCSK9 nucleic acid may be the sequence set forth in SEQ ID NO: 1.

[0021] Antisense compounds targeted to a PCSK9 nucleic acid may be 8 to 80 subunits in length, 12 to 50 subunits in length, 12 to 30 subunits in length, 15 to 30 subunits in length, 18 to 24 subunits in length, 19 to 22 subunits in length, or 20 subunits in length. Antisense oligonucleotides targeted to a PCSK9 nucleic acid may be 8 to 80 nucleotides in length, 12 to 50 nucleotides in length, 12 to 30 nucleotides in length, 15 to 30 nucleotides in length, 18 to 24 nucleotides in length, 19 to 22 nucleotides in length, or 20 nucleotides in length.

[0022] Moreover, the antisense compound may be a gapmer antisense oligonucleotide. The gapmer antisense oligonucleotide may comprise a gap segment of ten 2′-deoxynucleotides positioned between wing segments of five 2′-MOE nucleotides. The gapmer antisense oligonucleotide may be a gap-widened antisense oligonucleotide, comprising a gap segment of fourteen 2′-deoxynucleotides positioned between wing segments of three 2′-MOE nucleotides.

[0023] Antisense compounds, including antisense oligonucleotides, targeted to a PCSK9 nucleic acid may have at least one modified internucleoside linkage. Additionally, each internucleoside linkage may be a phosphorothioate internucleoside linkage. Each cytosine may be a 5-methyl cytosine.

[0024] Antisense compounds, including antisense oligonucleotides, targeted to a PCSK9 nucleic acid may have at least one modified nucleoside. In certain embodiments, a modified nucleoside is a sugar-modified nucleoside. In certain such embodiments, the sugar-modified nucleosides can further comprise a natural or modified heterocyclic base moiety and/or a natural or modified internucleoside linkage and may include further modifications independent from the sugar modification. In certain embodiments, a sugar modified nucleoside is a 2′-modified nucleoside, wherein the sugar ring is modified at the 2′ carbon from natural ribose or 2′-deoxy-ribose.

[0025] In certain embodiments, a 2′ modified nucleoside has a 2′-F, 2′-OCH₂ (2′-OMe) or a 2′-O(CH₂)₂—OCH₃ (2′-O-methoxyethyl or 2′-MOE) substituent group

[0026] In certain embodiments, a 2′-modified nucleoside has a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety is a D sugar in the alpha configuration. In certain such embodiments, the bicyclic sugar moiety is a D sugar in the beta configuration. In certain such embodiments, the bicyclic sugar moiety is an L sugar in the alpha configuration. In certain such embodiments, the bicyclic sugar moiety is an L sugar in the beta configuration.

[0027] In certain embodiments, the bicyclic sugar moiety comprises a bridge group between the 2′ and the 4′-carbon atoms. In certain such embodiments, the bridge group comprises from 1 to 8 linked biradical groups. In certain embodiments, the bicyclic sugar moiety comprises from 1 to 4 linked biradical groups. In certain embodiments, the bicyclic sugar moiety comprises 2 or 3 linked biradical groups. In certain embodiments, the bicyclic sugar moiety comprises 2 linked biradical groups. In certain embodiments, a linked biradical group is selected from —O—, —S—, —N(R1)-, —C(R1)(R2)-, —C(R1)=C(R1)-, —C(R1)=N—, —C(═NR1)-, —Si(R1)(R2)-, —S(═O)2-, —S(═O)—, —C(═O)— and —C(═S)—; where each R1 and R2 is, independently, H, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, a heterocycle radical, a substituted hetero-cycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, substituted oxy (—O—), amino, substituted amino, azido, carboxyl, substituted carboxyl, acyl, substituted acyl, CN, thiol, substituted thiol, sulfonyl (S(═O)2-H), substituted sulfonyl, sulfoxyl (S(═O)—H) or substituted sulfoxyl; and each substituent group is, independently, halogen, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, amino, substituted amino, acyl, substituted acyl, C1-C12 aminoalkyl, C1-C12 aminoalkoxy, substituted C1-C12 aminoalkyl, substituted C1-C12 aminoalkoxy or a protecting group.

[0028] In some embodiments, the bicyclic sugar moiety is bridged between the 2′ and 4′ carbon atoms with a biradical group selected from —O—(CH2)p-, —O—CH2-, —O—CH2CH2-, —O—CH(alkyl)-, —NH—(CH2)p-, —N(alkyl)-(CH2)p-, —O—CH(alkyl)-, —(CH(alkyl))-(CH2)p-, —NH—O—(CH2)p-, —N(alkyl)-O—(CH2)p-, or —O—N(alkyl)-(CH2)p-, wherein p is 1, 2, 3, 4 or 5 and each alkyl group can be further substituted. In certain embodiments, p is 1, 2 or 3.

[0029] In one aspect, each of said bridges is, independently, —[C(R1)(R2)]n-, —[C(R1)(R2)]n-O—, —C(R1R2)-N(R1)-O— or —C(R1R2)-O—N(R1)-. In another aspect, each of said bridges is, independently, 4′-(CH2)3-2′, 4′-(CH2)2-2′, 4′-CH2-O-2′, 4′-(CH2)2-O-2′, 4′-CH2-O—N(R1)-2′ and 4′-CH2-N(R1)-O-2′- wherein each R1 is, independently, H, a protecting group or C1-C12 alkyl.

[0030] The present disclosure further discloses subsets of the antisense compounds above that show superior properties relating to pharmacodynamics and/or pharmacokinetics, among other properties. Exemplary antisense compounds reduce PCSK9 expression in a cultured cell model system, e.g., Hep3B cells, cynomolgus monkey hepatocytes, or human primary hepatocytes. In particular embodiments, antisense compounds reduce PCSK9 expression in a dose-dependent manner in a cell culture system, where the antisense compounds have an IC₅₀ in the nanomolar range.

[0031] Exemplary antisense compounds also reduce the expression of a PCSK9 nucleic acid in an animal model, such as a transgenic mouse that expresses a human PCSK9 nucleic acid (HuPCSK9Tg mice). In particular embodiments, antisense compounds include those that reduce PCSK9 express in an animal model with a physiology that correlates closely with humans, such as monkey, e.g., the cynomolgus monkey.

[0032] Exemplary antisense compounds display minimal side effects. Side effects include responses to the administration of the antisense compound unrelated to the targeting of a PCSK9 nucleic acid, such as an inflammatory response in the host individual. Exemplary antisense compounds are well tolerated by the host individual. Tolerability may be determined though histological analysis of various cell types, and includes examination of such markers as cytoplasmic swelling from multifocal apoptosis in liver cells, follicular hyperplasia in spleen cells, macrophage infiltration, and the like. In particular embodiments, antisense compounds produce minimal signs of host intolerance.

[0033] Exemplary antisense compounds further display favorable pharmacokinetics. In particular embodiments, antisense compounds accumulate at a relatively high ratio in the liver versus other sensitive organs, such as kidneys. In particular embodiments, antisense compounds exhibit relatively high half-lives in relevant biological fluids or tissues, e.g., liver tissue, reflecting higher stability and resistance to nucleases, for example.

[0034] In particular embodiments, antisense compounds target identical sequences in human and animal PCSK9 sequences. In other embodiments, antisense compounds target PCSK9 sequences without a known single nucleotide polymorphism (SNP), have a high relative G-content, have minimal secondary structure, have greatly reduced effects on the host's serum chemistry, body weight, or histopathology, compared to other antisense compounds, and/or induce a minimal adverse immunohistocompatibility reaction.

[0035] In a particular embodiment, the antisense compound is selected from the group of Isis 405881, Isis 399819, Isis 395165, Isis 405879, Isis 406008, Isis 405891, Isis 395186, Isis 405988, Isis 405994, Isis 406023, Isis 395187, Isis 395185, Isis 406033, Isis 405923, Isis 399900, Isis 405995, Isis 405991, Isis 406005, Isis 399793, and Isis 395152. The aforementioned antisense compounds effectively repress PCSK9 expression in a Hep3B cell culture system. In another embodiment, antisense compounds effectively inhibit PCSK9 expression with a low IC₅₀ in a battery of cell culture systems. Exemplary antisense compounds include those selected from the group of Isis 395165, Isis 395185, Isis 395186, Isis 395187, Isis 405879, Isis 405881, Isis 405891, Isis 405988, Isis 405994, and Isis 406008. In yet another embodiment, antisense compounds exhibit minimal or no adverse histopathological effects when administered at particularly high doses to an individual. Exemplary antisense compounds include those selected from the group consisting of Isis 395185, Isis 395186, Isis 395187, Isis 405879, Isis 405891, and Isis 405988. In yet another embodiment, antisense compounds affect therapeutic end-points, e.g., reduction of plasma LDL-C, liver TG, or liver PCSK9 mRNA, in an animal model. Exemplary antisense compounds include Isis 405879. In yet another embodiment, antisense compounds display combinations of the characteristics above and reduce liver PCSK9 mRNA expression in an animal model with high efficiency, e.g., Isis 405879.

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by on of skill in the art to which the invention(s) belong. Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of subjects. Certain such techniques and procedures may be found for example in “Antisense Drug Technology: Principles, Strategies, and Applications.” by Stanley Crooke, Boca Raton: Taylor & Francis Group, 2008; “Carbohydrate Modifications in Antisense Research” Edited by Sangvi and Cook, American Chemical Society, Washington D.C., 1994; and “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 18th edition, 1990; and which is hereby incorporated by reference for any purpose. Where permitted, all patents, patent applications, published applications and publications, GENBANK sequences, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. All of the GENBANK® Accession Nos. along with their associated sequence and structural data pertaining to such sequences including gene organization and structural elements and SNP information that may be found in sequence databases such as the National Center for Biotechnology Information (NCBI) are incorporated herein by reference in their entirety. In the event that there is a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0037] A “pharmaceutical composition” means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition may comprise an antisense oligonucleotide and a sterile aqueous solution.

[0038] “Administering” means providing a pharmaceutical agent to an individual, and includes, but is not limited to administering by a medical professional and self-administering.

[0039] “Individual” means a human or non-human animal selected for treatment or therapy.

[0040] “Duration” means the period of time during which an activity or event continues. In certain embodiments, the duration of treatment is the period of time during which doses of a pharmaceutical agent are administered.

[0041] “Parenteral administration” means administration through injection or infusion. Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, or intramuscular administration.

[0042] “Subcutaneous administration” means administration just below the skin. “Intravenous administration” means administration into a vein.

[0043] “Dose” means a specified quantity of a pharmaceutical agent provided in a single administration. In certain embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments, where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose may be administered in two or more injections to minimize injection site reaction in a individual.

[0044] “Dosage unit” means a form in which a pharmaceutical agent is provided. In certain embodiments, a dosage unit is a vial containing lyophilized antisense oligonucleotide. In certain embodiments, a dosage unit is a vial containing reconstituted antisense oligonucleotide.

[0045] “Pharmaceutical agent” means a substance provides a therapeutic benefit when administered to a individual. For example, in certain embodiments, an antisense oligonucleotide targeted to PCSK9 is pharmaceutical agent.

[0046] “Diluent” means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable. For example, in drugs that are injected the diluent may be a liquid, e.g., saline solution.

[0047] “Active pharmaceutical ingredient” means the substance in a pharmaceutical composition that provides a desired effect.

[0048] “Pharmaceutically acceptable carrier” means a medium or diluent that does not interfere with the structure of the oligonucleotide. Certain of such carriers enable pharmaceutical compositions to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspension and lozenges for the oral ingestion by a subject.

[0049] “Therapeutically effective amount” means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual. In certain embodiments, a therapeutically effective amount of antisense compound targeted to a PCSK9 nucleic acid is an amount that decreases LDL-C in the individual.

[0050] “Hypercholesterolemia” means a condition characterized by elevated serum cholesterol.

[0051] “Hyperlipidemia” means a condition characterized by elevated serum lipids.

[0052] “Hypertriglyceridemia” means a condition characterized by elevated serum triglyceride levels.

[0053] “Non-familial hypercholesterolemia” means a condition characterized by elevated serum cholesterol that is not the result of a single gene mutation.

[0054] “Polygenic hypercholesterolemia” means a condition characterized by elevated cholesterol that results from the influence of a variety of genetic factors. In certain embodiments, polygenic hypercholesterolemia may be exacerbated by dietary intake of lipids.

[0055] “Familial hypercholesterolemia (FH)” means an autosomal dominant metabolic disorder characterized by a mutation in the LDL-receptor (LDL-R) gene, markedly elevated LDL-C and premature onset of atherosclerosis. A diagnosis of familial hypercholesterolemia is made when an individual meets one or more of the following criteria: genetic testing confirming 2 mutated LDL-receptor genes; genetic testing confirming one mutated LDL-receptor gene; document history of untreated serum LDL-cholesterol greater than 500 mg/dL; tendinous and/or cutaneous xanthoma prior to age 10 years; or, both parents have documented elevated serum LDL-cholesterol prior to lipid-lowering therapy consistent with heterozygous familial hypercholesterolemia.

[0056] “Homozygous familial hypercholesterolemia” or “HoFH” means a condition characterized by a mutation in both maternal and paternal LDL-R genes.

[0057] “Heterozygous familial hypercholesterolemia” or “HeFH” means a condition characterized by a mutation in either the maternal or paternal LDL-R gene.

[0058] “Mixed dyslipidemia” means a condition characterized by elevated serum cholesterol and elevated serum triglycerides.

[0059] “Diabetic dyslipidemia” or “Type II diabetes with dyslipidemia” means a condition characterized by Type II diabetes, reduced HDL-C, elevated serum triglycerides, and elevated small, dense LDL particles.

[0060] “CHD risk equivalents,” means indicators of clinical atherosclerotic disease that confer a high risk for coronary heart disease, and include clinical coronary heart disease, symptomatic carotid artery disease, peripheral arterial disease, and/or abdominal aortic aneurysm.

[0061] “Metabolic syndrome” means a condition characterized by a clustering of lipid and non-lipid cardiovascular risk factors of metabolic origin. In certain embodiments, metabolic syndrome is identified by the presence of any 3 of the following factors: waist circumference of greater than 102 cm in men or greater than 88 cm in women; serum triglyceride of at least 150 mg/dL; HDL-C less than 40 mg/dL in men or less than 50 mg/dL in women; blood pressure of at least 130/85 mmHg; and fasting glucose of at least 110 mg/dL.

[0062] “Non-alcoholic fatty liver disease (NAFLD)” means a condition characterized by fatty inflammation of the liver that is not due to excessive alcohol use (for example, alcohol consumption of over 20 g/day). In certain embodiments, NAFLD is related to insulin resistance and the metabolic syndrome.

[0063] “Non-alcoholic steatohepatitis (NASH)” means a condition characterized by inflammation and the accumulation of fat and fibrous tissue in the liver, that is not due to excessive alcohol use. NASH is an extreme form of NAFLD.

[0064] “Major risk factors” mean factors that contribute to a high risk for coronary heart disease, and include without limitation cigarette smoking, hypertension, low HDL-C, family history of coronary heart disease, and age.

[0065] “CHD risk factors” mean CHD risk equivalents and major risk factors.

[0066] “Coronary heart disease (CHD)” means a narrowing of the small blood vessels that supply blood and oxygen to the heart, which is often a result of atherosclerosis.

[0067] “Reduced coronary heart disease risk” means a reduction in the likelihood that an individual will develop coronary heart disease. In certain embodiments, a reduction in coronary heart disease risk is measured by an improvement in one or more CHD risk factors, for example, a decrease in LDL-C levels.

[0068] “Atherosclerosis” means a hardening of the arteries affecting large and medium-sized arteries and is characterized by the presence of fatty deposits. The fatty deposits are called “atheromas” or “plaques,” which consist mainly of cholesterol and other fats, calcium and scar tissue, and damage the lining of arteries.

[0069] “History of coronary heart disease” means the occurrence of clinically evident coronary heart disease in the medical history of an individual or a individual's family member.

[0070] “Early onset coronary heart disease” means a diagnosis of coronary heart disease prior to age 50.

[0071] “Statin intolerant individual” means an individual who as a result of statin therapy experiences one or more of creatine kinase increases, liver function test abnormalities, muscle aches, or central nervous system side effects.

[0072] “Efficacy” means the ability to produce a desired effect. For example, efficacy of a lipid-lowering therapy may be reduction in the concentration of one or more of LDL-C, VLDL-C, IDL-C, non-HDL-C, ApoB, lipoprotein(a), or triglycerides.

[0073] “Acceptable safety profile” means a pattern of side effects that is within clinically acceptable limits.

[0074] “Side effects” means physiological responses attributable to a treatment other than desired effects. In certain embodiments, side effects include, without limitation, injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, and myopathies. For example, increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality. For example, increased bilirubin may indicate liver toxicity or liver function abnormality.

[0075] “Injection site reaction” means inflammation or abnormal redness of skin at a site of injection in an individual.

[0076] “Individual compliance” means adherence to a recommended or prescribed therapy by an individual.

[0077] “Lipid-lowering therapy” means a therapeutic regimen provided to an individual to reduce one or more lipids in a individual. In certain embodiments, a lipid-lowering therapy is provide to reduce one or more of ApoB, total cholesterol, LDL-C, VLDL-C, IDL-C, non-HDL-C, triglycerides, small dense LDL particles, and Lp(a) in a individual.

[0078] “Lipid-lowering agent” means a pharmaceutical agent provided to an individual to achieve a lowering of lipids in the individual. For example, in certain embodiments, a lipid-lowering agent is provided to an individual to reduce one or more of ApoB, LDL-C, total cholesterol, and triglycerides.

[0079] “LDL-C target” means an LDL-C level that is desired following lipid-lowering therapy.

[0080] “Comply” means the adherence with a recommended therapy by a individual.

[0081] “Recommended therapy” means a therapeutic regimen recommended by a medical professional for the treatment, amelioration, or prevention of a disease.

[0082] “Low LDL-receptor activity” means LDL-receptor activity that is not sufficiently high to maintain clinically acceptable levels of LDL-C in the bloodstream.

[0083] “Cardiovascular outcome” means the occurrence of major adverse cardiovascular events.

[0084] “Improved cardiovascular outcome” means a reduction in the occurrence of major adverse cardiovascular events, or the risk thereof. Examples of major adverse cardiovascular events include, without limitation, death, reinfarction, stroke, cardiogenic shock, pulmonary edema, cardiac arrest, and atrial dysrhythmia.

[0085] “Surrogate markers of cardiovascular outcome” means indirect indicators of cardiovascular events, or the risk thereof. For example, surrogate markers of cardiovascular outcome include carotid intimal media thickness (CIMT). Another example of a surrogate marker of cardiovascular outcome includes atheroma size. Atheroma size may be determined by intravascular ultrasound (IVUS). Surrogate markers also include increased HDL-cholesterol, or any combination of the markers above.

[0086] “Increased HDL-C” means an increase in serum HDL-C in an individual over time.

[0087] “Lipid-lowering” means a reduction in one or more serum lipids in an individual over time.

[0088] “Co-administration” means administration of two or more pharmaceutical agents to a individual. The two or more pharmaceutical agents may be in a single pharmaceutical composition, or may be in separate pharmaceutical compositions. Each of the two or more pharmaceutical agents may be administered through the same or different routes of administration. Co-administration encompasses administration in parallel or sequentially.

[0089] “Administered concomitantly” refers to the administration of two agents at the same therapeutic time frame, in any manner in which the pharmacological effects of both are manifest in the patient at the same time. Concomitant administration does not require that both agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration.

[0090] “Therapeutic lifestyle change” means dietary and lifestyle changes intended to lower cholesterol and reduce the risk of developing heart disease, and includes recommendations for dietary intake of total daily calories, total fat, saturated fat, polyunsaturated fat, monounsaturated fat, carbohydrate, protein, cholesterol, insoluble fiber, as well as recommendations for physical activity.

[0091] “Statin” means a pharmaceutical agent that inhibits the activity of HMG-CoA reductase.

[0092] “HMG-CoA reductase inhibitor” means a pharmaceutical agent that acts through the inhibition of the enzyme HMG-CoA reductase.

[0093] “Cholesterol absorption inhibitor” means a pharmaceutical agent that inhibits the absorption of exogenous cholesterol obtained from diet.

[0094] “LDL apheresis” means a form of apheresis by which LDL-C is removed from blood. Typically, a individual's blood is removed from a vein, and separated into red cells and plasma. LDL-C is filtered out of the plasma prior to return of the plasma and red blood cells to the individual.

[0095] “MTP inhibitor” means a pharmaceutical agent that inhibits the enzyme microsomal triglyceride transfer protein.

[0096] “Low density lipoprotein-cholesterol (LDL-C)” means cholesterol carried in low density lipoprotein particles. Concentration of LDL-C in serum (or plasma) is typically quantified in mg/dL or nmol/L. “Serum LDL-C” and “plasma LDL-C” mean LDL-C in the serum and plasma, respectively.

