Methods and compositions for treating proprotein convertase subtilisin / kexin (PCSK9) gene-associated disorders

Abstract

Provided is an effective method for treating PCSK9-related diseases, such as hyperlipidemia, for example, hypercholesterolemia. [Solution] A method is provided for inhibiting expression of the PCSK9 gene in a subject, comprising administering to the subject a fixed dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (RNAi) agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, and the antisense strand comprises a complementary region comprising at least 15 contiguous nucleotides that differ from nucleotides 3544 to 3623 of a specific nucleotide sequence by no more than 3 nucleotides.

Description

[Technical Field] 【0001】 Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 209,526, filed August 25, 2015, the entire contents of which are incorporated herein by reference. 【0002】 This application is related to U.S. Provisional Patent Application No. 61 / 733,518, filed December 5, 2012; U.S. Provisional Patent Application No. 61 / 793,530, filed March 15, 2013; U.S. Provisional Patent Application No. 61 / 886,916, filed October 4, 2013; U.S. Provisional Patent Application No. 61 / 892,188, filed October 17, 2013; PCT Application No. PCT / US2013 / 073349, filed December 5, 2013; and U.S. Patent Application No. 14 / 650,128, filed June 5, 2015. The entire contents of each of the foregoing patent applications are hereby incorporated by reference herein. 【0003】 Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on August 24, 2016, is named 121301-04420_SL.txt and is 188,218 bytes in size. [Background technology] 【0004】 Proprotein convertase subtilisin / kexin type 9 (PCSK9) is a member of the subtilisin serine protease family. The other eight mammalian subtilisin proteases, PCSK1 to PCSK8 (also called PC1 / 3, PC2, furin, PC4, PC5 / 6, PACE4, PC7, and S1P / SKI-1), are proprotein convertases that process a wide variety of proteins in the secretory pathway and play roles in diverse biological processes (Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4, and Non-Patent Document 5). 【0005】 PCSK9 has been proposed to play a role in cholesterol metabolism. Similar to cholesterol biosynthetic enzymes and the low-density lipoprotein receptor (LDLR), PCSK9 mRNA expression is downregulated by dietary cholesterol feeding in mice (Non-Patent Document 6), upregulated by statins in HepG2 cells (Non-Patent Document 7), and upregulated in sterol regulatory element-binding protein (SREBP) transgenic mice (Non-Patent Document 8). Furthermore, missense mutations in PCSK9 have been found to be associated with a form of autosomal dominant hypercholesterolemia (Hchola3) (Non-Patent Document 9, Non-Patent Document 10, Non-Patent Document 11). Because single nucleotide polymorphisms (SNPs) are associated with cholesterol levels in the Japanese population, PCSK9 may also play a role in determining LDL cholesterol levels in the general population (Non-Patent Document 12). 【0006】 Autosomal dominant hypercholesterolemia (ADH) is a monogenic disease in which patients present with elevated total and LDL cholesterol levels, tendon xanthomas, and premature atherosclerosis (Non-Patent Document 13). The pathogenesis of ADH and the recessive form of autosomal recessive hypercholesterolemia (ARH) (Non-Patent Document 14) is due to abnormalities in LDL uptake by the liver. ADH can be caused by LDLR mutations that block LDL uptake or mutations in apolipoprotein B, a protein on LDL that binds to LDLR. ARH is caused by mutations in the ARH protein, which is necessary for endocytosis of the LDLR-LDL complex via its interaction with clathrin. Therefore, if PCSK9 mutations are the cause in the Hchola3 family, it seems likely that PCSK9 plays a role in receptor-mediated LDL uptake. 