[0097] “Very low density lipoprotein-cholesterol (VLDL-C)” means cholesterol associated with very low density lipoprotein particles. Concentration of VLDL-C in serum (or plasma) is typically quantified in mg/dL or nmol/L. “Serum VLDL-C” and “plasma VLDL-C” mean VLDL-C in the serum or plasma, respectively.

[0098] “Intermediate low density lipoprotein-cholesterol (IDL-C)” means cholesterol associated with intermediate density lipoprotein. Concentration of IDL-C in serum (or plasma) is typically quantified in mg/dL or nmol/L. “Serum IDL-C” and “plasma IDL-C” mean IDL-C in the serum or plasma, respectively.

[0099] “Non-high density lipoprotein-cholesterol (Non-HDL-C)” means cholesterol associated with lipoproteins other than high density lipoproteins, and includes, without limitation, LDL-C, VLDL-C, and IDL-C.

[0100] “High density lipoprotein-C (HDL-C)” means cholesterol associated with high density lipoprotein particles. Concentration of HDL-C in serum (or plasma) is typically quantified in mg/dL or nmol/L. “Serum HDL-C” and “plasma HDL-C” mean HDL-C in the serum and plasma, respectively.

[0101] “Total cholesterol” means all types of cholesterol, including, but not limited to, LDL-C, HDL-C, IDL-C and VLDL-C. Concentration of total cholesterol in serum (or plasma) is typically quantified in mg/dL or nmol/L.

[0102] “Lipoprotein(a)” or “Lp(a)” means a lipoprotein particle that is comprised of LDL-C, an apolipoprotein(a) particle, and an apolipoproteinB-100 particle.

[0103] “ApoA1” means apolipoprotein-A1 protein in serum. Concentration of ApoA1 in serum is typically quantified in mg/dL or nmol/L.

[0104] “ApoB:ApoA1 ratio” means the ratio of ApoB concentration to ApoA1 concentration.

[0105] “ApoB-containing lipoprotein” means any lipoprotein that has apolipoprotein B as its protein component, and is understood to include LDL, VLDL, IDL, and lipoprotein(a).

[0106] “Small LDL particle” means a subclass of LDL particles characterized by a smaller, denser size compared to other LDL particles. In certain embodiments, large LDL particles are 23-27 nm in diameter. In certain embodiments, intermediate LDL particles are 21.2-23 nm in diameter. In certain embodiments, small LDL particles are 18-21.2 nm in diameter. In certain embodiments, particle size is measured by nuclear magnetic resonance analysis.

[0107] “Small VLDL particle” means a subclass of VLDL particles characterized by a smaller, denser size compared to other VLDL particles. In certain embodiments, large VLDL particles are greater than 60 nm in diameter. In certain embodiments, medium VLDL particles are 35-60 nm in diameter. In certain embodiments, small VLDL particles are 27-35 nm in diameter. In certain embodiments, particle size is measured by nuclear magnetic resonance analysis.

[0108] “Triglycerides” means lipids that are the triesters of glycerol. “Serum triglycerides” mean triglycerides present in serum. “Liver triglycerides” mean triglycerides present in liver tissue.

[0109] “Serum lipids” mean cholesterol and triglycerides in the serum.

[0110] “Elevated total cholesterol” means total cholesterol at a concentration in an individual at which lipid-lowering therapy is recommended, and includes, without limitation, elevated LDL-C”, “elevated VLDL-C,” “elevated IDL-C,” and “elevated non-HDL-C.” In certain embodiments, total cholesterol concentrations of less than 200 mg/dL, 200-239 mg/dL, and greater than 240 mg/dL are considered desirable, borderline high, and high, respectively. In certain embodiments, LDL-C concentrations of 100 mg/dL, 100-129 mg/dL, 130-159 mg/dL, 160-189 mg/dL, and greater than 190 mg/dL are considered optimal, near optimal/above optimal, borderline high, high, and very high, respectively.

[0111] “Elevated triglyceride” means concentrations of triglyceride in the serum or liver at which lipid-lowering therapy is recommended, and includes “elevated serum triglyceride” and “elevated liver triglyceride.” In certain embodiments, serum triglyceride concentration of 150-199 mg/dL, 200-499 mg/dL, and greater than or equal to 500 mg/dL is considered borderline high, high, and very high, respectively.

[0112] “Elevated small LDL particles” means a concentration of small LDL particles in an individual at which lipid-lowering therapy is recommended.

[0113] “Elevated small VLDL particles” means a concentration of small VLDL particles in an individual at which lipid-lowering therapy is recommended.

[0114] “Elevated lipoprotein(a)” means a concentration of lipoprotein(a) in an individual at which lipid-lowering therapy is recommended.

[0115] “Low HDL-C” means a concentration of HDL-C in an individual at which lipid-lowering therapy is recommended. In certain embodiments lipid-lowering therapy is recommended when low HDL-C is accompanied by elevations in non-HDL-C and/or elevations in triglyceride. In certain embodiments, HDL-C concentrations of less than 40 mg/dL are considered low. In certain embodiments, HDL-C concentrations of less than 50 mg/dL are considered low.

[0116] “ApoB” means apolipoprotein B-100 protein. Concentration of ApoB in serum (or plasma) is typically quantified in mg/dL or nmol/L. “Serum ApoB” and “plasma ApoB” mean ApoB in the serum and plasma, respectively.

[0117] “LDL/HDL ratio” means the ratio of LDL-C to HDL-C.

[0118] “Oxidized-LDL” or “Ox-LDL-C” means LDL-C that is oxidized following exposure to free radicals.

[0119] “Individual having elevated LDL-C levels” means an individual who has been identified by a medical professional (e.g., a physician) as having LDL-C levels near or above the level at which therapeutic intervention is recommended, according to guidelines recognized by medical professionals. Such an individual may also be considered “in need of treatment” to decrease LDL-C levels.

[0120] “Individual having elevated apoB-100 levels” means an individual who has been identified as having apoB-100 levels near or above the level at which therapeutic intervention is recommended, according to guidelines recognized by medical professionals. Such an individual may also be considered “in need of treatment” to decrease apoB-100 levels.

[0121] “Treatment of elevated LDL-C levels” means administration of an antisense compound targeted to a PCSK9 nucleic acid to an individual having elevated LDL-C levels.

[0122] “Treatment of atherosclerosis” means administration of an antisense compound targeted to a PCSK9 nucleic acid to an individual who, based upon a physician's assessment, has or is likely to have atherosclerosis. “Prevention of atherosclerosis” means administration of an antisense compound targeted to a PCSK9 nucleic acid to an individual who, based upon a physician's assessment, is susceptible to atherosclerosis.

[0123] “Ameliorate” means to make better or improve a detrimental condition in an individual.

[0124] “Target nucleic acid,” “target RNA,” “target RNA transcript” and “nucleic acid target” all mean any nucleic acid capable of being targeted by antisense compounds.

[0125] “PCSK9 nucleic acid” means any nucleic acid encoding PCSK9. For example, in certain embodiments, a PCSK9 nucleic acid includes, without limitation, a DNA sequence encoding PCSK9, an RNA sequence transcribed from DNA encoding PCSK9, and an mRNA sequence encoding PCSK9. “PCSK9 mRNA” means an mRNA encoding a PCSK9 protein.

[0126] “Targeting” means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.

[0127] “Targeted” means having a nucleobase sequence that will allow specific hybridization of an antisense compound to a target nucleic acid to induce a desired effect. In certain embodiments, a desired effect is reduction of a target nucleic acid. In certain such embodiments, a desired effect is reduction of PCSK9 mRNA.

[0128] “Antisense inhibition” means reduction of a target nucleic acid levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels in the absence of the antisense compound.

[0129] “Target region” means a fragment of a target nucleic acid to which one or more antisense compounds is targeted.

[0130] “Target segment” means the sequence of nucleotides of a target nucleic acid to which an antisense compound is targeted. “5′ target site” refers to the 5′-most nucleotide of a target segment. “3′ target site” refers to the 3′-most nucleotide of a target segment.

[0131] “Active target region” means a target region to which one or more active antisense compounds is targeted. “Active antisense compounds” means antisense compounds that reduce target nucleic acid levels.

[0132] “Hybridization” means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense compound and a nucleic acid target. In certain such embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense oligonucleotide and a nucleic acid target

[0133] “Stringent hybridization conditions” means conditions under which a nucleic acid molecule, such as an antisense compound, will hybridize to a target nucleic acid sequence, but to a minimal number of other sequences.

[0134] “Specifically hybridizable” means an antisense compound that hybridizes to a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids.

[0135] “Complementarity” means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.

[0136] “Fully complementary” means each nucleobase of a first nucleic acid is capable of pairing with each nucleobase of a second nucleic acid. In certain embodiments, a first nucleic acid is an antisense compound and a target nucleic acid is a second nucleic acid. In certain such embodiments, an antisense oligonucleotide is a first nucleic acid and a target nucleic acid is a second nucleic acid.

[0137] “Non-complementary nucleobase” means a nucleobase of first nucleic acid that is not capable of pairing with the corresponding nucleobase of a target nucleic acid. “Mismatch” a nucleobase of first nucleic acid that is not capable of pairing with the corresponding nucleobase of a target nucleic acid.

[0138] “Portion” means a defined number of contiguous (i.e., linked) nucleobases. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound.

[0139] “Oligomeric compound” means a polymer of linked monomeric subunits which is capable of hybridizing to at least a region of an RNA molecule.

[0140] “Antisense compound” means an oligomeric compound that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.

[0141] “Oligonucleotide” means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another.

[0142] “Oligonucleoside” means an oligonucleotide in which the internucleoside linkages do not contain a phosphorus atom.

[0143] “Unmodified nucleotide” means a nucleotide composed of naturally occurring nucleobases, sugar moieties and internucleoside linkages. In certain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e., β-D-ribonucleosides) or a DNA nucleotide (i.e., β-D-deoxyribonucleoside).

[0144] “Antisense oligonucleotide” means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region of a target nucleic acid.

[0145] “Motif” means the pattern of unmodified and modified nucleosides in an antisense compound.

[0146] “Chimeric antisense compound” means an antisense compound that has at least 2 chemically distinct regions, each region having a plurality of subunits.

[0147] A “gapmer” means an antisense compound in which an internal position having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having one or more nucleotides that are chemically distinct from the nucleosides of the internal region.

[0148] A “gap segment” means the plurality of nucleotides that make up the internal region of a gapmer.

[0149] A “wing segment” means the external region of a gapmer.

[0150] “Gap-widened” means an antisense compound has a gap segment of 12 or more contiguous 2′-deoxyribonucleotides positioned between 5′ and 3′ wing segments having from one to six nucleotides having modified sugar moieties.

[0151] “Nucleoside” means a nucleobase linked to a sugar.

[0152] “Nucleobase” means a heterocyclic base moiety capable of pairing with a base of another nucleic acid.

[0153] “Nucleotide” means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.

[0154] “Nucleobase sequence” means the order of contiguous nucleobases independent of any sugar, linkage, and/or nucleobase modification.

[0155] “Modified nucleotide” means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, or modified nucleobase.

[0156] “Modified nucleoside” means a nucleotide having, independently, a modified sugar moiety or modified nucleobase.

[0157] “Contiguous nucleobases” means nucleobases immediately adjacent to each other.

[0158] “Modified oligonucleotide” means an oligonucleotide comprising a modified internucleoside linkage, a modified sugar, and/or a modified nucleobase.

[0159] “Single-stranded modified oligonucleotide” means a modified oligonucleotide which is not hybridized to a complementary strand.

[0160] “Internucleoside linkage” refers to the chemical bond between nucleosides.

[0161] “Linked nucleoside” means adjacent nucleosides which are bonded together.

[0162] “Linked deoxynucleoside” means a nucleic acid base (A, G, C, T, U) substituted by deoxyribose linked by a phosphate ester to form a nucleotide.

[0163] “Naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage.

[0164] “Modified internucleoside linkage” means substitution and/or any change from a naturally occurring internucleoside bond. In certain instances, the modified internucleoside linkage refers to a phosphodiester internucleoside bond.

[0165] “Phosphorothioate linkage” means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.

[0166] “Modified sugar moiety” means substitution and/or any change from a natural sugar moiety. For the purposes of this disclosure, a “natural sugar moiety” is a sugar moiety found in DNA (2′-H) or RNA (2′-OH).

[0167] “Modified sugar” refers to a substitution and/or any change from a natural sugar.

[0168] “Bicyclic sugar” means a furosyl ring modified by the bridging of the two non-geminal ring atoms. A bicyclic sugar is a modified sugar.

[0169] “Modified nucleobase” means any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. An “unmodified nucleobase” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

[0170] “Modified sugar moiety” means a sugar moiety having a substitution and/or any change from a natural sugar moiety.

[0171] “Natural sugar moiety” means a sugar moiety found in DNA (2′-H) or RNA (2′-OH).

[0172] “2′-O-methoxyethyl sugar moiety” means a 2′-substituted furosyl ring having a 2′-O(CH2)₂-OCH₃ (2′-O-methoxyethyl or 2′-MOE) substituent group.

[0173] “2′-O-methoxyethyl nucleotide” means a nucleotide comprising a 2′-O-methoxyethyl modified sugar moiety.

[0174] “2′-O-methoxyethyl” refers to an O-methoxy-ethyl modification of the 2′ position of a furosyl ring. A 2′-O-methoxyethyl modified sugar is a modified sugar.

[0175] “Bicyclic nucleic acid sugar moiety” means a furosyl ring modified by the bridging of two non-geminal ring atoms.

[0176] “5-methylcytosine” means a cytosine modified with a methyl group attached to the 5′ position. A 5-methylcytosine is a modified nucleobase.

[0177] “Prodrug” means a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.

[0178] “Pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.

[0179] “Salts” refers to physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.

[0180] “Cures” means a method or course that restores health or a prescribed treatment for an illness.

[0181] “Slows progression” means decrease in the development of the said disease.

[0182] “Cap structure” or “terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an antisense compound.

[0183] “ISIS 301012” means a lipid-lowering agent that is an antisense oligonucleotide having the sequence “GCCTCAGTCTGCTTCGCACC” (SEQ ID NO: 457), where each internucleoside linkage is a phosphorothioate internucleoside linkage, each cytosine is a 5-methylcytosine, nucleotides 6-15 are 2′-deoxynucleotides, and nucleotides 1-5 and 16-20 are 2′-O-methoxyethyl nucleotides. ISIS 301012 does not target PCSK9. It is used herein as a non-PCSK9 control.

Overview

[0214] Elevated levels of LDL-cholesterol (HDL-C) are recognized as a major independent risk factor for coronary heart disease (CHD). Even in individuals undergoing aggressive treatment with currently available cholesterol-lowering agents to reduce LDL-cholesterol (LDL-C) levels, coronary events still occur, and elevated LDL-C levels remain a major risk factor for coronary heart disease in these individuals. Furthermore, many individuals undergoing LDL-lowering therapy do not reach their target LDL-C levels, and thus remain at risk for CHD. Accordingly, there is a need for additional LDL-C lowering agents.

[0215] The antisense compounds and methods provided herein are useful for the treatment of hypercholesterolemia. Treatment of hypercholesterolemia encompasses a therapeutic regimen that results in a clinically desirable outcome. For example, the antisense compounds and methods provided herein are useful for the treatment of elevated cholesterol, such as elevated LDL-C. In addition, the antisense compounds and methods provided herein may be used to reduce the risk of CHD, in individuals exhibiting one or more risk factors for CHD. Furthermore, the antisense compounds and methods provided herein may be used to treat and/or prevent atherosclerosis.

[0216] As illustrated herein, administration of an antisense oligonucleotide targeted to PCSK9 to animals fed a high-fat diet (an experimental model of hyperlipidemia) resulted in antisense inhibition of PCSK9, upregulation of the LDL-R, reduction of LDL-C levels, and reduction of liver triglycerides. Thus, it is demonstrated that in an experimental model of hyperlipidemia, antisense inhibition of PCSK9 results in lowering LDL-C levels. Accordingly, provided herein are methods for the treatment of reducing LDL-C levels through the administration of an antisense compound targeted to a PCSK9 nucleic acid. Increased LDL-C levels are considered a risk factor for CHD, and are also linked to atherosclerosis. Accordingly, also provided herein are methods for the reduction of CHD risk, and for the prevention and/or treatment of atherosclerosis. Also provided herein are methods for the treatment of conditions characterized by elevated liver triglycerides, such as hepatic steatosis.

[0217] In a particular embodiment, methods comprise the use of antisense compounds targeted to particularly advantageous sequences within a PCSK9 nucleic acid. The PCSK9 target sequences are selected on the basis of superior results shown in one or more selection criteria. The presently disclosed antisense compounds all exhibit high in vitro efficacy, determined using a battery of cell models to measure antisense regulation of PCSK9 expression. Further, preferred antisense compounds exhibit relatively high in vivo efficacy, determined using a transgenic mouse model that expresses human PCSK9 or a cynomolgus (“cyno”) monkey model. The antisense compound ISIS 405879 (SEQ ID NO: 248) displays superior results in suppressing PCSK9 mRNA expression in the liver of a host individual.

[0218] The present antisense compounds are tolerated to different extents when administered to a host individual. Increased tolerability can depend on a number of factors, including, but not limited to, the nucleotide sequence of the antisense compound, chemical modifications to the nucleotides, the particular motif of unmodified and modified nucleosides in the antisense compound, or combinations thereof. Antisense compounds that exhibit increased or superior tolerability are particularly preferred in the present methods. The present antisense compounds also can show different pharmacokinetic properties, depending on their nucleotide sequence, chemical modifications, motifs, or combinations thereof. Particularly preferred antisense compounds also exhibit favorable pharmacokinetics when administered to a host individual.

Certain Indications

[0219] In certain embodiments, the invention provides methods of treating an individual comprising administering one or more pharmaceutical compositions of the present invention. In certain embodiments, the individual has hypercholesterolemia, mixed dyslipidemia, atherosclerosis, a risk of developing atherosclerosis, coronary heart disease, a history of coronary heart disease, early onset coronary heart disease, acute coronary syndrome, one or more risk factors for coronary heart disease, type II diabetes, type II diabetes with dyslipidemia, dyslipidemia, hypertriglyceridemia, hyperlipidemia, hyperfattyacidemia, hepatic steatosis, non-alcoholic steatohepatitis, or non-alcoholic fatty liver disease.

[0220] Guidelines for lipid-lowering therapy were established in 2001 by Adult Treatment Panel III (ATP III) of the National Cholesterol Education Program (NCEP), and updated in 2004 (Grundy et al., Circulation, 2004, 110, 227-239). The guidelines include obtaining a complete lipoprotein profile, typically after a 9 to 12 hour fast, for determination of LDL-C, total cholesterol, and HDL-C levels. According to the most recently established guidelines, LDL-C levels of 130-159 mg/dL, 160-189 mg/dL, and greater than or equal to 190 mg/dL are considered borderline high, high, and very high, respectively. Total cholesterol levels of 200-239 and greater than or equal to 240 mg/dL are considered borderline high and high, respectively. HDL-C levels of less than 40 mg/dL are considered low.

[0221] In certain embodiments, the individual has been identified as in need of lipid-lowering therapy. In certain such embodiments, the individual has been identified as in need of lipid-lowering therapy according to the guidelines established in 2001 by Adult Treatment Panel III (ATP III) of the National Cholesterol Education Program (NCEP), and updated in 2004 (Grundy et al., Circulation, 2004, 110, 227-239). In certain such embodiments, the individual in need of lipid-lowering therapy has LDL-C above 190 mg/dL. In certain such embodiments, the individual in need of lipid-lowering therapy has LDL-C above 160 mg/dL. In certain such embodiments, the individual in need of lipid-lowering therapy has LDL-C above 130 mg/dL. In certain such embodiments, the individual in need of lipid-lowering therapy has LDL-C above 100 mg/dL. In certain such embodiments, the individual in need of lipid-lowering therapy should maintain LDL-C below 160 mg/dL. In certain such embodiments, the individual in need of lipid-lowering therapy should maintain LDL-C below 130 mg/dL. In certain such embodiments, the individual in need of lipid-lowering therapy should maintain LDL-C below 100 mg/dL. In certain such embodiments, the individual should maintain LDL-C below 70 mg/dL or even below 50 mg/dL.

[0222] In certain embodiments, the invention provides methods for reducing ApoB in an individual. In certain embodiments, the invention provides methods for reducing ApoB-containing lipoprotein in an individual. In certain embodiments, the invention provides methods for reducing LDL-C in an individual. In certain embodiments, the invention provides methods for reducing VLDL-C in an individual. In certain embodiments, the invention provides methods for reducing IDL-C in an individual. In certain embodiments, the invention provides methods for reducing non-HDL-C in an individual. In certain embodiments the invention provides methods for reducing Lp(a) in an individual. In certain embodiments, the invention provides methods for reducing serum triglyceride in an individual. In certain embodiments, the invention provides methods for reducing liver triglyceride in an individual. In certain embodiments, the invention provides methods for reducing Ox-LDL-C in an individual. In certain embodiments, the invention provides methods for reducing small LDL particles in an individual. In certain embodiments, the invention provides methods for reducing small VLDL particles in an individual. In certain embodiments, the invention provides methods for reducing phospholipids in an individual. In certain embodiments, the invention provides methods for reducing oxidized phospholipids in an individual.

[0223] In certain embodiments, the methods provided by the present invention do not lower HDL-C. In certain embodiments, the methods provided by the present invention do not result in accumulation of lipids in the liver.

[0224] In one embodiment are methods for decreasing LDL-C levels, or alternatively methods for treating hypercholesterolemia, by administering to an individual suffering from elevated LDL-C levels a therapeutically effective amount of an antisense compound targeted to a PCSK9 nucleic acid. In another embodiment, a method of decreasing LDL-C levels comprises selecting an individual in need of a decrease in LDL-C levels, and administering to the individual a therapeutically effective amount of an antisense compound targeted to a PCSK9 nucleic acid. In a further embodiment, a method of reducing coronary heart disease risk includes selecting an individual having elevated LDL-C levels and one or more additional indicators of coronary heart disease risk, and administering to the individual a therapeutically effective amount of an antisense compound targeted to a PCSK9 nucleic acid.

[0225] In other embodiments, the LDL-C level is from 100-129 mg/dL, from 130 to 159 mg/dL, from 160-189 mg/dL, or greater than or equal to 190 mg/dL.

[0226] In one embodiment, administration of a therapeutically effective amount of an antisense compound targeted to a PCSK9 nucleic acid is accompanied by monitoring of LDL-C levels in the serum of an individual, to determine an individual's response to administration of the antisense compound. An individual's response to administration of the antisense compound is used by a physician to determine the amount and duration of therapeutic intervention.

[0227] In one embodiment, administration of an antisense compound targeted to a PCSK9 nucleic acid results in LDL-C levels below 190 mg/dL, below 160 mg/dL, below 130 mg/dL, below 100 mg/dL, below 70 mg/dL, or below 50 mg/dL. In another embodiment, administration of an antisense compound targeted to a PCSK9 nucleic acid decreases LDL-C by at least 15%, by at least 25%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, or by at least 95%.

[0228] An individual having elevated LDL-C levels may also exhibit reduced HDL-C levels and/or elevated total cholesterol levels. Accordingly, in one embodiment a therapeutically effective amount of an antisense compound targeted to a PCSK9 nucleic acid is administered to an individual having elevated LDL-C levels, who also has reduced HDL-C levels and/or elevated total cholesterol levels.

[0229] Individuals having elevated LDL-C levels may also exhibit elevated triglyceride levels. Accordingly, in one embodiment a therapeutically effective amount of an antisense compound targeted to a PCSK9 nucleic acid is administered to an individual having elevated LDL-C levels, and also having elevated triglyceride levels.

[0230] Atherosclerosis can lead to coronary heart disease, stroke, or peripheral vascular disease. Elevated LDL-C levels are considered a risk factor in the development and progression of atherosclerosis. Accordingly, in one embodiment, a therapeutically effective amount of an antisense compound targeted to a PCSK9 nucleic acid is administered to an individual having atherosclerosis. In a further embodiment, a therapeutically effective amount of antisense compound targeted to a PCSK9 nucleic acid is administered to an individual susceptible to atherosclerosis. Atherosclerosis is assessed directly through routine imaging techniques such as, for example, ultrasound imaging techniques that reveal carotid intimomedial thickness. Accordingly, treatment and/or prevention of atherosclerosis further include monitoring atherosclerosis through routine imaging techniques. In one embodiment, administration of an antisense compound targeted to a PCSK9 nucleic acid leads to a lessening of the severity of atherosclerosis, as indicated by, for example, a reduction of carotid intimomedial thickness in arteries.

[0231] Measurements of cholesterol, lipoproteins and triglycerides are obtained using serum or plasma collected from an individual. Methods of obtaining serum or plasma samples are routine, as are methods of preparation of the serum samples for analysis of cholesterol, triglycerides, and other serum markers.

[0232] A physician may determine the need for therapeutic intervention for individuals in cases where more or less aggressive LDL-lowering therapy is needed. The practice of the methods herein may be applied to any altered guidelines provided by the NCEP, or other entities that establish guidelines for physicians used in treating any of the diseases or conditions listed herein, for determining coronary heart disease risk and diagnosing metabolic syndrome.

[0233] In one embodiment, administration of an antisense compound targeted to a PCSK9 nucleic acid is parenteral administration. Parenteral administration may be intravenous or subcutaneous administration. Accordingly, in another embodiment, administration of an antisense compound targeted to a PCSK9 nucleic acid is intravenous or subcutaneous administration. Administration may include multiple doses of an antisense compound targeted to a PCSK9 nucleic acid.

[0234] In certain embodiments, a pharmaceutical composition comprising an antisense compound targeted to PCSK9 is for use in therapy. In certain embodiments, the therapy is the reduction of LDL-C, ApoB, VLDL-C, IDL-C, non-HDL-C, Lp(a), serum triglyceride, liver triglyceride, Ox-LDL-C, small LDL particles, small VLDL, phospholipids, or oxidized phospholipids in an individual. In certain embodiments, the therapy is the treatment of hypercholesterolemia, mixed dyslipidemia, atherosclerosis, a risk of developing atherosclerosis, coronary heart disease, acute coronary syndrome, a history of coronary heart disease, early onset coronary heart disease, one or more risk factors for coronary heart disease, type II diabetes, type II diabetes with dyslipidemia, dyslipidemia, hypertriglyceridemia, hyperlipidemia, hyperfattyacidemia, hepatic steatosis, non-alcoholic steatohepatitis, or non-alcoholic fatty liver disease. In additional embodiments, the therapy is the reduction of CHD risk. In certain aspects, the therapy is prevention of atherosclerosis. In certain embodiments, the therapy is the prevention of coronary heart disease.

[0235] In certain embodiments, a pharmaceutical composition comprising an antisense compound targeted to PCSK9 is used for the preparation of a medicament for reducing LDL-C, ApoB, VLDL-C, IDL-C, non-HDL-C, Lp(a), serum triglyceride, liver triglyceride, Ox-LDL-C, small LDL particles, small VLDL, phospholipids, or oxidized phospholipids in an individual. In certain embodiments pharmaceutical composition comprising an antisense compound targeted to PCKS9 is used for the preparation of a medicament for reducing coronary heart disease risk. In certain embodiments, an antisense compound targeted to PCSK9 is used for the preparation of a medicament for the treatment of hypercholesterolemia, mixed dyslipidemia, atherosclerosis, a risk of developing atherosclerosis, coronary heart disease, a history of coronary heart disease, early onset coronary heart disease, one or more risk factors for coronary heart disease, type II diabetes, type II diabetes with dyslipidemia, dyslipidemia, hypertriglyceridemia, hyperlipidemia, hyperfattyacidemia, hepatic steatosis, non-alcoholic steatohepatitis, or non-alcoholic fatty liver disease.

Certain Combination Therapies

[0236] In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with one or more other pharmaceutical agents. In certain embodiments, such one or more other pharmaceutical agents are designed to treat the same disease or condition as the one or more pharmaceutical compositions of the present invention. In certain embodiments, such one or more other pharmaceutical agents are designed to treat a different disease or condition as the one or more pharmaceutical compositions of the present invention. In certain embodiments, such one or more other pharmaceutical agents are designed to treat an undesired effect of one or more pharmaceutical compositions of the present invention. In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with another pharmaceutical agent to treat an undesired effect of that other pharmaceutical agent. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at the same time. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at different times. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared together in a single formulation. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared separately. For example, a composition may comprise a pharmaceutical agent for separate, sequential, or simultaneous administration with an antisense compound.

[0237] In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include lipid-lowering agents or LXR agonists. In certain such embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include, but are not limited to atorvastatin, simvastatin, rosuvastatin, and ezetimibe. In certain such embodiments, the lipid-lowering agent is administered prior to administration of a pharmaceutical composition of the present invention. In certain such embodiments, the lipid-lowering agent is administered following administration of a pharmaceutical composition of the present invention. In certain such embodiments the lipid-lowering agent is administered at the same time as a pharmaceutical composition of the present invention. In certain such embodiments the dose of a co-administered lipid-lowering agent is the same as the dose that would be administered if the lipid-lowering agent was administered alone. In certain such embodiments the dose of a co-administered lipid-lowering agent is lower than the dose that would be administered if the lipid-lowering agent was administered alone. In certain such embodiments the dose of a co-administered lipid-lowering agent is greater than the dose that would be administered if the lipid-lowering agent was administered alone.

[0238] In certain embodiments, a co-administered lipid-lowering agent is a HMG-CoA reductase inhibitor. In certain such embodiments the HMG-CoA reductase inhibitor is a statin. In certain such embodiments, the statin is selected from, for example, atorvastatin, simvastatin, pravastatin, fluvastatin, and rosuvastatin.

[0239] In certain embodiments, a co-administered lipid-lowering agent is a cholesterol absorption inhibitor. In certain such embodiments, cholesterol absorption inhibitor is ezetimibe.

[0240] In certain embodiments, a co-administered lipid-lowering agent is a co-formulated HMG-CoA reductase inhibitor and cholesterol absorption inhibitor. In certain such embodiments the co-formulated lipid-lowering agent is ezetimibe/simvastatin.

[0241] In certain embodiments, a co-administered lipid-lowering agent is a microsomal triglyceride transfer protein inhibitor (MTP inhibitor).

[0242] In certain embodiments, a co-administered lipid-lowering agent is an oligonucleotide targeted to ApoB.

[0243] In certain embodiments, a co-administered pharmaceutical agent is a bile acid sequestrant. In certain such embodiments, the bile acid sequestrant is selected from cholestyramine, colestipol, and colesevelam.

[0244] In certain embodiments, a co-administered pharmaceutical agent is a nicotinic acid. In certain such embodiments, the nicotinic acid is selected from immediate release nicotinic acid, extended release nicotinic acid, and sustained release nicotinic acid.

[0245] In certain embodiments, a co-administered pharmaceutical agent is a fibric acid. In certain such embodiments, a fibric acid is selected from gemfibrozil, fenofibrate, clofibrate, bezafibrate, and ciprofibrate.

[0246] Further examples of pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include, but are not limited to, corticosteroids, including but not limited to prednisone; LXR agonists; immunoglobulins, including, but not limited to intravenous immunoglobulin (IVIg); analgesics (e.g., acetaminophen); anti-inflammatory agents, including, but not limited to non-steroidal anti-inflammatory drugs (e.g., ibuprofen, COX-1 inhibitors, and COX-2, inhibitors); salicylates; antibiotics; antivirals; antifungal agents; antidiabetic agents (e.g., biguanides, glucosidase inhibitors, insulins, sulfonylureas, and thiazolidenediones); adrenergic modifiers; diuretics; hormones (e.g., anabolic steroids, androgen, estrogen, calcitonin, progestin, somatostan, and thyroid hormones); immunomodulators; muscle relaxants; antihistamines; osteoporosis agents (e.g., biphosphonates, calcitonin, and estrogens); prostaglandins, antineoplastic agents; psychotherapeutic agents; sedatives; poison oak or poison sumac products; antibodies; and vaccines.

[0247] In certain embodiments, the pharmaceutical compositions of the present invention may be administered in conjunction with a lipid-lowering therapy. In certain such embodiments, a lipid-lowering therapy is therapeutic lifestyle change. In certain such embodiments, a lipid-lowering therapy is LDL apheresis.

Antisense Compounds

[0248] Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs. An oligomeric compound may be “antisense” to a target nucleic acid, meaning that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.

[0249] In certain embodiments, an antisense compound has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted. In certain such embodiments, an antisense oligonucleotide has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.

[0250] In certain embodiments, an antisense compound targeted to a PCSK9 nucleic acid is 8 to 80, 12 to 50, 12 to 30 or 15 to 30 subunits in length. In other words, antisense compounds are from 8 to 80, 12 to 50, 12 to 30 or 15 to 30 linked subunits. In certain such embodiments, the antisense compounds are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or subunits in length.

[0251] In certain embodiments, an antisense oligonucleotide targeted to a PCSK9 nucleic acid is 12 to 30 nucleotides in length. In certain such embodiments, an antisense oligonucleotide targeted to a PCSK9 nucleic acid is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0252] In certain embodiment, an antisense compound targeted to a PCSK9 nucleic acid is 15 to 30 subunits in length. In other words, antisense compounds are from 15 to 30 linked subunits. In certain such embodiments, the antisense compounds are 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 subunits in length.

[0253] In certain embodiments, an antisense oligonucleotide targeted to a PCSK9 nucleic acid is 15 to 30 nucleotides in length. In certain such embodiments, an antisense oligonucleotide targeted to a PCSK9 nucleic acid is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0254] In certain embodiments, an antisense compound targeted to a PCSK9 nucleic acid is 18 to 24 subunits in length. In other words, antisense compounds are from 18 to 24 linked subunits. In one embodiment, the antisense compounds are 18, 19, 20, 21, 22, 23, or 24 subunits in length.

[0255] In certain embodiments, an antisense oligonucleotide targeted to a PCSK9 nucleic acid is 18 to 24 nucleotides in length. In certain such embodiments, an antisense oligonucleotide targeted to a PCSK9 nucleic acid is 18, 19, 20, 21, 22, 23, or 24 nucleotides in length.

[0256] In certain embodiments, an antisense compound targeted to a PCSK9 nucleic acid is 19 to 22 subunits in length. In other words, antisense compounds are from 19 to 22 linked subunits. This embodies antisense compounds of 19, 20, 21, or 22 subunits in length.

[0257] In certain embodiments, an antisense oligonucleotide targeted to a PCSK9 nucleic acid is 19 to 22 nucleotides in length. In certain such embodiments, an antisense oligonucleotide targeted to a PCSK9 nucleic acid is 19, 20, 21, or 22 nucleotides in length.

[0258] In certain embodiments, an antisense compound targeted to a PCSK9 nucleic acid is 20 subunits in length. In certain such embodiments, antisense compounds are 20 linked subunits in length.

[0259] In certain embodiments, an antisense oligonucleotide targeted to a PCSK9 nucleic acid is 20 nucleotides in length. In certain such embodiments, an antisense oligonucleotide targeted to an PCSK9 nucleic acid is 20 linked nucleotides in length.

[0260] In certain embodiments, antisense compounds target a range of a PCSK9 nucleic acid. In certain embodiment, such compounds contain at least an 8 nucleotide core sequence in common.

[0261] In certain embodiments, such compounds sharing at least an 8 nucleotide core sequence target a region identified herein.

[0262] In certain embodiments, an antisense compound targeted to a PCSK9 nucleic acid may target the following nucleotide regions of SEQ ID NO: 1: 294-317, 406-440, 406-526, 410-436, 410-499, 446-526, 545-581, 591-619, 591-704, 591-743, 595-622, 600-626, 600-639, 600-670, 601-628, 602-628, 603-630, 611-636, 620-647, 638-665, 648-674, 657-684, 705-743, 782-810, 821-859, 835-859, 835-917, 835-942, 860-887, 860-899, 860-909, 860-917, 869-895, 878-905, 888-909, 923-952, 960-1034, 960-1173, 960-986, 967-991, 970-1023, 970-1064, 970-1117, 970-996, 977-1004, 985-1011, 989-1016, 992-1019, 997-1024, 997-1024, 998-1025, 999-1026, 1000-1027, 1001-1028, 1002-1029, 1003-1029, 1004-1029, 1005-1029, 1006-1029, 1007-1034, 1036-1061, 1045-1072, 1076-1096, 1088-1115, 1098-1123, 1200-1251, 1210-1237, 1219-1245, 1228-1251, 1273-1444, 1295-1316, 1318-1345, 1328-1354, 1337-1361, 1344-1371, 1354-1377, 1380-1406, 1389-1416, 1400-1426, 1409-1434, 1465-1491, 1465-1602, 1474-1499, 1482-1519, 1513-1540, 1523-1549, 1526-1602, 1526-1624, 1532-1558, 1541-1568, 1552-1579, 1560-1587, 1561-1589, 1564-1591, 1565-1592, 1566-1592, 1567-1592, 1570-1597, 1571-1599, 1605-1706, 1628-1706, 1640-1666, 1672-1698, 1681-1706, 1735-1761, 1735-1765, 1740-1765, 1849-1876, 1849-1879, 1850-1877, 1851-1877, 1852-1878, 1852-1879, 1853-1879, 1854-1879, 1905-1955, 1915-1942, 1916-1943, 1917-1944, 1918-1945, 1919-1946, 1920-1939, 1920-1947, 1921-1948, 1922-1949, 1923-1950, 1924-1951, 1925-1952, 1926-1952, 1927-1952, 1928-1955, 1962-2059, 2040-2126, 2100-2126, 2100-2139, 2100-2206, 2101-2126, 2305-2332, 2305-2354, 2306-2333, 2307-2334, 2308-2334, 2309-2334, 2310-2334, 2410-2434, 2504-2528, 2509-2528, 2582-2625, 2606-2668, 2828-2855, 2832-2851, 2900-2927, 2900-2929, 2902-2927, 2983-3007, 2983-3013, 3227-3252, 3227-3456, 3472-3496, or 3543-3569.

[0263] In certain embodiments, antisense compounds target a range of a PCSK9 nucleic acid. In certain embodiment, such compounds contain at least an 8 nucleotide core sequence in common. In certain embodiments, such compounds sharing at least an 8 nucleotide core sequence targets the following nucleotide regions of SEQ ID NO: 1: 294-317, 406-440, 406-526, 410-436, 410-499, 446-526, 545-581, 591-619, 591-704, 591-743, 595-622, 600-626, 600-639, 600-670, 601-628, 602-628, 603-630, 611-636, 620-647, 638-665, 648-674, 657-684, 705-743, 782-810, 821-859, 835-859, 835-917, 835-942, 860-887, 860-899, 860-909, 860-917, 869-895, 878-905, 888-909, 923-952, 960-1034, 960-1173, 960-986, 967-991, 970-1023, 970-1064, 970-1117, 970-996, 977-1004, 985-1011, 989-1016, 992-1019, 997-1024, 997-1024, 998-1025, 999-1026, 1000-1027, 1001-1028, 1002-1021, 1002-1029, 1003-1029, 1004-1029, 1005-1029, 1006-1029, 1007-1034, 1036-1061, 1045-1072, 1076-1096, 1088-1115, 1098-1123, 1200-1251, 1210-1237, 1219-1245, 1228-1251, 1273-1444, 1295-1316, 1318-1345, 1328-1354, 1337-1361, 1344-1371, 1354-1377, 1380-1406, 1389-1416, 1400-1426, 1409-1434, 1465-1491, 1465-1602, 1474-1499, 1482-1519, 1513-1540, 1523-1549, 1526-1602, 1526-1624, 1532-1558, 1541-1568, 1552-1579, 1560-1587, 1561-1589, 1564-1591, 1565-1592, 1566-1592, 1567-1592, 1570-1597, 1571-1599, 1605-1706, 1628-1706, 1640-1666, 1672-1698, 1681-1706, 1735-1761, 1735-1765, 1740-1765, 1849-1876, 1849-1879, 1850-1877, 1851-1877, 1852-1878, 1852-1879, 1853-1879, 1854-1879, 1905-1955, 1915-1942, 1916-1943, 1917-1944, 1918-1945, 1919-1946, 1920-1939, 1920-1947, 1921-1948, 1922-1949, 1923-1950, 1924-1951, 1925-1952, 1926-1952, 1927-1952, 1928-1955, 1962-2059, 2040-2126, 2100-2126, 2100-2139, 2100-2206, 2101-2126, 2305-2332, 2305-2354, 2306-2333, 2307-2334, 2308-2334, 2309-2334, 2310-2334, 2410-2434, 2504-2528, 2509-2528, 2582-2625, 2606-2668, 2828-2855, 2832-2851, 2900-2927, 2900-2929, 2902-2927, 2983-3007, 2983-3013, 3227-3252, 3227-3456, 3472-3496, or 3543-3569.

[0264] In certain embodiments, an antisense compound targeted to a PCSK9 nucleic acid may target the following nucleotide regions of SEQ ID NO: 2: 2274-2400, 2274-2575, 2433-2570, 2433-2579, 2549-2575, 2552-2579, 2585-2638, 2605-2638, 3056-3075, 4150-5159, 4306-4325, 5590-5618, 5667-5686, 6444-6463, 6482-6518, 6492-6518, 6528-6555, 6528-6623, 6534-6561, 6535-6562, 6536-6563, 6537-6563, 6538-6565, 6539-6565, 6540-6567, 6541-6567, 6542-6569, 6546-6573, 6557-6584, 6575-6602, 6585-6611, 6594-6621, 6596-6623, 6652-6671, 7099-7118, 7556-7584, 8836-8855, 8948-8967, 9099-9118, 9099-9168, 9130-9168, 9207-9233, 9207-9235, 9209-9235, 10252-10271, 10633-10652, 11308-11491, 12715-12734, 12928-12947, 13681-13700, 13746-13779, 13816-13847, 13903-13945, 13977-14141, 14179-14198, 14267-14286, 14397-14423, 14441-14460, 14494-14513, 14494-14543, 14524-14543, 14601-14650, 14670-14700, 14675-14700, 14801-14828, 14877-14912, 14877-14915, 14877-14973, 14916-14943, 14916-14973, 14925-14951, 14934-14963, 14946-14973, 14979-14998, 15254-15280, 15254-15328, 15264-15290, 15279-15305, 15291-15318, 15292-15319, 15293-15320, 15294-15321, 15294-15321, 15295-15322, 15296-15323, 15297-15323, 15298-15323, 15299-15323, 15300-15323, 15301-15328, 15330-15355, 15330-15490, 15339-15366, 15358-15490, 16134-16153, 16668-16687, 17267-17286, 18377-18427, 18561-18580, 18591-18618, 18591-18646, 18591-18668, 18695-18746, 18705-18730, 18709-18736, 18719-18746, 19203-20080, 19931-19952, 19954-19981, 19964-19990, 19973-19999, 19982-20009, 19992-20016, 20016-20042, 20025-20052, 20036-20062, 20045-20070, 20100-20119, 20188-20207, 20624-20650, 20624-20759, 20629-20804, 20633-20660, 20635-20781, 20643-20662, 20657-20676, 20670-20697, 20680-20706, 20683-20781, 20689-20715, 20698-20725, 20709-20736, 20717-20744, 20718-20745, 20719-20746, 20720-20747, 20721-20748, 20722-20749, 20727-20752, 20735-20759, 20762-21014, 20785-21014, 21082-21107, 21082-21152, 21091-21114, 21118-21144, 21127-21152, 21181-21209, 21181-21211, 21183-21211, 21481-21500, 21589-21608, 21692-21719, 22000-22227, 22096-22115, 22096-22223, 22096-22311, 22133-22160, 22133-22163, 22134-22161, 22135-22162, 22136-22163, 22137-22163, 22138-22163, 22189-22239, 22199-22226, 22199-22227, 22200-22227, 22201-22228, 22202-22229, 22203-22230, 22204-22231, 22205-22232, 22206-22233, 22207-22234, 22208-22235, 22209-22236, 22210-22236, 22210-22239, 22211-22236, 22212-22239, 22292-22311, 23985-24054, 24035-24134, 24095-24121, 24858-24877, 24907-24926, 25413-25432, 25994-26013, 26112-26139, 26112-26161, 26112-27303, 26113-26140, 26114-26141, 26115-26141, 26116-26141, 26117-26141, 26117-26475, 26118-26141, 26120-26141, 26132-26151, 26142-26161, 26217-26241, 26311-26335, 26389-26432, 26456-26576, 26635-26662, 26707-26734, 26707-26736, 26790-26820, 27034-27263, 27279-27303, or 27350-27376.

[0265] In certain embodiments, antisense compounds target a range of a PCSK9 nucleic acid. In certain embodiment, such compounds contain at least an 8 nucleotide core sequence in common. In certain embodiments, such compounds sharing at least an 8 nucleotide core sequence targets the following nucleotide regions of SEQ ID NO: 2: 2274-2400, 2274-2575, 2433-2570, 2433-2579, 2549-2575, 2552-2579, 2585-2638, 2605-2638, 3056-3075, 4150-5159, 4306-4325, 5590-5618, 5667-5686, 6444-6463, 6482-6518, 6492-6518, 6528-6555, 6528-6623, 6534-6561, 6535-6562, 6536-6563, 6537-6563, 6538-6565, 6539-6565, 6540-6567, 6541-6567, 6542-6569, 6546-6573, 6557-6584, 6575-6602, 6585-6611, 6594-6621, 6596-6623, 6652-6671, 7099-7118, 7556-7584, 8836-8855, 8948-8967, 9099-9118, 9099-9168, 9130-9168, 9207-9233, 9207-9235, 9209-9235, 10252-10271, 10633-10652, 11308-11491, 12715-12734, 12928-12947, 13681-13700, 13746-13779, 13816-13847, 13903-13945, 13977-14141, 14179-14198, 14267-14286, 14397-14423, 14441-14460, 14494-14513, 14494-14543, 14524-14543, 14601-14650, 14670-14700, 14675-14700, 14801-14828, 14877-14912, 14877-14915, 14877-14973, 14916-14943, 14916-14973, 14925-14951, 14934-14963, 14946-14973, 14979-14998, 15254-15280, 15254-15328, 15264-15290, 15279-15305, 15291-15318, 15292-15319, 15293-15320, 15294-15321, 15294-15321, 15295-15322, 15296-15323, 15297-15323, 15298-15323, 15299-15323, 15300-15323, 15301-15328, 15330-15355, 15330-15490, 15339-15366, 15358-15490, 16134-16153, 16668-16687, 17267-17286, 18377-18427, 18561-18580, 18591-18618, 18591-18646, 18591-18668, 18695-18746, 18705-18730, 18709-18736, 18719-18746, 19203-20080, 19931-19952, 19954-19981, 19964-19990, 19973-19999, 19982-20009, 19992-20016, 20016-20042, 20025-20052, 20036-20062, 20045-20070, 20100-20119, 20188-20207, 20624-20650, 20624-20759, 20629-20804, 20633-20660, 20635-20781, 20643-20662, 20657-20676, 20670-20697, 20680-20706, 20683-20781, 20689-20715, 20698-20725, 20709-20736, 20717-20744, 20718-20745, 20719-20746, 20720-20747, 20721-20748, 20722-20749, 20727-20752, 20735-20759, 20762-21014, 20785-21014, 21082-21107, 21082-21152, 21091-21114, 21118-21144, 21127-21152, 21181-21209, 21181-21211, 21183-21211, 21481-21500, 21589-21608, 21692-21719, 22000-22227, 22096-22115, 22096-22223, 22096-22311, 22133-22160, 22133-22163, 22134-22161, 22135-22162, 22136-22163, 22137-22163, 22138-22163, 22189-22239, 22199-22226, 22199-22227, 22200-22227, 22201-22228, 22202-22229, 22203-22230, 22204-22231, 22205-22232, 22206-22233, 22207-22234, 22208-22235, 22209-22236, 22210-22236, 22210-22239, 22211-22236, 22212-22239, 22292-22311, 23985-24054, 24035-24134, 24095-24121, 24858-24877, 24907-24926, 25413-25432, 25994-26013, 26112-26139, 26112-26161, 26112-27303, 26113-26140, 26114-26141, 26115-26141, 26116-26141, 26117-26141, 26117-26475, 26118-26141, 26120-26141, 26132-26151, 26142-26161, 26217-26241, 26311-26335, 26389-26432, 26456-26576, 26635-26662, 26707-26734, 26707-26736, 26790-26820, 27034-27263, 27279-27303, or 27350-27376.

[0266] In certain embodiments, an antisense compound targeted to a PCSK9 nucleic acid may target the following nucleotide regions of SEQ ID NO: 3: 220-253, 290-321, 377-419, 451-615, 653-672, 741-760, 871-897, 915-934, 968-1017, 1075-1124, 1075-1174, 1144-1174, 1275-1302, 1315-1341, 1351-1389, 1351-1447, 1365-1439, 1390-1417, 1390-1429, 1390-1439, 1399-1425, 1408-1435, 1420-1447, 1453-1482, 1490-1516, 1490-1564, 1500-1526, 1515-1541, 1527-1553, 1527-1554, 1528-1554, 1529-1555, 1529-1556, 1530-1556, 1530-1557, 1531-1557, 1532-1558, 1533-1559, 1534-1559, 1535-1559, 1536-1559, 1537-1564, 1566-1602, 1566-1681, 1606-1626, 1618-1645, 1626-1653, 1684-1703, 1730-1781, 1740-1767, 1749-1775, 1758-1781, 1820-1847, 1820-1877, 1822-2198, 1830-1856, 1839-1865, 1840-1867, 1898-1924, 1898-2035, 1903-2127, 1907-1934, 1911-1938, 1946-1971, 1954-1980, 1959-2035, 1959-2057, 1963-1988, 1967-2035, 1972-1999, 1982-2008, 1991-2018, 1993-2019, 1995-2022, 1996-2023, 1997-2024, 1998-2025, 1999-2025, 2000-2025, 2009-2035, 2038-2139, 2061-2139, 2073-2099, 2078-2104, 2105-2131, 2112-2139, 2168-2198, 2170-2177, 2245-2284, 2295-2394, 2355-2381, 2355-2394, 2405-2461, 2560-2587, 2560-2609, 2561-2588, 2562-2589, 2563-2589, 2564-2589, 2565-2589, 2566-2589, 2567-2589, 2568-2589, 2665-2689, 2759-2783, 2837-2880, 2904-2923, 3005-3024, 3005-3174, 3083-3110, 3155-3184, 3238-3268, 3482-3711, 3727-3751, or 3798-3824.

[0267] In certain embodiments, antisense compounds target a range of a PCSK9 nucleic acid. In certain embodiment, such compounds contain at least an 8 nucleotide core sequence in common. In certain embodiments, such compounds sharing at least an 8 nucleotide core sequence targets the following nucleotide regions of SEQ ID NO: 3: 220-253, 290-321, 377-419, 451-615, 653-672, 741-760, 871-897, 915-934, 968-1017, 1075-1124, 1075-1174, 1144-1174, 1275-1302, 1315-1341, 1351-1389, 1351-1447, 1365-1439, 1390-1417, 1390-1429, 1390-1439, 1399-1425, 1408-1435, 1420-1447, 1453-1482, 1490-1516, 1490-1564, 1500-1526, 1515-1541, 1527-1553, 1527-1554, 1528-1554, 1529-1555, 1529-1556, 1530-1556, 1530-1557, 1531-1557, 1532-1558, 1533-1559, 1534-1559, 1535-1559, 1536-1559, 1537-1564, 1566-1602, 1566-1681, 1606-1626, 1618-1645, 1626-1653, 1684-1703, 1730-1781, 1740-1767, 1749-1775, 1758-1781, 1820-1847, 1820-1877, 1822-2198, 1830-1856, 1839-1865, 1840-1867, 1898-1924, 1898-2035, 1903-2127, 1907-1934, 1911-1938, 1946-1971, 1954-1980, 1959-2035, 1959-2057, 1963-1988, 1967-2035, 1972-1999, 1982-2008, 1991-2018, 1993-2019, 1995-2022, 1996-2023, 1997-2024, 1998-2025, 1999-2025, 2000-2025, 2009-2035, 2038-2139, 2061-2139, 2073-2099, 2078-2104, 2105-2131, 2112-2139, 2168-2198, 2170-2177, 2245-2284, 2295-2394, 2355-2381, 2355-2394, 2405-2461, 2560-2587, 2560-2609, 2561-2588, 2562-2589, 2563-2589, 2564-2589, 2565-2589, 2566-2589, 2567-2589, 2568-2589, 2665-2689, 2759-2783, 2837-2880, 2904-2923, 3005-3024, 3005-3174, 3083-3110, 3155-3184, 3238-3268, 3482-3711, 3727-3751, or 3798-3824.

[0268] In certain embodiments, an antisense compound targeted to a PCSK9 nucleic acid may target the following nucleotide regions of SEQ ID NO: 1: 320-405, 441-445, 527-544, 582-590, 744-781, 811-820, 918-922, 953-959, 1034-1036, 1152-1153, 1174-1199, 1251-1272, 1445-1464, 1603-1604, 1625-1627, 1707-1734, 1766-1811, 1832-1848, 1880-1904, 1956-1961, 1982-1939, 2030-2039, 2060-2099, 2140-2149, 2170-2186, 2207-2304, 2355-2409, 2435-2503, 2529-2581, 2626-2648, 2669-2749, 2770-2827, 2856-2876, 2891-2899, 2930-2982, 3014-3226, 3253-3436, 3457-3471, or 3497-3542.

[0269] In certain embodiments, antisense compounds target a range of a PCSK9 nucleic acid. In certain embodiment, such compounds contain at least an 8 nucleotide core sequence in common.

[0270] In certain embodiments, such compounds sharing at least an 8 nucleotide core sequence targets the following nucleotide regions of SEQ ID NO: 1: 320-405, 441-445, 527-544, 582-590, 744-781, 811-820, 918-922, 953-959, 1034-1036, 1152-1153, 1174-1199, 1251-1272, 1445-1464, 1603-1604, 1625-1627, 1707-1734, 1766-1811, 1832-1848, 1880-1904, 1956-1961, 1982-1939, 2030-2039, 2060-2099, 2140-2149, 2170-2186, 2207-2304, 2355-2409, 2435-2503, 2529-2581, 2626-2648, 2669-2749, 2770-2827, 2856-2876, 2891-2899, 2930-2982, 3014-3226, 3253-3436, 3457-3471, or 3497-3542.

[0271] In certain embodiments, a shortened or truncated antisense compound targeted to a PCSK9 nucleic acid has a single subunit deleted from the 5′ end (5′ truncation), or alternatively from the 3′ end (3′ truncation). A shortened or truncated antisense compound targeted to a PCSK9 nucleic acid may have two subunits deleted from the 5′ end, or alternatively may have two subunits deleted from the 3′ end, of the antisense compound. Alternatively, the deleted nucleosides may be dispersed throughout the antisense compound, for example, in an antisense compound having one nucleoside deleted from the 5′ end and one nucleoside deleted from the 3′ end.

[0272] When a single additional subunit is present in a lengthened antisense compound, the additional subunit may be located at the 5′ or 3′ end of the antisense compound. When two are more additional subunits are present, the added subunits may be adjacent to each other, for example, in an antisense compound having two subunits added to the 5′ end (5′ addition), or alternatively to the 3′ end (3′ addition), of the antisense compound. Alternatively, the added subunits may be dispersed throughout the antisense compound, for example, in an antisense compound having one subunit added to the 5′ end and one subunit added to the 3′ end.

[0273] It is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Antisense oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.

[0274] Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent anti-tumor activity in vivo.

[0275] Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a series of tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42 nucleobase antisense oligonucleotides comprised of the sequence of two or three of the tandem antisense oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase antisense oligonucleotides alone was able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase antisense oligonucleotides.

[0276] PCT/US2007/068404 describes incorporation of chemically-modified high-affinity nucleotides into short antisense compounds about 8-16 nucleobases in length and that such compounds are useful in the reduction of target RNAs in animals with increased potency and improved therapeutic index.

[0277] In certain embodiments, antisense compounds targeted to a PCSK9 nucleic acid are short antisense compounds. In certain embodiments, such short antisense compounds are oligonucleotide compounds. In certain embodiments, such short antisense compounds are about 8 to 16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to 14 nucleotides in length and comprises a gap region flanked on each side by a wing, wherein each wing independently consists of 1 to 3 nucleotides. Preferred motifs include but are not limited to wing-deoxy gap-wing motifs selected from 3-10-3, 2-10-3, 2-10-2, 1-10-1, 2-8-2, 1-8-1, 3-6-3 or 1-6-1.

[0278] Antisense compounds targeted to a PCSK9 nucleic acid are synthesized in vitro and do not include antisense compositions of biological origin, or genetic vector constructs designed to direct the in vivo synthesis of antisense molecules.

[0279] In certain embodiments, an antisense compound is targeted to a region of a PCSK9 nucleic acid that does not contain a single nucleotide polymorphism (SNPs). In certain embodiments, an antisense compound is targeted to a region of a PCSK9 nucleic acid that does contain a single nucleotide polymorph (SNPs). A single nucleotide polymorphism refers to polymorphisms that are the result of a single nucleotide alteration or the existence of two or more alternative sequences which can be, for example, different allelic forms of a gene. A polymorphism may comprise one or more base changes including, for example, an insertion, a repeat, or a deletion. In certain embodiments, an antisense oligonucleotide targeted to a PCSK9 nucleic acid overlaps with a SNP at the following positions: 428, 432, 449, 996, 1011, 1044, 1317, 1565, 1617, 1618, 1671, 1711, 1722, 1836, 1911. In certain embodiments, the compounds provided herein that target a region of a PCSK9 nucleic acid that contains one or more SNPs will contain the appropriate base substitution, insertion, repeat or deletion such that the compound is fully complementary to the altered PCSK9 nucleic acid sequence.

[0280] A subgroup of these antisense compounds were selected for further characterization, based on the ability of the selected antisense compounds to inhibit PCSK9 expression selectively and effectively in cell culture assays, set forth in the Examples. The antisense compounds selected for further evaluation are listed in Table 1. The 5′ and 3′ boundaries of the PCSK9 target sequence are shown for each antisense compound, with reference to the nucleotide positions of SEQ ID NO: 1.

[0281] “Motif” in Table 1 refers to the use of chemically modified nucleosides at the 5′ and 3′ ends of the antisense compounds. In a “5-10-5” motif, for example, five chemical modified nucleosides at both the 5′ and 3′ ends flank ten unmodified nucleosides in the center of the antisense compound. As disclosed in more detail below, modified nucleosides may make the antisense compounds more resistant to nucleases, among other things, which generally improves the pharmacodynamic and pharmacokinetic properties of the antisense compounds when administered to an individual.

[0282] Table 1 further discloses the nucleobase sequence of the antisense compounds. In certain embodiments, an antisense compound has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.

[0283]

TABLE 1
	5′ Target	3′ Target			SEQ ID
Isis No.	Site	Site	Sequence 5′-3′	Motif	NO
405881	1005	1024	CACCCTTGGCCACGCCGGCA	5-10-5	250
399819	2509	2528	CCCACTCAAGGGCCAGGCCA	5-10-5	65
395165	1004	1023	ACCCTTGGCCACGCCGGCAT	5-10-5	28
405879	1002	1021	CCTTGGCCACGCCGGCATCC	5-10-5	248
406008	602	621	CTTGGTGAGGTATCCCCGGC	5-10-5	188
405891	1567	1586	TCCTCAGGGAACCAGGCCTC	5-10-5	352
395186	2100	2119	CTTTGCATTCCAGACCTGGG	5-10-5	60
405988	1854	1873	GGCAGCACCTGGCAATGGCG	5-10-5	381
405994	1928	1947	GCAGTGGACACGGGTCCCCA	5-10-5	400
406023	787	806	TGGTATTCATCCGCCCGGTA	5-10-5	212
395187	2310	2329	GGCAGCAGATGGCAACGGCT	5-10-5	62
395185	1920	1939	CACGGGTCCCCATGCTGGCC	5-10-5	59
406033	967	986	CCTGCCAGGTGGGTGCCATG	5-10-5	237
405923	1295	1314	GGCATTGGTGGCCCCAACTG	5-10-5	288
399900	1569	1588	GGTCCTCAGGGAACCAGGCC	3-14-3	50
405995	1930	1949	TGGCAGTGGACACGGGTCCC	5-10-5	402
405991	1922	1941	GACACGGGTCCCCATGCTGG	5-10-5	394
406005	559	578	CGGGCAGTGCGCTCTGACTG	5-10-5	180
399793	417	436	CCTCGGAACGCAAGGCTAGC	5-10-5	8
395152	410	429	ACGCAAGGCTAGCACCAGCT	5-10-5	7

Antisense Compound Motifs

[0284] In certain embodiments, antisense compounds targeted to a PCSK9 nucleic acid have chemically modified subunits arranged in patterns, or motifs, to confer to the antisense compounds properties such as enhanced the inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.

[0285] Chimeric antisense compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and/or increased inhibitory activity. A second region of a chimeric antisense compound may serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.

[0286] Antisense compounds having a gapmer motif are considered chimeric antisense compounds. In a gapmer an internal position having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region. In the case of an antisense oligonucleotide having a gapmer motif, the gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides. The regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region. The types of sugar moieties that are used to differentiate the regions of a gapmer may in some embodiments include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modified nucleosides (such 2′-modified nucleosides may include 2′-MOE, and 2′-O—CH₃, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a 4′-(CH2)n-O-2′ bridge, where n=1 or n=2). In general, each distinct region comprises uniform sugar moieties. The wing-gap-wing motif is frequently described as “X-Y-Z”, where “X” represents the length of the 5′ wing region, “Y” represents the length of the gap region, and “Z” represents the length of the 3′ wing region.

[0287] In some embodiments, an antisense compound targeted to a PCSK9 nucleic acid has a gap-widened motif. In other embodiments, an antisense oligonucleotide targeted to a PCSK9 nucleic acid has a gap-widened motif.

[0288] The gap-widened antisense oligonucleotides described herein may have various wing-gap-wing motifs selected from: 1-16-1, 2-15-1, 1-15-2, 1-14-3, 3-14-1, 2-14-2, 1-13-4, 4-13-1, 2-13-3, 3-13-2, 1-12-5, 5-12-1, 2-12-4, 4-12-2, 3-12-3, 1-11-6, 6-11-1, 2-11-5, 5-11-2, 3-11-4, 4-11-3, 1-17-1, 2-16-1, 1-16-2, 1-15-3, 3-15-1, 2-15-2, 1-14-4, 4-14-1, 2-14-3, 3-14-2, 1-13-5, 5-13-1, 2-13-4, 4-13-2, 3-13-3, 1-12-6, 6-12-1, 2-12-5, 5-12-2, 3-12-4, 4-12-3, 1-11-7, 7-11-1, 2-11-6, 6-11-2, 3-11-5, 5-11-3, 4-11-4, 1-18-1, 1-17-2, 2-17-1, 1-16-3, 1-16-3, 2-16-2, 1-15-4, 4-15-1, 2-15-3, 3-15-2, 1-14-5, 5-14-1, 2-14-4, 4-14-2, 3-14-3, 1-13-6, 6-13-1, 2-13-5, 5-13-2, 3-13-4, 4-13-3, 1-12-7, 7-12-1, 2-12-6, 6-12-2, 3-12-5, 5-12-3, 4-12-4, 1-11-8, 8-11-1, 2-11-7, 7-11-2, 3-11-6, 6-11-3, 4-11-5, 5-11-4, 1-18-1, 1-17-2, 2-17-1, 1-16-3, 3-16-1, 2-16-2, 1-15-4, 4-15-1, 2-15-3, 3-15-2, 1-14-5, 2-14-4, 4-14-2, 3-14-3, 1-13-6, 6-13-1, 2-13-5, 5-13-2, 3-13-4, 4-13-3, 1-12-7, 7-12-1, 2-12-6, 6-12-2, 3-12-5, 5-12-3, 4-12-4, 1-11-8, 8-11-1, 2-11-7, 7-11-2, 3-11-6, 6-11-3, 4-11-5, 5-11-4, 1-19-1, 1-18-2, 2-18-1, 1-17-3, 3-17-1, 2-17-2, 1-16-4, 4-16-1, 2-16-3, 3-16-2, 1-15-5, 2-15-4, 4-15-2, 3-15-3, 1-14-6, 6-14-1, 2-14-5, 5-14-2, 3-14-4, 4-14-3, 1-13-7, 7-13-1, 2-13-6, 6-13-2, 3-13-5, 5-13-3, 4-13-4, 1-12-8, 8-12-1, 2-12-7, 7-12-2, 3-12-6, 6-12-3, 4-12-5, 5-12-4, 2-11-8, 8-11-2, 3-11-7, 7-11-3, 4-11-6, 6-11-4, 5-11-5, 1-20-1, 1-19-2, 2-19-1, 1-18-3, 3-18-1, 2-18-2, 1-17-4, 4-17-1, 2-17-3, 3-17-2, 1-16-5, 2-16-4, 4-16-2, 3-16-3, 1-15-6, 6-15-1, 2-15-5, 5-15-2, 3-15-4, 4-15-3, 1-14-7, 7-14-1, 2-14-6, 6-14-2, 3-14-5, 5-14-3, 4-14-4, 1-13-8, 8-13-1, 2-13-7, 7-13-2, 3-13-6, 6-13-3, 4-13-5, 5-13-4, 2-12-8, 8-12-2, 3-12-7, 7-12-3, 4-12-6, 6-12-4, 5-12-5, 3-11-8, 8-11-3, 4-11-7, 7-11-4, 5-11-6, 6-11-5, 1-21-1, 1-20-2, 2-20-1, 1-20-3, 3-19-1, 2-19-2, 1-18-4, 4-18-1, 2-18-3, 3-18-2, 1-17-5, 2-17-4, 4-17-2, 3-17-3, 1-16-6, 6-16-1, 2-16-5, 5-16-2, 3-16-4, 4-16-3, 1-15-7, 7-15-1, 2-15-6, 6-15-2, 3-15-5, 5-15-3, 4-15-4, 1-14-8, 8-14-1, 2-14-7, 7-14-2, 3-14-6, 6-14-3, 4-14-5, 5-14-4, 2-13-8, 8-13-2, 3-13-7, 7-13-3, 4-13-6, 6-13-4, 5-13-5, 1-12-10, 10-12-1, 2-12-9, 9-12-2, 3-12-8, 8-12-3, 4-12-7, 7-12-4, 5-12-6, 6-12-5, 4-11-8, 8-11-4, 5-11-7, 7-11-5, 6-11-6, 1-22-1, 1-21-2, 2-21-1, 1-21-3, 3-20-1, 2-20-2, 1-19-4, 4-19-1, 2-19-3, 3-19-2, 1-18-5, 2-18-4, 4-18-2, 3-18-3, 1-17-6, 6-17-1, 2-17-5, 5-17-2, 3-17-4, 4-17-3, 1-16-7, 7-16-1, 2-16-6, 6-16-2, 3-16-5, 5-16-3, 4-16-4, 1-15-8, 8-15-1, 2-15-7, 7-15-2, 3-15-6, 6-15-3, 4-15-5, 5-15-4, 2-14-8, 8-14-2, 3-14-7, 7-14-3, 4-14-6, 6-14-4, 5-14-5, 3-13-8, 8-13-3, 4-13-7, 7-13-4, 5-13-6, 6-13-5, 4-12-8, 8-12-4, 5-12-7, 7-12-5, 6-12-6, 5-11-8, 8-11-5, 6-11-7, or 7-11-6. In certain preferred embodiments, a gap-widened motif includes, but is not limited to, 2-13-5, 3-14-3, 3-14-4 gapmer motif.

[0289] In one embodiment, a gap-widened antisense oligonucleotide targeted to a PCSK9 nucleic acid has a gap segment of fourteen 2′-deoxyribonucleotides positioned between wing segments of three chemically modified nucleosides. In one embodiment, the chemical modification comprises a 2′-sugar modification. In another embodiment, the chemical modification comprises a 2′-MOE sugar modification.

[0290] In one embodiment, antisense compounds targeted to a PCSK9 nucleic acid possess a 5-10-5 gapmer motif.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

[0291] Nucleotide sequences that encode PCSK9 include, without limitation, the following: GENBANK® Accession No. NM_—174936.2, first deposited with GENBANK® on Jun. 1, 2003, and incorporated herein as SEQ ID NO: 1; nucleotides 25475000 to 25504000 of GENBANK® Accession No. NT_—032977.8, first deposited with GENBANK® on Feb. 26, 2006, and incorporated herein as SEQ ID NO: 2; and GENBANK® Accession No. AK124635.1, first deposited with GENBANK® on Sep. 8, 2003, and incorporated herein as SEQ ID NO: 3.

[0292] It is noted that some portions of these nucleotide sequences share identical sequence. For example, portions of SEQ ID NO: 1 are identical to portions of SEQ ID NO: 2; portions of SEQ ID NO: 1 are identical to portions of SEQ ID NO: 3; and portions of SEQ ID NO: 2 are identical to portions of SEQ ID NO: 3. Accordingly, antisense compounds targeted to SEQ ID NO: 1 may also target SEQ ID NO: 2 and/or SEQ ID NO: 3; antisense compounds targeted to SEQ ID NO: 2 may also target SEQ ID NO: 1 and/or SEQ ID NO: 3; and antisense compounds targeted to SEQ ID NO: 3 may also target SEQ ID NO: 1 and/or SEQ ID NO: 2. Examples of such antisense compounds are shown in the following tables.

[0293] In certain embodiments, antisense compounds target a PCSK9 nucleic acid having the sequence of GENBANK® Accession No. NM_—174936.2, first deposited with GENBANK® on Jun. 1, 2003, and incorporated herein as SEQ ID NO: 1. In certain such embodiments, an antisense oligonucleotide targets SEQ ID NO: 1. In certain such embodiments, an antisense oligonucleotide that is targeted to SEQ ID NO: 1 is at least 90% complementary to SEQ ID NO: 1. In certain such embodiments, an antisense oligonucleotide that is targeted to SEQ ID NO: 1 is at least 95% complementary to SEQ ID NO: 1. In certain such embodiments, an antisense oligonucleotide that is targeted to SEQ ID NO: 1 is 100% complementary to SEQ ID NO: 1. In certain embodiments, an antisense oligonucleotide targeted to SEQ ID NO: 1 comprises a nucleotide sequence selected from the nucleotide sequences set forth in Table 2.

[0294]

TABLE 2
Nucleotide sequences targeted to
NM_174936.2 (SEQ ID NO: 1)
	5′ Target	3′ Target
	Site on	Site on
SEQ ID	SEQ ID	SEQ ID
NO	NO: 1	NO: 1	Sequence (5′-3′)
4	135	154	GCGCGGAATCCTGGCTGGGA
5	242	261	GAGGAGACCTAGAGGCCGTG
159	294	313	GCCTGGAGCTGACGGTGCCC
160	298	317	GACCGCCTGGAGCTGACGGT
6	300	319	AGGACCGCCTGGAGCTGACG
162	406	425	AAGGCTAGCACCAGCTCCTC
163	407	426	CAAGGCTAGCACCAGCTCCT
164	408	427	GCAAGGCTAGCACCAGCTCC
165	409	428	CGCAAGGCTAGCACCAGCTC
7	410	429	ACGCAAGGCTAGCACCAGCT
166	411	430	AACGCAAGGCTAGCACCAGC
167	412	431	GAACGCAAGGCTAGCACCAG
168	413	432	GGAACGCAAGGCTAGCACCA
169	414	433	CGGAACGCAAGGCTAGCACC
8	417	436	CCTCGGAACGCAAGGCTAGC
170	421	440	TCCTCCTCGGAACGCAAGGC
171	446	465	GTGCTCGGGTGCTTCGGCCA
172	466	485	TGGAAGGTGGCTGTGGTTCC
9	480	499	CCTTGGCGCAGCGGTGGAAG
173	482	501	ATCCTTGGCGCAGCGGTGGA
174	484	503	GGATCCTTGGCGCAGCGGTG
175	488	507	CCACGGATCCTTGGCGCAGC
176	507	526	CGTAGGTGCCAGGCAACCTC
177	545	564	TGACTGCGAGAGGTGGGTCT
178	555	574	CAGTGCGCTCTGACTGCGAG
179	557	576	GGCAGTGCGCTCTGACTGCG
180	559	578	CGGGCAGTGCGCTCTGACTG
10	561	580	GGCGGGCAGTGCGCTCTGAC
181	562	581	CGGCGGGCAGTGCGCTCTGA
182	591	610	ATCCCCGGCGGGCAGCCTGG
183	595	614	AGGTATCCCCGGCGGGCAGC
184	597	616	TGAGGTATCCCCGGCGGGCA
185	598	617	GTGAGGTATCCCCGGCGGGC
186	599	618	GGTGAGGTATCCCCGGCGGG
11	600	619	TGGTGAGGTATCCCCGGCGG
187	601	620	TTGGTGAGGTATCCCCGGCG
188	602	621	CTTGGTGAGGTATCCCCGGC
189	603	622	TCTTGGTGAGGTATCCCCGG
190	604	623	ATCTTGGTGAGGTATCCCCG
191	605	624	GATCTTGGTGAGGTATCCCC
12	606	625	GGATCTTGGTGAGGTATCCC
192	607	626	AGGATCTTGGTGAGGTATCC
193	609	628	GCAGGATCTTGGTGAGGTAT
194	611	630	ATGCAGGATCTTGGTGAGGT
195	613	632	ACATGCAGGATCTTGGTGAG
13	615	634	AGACATGCAGGATCTTGGTG
196	617	636	GAAGACATGCAGGATCTTGG
14	620	639	ATGGAAGACATGCAGGATCT
197	628	647	AGAAGGCCATGGAAGACATG
198	638	657	GAAGCCAGGAAGAAGGCCAT
15	646	665	TTCACCAGGAAGCCAGGAAG
199	648	667	TCTTCACCAGGAAGCCAGGA
16	651	670	TCATCTTCACCAGGAAGCCA
200	653	672	ACTCATCTTCACCAGGAAGC
201	655	674	CCACTCATCTTCACCAGGAA
202	657	676	CGCCACTCATCTTCACCAGG
203	659	678	GTCGCCACTCATCTTCACCA
204	661	680	AGGTCGCCACTCATCTTCAC
205	663	682	GCAGGTCGCCACTCATCTTC
206	665	684	CAGCAGGTCGCCACTCATCT
207	667	686	TCCAGCAGGTCGCCACTCAT
208	685	704	GGCAACTTCAAGGCCAGCTC
17	705	724	CCTCGATGTAGTCGACATGG
209	724	743	GCAAAGACAGAGGAGTCCTC
210	782	801	TTCATCCGCCCGGTACCGTG
211	784	803	TATTCATCCGCCCGGTACCG
18	785	804	GTATTCATCCGCCCGGTACC
212	787	806	TGGTATTCATCCGCCCGGTA
213	789	808	GCTGGTATTCATCCGCCCGG
214	791	810	GGGCTGGTATTCATCCGCCC
215	821	840	ATACACCTCCACCAGGCTGC
216	832	851	GTGTCTAGGAGATACACCTC
19	835	854	CTGGTGTCTAGGAGATACAC
217	837	856	TGCTGGTGTCTAGGAGATAC
20	840	859	GTATGCTGGTGTCTAGGAGA
21	860	879	GATTTCCCGGTGGTCACTCT
218	862	881	TCGATTTCCCGGTGGTCACT
219	863	882	CTCGATTTCCCGGTGGTCAC
220	864	883	CCTCGATTTCCCGGTGGTCA
221	865	884	CCCTCGATTTCCCGGTGGTC
22	866	885	GCCCTCGATTTCCCGGTGGT
222	867	886	TGCCCTCGATTTCCCGGTGG
223	868	887	CTGCCCTCGATTTCCCGGTG
224	869	888	CCTGCCCTCGATTTCCCGGT
225	870	889	CCCTGCCCTCGATTTCCCGG
226	874	893	ATGACCCTGCCCTCGATTTC
227	876	895	CCATGACCCTGCCCTCGATT
228	878	897	GACCATGACCCTGCCCTCGA
23	880	899	GTGACCATGACCCTGCCCTC
229	882	901	CGGTGACCATGACCCTGCCC
230	884	903	GTCGGTGACCATGACCCTGC
231	886	905	AAGTCGGTGACCATGACCCT
232	888	907	CGAAGTCGGTGACCATGACC
24	890	909	CTCGAAGTCGGTGACCATGA
233	898	917	GGCACATTCTCGAAGTCGGT
25	923	942	GTGGAAGCGGGTCCCGTCCT
234	933	952	TGGCCTGTCTGTGGAAGCGG
235	960	979	GGTGGGTGCCATGACTGTCA
236	963	982	CCAGGTGGGTGCCATGACTG
237	967	986	CCTGCCAGGTGGGTGCCATG
26	970	989	ACCCCTGCCAGGTGGGTGCC
238	972	991	CCACCCCTGCCAGGTGGGTG
27	975	994	TGACCACCCCTGCCAGGTGG
239	977	996	GCTGACCACCCCTGCCAGGT
240	985	1004	TCCCGGCCGCTGACCACCCC
241	989	1008	GGCATCCCGGCCGCTGACCA
242	992	1011	GCCGGCATCCCGGCCGCTGA
243	997	1016	GCCACGCCGGCATCCCGGCC
244	998	1017	GGCCACGCCGGCATCCCGGC
245	999	1018	TGGCCACGCCGGCATCCCGG
246	1000	1019	TTGGCCACGCCGGCATCCCG
247	1001	1020	CTTGGCCACGCCGGCATCCC
248	1002	1021	CCTTGGCCACGCCGGCATCC
249	1003	1022	CCCTTGGCCACGCCGGCATC
28	1004	1023	ACCCTTGGCCACGCCGGCAT
447	1004	1023	ACCCTTGGTCACGCCGGCAT
250	1005	1024	CACCCTTGGCCACGCCGGCA
251	1006	1025	GCACCCTTGGCCACGCCGGC
252	1007	1026	GGCACCCTTGGCCACGCCGG
253	1008	1027	TGGCACCCTTGGCCACGCCG
254	1009	1028	CTGGCACCCTTGGCCACGCC
255	1010	1029	GCTGGCACCCTTGGCCACGC
256	1015	1034	CGCATGCTGGCACCCTTGGC
257	1036	1055	CAGTTGAGCACGCGCAGGCT
258	1038	1057	GGCAGTTGAGCACGCGCAGG
29	1040	1059	TTGGCAGTTGAGCACGCGCA
259	1042	1061	CCTTGGCAGTTGAGCACGCG
30	1045	1064	TTCCCTTGGCAGTTGAGCAC
260	1047	1066	CCTTCCCTTGGCAGTTGAGC
261	1051	1070	GTGCCCTTCCCTTGGCAGTT
262	1053	1072	CCGTGCCCTTCCCTTGGCAG
263	1064	1083	GGTGCCGCTAACCGTGCCCT
264	1076	1095	CAGGCCTATGAGGGTGCCGC
31	1077	1096	CCAGGCCTATGAGGGTGCCG
458	1079	1092	GCCTATGAGGGTGC
459	1084	1097	TCCAGGCCTATGAG
265	1088	1107	CCGAATAAACTCCAGGCCTA
266	1096	1115	TGGCTTTTCCGAATAAACTC
32	1098	1117	GCTGGCTTTTCCGAATAAAC
267	1100	1119	CAGCTGGCTTTTCCGAATAA
268	1102	1121	ACCAGCTGGCTTTTCCGAAT
269	1104	1123	GGACCAGCTGGCTTTTCCGA
270	1108	1127	GGCTGGACCAGCTGGCTTTT
271	1119	1138	GTGGCCCCACAGGCTGGACC
272	1132	1151	AGCAGCACCACCAGTGGCCC
273	1154	1173	GCTGTACCCACCCGCCAGGG
274	1200	1219	CGACCCCAGCCCTCGCCAGG
33	1210	1229	GTGACCAGCACGACCCCAGC
275	1212	1231	CGGTGACCAGCACGACCCCA
276	1214	1233	AGCGGTGACCAGCACGACCC
277	1216	1235	GCAGCGGTGACCAGCACGAC
278	1218	1237	CGGCAGCGGTGACCAGCACG
279	1219	1238	CCGGCAGCGGTGACCAGCAC
280	1222	1241	TTGCCGGCAGCGGTGACCAG
281	1224	1243	AGTTGCCGGCAGCGGTGACC
282	1226	1245	GAAGTTGCCGGCAGCGGTGA
283	1228	1247	CGGAAGTTGCCGGCAGCGGT
284	1230	1249	CCCGGAAGTTGCCGGCAGCG
285	1232	1251	GTCCCGGAAGTTGCCGGCAG
286	1273	1292	ATGACCTCGGGAGCTGAGGC
287	1283	1302	CCCAACTGTGATGACCTCGG
288	1295	1314	GGCATTGGTGGCCCCAACTG
149	1297	1316	TGGGCATTGGTGGCCCCAAC
289	1305	1324	GCTGGTCTTGGGCATTGGTG
290	1318	1337	CCCAGGGTCACCGGCTGGTC
291	1320	1339	TCCCCAGGGTCACCGGCTGG
292	1322	1341	AGTCCCCAGGGTCACCGGCT
293	1324	1343	AAAGTCCCCAGGGTCACCGG
34	1326	1345	CCAAAGTCCCCAGGGTCACC
294	1328	1347	CCCCAAAGTCCCCAGGGTCA
128	1330	1349	GTCCCCAAAGTCCCCAGGGT
295	1333	1352	TTGGTCCCCAAAGTCCCCAG
35	1335	1354	AGTTGGTCCCCAAAGTCCCC
296	1337	1356	AAAGTTGGTCCCCAAAGTCC
36	1340	1359	GCCAAAGTTGGTCCCCAAAG
297	1342	1361	CGGCCAAAGTTGGTCCCCAA
298	1344	1363	AGCGGCCAAAGTTGGTCCCC
299	1346	1365	ACAGCGGCCAAAGTTGGTCC
300	1348	1367	ACACAGCGGCCAAAGTTGGT
301	1350	1369	CCACACAGCGGCCAAAGTTG
37	1352	1371	GTCCACACAGCGGCCAAAGT
302	1354	1373	AGGTCCACACAGCGGCCAAA
303	1356	1375	AGAGGTCCACACAGCGGCCA
304	1358	1377	AAAGAGGTCCACACAGCGGC
38	1361	1380	GGCAAAGAGGTCCACACAGC
305	1380	1399	CAATGATGTCCTCCCCTGGG
306	1387	1406	GAGGCACCAATGATGTCCTC
39	1389	1408	TGGAGGCACCAATGATGTCC
307	1391	1410	GCTGGAGGCACCAATGATGT
308	1393	1412	TCGCTGGAGGCACCAATGAT
309	1395	1414	AGTCGCTGGAGGCACCAATG
310	1397	1416	GCAGTCGCTGGAGGCACCAA
40	1400	1419	GCTGCAGTCGCTGGAGGCAC
311	1402	1421	GTGCTGCAGTCGCTGGAGGC
312	1404	1423	AGGTGCTGCAGTCGCTGGAG
313	1406	1425	GCAGGTGCTGCAGTCGCTGG
314	1407	1426	AGCAGGTGCTGCAGTCGCTG
315	1409	1428	AAAGCAGGTGCTGCAGTCGC
41	1411	1430	ACAAAGCAGGTGCTGCAGTC
316	1413	1432	ACACAAAGCAGGTGCTGCAG
317	1415	1434	TGACACAAAGCAGGTGCTGC
318	1425	1444	TCCCACTCTGTGACACAAAG
101	1465	1484	ATGGCTGCAATGCCAGCCAC
319	1467	1486	TCATGGCTGCAATGCCAGCC
42	1470	1489	GCATCATGGCTGCAATGCCA
320	1472	1491	CAGCATCATGGCTGCAATGC
321	1474	1493	GACAGCATCATGGCTGCAAT
322	1476	1495	CAGACAGCATCATGGCTGCA
43	1478	1497	GGCAGACAGCATCATGGCTG
323	1480	1499	TCGGCAGACAGCATCATGGC
324	1482	1501	GCTCGGCAGACAGCATCATG
325	1484	1503	CGGCTCGGCAGACAGCATCA
326	1486	1505	TCCGGCTCGGCAGACAGCAT
327	1500	1519	CGGCCAGGGTGAGCTCCGGC
328	1513	1532	CTCTGCCTCAACTCGGCCAG
329	1515	1534	GTCTCTGCCTCAACTCGGCC
330	1517	1536	CAGTCTCTGCCTCAACTCGG
331	1519	1538	ATCAGTCTCTGCCTCAACTC
332	1521	1540	GGATCAGTCTCTGCCTCAAC
333	1523	1542	GTGGATCAGTCTCTGCCTCA
334	1525	1544	AAGTGGATCAGTCTCTGCCT
44	1526	1545	GAAGTGGATCAGTCTCTGCC
335	1528	1547	GAGAAGTGGATCAGTCTCTG
336	1530	1549	CAGAGAAGTGGATCAGTCTC
337	1532	1551	GGCAGAGAAGTGGATCAGTC
45	1534	1553	TTGGCAGAGAAGTGGATCAG
338	1536	1555	CTTTGGCAGAGAAGTGGATC
46	1539	1558	CATCTTTGGCAGAGAAGTGG
339	1541	1560	GACATCTTTGGCAGAGAAGT
340	1543	1562	ATGACATCTTTGGCAGAGAA
47	1545	1564	TGATGACATCTTTGGCAGAG
341	1547	1566	ATTGATGACATCTTTGGCAG
342	1549	1568	TCATTGATGACATCTTTGGC
48	1552	1571	GCCTCATTGATGACATCTTT
343	1554	1573	AGGCCTCATTGATGACATCT
344	1556	1575	CCAGGCCTCATTGATGACAT
345	1558	1577	AACCAGGCCTCATTGATGAC
346	1560	1579	GGAACCAGGCCTCATTGATG
347	1561	1580	GGGAACCAGGCCTCATTGAT
348	1562	1581	AGGGAACCAGGCCTCATTGA
349	1563	1582	CAGGGAACCAGGCCTCATTG
49	1564	1583	TCAGGGAACCAGGCCTCATT
49	1564	1583	TCAGGGAACCAGGCCTCATT
350	1565	1584	CTCAGGGAACCAGGCCTCAT
351	1566	1585	CCTCAGGGAACCAGGCCTCA
352	1567	1586	TCCTCAGGGAACCAGGCCTC
353	1568	1587	GTCCTCAGGGAACCAGGCCT
50	1569	1588	GGTCCTCAGGGAACCAGGCC
354	1570	1589	TGGTCCTCAGGGAACCAGGC
355	1571	1590	CTGGTCCTCAGGGAACCAGG
356	1572	1591	GCTGGTCCTCAGGGAACCAG
357	1573	1592	CGCTGGTCCTCAGGGAACCA
87	1576	1595	ACCCGCTGGTCCTCAGGGAA
358	1578	1597	GTACCCGCTGGTCCTCAGGG
359	1580	1599	CAGTACCCGCTGGTCCTCAG
51	1583	1602	GGTCAGTACCCGCTGGTCCT
119	1605	1624	GCAGGGCGGCCACCAGGTTG
360	1628	1647	ACCTGCCCCATGGGTGCTGG
52	1640	1659	AAACAGCTGCCAACCTGCCC
361	1642	1661	CAAAACAGCTGCCAACCTGC
53	1645	1664	CTGCAAAACAGCTGCCAACC
362	1647	1666	TCCTGCAAAACAGCTGCCAA
363	1649	1668	AGTCCTGCAAAACAGCTGCC
364	1660	1679	GCTGACCATACAGTCCTGCA
365	1672	1691	GGCCCCGAGTGTGCTGACCA
54	1675	1694	GTAGGCCCCGAGTGTGCTGA
366	1677	1696	GTGTAGGCCCCGAGTGTGCT
367	1679	1698	CCGTGTAGGCCCCGAGTGTG
368	1681	1700	ATCCGTGTAGGCCCCGAGTG
369	1683	1702	CCATCCGTGTAGGCCCCGAG
370	1685	1704	GGCCATCCGTGTAGGCCCCG
371	1687	1706	GTGGCCATCCGTGTAGGCCC
372	1735	1754	CTGGAGCAGCTCAGCAGCTC
460	1735	1748	CAGCTCAGCAGCTC
373	1737	1756	AACTGGAGCAGCTCAGCAGC
55	1740	1759	AGAAACTGGAGCAGCTCAGC
374	1742	1761	GGAGAAACTGGAGCAGCTCA
375	1744	1763	CTGGAGAAACTGGAGCAGCT
56	1746	1765	TCCTGGAGAAACTGGAGCAG
57	1812	1831	CGTTGTGGGCCCGGCAGACC
376	1849	1868	CACCTGGCAATGGCGTAGAC
377	1850	1869	GCACCTGGCAATGGCGTAGA
378	1851	1870	AGCACCTGGCAATGGCGTAG
379	1852	1871	CAGCACCTGGCAATGGCGTA
380	1853	1872	GCAGCACCTGGCAATGGCGT
381	1854	1873	GGCAGCACCTGGCAATGGCG
382	1855	1874	AGGCAGCACCTGGCAATGGC
383	1856	1875	CAGGCAGCACCTGGCAATGG
384	1857	1876	GCAGGCAGCACCTGGCAATG
58	1858	1877	AGCAGGCAGCACCTGGCAAT
385	1859	1878	TAGCAGGCAGCACCTGGCAA
386	1860	1879	GTAGCAGGCAGCACCTGGCA
387	1905	1924	TGGCCTCAGCTGGTGGAGCT
388	1915	1934	GTCCCCATGCTGGCCTCAGC
389	1916	1935	GGTCCCCATGCTGGCCTCAG
390	1917	1936	GGGTCCCCATGCTGGCCTCA
391	1918	1937	CGGGTCCCCATGCTGGCCTC
392	1919	1938	ACGGGTCCCCATGCTGGCCT
59	1920	1939	CACGGGTCCCCATGCTGGCC
59	1920	1939	CACGGGTCCCCATGCTGGCC
393	1921	1940	ACACGGGTCCCCATGCTGGC
394	1922	1941	GACACGGGTCCCCATGCTGG
395	1923	1942	GGACACGGGTCCCCATGCTG
396	1924	1943	TGGACACGGGTCCCCATGCT
397	1925	1944	GTGGACACGGGTCCCCATGC
398	1926	1945	AGTGGACACGGGTCCCCATG
399	1927	1946	CAGTGGACACGGGTCCCCAT
400	1928	1947	GCAGTGGACACGGGTCCCCA
401	1929	1948	GGCAGTGGACACGGGTCCCC
402	1930	1949	TGGCAGTGGACACGGGTCCC
403	1931	1950	GTGGCAGTGGACACGGGTCC
404	1932	1951	GGTGGCAGTGGACACGGGTC
405	1933	1952	TGGTGGCAGTGGACACGGGT
406	1936	1955	TGTTGGTGGCAGTGGACACG
407	1962	1981	AGCTGCAGCCTGTGAGGACG
408	1990	2009	GTGCCAAGGTCCTCCACCTC
409	2010	2029	TCAGCACAGGCGGCTTGTGG
410	2040	2059	CCACGCACTGGTTGGGCTGA
60	2100	2119	CTTTGCATTCCAGACCTGGG
411	2101	2120	ACTTTGCATTCCAGACCTGG
412	2102	2121	GACTTTGCATTCCAGACCTG
413	2103	2122	TGACTTTGCATTCCAGACCT
414	2104	2123	TTGACTTTGCATTCCAGACC
61	2105	2124	CTTGACTTTGCATTCCAGAC
415	2107	2126	TCCTTGACTTTGCATTCCAG
416	2120	2139	CGGGATTCCATGCTCCTTGA
417	2150	2169	GCAGGCCACGGTCACCTGCT
418	2187	2206	GGAGGGCACTGCAGCCAGTC
419	2305	2324	CAGATGGCAACGGCTGTCAC
420	2306	2325	GCAGATGGCAACGGCTGTCA
421	2307	2326	AGCAGATGGCAACGGCTGTC
422	2308	2327	CAGCAGATGGCAACGGCTGT
423	2309	2328	GCAGCAGATGGCAACGGCTG
62	2310	2329	GGCAGCAGATGGCAACGGCT
424	2311	2330	CGGCAGCAGATGGCAACGGC
425	2312	2331	CCGGCAGCAGATGGCAACGG
426	2313	2332	TCCGGCAGCAGATGGCAACG
427	2314	2333	CTCCGGCAGCAGATGGCAAC
428	2315	2334	GCTCCGGCAGCAGATGGCAA
429	2325	2344	CCAGGTGCCGGCTCCGGCAG
430	2335	2354	GAGGCCTGCGCCAGGTGCCG
154	2410	2429	TTTTAAAGCTCAGCCCCAGC
63	2415	2434	AACCATTTTAAAGCTCAGCC
64	2504	2523	TCAAGGGCCAGGCCAGCAGC
65	2509	2528	CCCACTCAAGGGCCAGGCCA
122	2582	2601	GGAGGGAGCTTCCTGGCACC
66	2597	2616	ATGCCCCACAGTGAGGGAGG
67	2606	2625	AATGGTGAAATGCCCCACAG
153	2649	2668	TTGGGAGCAGCTGGCAGCAC
68	2750	2769	CATGGGAAGAATCCTGCCTC
431	2828	2847	ATGAGGGCCATCAGCACCTT
432	2829	2848	GATGAGGGCCATCAGCACCT
433	2830	2849	AGATGAGGGCCATCAGCACC
434	2831	2850	GAGATGAGGGCCATCAGCAC
69	2832	2851	GGAGATGAGGGCCATCAGCA
435	2833	2852	TGGAGATGAGGGCCATCAGC
436	2834	2853	CTGGAGATGAGGGCCATCAG
437	2835	2854	GCTGGAGATGAGGGCCATCA
438	2836	2855	AGCTGGAGATGAGGGCCATC
461	2877	2890	TTAATCAGGGAGCC
70	2900	2919	TAGATGCCATCCAGAAAGCT
439	2902	2921	GCTAGATGCCATCCAGAAAG
440	2903	2922	GGCTAGATGCCATCCAGAAA
441	2904	2923	TGGCTAGATGCCATCCAGAA
442	2905	2924	CTGGCTAGATGCCATCCAGA
71	2906	2925	TCTGGCTAGATGCCATCCAG
443	2907	2926	CTCTGGCTAGATGCCATCCA
444	2908	2927	CCTCTGGCTAGATGCCATCC
445	2909	2928	GCCTCTGGCTAGATGCCATC
446	2910	2929	AGCCTCTGGCTAGATGCCAT
72	2983	3002	GGCATAGAGCAGAGTAAAGG
73	2988	3007	AGCCTGGCATAGAGCAGAGT
135	2994	3013	TAGCACAGCCTGGCATAGAG
112	3227	3246	GAAGAGGCTTGGCTTCAGAG
74	3233	3252	AAGTAAGAAGAGGCTTGGCT
75	3437	3456	GCTCAAGGAGGGACAGTTGT
76	3472	3491	AAAGATAAATGTCTGCTTGC
77	3477	3496	ACCCAAAAGATAAATGTCTG
78	3543	3562	TCTTCAAGTTACAAAAGCAA
99	3550	3569	ATAAATATCTTCAAGTTACA

[0295] In certain embodiments, gapmer antisense compounds are targeted to a PCSK9 nucleic acid. In certain such embodiments, gapmer antisense compounds are targeted to SEQ ID NO: 1. In certain such embodiments, the nucleotide sequences illustrated in Table 2 have a 5-10-5 gapmer motif. Table 3 illustrates gapmer antisense compounds targeted to SEQ ID NO: 1, having a 5-10-5 motif, where the gap segment comprises 2′-deoxynucleotides and each wing segment comprises nucleotides comprising a 2′-O-methoxyethyl sugar modification. Internucleoside linkages are phosphorthioate, and cytidines are 5-methylcytidines.

[0296]

TABLE 3
Gapmer antisense compounds having a 5-10-5
motif targeted to SEQ ID NO: 1
			5′ Target	3′ Target
		SEQ	Site on	Site on
		ID	SEQ ID	SEQ ID
Isis No	Motif	NO	NO: 1	NO: 1	Mismatches	Sequence (5′-3′)
395149	5-10-5	4	135	154	0	GCGCGGAATCCTGGCTGGGA
395150	5-10-5	5	242	261	0	GAGGAGACCTAGAGGCCGTG
395151	5-10-5	6	300	319	0	AGGACCGCCTGGAGCTGACG
395152	5-10-5	7	410	429	0	ACGCAAGGCTAGCACCAGCT
399793	5-10-5	8	417	436	0	CCTCGGAACGCAAGGCTAGC
395153	5-10-5	9	480	499	0	CCTTGGCGCAGCGGTGGAAG
395154	5-10-5	10	561	580	0	GGCGGGCAGTGCGCTCTGAC
395155	5-10-5	11	600	619	0	TGGTGAGGTATCCCCGGCGG
399794	5-10-5	12	606	625	0	GGATCTTGGTGAGGTATCCC
399795	5-10-5	13	615	634	0	AGACATGCAGGATCTTGGTG
395156	5-10-5	14	620	639	0	ATGGAAGACATGCAGGATCT
395157	5-10-5	15	646	665	0	TTCACCAGGAAGCCAGGAAG
399796	5-10-5	16	651	670	0	TCATCTTCACCAGGAAGCCA
395158	5-10-5	17	705	724	0	CCTCGATGTAGTCGACATGG
395159	5-10-5	18	785	804	0	GTATTCATCCGCCCGGTACC
395160	5-10-5	19	835	854	0	CTGGTGTCTAGGAGATACAC
399797	5-10-5	20	840	859	0	GTATGCTGGTGTCTAGGAGA
395161	5-10-5	21	860	879	0	GATTTCCCGGTGGTCACTCT
399798	5-10-5	22	866	885	0	GCCCTCGATTTCCCGGTGGT
399799	5-10-5	23	880	899	0	GTGACCATGACCCTGCCCTC
395162	5-10-5	24	890	909	0	CTCGAAGTCGGTGACCATGA
395163	5-10-5	25	923	942	0	GTGGAAGCGGGTCCCGTCCT
395164	5-10-5	26	970	989	0	ACCCCTGCCAGGTGGGTGCC
399800	5-10-5	27	975	994	0	TGACCACCCCTGCCAGGTGG
395165	5-10-5	28	1004	1023	0	ACCCTTGGCCACGCCGGCAT
395166	5-10-5	29	1040	1059	0	TTGGCAGTTGAGCACGCGCA
399801	5-10-5	30	1045	1064	0	TTCCCTTGGCAGTTGAGCAC
395167	5-10-5	31	1077	1096	0	CCAGGCCTATGAGGGTGCCG
395168	5-10-5	32	1098	1117	0	GCTGGCTTTTCCGAATAAAC
395169	5-10-5	33	1210	1229	0	GTGACCAGCACGACCCCAGC
395170	5-10-5	149	1297	1316	0	TGGGCATTGGTGGCCCCAAC
395171	5-10-5	34	1326	1345	0	CCAAAGTCCCCAGGGTCACC
395172	5-10-5	128	1330	1349	0	GTCCCCAAAGTCCCCAGGGT
399802	5-10-5	35	1335	1354	0	AGTTGGTCCCCAAAGTCCCC
395173	5-10-5	36	1340	1359	0	GCCAAAGTTGGTCCCCAAAG
399803	5-10-5	37	1352	1371	0	GTCCACACAGCGGCCAAAGT
395174	5-10-5	38	1361	1380	0	GGCAAAGAGGTCCACACAGC
395175	5-10-5	39	1389	1408	0	TGGAGGCACCAATGATGTCC
399804	5-10-5	40	1400	1419	0	GCTGCAGTCGCTGGAGGCAC
399805	5-10-5	41	1411	1430	0	ACAAAGCAGGTGCTGCAGTC
395176	5-10-5	101	1465	1484	0	ATGGCTGCAATGCCAGCCAC
399806	5-10-5	42	1470	1489	0	GCATCATGGCTGCAATGCCA
399807	5-10-5	43	1478	1497	0	GGCAGACAGCATCATGGCTG
399808	5-10-5	44	1526	1545	0	GAAGTGGATCAGTCTCTGCC
395177	5-10-5	45	1534	1553	0	TTGGCAGAGAAGTGGATCAG
399809	5-10-5	46	1539	1558	0	CATCTTTGGCAGAGAAGTGG
399810	5-10-5	47	1545	1564	0	TGATGACATCTTTGGCAGAG
399811	5-10-5	48	1552	1571	0	GCCTCATTGATGACATCTTT
399812	5-10-5	49	1564	1583	0	TCAGGGAACCAGGCCTCATT
395178	5-10-5	50	1569	1588	0	GGTCCTCAGGGAACCAGGCC
395179	5-10-5	87	1576	1595	0	ACCCGCTGGTCCTCAGGGAA
399813	5-10-5	51	1583	1602	0	GGTCAGTACGCGCTGGTCCT
395180	5-10-5	119	1605	1624	0	GCAGGGCGGCCACCAGGTTG
395181	5-10-5	52	1640	1659	0	AAACAGCTGCCAACCTGCCC
399814	5-10-5	53	1645	1664	0	CTGCAAAACAGCTGCCAACC
395182	5-10-5	54	1675	1694	0	GTAGGCCCCGAGTGTGCTGA
399815	5-10-5	55	1740	1759	0	AGAAACTGGAGCAGCTCAGC
399816	5-10-5	56	1746	1765	0	TCCTGGAGAAACTGGAGCAG
395183	5-10-5	57	1812	1831	0	CGTTGTGGGCCCGGCAGACC
395184	5-10-5	58	1858	1877	0	AGCAGGCAGCACCTGGCAAT
395185	5-10-5	59	1920	1939	0	CACGGGTCCCCATGCTGGCC
395186	5-10-5	60	2100	2119	0	CTTTGCATTCCAGACCTGGG
399817	5-10-5	61	2105	2124	0	CTTGACTTTGCATTCCAGAC
395187	5-10-5	62	2310	2329	0	GGCAGCAGATGGCAACGGCT
395188	5-10-5	154	2410	2429	0	TTTTAAAGCTCAGCCCCAGC
399818	5-10-5	63	2415	2434	0	AACCATTTTAAAGCTCAGCC
395189	5-10-5	64	2504	2523	0	TCAAGGGCCAGGCCAGCAGC
399819	5-10-5	65	2509	2528	0	CCCACTCAAGGGCCAGGCCA
399820	5-10-5	122	2582	2601	0	GGAGGGAGCTTCCTGGCACC
395190	5-10-5	66	2597	2616	0	ATGCCCCACAGTGAGGGAGG
395191	5-10-5	67	2606	2625	0	AATGGTGAAATGCCCCACAG
395192	5-10-5	153	2649	2668	0	TTGGGAGCAGCTGGCAGCAC
395193	5-10-5	68	2750	2769	0	CATGGGAAGAATCCTGCCTC
395194	5-10-5	69	2832	2851	0	GGAGATGAGGGCCATCAGCA
395195	5-10-5	70	2900	2919	0	TAGATGCCATCCAGAAAGCT
399821	5-10-5	71	2906	2925	0	TCTGGCTAGATGCCATCCAG
395196	5-10-5	72	2983	3002	0	GGCATAGAGCAGAGTAAAGG
399822	5-10-5	73	2988	3007	0	AGCCTGGCATAGAGCAGAGT
399823	5-10-5	135	2994	3013	0	TAGCACAGCCTGGCATAGAG
395197	5-10-5	112	3227	3246	0	GAAGAGGCTTGGCTTCAGAG
399824	5-10-5	74	3233	3252	0	AAGTAAGAAGAGGCTTGGCT
395198	5-10-5	75	3437	3456	0	GCTCAAGGAGGGACAGTTGT
395199	5-10-5	76	3472	3491	0	AAAGATAAATGTCTGCTTGC
399825	5-10-5	77	3477	3496	0	ACCCAAAAGATAAATGTCTG
395200	5-10-5	78	3543	3562	0	TCTTCAAGTTACAAAAGCAA
399826	5-10-5	99	3550	3569	0	ATAAATATCTTCAAGTTACA
405861	5-10-5	162	406	425	0	AAGGCTAGCACCAGCTCCTC
405862	5-10-5	163	407	426	0	CAAGGCTAGCACCAGCTCCT
405863	5-10-5	164	408	427	0	GCAAGGCTAGCACCAGCTCC
405864	5-10-5	165	409	428	0	CGCAAGGCTAGCACCAGCTC
405865	5-10-5	166	411	430	0	AACGCAAGGCTAGCACCAGC
405866	5-10-5	167	412	431	0	GAACGCAAGGCTAGCACCAG
405867	5-10-5	168	413	432	0	GGAACGCAAGGCTAGCACCA
405868	5-10-5	169	414	433	0	CGGAACGCAAGGCTAGCACC
405869	5-10-5	218	862	881	0	TCGATTTCCCGGTGGTCACT
405870	5-10-5	219	863	882	0	CTCGATTTCCCGGTGGTCAC
405871	5-10-5	220	864	883	0	CCTCGATTTCCCGGTGGTCA
405872	5-10-5	221	865	884	0	CCCTCGATTTCCCGGTGGTC
405873	5-10-5	222	867	886	0	TGCCCTCGATTTCCCGGTGG
405874	5-10-5	223	868	887	0	CTGCCCTCGATTTCCCGGTG
405875	5-10-5	224	869	888	0	CCTGCCCTCGATTTCCCGGT
405876	5-10-5	225	870	889	0	CCCTGCCCTCGATTTCCCGG
405877	5-10-5	246	1000	1019	0	TTGGCCACGCCGGCATCCCG
405878	5-10-5	247	1001	1020	0	CTTGGCCACGCCGGCATCCC
405879	5-10-5	248	1002	1021	0	CCTTGGCCACGCCGGCATCC
405880	5-10-5	249	1003	1022	0	CCCTTGGCCACGCCGGCATC
405881	5-10-5	250	1005	1024	0	CACCCTTGGCCACGCCGGCA
405882	5-10-5	251	1006	1025	0	GCACCCTTGGCCACGCCGGC
405883	5-10-5	252	1007	1026	0	GGCACCCTTGGCCACGCCGG
405884	5-10-5	253	1008	1027	0	TGGCACCCTTGGCCACGCCG
405885	5-10-5	346	1560	1579	0	GGAACCAGGCCTCATTGATG
405886	5-10-5	347	1561	1580	0	GGGAACCAGGCCTCATTGAT
405887	5-10-5	348	1562	1581	0	AGGGAACCAGGCCTCATTGA
405888	5-10-5	349	1563	1582	0	CAGGGAACCAGGCCTCATTG
405889	5-10-5	350	1565	1584	0	CTCAGGGAACCAGGCCTCAT
405890	5-10-5	351	1566	1585	0	CCTCAGGGAACCAGGCCTCA
405891	5-10-5	352	1567	1586	0	TCCTCAGGGAACCAGGCCTC
405892	5-10-5	353	1568	1587	0	GTCCTCAGGGAACCAGGCCT
405893	5-10-5	431	2828	2847	0	ATGAGGGCCATCAGCACCTT
405894	5-10-5	432	2829	2848	0	GATGAGGGCCATCAGCACCT
405895	5-10-5	433	2830	2849	0	AGATGAGGGCCATCAGCACC
405896	5-10-5	434	2831	2850	0	GAGATGAGGGCCATCAGCAC
405897	5-10-5	435	2833	2852	0	TGGAGATGAGGGCCATCAGC
405898	5-10-5	436	2834	2853	0	CTGGAGATGAGGGCCATCAG
405899	5-10-5	437	2835	2854	0	GCTGGAGATGAGGGCCATCA
405900	5-10-5	438	2836	2855	0	AGCTGGAGATGAGGGCCATC
405901	5-10-5	439	2902	2921	0	GCTAGATGCCATCCAGAAAG
405902	5-10-5	440	2903	2922	0	GGCTAGATGCCATCCAGAAA
405903	5-10-5	441	2904	2923	0	TGGCTAGATGCCATCCAGAA
405904	5-10-5	442	2905	2924	0	CTGGCTAGATGCCATCCAGA
405905	5-10-5	443	2907	2926	0	CTCTGGCTAGATGCCATCCA
405906	5-10-5	444	2908	2927	0	CCTCTGGCTAGATGCCATCC
405907	5-10-5	445	2909	2928	0	GCCTCTGGCTAGATGCCATC
405908	5-10-5	446	2910	2929	0	AGCCTCTGGCTAGATGCCAT
405909	5-10-5	267	1100	1119	0	CAGCTGGCTTTTCCGAATAA
405910	5-10-5	268	1102	1121	0	ACCAGCTGGCTTTTCCGAAT
405911	5-10-5	269	1104	1123	0	GGACCAGCTGGCTTTTCCGA
405912	5-10-5	270	1108	1127	0	GGCTGGACCAGCTGGCTTTT
405913	5-10-5	275	1212	1231	0	CGGTGACCAGCACGACCCCA
405914	5-10-5	276	1214	1233	0	AGCGGTGACCAGCACGACCC
405915	5-10-5	277	1216	1235	0	GCAGCGGTGACCAGCACGAC
405916	5-10-5	278	1218	1237	0	CGGCAGCGGTGACCAGCACG
405917	5-10-5	280	1222	1241	0	TTGCCGGCAGCGGTGACCAG
405918	5-10-5	281	1224	1243	0	AGTTGCCGGCAGCGGTGACC
405919	5-10-5	282	1226	1245	0	GAAGTTGCCGGCAGCGGTGA
405920	5-10-5	283	1228	1247	0	CGGAAGTTGCCGGCAGCGGT
405921	5-10-5	284	1230	1249	0	CCCGGAAGTTGCCGGCAGCG
405922	5-10-5	285	1232	1251	0	GTCCCGGAAGTTGCCGGCAG
405923	5-10-5	288	1295	1314	0	GGCATTGGTGGCCCCAACTG
405924	5-10-5	290	1318	1337	0	CCCAGGGTCACCGGCTGGTC
405925	5-10-5	292	1322	1341	0	AGTCCCCAGGGTCACCGGCT
405926	5-10-5	293	1324	1343	0	AAAGTCCCCAGGGTCACCGG
405927	5-10-5	294	1328	1347	0	CCCCAAAGTCCCCAGGGTCA
405928	5-10-5	295	1333	1352	0	TTGGTCCCCAAAGTCCCCAG
405929	5-10-5	296	1337	1356	0	AAAGTTGGTCCCCAAAGTCC
405930	5-10-5	297	1342	1361	0	CGGCCAAAGTTGGTCCCCAA
405931	5-10-5	298	1344	1363	0	AGCGGCCAAAGTTGGTCCCC
405932	5-10-5	299	1346	1365	0	ACAGCGGCCAAAGTTGGTCC
405933	5-10-5	300	1348	1367	0	ACACAGCGGCCAAAGTTGGT
405934	5-10-5	301	1350	1369	0	CCACACAGCGGCCAAAGTTG
405935	5-10-5	302	1354	1373	0	AGGTCCACACAGCGGCCAAA
405936	5-10-5	304	1358	1377	0	AAAGAGGTCCACACAGCGGC
405937	5-10-5	306	1387	1406	0	GAGGCACCAATGATGTCCTC
405938	5-10-5	307	1391	1410	0	GCTGGAGGCACCAATGATGT
405939	5-10-5	308	1393	1412	0	TCGCTGGAGGCACCAATGAT
405940	5-10-5	309	1395	1414	0	AGTCGCTGGAGGCACCAATG
405941	5-10-5	310	1397	1416	0	GCAGTCGCTGGAGGCACCAA
405942	5-10-5	311	1402	1421	0	GTGCTGCAGTCGCTGGAGGC
405943	5-10-5	312	1404	1423	0	AGGTGCTGCAGTCGCTGGAG
405944	5-10-5	314	1407	1426	0	AGCAGGTGCTGCAGTCGCTG
405945	5-10-5	315	1409	1428	0	AAAGCAGGTGCTGCAGTCGC
405946	5-10-5	316	1413	1432	0	ACACAAAGCAGGTGCTGCAG
405947	5-10-5	317	1415	1434	0	TGACACAAAGCAGGTGCTGC
405948	5-10-5	319	1467	1486	0	TCATGGCTGCAATGCCAGCC
405949	5-10-5	320	1472	1491	0	CAGCATCATGGCTGCAATGC
405950	5-10-5	321	1474	1493	0	GACAGCATCATGGCTGCAAT
405951	5-10-5	322	1476	1495	0	CAGACAGCATCATGGCTGCA
405952	5-10-5	323	1480	1499	0	TCGGCAGACAGCATCATGGC
405953	5-10-5	325	1484	1503	0	CGGCTCGGCAGACAGCATCA
405954	5-10-5	326	1486	1505	0	TCCGGCTCGGCAGACAGCAT
405955	5-10-5	328	1513	1532	0	CTCTGCCTCAACTCGGCCAG
405956	5-10-5	329	1515	1534	0	GTCTCTGCCTCAACTCGGCC
405957	5-10-5	330	1517	1536	0	CAGTCTCTGCCTCAACTCGG
405958	5-10-5	331	1519	1538	0	ATCAGTCTCTGCCTCAACTC
405959	5-10-5	332	1521	1540	0	GGATCAGTCTCTGCCTCAAC
405960	5-10-5	333	1523	1542	0	GTGGATCAGTCTCTGCCTCA
405961	5-10-5	334	1525	1544	0	AAGTGGATCAGTCTCTGCCT
405962	5-10-5	335	1528	1547	0	GAGAAGTGGATCAGTCTCTG
405963	5-10-5	337	1532	1551	0	GGCAGAGAAGTGGATCAGTC
405964	5-10-5	338	1536	1555	0	CTTTGGCAGAGAAGTGGATC
405965	5-10-5	339	1541	1560	0	GACATCTTTGGCAGAGAAGT
405966	5-10-5	340	1543	1562	0	ATGACATCTTTGGCAGAGAA
405967	5-10-5	341	1547	1566	0	ATTGATGACATCTTTGGCAG
405968	5-10-5	342	1549	1568	0	TCATTGATGACATCTTTGGC
405969	5-10-5	343	1554	1573	0	AGGCCTCATTGATGACATCT
405970	5-10-5	344	1556	1575	0	CCAGGCCTCATTGATGACAT
405971	5-10-5	355	1571	1590	0	CTGGTCCTCAGGGAACCAGG
405972	5-10-5	357	1573	1592	0	CGCTGGTCCTCAGGGAACCA
405973	5-10-5	358	1578	1597	0	GTACCCGCTGGTCCTCAGGG
405974	5-10-5	359	1580	1599	0	CAGTACCCGCTGGTCCTCAG
405975	5-10-5	361	1642	1661	0	CAAAACAGCTGCCAACCTGC
405976	5-10-5	362	1647	1666	0	TCCTGCAAAACAGCTGCCAA
405977	5-10-5	363	1649	1668	0	AGTCCTGCAAAACAGCTGCC
405978	5-10-5	365	1672	1691	0	GGCCCCGAGTGTGCTGACCA
405979	5-10-5	366	1677	1696	0	GTGTAGGCCCCGAGTGTGCT
405980	5-10-5	367	1679	1698	0	CCGTGTAGGCCCCGAGTGTG
405981	5-10-5	368	1681	1700	0	ATCCGTGTAGGCCCCGAGTG
405982	5-10-5	370	1685	1704	0	GGCCATCCGTGTAGGCCCCG
405983	5-10-5	371	1687	1706	0	GTGGCCATCCGTGTAGGCCC
405984	5-10-5	372	1735	1754	0	CTGGAGCAGCTCAGCAGCTC
405985	5-10-5	373	1737	1756	0	AACTGGAGCAGCTCAGCAGC
405986	5-10-5	374	1742	1761	0	GGAGAAACTGGAGCAGCTCA
405987	5-10-5	375	1744	1763	0	CTGGAGAAACTGGAGCAGCT
405988	5-10-5	381	1854	1873	0	GGCAGCACCTGGCAATGGCG
405989	5-10-5	383	1856	1875	0	CAGGCAGCACCTGGCAATGG
405990	5-10-5	386	1860	1879	0	GTAGCAGGCAGCACCTGGCA
405991	5-10-5	394	1922	1941	0	GACACGGGTCCCCATGCTGG
405992	5-10-5	396	1924	1943	0	TGGACACGGGTCCCCATGCT
405993	5-10-5	398	1926	1945	0	AGTGGACACGGGTCCCCATG
405994	5-10-5	400	1928	1947	0	GCAGTGGACACGGGTCCCCA
405995	5-10-5	402	1930	1949	0	TGGCAGTGGACACGGGTCCC
405996	5-10-5	412	2102	2121	0	GACTTTGCATTCCAGACCTG
405997	5-10-5	415	2107	2126	0	TCCTTGACTTTGCATTCCAG
405998	5-10-5	426	2313	2332	0	TCCGGCAGCAGATGGCAACG
405999	5-10-5	160	298	317	0	GACCGCCTGGAGCTGACGGT
406000	5-10-5	173	482	501	0	ATCCTTGGCGCAGCGGTGGA
406001	5-10-5	174	484	503	0	GGATCCTTGGCGCAGCGGTG
406002	5-10-5	175	488	507	0	CCACGGATCCTTGGCGCAGC
406003	5-10-5	178	555	574	0	CAGTGCGCTCTGACTGCGAG
406004	5-10-5	179	557	576	0	GGCAGTGCGCTCTGACTGCG
406005	5-10-5	180	559	578	0	CGGGCAGTGCGCTCTGACTG
406006	5-10-5	181	562	581	0	CGGCGGGCAGTGCGCTCTGA
406007	5-10-5	183	595	614	0	AGGTATCCCCGGCGGGCAGC
406008	5-10-5	188	602	621	0	CTTGGTGAGGTATCCCCGGC
406009	5-10-5	190	604	623	0	ATCTTGGTGAGGTATCCCCG
406010	5-10-5	193	609	628	0	GCAGGATCTTGGTGAGGTAT
406011	5-10-5	194	611	630	0	ATGCAGGATCTTGGTGAGGT
406012	5-10-5	195	613	632	0	ACATGCAGGATCTTGGTGAG
406013	5-10-5	196	617	636	0	GAAGACATGCAGGATCTTGG
406014	5-10-5	199	648	667	0	TCTTCACCAGGAAGCCAGGA
406015	5-10-5	200	653	672	0	ACTCATCTTCACCAGGAAGC
406016	5-10-5	201	655	674	0	CCACTCATCTTCACCAGGAA
406017	5-10-5	203	659	678	0	GTCGCCACTCATCTTCACCA
406018	5-10-5	204	661	680	0	AGGTCGCCACTCATCTTCAC
406019	5-10-5	205	663	682	0	GCAGGTCGCCACTCATCTTC
406020	5-10-5	206	665	684	0	CAGCAGGTCGCCACTCATCT
406021	5-10-5	210	782	801	0	TTCATCCGCCCGGTACCGTG
406022	5-10-5	211	784	803	0	TATTCATCCGCCCGGTACCG
406023	5-10-5	212	787	806	0	TGGTATTCATCCGCCCGGTA
406024	5-10-5	213	789	808	0	GCTGGTATTCATCCGCCCGG
406025	5-10-5	216	832	851	0	GTGTCTAGGAGATACACCTC
406026	5-10-5	217	837	856	0	TGCTGGTGTCTAGGAGATAC
406027	5-10-5	226	874	893	0	ATGACCCTGCCCTCGATTTC
406028	5-10-5	227	876	895	0	CCATGACCCTGCCCTCGATT
406029	5-10-5	228	878	897	0	GACCATGACCCTGCCCTCGA
406030	5-10-5	229	882	901	0	CGGTGACCATGACCCTGCCC
406031	5-10-5	230	884	903	0	GTCGGTGACCATGACCCTGC
406032	5-10-5	232	888	907	0	CGAAGTCGGTGACCATGACC
406033	5-10-5	237	967	986	0	CCTGCCAGGTGGGTGCCATG
406034	5-10-5	238	972	991	0	CCACCCCTGCCAGGTGGGTG
406035	5-10-5	239	977	996	0	GCTGACCACCCCTGCCAGGT
406036	5-10-5	241	989	1008	0	GGCATCCCGGCCGCTGACCA
406037	5-10-5	242	992	1011	0	GCCGGCATCCCGGCCGCTGA
406038	5-10-5	245	999	1018	0	TGGCCACGCCGGCATCCCGG
406039	5-10-5	257	1036	1055	0	CAGTTGAGCACGCGCAGGCT
406040	5-10-5	258	1038	1057	0	GGCAGTTGAGCACGCGCAGG
406041	5-10-5	259	1042	1061	0	CCTTGGCAGTTGAGCACGCG
406042	5-10-5	260	1047	1066	0	CCTTCCCTTGGCAGTTGAGC
406043	5-10-5	261	1051	1070	0	GTGCCCTTCCCTTGGCAGTT
406044	5-10-5	262	1053	1072	0	CCGTGCCCTTCCCTTGGCAG
406045	5-10-5	266	1096	1115	0	TGGCTTTTCCGAATAAACTC
408642	5-10-5	264	1076	1095	0	CAGGCCTATGAGGGTGCCGC
408653	5-10-5	354	1570	1589	0	TGGTCCTCAGGGAACCAGGC
409126	5-10-5	447	1004	1023	1	ACCCTTGGTCACGCCGGCAT
410529	5-10-5	184	597	616	0	TGAGGTATCCCCGGCGGGCA
410530	5-10-5	185	598	617	0	GTGAGGTATCCCCGGCGGGC
410531	5-10-5	186	599	618	0	GGTGAGGTATCCCCGGCGGG
410532	5-10-5	187	601	620	0	TTGGTGAGGTATCCCCGGCG
410533	5-10-5	189	603	622	0	TCTTGGTGAGGTATCCCCGG
410534	5-10-5	191	605	624	0	GATCTTGGTGAGGTATCCCC
410535	5-10-5	192	607	626	0	AGGATCTTGGTGAGGTATCC
410536	5-10-5	243	997	1016	0	GCCACGCCGGCATCCCGGCC
410537	5-10-5	244	998	1017	0	GGCCACGCCGGCATCCCGGC
410538	5-10-5	254	1009	1028	0	CTGGCACCCTTGGCCACGCC
410539	5-10-5	255	1010	1029	0	GCTGGCACCCTTGGCCACGC
410540	5-10-5	356	1572	1591	0	GCTGGTCCTCAGGGAACCAG
410541	5-10-5	376	1849	1868	0	CACCTGGCAATGGCGTAGAC
410542	5-10-5	377	1850	1869	0	GCACCTGGCAATGGCGTAGA
410543	5-10-5	378	1851	1870	0	AGCACCTGGCAATGGCGTAG
410544	5-10-5	379	1852	1871	0	CAGCACCTGGCAATGGCGTA
410545	5-10-5	380	1853	1872	0	GCAGCACCTGGCAATGGCGT
410546	5-10-5	382	1855	1874	0	AGGCAGCACCTGGCAATGGC
410547	5-10-5	384	1857	1876	0	GCAGGCAGCACCTGGCAATG
410548	5-10-5	385	1859	1878	0	TAGCAGGCAGCACCTGGCAA
410549	5-10-5	388	1915	1934	0	GTCCCCATGCTGGCCTCAGC
410550	5-10-5	389	1916	1935	0	GGTCCCCATGCTGGCCTCAG
410551	5-10-5	390	1917	1936	0	GGGTCCCCATGCTGGCCTCA
410552	5-10-5	391	1918	1937	0	CGGGTCCCCATGCTGGCCTC
410553	5-10-5	392	1919	1938	0	ACGGGTCCCCATGCTGGCCT
410554	5-10-5	393	1921	1940	0	ACACGGGTCCCCATGCTGGC
410555	5-10-5	395	1923	1942	0	GGACACGGGTCCCCATGCTG
410556	5-10-5	397	1925	1944	0	GTGGACACGGGTCCCCATGC
410557	5-10-5	399	1927	1946	0	CAGTGGACACGGGTCCCCAT
410558	5-10-5	401	1929	1948	0	GGCAGTGGACACGGGTCCCC
410559	5-10-5	403	1931	1950	0	GTGGCAGTGGACACGGGTCC
410560	5-10-5	404	1932	1951	0	GGTGGCAGTGGACACGGGTC
410561	5-10-5	405	1933	1952	0	TGGTGGCAGTGGACACGGGT
410562	5-10-5	411	2101	2120	0	ACTTTGCATTCCAGACCTGG
410563	5-10-5	413	2103	2122	0	TGACTTTGCATTCCAGACCT
410564	5-10-5	414	2104	2123	0	TTGACTTTGCATTCCAGACC
410565	5-10-5	419	2305	2324	0	CAGATGGCAACGGCTGTCAC
410566	5-10-5	420	2306	2325	0	GCAGATGGCAACGGCTGTCA
410567	5-10-5	421	2307	2326	0	AGCAGATGGCAACGGCTGTC
410568	5-10-5	422	2308	2327	0	CAGCAGATGGCAACGGCTGT
410569	5-10-5	423	2309	2328	0	GCAGCAGATGGCAACGGCTG
410570	5-10-5	424	2311	2330	0	CGGCAGCAGATGGCAACGGC
410571	5-10-5	425	2312	2331	0	CCGGCAGCAGATGGCAACGG
410572	5-10-5	427	2314	2333	0	CTCCGGCAGCAGATGGCAAC
410573	5-10-5	428	2315	2334	0	GCTCCGGCAGCAGATGGCAA
410730	5-10-5	202	657	676	0	CGCCACTCATCTTCACCAGG
410731	5-10-5	207	667	686	0	TCCAGCAGGTCGCCACTCAT
410732	5-10-5	214	791	810	0	GGGCTGGTATTCATCCGCCC
410733	5-10-5	231	886	905	0	AAGTCGGTGACCATGACCCT
410734	5-10-5	279	1219	1238	0	CCGGCAGCGGTGACCAGCAC
410735	5-10-5	291	1320	1339	0	TCCCCAGGGTCACCGGCTGG
410736	5-10-5	303	1356	1375	0	AGAGGTCCACACAGCGGCCA
410737	5-10-5	313	1406	1425	0	GCAGGTGCTGCAGTCGCTGG
410738	5-10-5	324	1482	1501	0	GCTCGGCAGACAGCATCATG
410739	5-10-5	336	1530	1549	0	CAGAGAAGTGGATCAGTCTC
410740	5-10-5	345	1558	1577	0	AACCAGGCCTCATTGATGAC
410741	5-10-5	369	1683	1702	0	CCATCCGTGTAGGCCCCGAG
410742	5-10-5	159	294	313	0	GCCTGGAGCTGACGGTGCCC
410743	5-10-5	170	421	440	0	TCCTCCTCGGAACGCAAGGC
410744	5-10-5	171	446	465	0	GTGCTCGGGTGCTTCGGCCA
410745	5-10-5	172	466	485	0	TGGAAGGTGGCTGTGGTTCC
410746	5-10-5	176	507	526	0	CGTAGGTGCCAGGCAACCTC
410747	5-10-5	177	545	564	0	TGACTGCGAGAGGTGGGTCT
410748	5-10-5	182	591	610	0	ATCCCCGGCGGGCAGCCTGG
410749	5-10-5	197	628	647	0	AGAAGGCCATGGAAGACATG
410750	5-10-5	198	638	657	0	GAAGCCAGGAAGAAGGCCAT
410751	5-10-5	208	685	704	0	GGCAACTTCAAGGCCAGCTC
410752	5-10-5	209	724	743	0	GCAAAGACAGAGGAGTCCTC
410753	5-10-5	215	821	840	0	ATACACCTCCACCAGGCTGC
410754	5-10-5	233	898	917	0	GGCACATTCTCGAAGTCGGT
410755	5-10-5	234	933	952	0	TGGCCTGTCTGTGGAAGCGG
410756	5-10-5	235	960	979	0	GGTGGGTGCCATGACTGTCA
410757	5-10-5	240	985	1004	0	TCCCGGCCGCTGACCACCCC
410758	5-10-5	256	1015	1034	0	CGCATGCTGGCACCCTTGGC
410759	5-10-5	263	1064	1083	0	GGTGCCGCTAACCGTGCCCT
410760	5-10-5	265	1088	1107	0	CCGAATAAACTCCAGGCCTA
410761	5-10-5	271	1119	1138	0	GTGGCCCCACAGGCTGGACC
410762	5-10-5	272	1132	1151	0	AGCAGCACCACCAGTGGCCC
410763	5-10-5	273	1154	1173	0	GCTGTACCCACCCGCCAGGG
410764	5-10-5	274	1200	1219	0	CGACCCCAGCCCTCGCCAGG
410765	5-10-5	286	1273	1292	0	ATGACCTCGGGAGCTGAGGC
410766	5-10-5	287	1283	1302	0	CCCAACTGTGATGACCTCGG
410767	5-10-5	289	1305	1324	0	GCTGGTCTTGGGCATTGGTG
410768	5-10-5	305	1380	1399	0	CAATGATGTCCTCCCCTGGG
410769	5-10-5	318	1425	1444	0	TCCCACTCTGTGACACAAAG
410770	5-10-5	327	1500	1519	0	CGGCCAGGGTGAGCTCCGGC
410771	5-10-5	360	1628	1647	0	ACCTGCCCCATGGGTGCTGG
410772	5-10-5	364	1660	1679	0	GCTGACCATACAGTCCTGCA
410773	5-10-5	387	1905	1924	0	TGGCCTCAGCTGGTGGAGCT
410774	5-10-5	406	1936	1955	0	TGTTGGTGGCAGTGGACACG
410775	5-10-5	407	1962	1981	0	AGCTGCAGCCTGTGAGGACG
410776	5-10-5	408	1990	2009	0	GTGCCAAGGTCCTCCACCTC
410777	5-10-5	409	2010	2029	0	TCAGCACAGGCGGCTTGTGG
410778	5-10-5	410	2040	2059	0	CCACGCACTGGTTGGGCTGA
410779	5-10-5	416	2120	2139	0	CGGGATTCCATGCTCCTTGA
410780	5-10-5	417	2150	2169	0	GCAGGCCACGGTCACCTGCT
410781	5-10-5	418	2187	2206	0	GGAGGGCACTGCAGCCAGTC
410782	5-10-5	429	2325	2344	0	CCAGGTGCCGGCTCCGGCAG
410783	5-10-5	430	2335	2354	0	GAGGCCTGCGCCAGGTGCCG

[0297] In certain embodiments, gap-widened antisense compounds are targeted to a PCSK9 nucleic acid. In certain such embodiments, gap-widened antisense compounds are targeted to SEQ ID NO: 1. In certain such embodiments, the nucleotide sequences illustrated in Table 2 have a 3-14-3 gap-widened motif. Table 4 illustrates gap-widened antisense compounds targeted to SEQ ID NO: 1, having a 3-14-3 motif, where the gap segment comprises 2′-deoxynucleotides and each wing segment comprises nucleotides comprising a 2′-O-methoxyethyl sugar modification. Internucleoside linkages are phosphorthioate, and cytidines are 5-methylcytidines.

[0298]

TABLE 4
Gapmer antisense compounds having a 3-14-3 motif targeted to SEQ ID NO: 1
			5′ Target Site	3′ Target Site
		SEQ	to SEQ ID	to SEQ ID
Isis No	Motif	ID NO	NO: 1	NO: 1	Mismatches	Sequence (5′-3′)
399871	3-14-3	4	135	154	0	GCGCGGAATCCTGGCTGGGA
399872	3-14-3	5	242	261	0	GAGGAGACCTAGAGGCCGTG
399873	3-14-3	6	300	319	0	AGGACCGCCTGGAGCTGACG
399874	3-14-3	7	410	429	0	ACGCAAGGCTAGCACCAGCT
399949	3-14-3	8	417	436	0	CCTCGGAACGCAAGGCTAGC
399875	3-14-3	9	480	499	0	CCTTGGCGCAGCGGTGGAAG
399876	3-14-3	10	561	580	0	GGCGGGCAGTGCGCTCTGAC
399877	3-14-3	11	600	619	0	TGGTGAGGTATCCCCGGCGG
399950	3-14-3	12	606	625	0	GGATCTTGGTGAGGTATCCC
399951	3-14-3	13	615	634	0	AGACATGCAGGATCTTGGTG
399878	3-14-3	14	620	639	0	ATGGAAGACATGCAGGATCT
399879	3-14-3	15	646	665	0	TTCACCAGGAAGCCAGGAAG
399952	3-14-3	16	651	670	0	TCATCTTCACCAGGAAGCCA
399880	3-14-3	17	705	724	0	CCTCGATGTAGTCGACATGG
399881	3-14-3	18	785	804	0	GTATTCATCCGCCCGGTACC
399882	3-14-3	19	835	854	0	CTGGTGTCTAGGAGATACAC
399953	3-14-3	20	840	859	0	GTATGCTGGTGTCTAGGAGA
399883	3-14-3	21	860	879	0	GATTTCCCGGTGGTCACTCT
399954	3-14-3	22	866	885	0	GCCCTCGATTTCCCGGTGGT
399955	3-14-3	23	880	899	0	GTGACCATGACCCTGCCCTC
399884	3-14-3	24	890	909	0	CTCGAAGTCGGTGACCATGA
399885	3-14-3	25	923	942	0	GTGGAAGCGGGTCCCGTCCT
399886	3-14-3	26	970	989	0	ACCCCTGCCAGGTGGGTGCC
399956	3-14-3	27	975	994	0	TGACCACCCCTGCCAGGTGG
399887	3-14-3	28	1004	1023	0	ACCCTTGGCCACGCCGGCAT
399888	3-14-3	29	1040	1059	0	TTGGCAGTTGAGCACGCGCA
399957	3-14-3	30	1045	1064	0	TTCCCTTGGCAGTTGAGCAC
399889	3-14-3	31	1077	1096	0	CCAGGCCTATGAGGGTGCCG
399890	3-14-3	32	1098	1117	0	GCTGGCTTTTCCGAATAAAC
399891	3-14-3	33	1210	1229	0	GTGACCAGCACGACCCCAGC
399892	3-14-3	149	1297	1316	0	TGGGCATTGGTGGCCCCAAC
399893	3-14-3	34	1326	1345	0	CCAAAGTCCCCAGGGTCACC
399894	3-14-3	128	1330	1349	0	GTCCCCAAAGTCCCCAGGGT
399958	3-14-3	35	1335	1354	0	AGTTGGTCCCCAAAGTCCCC
399895	3-14-3	36	1340	1359	0	GCCAAAGTTGGTCCCCAAAG
399959	3-14-3	37	1352	1371	0	GTCCACACAGCGGCCAAAGT
399896	3-14-3	38	1361	1380	0	GGCAAAGAGGTCCACACAGC
399897	3-14-3	39	1389	1408	0	TGGAGGCACCAATGATGTCC
399960	3-14-3	40	1400	1419	0	GCTGCAGTCGCTGGAGGCAC
399961	3-14-3	41	1411	1430	0	ACAAAGCAGGTGCTGCAGTC
399898	3-14-3	101	1465	1484	0	ATGGCTGCAATGCCAGCCAC
399962	3-14-3	42	1470	1489	0	GCATCATGGCTGCAATGCCA
399963	3-14-3	43	1478	1497	0	GGCAGACAGCATCATGGCTG
399964	3-14-3	44	1526	1545	0	GAAGTGGATCAGTCTCTGCC
399899	3-14-3	45	1534	1553	0	TTGGCAGAGAAGTGGATCAG
399965	3-14-3	46	1539	1558	0	CATCTTTGGCAGAGAAGTGG
399966	3-14-3	47	1545	1564	0	TGATGACATCTTTGGCAGAG
399967	3-14-3	48	1552	1571	0	GCCTCATTGATGACATCTTT
399968	3-14-3	49	1564	1583	0	TCAGGGAACCAGGCCTCATT
399900	3-14-3	50	1569	1588	0	GGTCCTCAGGGAACCAGGCC
399901	3-14-3	87	1576	1595	0	ACCCGCTGGTCCTCAGGGAA
399969	3-14-3	51	1583	1602	0	GGTCAGTACCCGCTGGTCCT
399902	3-14-3	119	1605	1624	0	GCAGGGCGGCCACCAGGTTG
399903	3-14-3	52	1640	1659	0	AAACAGCTGCCAACCTGCCC
399970	3-14-3	53	1645	1664	0	CTGCAAAACAGCTGCCAACC
399904	3-14-3	54	1675	1694	0	GTAGGCCCCGAGTGTGCTGA
399971	3-14-3	55	1740	1759	0	AGAAACTGGAGCAGCTCAGC
399972	3-14-3	56	1746	1765	0	TCCTGGAGAAACTGGAGCAG
399905	3-14-3	57	1812	1831	0	CGTTGTGGGCCCGGCAGACC
399906	3-14-3	58	1858	1877	0	AGCAGGCAGCACCTGGCAAT
399907	3-14-3	59	1920	1939	0	CACGGGTCCCCATGCTGGCC
399908	3-14-3	60	2100	2119	0	CTTTGCATTCCAGACCTGGG
399973	3-14-3	61	2105	2124	0	CTTGACTTTGCATTCCAGAC
399909	3-14-3	62	2310	2329	0	GGCAGCAGATGGCAACGGCT
399910	3-14-3	154	2410	2429	0	TTTTAAAGCTCAGCCCCAGC
399974	3-14-3	63	2415	2434	0	AACCATTTTAAAGCTCAGCC
399911	3-14-3	64	2504	2523	0	TCAAGGGCCAGGCCAGCAGC
399975	3-14-3	65	2509	2528	0	CCCACTCAAGGGCCAGGCCA
399976	3-14-3	122	2582	2601	0	GGAGGGAGCTTCCTGGCACC
399912	3-14-3	66	2597	2616	0	ATGCCCCACAGTGAGGGAGG
399913	3-14-3	67	2606	2625	0	AATGGTGAAATGCCCCACAG
399914	3-14-3	153	2649	2668	0	TTGGGAGCAGCTGGCAGCAC
399915	3-14-3	68	2750	2769	0	CATGGGAAGAATCCTGCCTC
399916	3-14-3	69	2832	2851	0	GGAGATGAGGGCCATCAGCA
399917	3-14-3	70	2900	2919	0	TAGATGCCATCCAGAAAGCT
399977	3-14-3	71	2906	2925	0	TCTGGCTAGATGCCATCCAG
399918	3-14-3	72	2983	3002	0	GGCATAGAGCAGAGTAAAGG
399978	3-14-3	73	2988	3007	0	AGCCTGGCATAGAGCAGAGT
399979	3-14-3	135	2994	3013	0	TAGCACAGCCTGGCATAGAG
399919	3-14-3	112	3227	3246	0	GAAGAGGCTTGGCTTCAGAG
399980	3-14-3	74	3233	3252	0	AAGTAAGAAGAGGCTTGGCT
399920	3-14-3	75	3437	3456	0	GCTCAAGGAGGGACAGTTGT
399921	3-14-3	76	3472	3491	0	AAAGATAAATGTCTGCTTGC
399981	3-14-3	77	3477	3496	0	ACCCAAAAGATAAATGTCTG
399922	3-14-3	78	3543	3562	0	TCTTCAAGTTACAAAAGCAA
399982	3-14-3	99	3550	3569	0	ATAAATATCTTCAAGTTACA
410574	3-14-3	184	597	616	0	TGAGGTATCCCCGGCGGGCA
410575	3-14-3	185	598	617	0	GTGAGGTATCCCCGGCGGGC
410576	3-14-3	186	599	618	0	GGTGAGGTATCCCCGGCGGG
410577	3-14-3	187	601	620	0	TTGGTGAGGTATCCCCGGCG
410578	3-14-3	188	602	621	0	CTTGGTGAGGTATCCCCGGC
410579	3-14-3	189	603	622	0	TCTTGGTGAGGTATCCCCGG
410580	3-14-3	190	604	623	0	ATCTTGGTGAGGTATCCCCG
410581	3-14-3	191	605	624	0	GATCTTGGTGAGGTATCCCC
410582	3-14-3	192	607	626	0	AGGATCTTGGTGAGGTATCC
405604	3-14-3	236	963	982	0	CCAGGTGGGTGCCATGACTG
410583	3-14-3	243	997	1016	0	GCCACGCCGGCATCCCGGCC
410584	3-14-3	244	998	1017	0	GGCCACGCCGGCATCCCGGC
410585	3-14-3	245	999	1018	0	TGGCCACGCCGGCATCCCGG
410586	3-14-3	246	1000	1019	0	TTGGCCACGCCGGCATCCCG
410587	3-14-3	247	1001	1020	0	CTTGGCCACGCCGGCATCCC
410588	3-14-3	248	1002	1021	0	CCTTGGCCACGCCGGCATCC
410589	3-14-3	249	1003	1022	0	CCCTTGGCCACGCCGGCATC
410590	3-14-3	250	1005	1024	0	CACCCTTGGCCACGCCGGCA
410591	3-14-3	251	1006	1025	0	GCACCCTTGGCCACGCCGGC
410592	3-14-3	252	1007	1026	0	GGCACCCTTGGCCACGCCGG
410593	3-14-3	253	1008	1027	0	TGGCACCCTTGGCCACGCCG
410594	3-14-3	254	1009	1028	0	CTGGCACCCTTGGCCACGCC
410595	3-14-3	255	1010	1029	0	GCTGGCACCCTTGGCCACGC
410596	3-14-3	348	1562	1581	0	AGGGAACCAGGCCTCATTGA
410597	3-14-3	349	1563	1582	0	CAGGGAACCAGGCCTCATTG
410598	3-14-3	350	1565	1584	0	CTCAGGGAACCAGGCCTCAT
410599	3-14-3	351	1566	1585	0	CCTCAGGGAACCAGGCCTCA
410600	3-14-3	352	1567	1586	0	TCCTCAGGGAACCAGGCCTC
410601	3-14-3	353	1568	1587	0	GTCCTCAGGGAACCAGGCCT
410602	3-14-3	354	1570	1589	0	TGGTCCTCAGGGAACCAGGC
410603	3-14-3	355	1571	1590	0	CTGGTCCTCAGGGAACCAGG
410604	3-14-3	356	1572	1591	0	GCTGGTCCTCAGGGAACCAG
405641	3-14-3	373	1737	1756	0	AACTGGAGCAGCTCAGCAGC
410605	3-14-3	376	1849	1868	0	CACCTGGCAATGGCGTAGAC
410606	3-14-3	377	1850	1869	0	GCACCTGGCAATGGCGTAGA
410607	3-14-3	378	1851	1870	0	AGCACCTGGCAATGGCGTAG
410608	3-14-3	379	1852	1871	0	CAGCACCTGGCAATGGCGTA
410609	3-14-3	380	1853	1872	0	GCAGCACCTGGCAATGGCGT
410610	3-14-3	381	1854	1873	0	GGCAGCACCTGGCAATGGCG
410611	3-14-3	382	1855	1874	0	AGGCAGCACCTGGCAATGGC
410612	3-14-3	383	1856	1875	0	CAGGCAGCACCTGGCAATGG
410613	3-14-3	384	1857	1876	0	GCAGGCAGCACCTGGCAATG
410614	3-14-3	385	1859	1878	0	TAGCAGGCAGCACCTGGCAA
410615	3-14-3	388	1915	1934	0	GTCCCCATGCTGGCCTCAGC
410616	3-14-3	389	1916	1935	0	GGTCCCCATGCTGGCCTCAG
410617	3-14-3	390	1917	1936	0	GGGTCCCCATGCTGGCCTCA
410618	3-14-3	391	1918	1937	0	CGGGTCCCCATGCTGGCCTC
410619	3-14-3	392	1919	1938	0	ACGGGTCCCCATGCTGGCCT
410620	3-14-3	393	1921	1940	0	ACACGGGTCCCCATGCTGGC
410621	3-14-3	394	1922	1941	0	GACACGGGTCCCCATGCTGG
410622	3-14-3	395	1923	1942	0	GGACACGGGTCCCCATGCTG
410623	3-14-3	396	1924	1943	0	TGGACACGGGTCCCCATGCT
410624	3-14-3	397	1925	1944	0	GTGGACACGGGTCCCCATGC
410625	3-14-3	398	1926	1945	0	AGTGGACACGGGTCCCCATG
410626	3-14-3	399	1927	1946	0	CAGTGGACACGGGTCCCCAT
410627	3-14-3	400	1928	1947	0	GCAGTGGACACGGGTCCCCA
410628	3-14-3	401	1929	1948	0	GGCAGTGGACACGGGTCCCC
410629	3-14-3	402	1930	1949	0	TGGCAGTGGACACGGGTCCC
410630	3-14-3	403	1931	1950	0	GTGGCAGTGGACACGGGTCC
410631	3-14-3	404	1932	1951	0	GGTGGCAGTGGACACGGGTC
410632	3-14-3	405	1933	1952	0	TGGTGGCAGTGGACACGGGT
410633	3-14-3	411	2101	2120	0	ACTTTGCATTCCAGACCTGG
410634	3-14-3	412	2102	2121	0	GACTTTGCATTCCAGACCTG
410635	3-14-3	413	2103	2122	0	TGACTTTGCATTCCAGACCT
410636	3-14-3	414	2104	2123	0	TTGACTTTGCATTCCAGACC
410637	3-14-3	419	2305	2324	0	CAGATGGCAACGGCTGTCAC
410638	3-14-3	420	2306	2325	0	GCAGATGGCAACGGCTGTCA
410639	3-14-3	421	2307	2326	0	AGCAGATGGCAACGGCTGTC
410640	3-14-3	422	2308	2327	0	CAGCAGATGGCAACGGCTGT
410641	3-14-3	423	2309	2328	0	GCAGCAGATGGCAACGGCTG
410642	3-14-3	424	2311	2330	0	CGGCAGCAGATGGCAACGGC
410643	3-14-3	425	2312	2331	0	CCGGCAGCAGATGGCAACGG
410644	3-14-3	426	2313	2332	0	TCCGGCAGCAGATGGCAACG
410645	3-14-3	427	2314	2333	0	CTCCGGCAGCAGATGGCAAC
410646	3-14-3	428	2315	2334	0	GCTCCGGCAGCAGATGGCAA