【0007】 Overexpression studies indicate a role for PCSK9 in controlling LDLR levels and, therefore, LDL uptake by the liver ((Non-Patent Document 15), (Non-Patent Document 16), (Non-Patent Document 17)). Adenovirus-mediated overexpression of mouse or human PCSK9 in mice for 3 or 4 days results in elevated total and LDL cholesterol levels; this effect is not observed in LDLR knockout animals ((Non-Patent Document 15), (Non-Patent Document 16), (Non-Patent Document 17)). Furthermore, overexpression of PCSK9 results in a dramatic reduction in hepatic LDLR protein without affecting LDLR mRNA levels, SREBP protein levels, or the nuclear-to-cytoplasmic ratio of SREBP protein. 【0008】 While hypercholesterolemia itself is asymptomatic, long-term elevation of serum cholesterol can lead to atherosclerosis.Over a period of several decades, chronically elevated serum cholesterol contributes to the formation of atheromatous plaque in arteries, which can lead to the progressive narrowing or even complete occlusion of the involved arteries.Furthermore, smaller plaques can rupture, cause the formation of blood clots, and block blood flow, resulting in, for example, myocardial infarction and / or stroke.When narrowing or blockage is formed slowly, the blood supply to tissues and organs gradually decreases, and organ function becomes impaired. [Prior art documents] [Non-patent literature] 【0009】 [Non-Patent Document 1] Bergeron, F. (2000) J. Mol. Endocrinol. 24, 1-22 [Non-patent document 2] Gensberg, K., (1998) Semin.Cell Dev.Biol.9,11-17 [Non-patent document 3] Seidah, NG (1999) Brain Res. 848, 45-62 [Non-patent document 4] Taylor,NA,(2003)FASEB J.17,1215-1227 【Non-licensed Document 5】 Zhou,A.,(1999)J.Biol.Chem.274,20745-20748 【Non-licensed Document 6】 Maxwell,KN,(2003)J.Lipid Res.44,2109-2119 【Non-licensed Document 7】 Dubuc,G.,(2004)Arterioscler.Thromb.Vasc.Biol.24,1454-1459 [Non-licensed document 8] Horton,JD,(2003)Proc.Natl.Acad.Sci.USA 100,12027-12032 【Non-licensed literature 9】 Abifadel,M.,et al.(2003)Nat.Genet.34,154-156 【Non-licensed literature 10】 Timms,KM,(2004)Hum.Genet.114,349-353 【Non-licensed Document 11】 Leren,TP(2004)Clin.Genet.65,419-422 【Non-licensed Document 12】 Shioji,K.,(2004)J.Hum.Genet.49,109-114 【Non-licensed Document 13】 Rader,DJ,(2003)J.Clin.Invest.111,1795-1803 【Non-licensed Document 14】 Cohen,JC,(2003)Curr.Opin.Lipidol.14,121-127 【Non-licensed Document 15】 Maxwell,KN(2004)Proc.Natl.Acad.Sci.USA 101,7100-7105 【Non-licensed Document 16】 Benjannet, S., et al. (2004) J. Biol. Chem. 279, 48865-48875 [Non-Patent Document 17] Park, SW, (2004) J. Biol. Chem. 279, 50630-50638 Summary of the Invention [Problem to be solved by the invention] 【0010】 Thus, there is a need in the art for effective treatments for PCSK9-related diseases, such as hyperlipidemia, eg, hypercholesterolemia. [Means for solving the problem] 【0011】 The present invention is based, at least in part, on the surprising discovery that double-stranded RNAi agents containing chemical modifications exhibit exceptional potency and durability in inhibiting PCSK9 expression at a single dose. Specifically, it is shown herein that RNAi agents targeting the human PCSK9 gene, e.g., nucleotides 3544-3623 of the human PCSK9 gene (nucleotides 3544-3623 of SEQ ID NO:1), e.g., nucleotides 3601-3623 of SEQ ID NO:1 (containing a GalNAc ligand), are exceptionally effective and durable in silencing the activity of the PCSK9 gene at a single fixed dose, e.g., a fixed dose of about 300 mg to about 500 mg. 【0012】 Thus, the present invention provides methods for inhibiting expression of the PCSK9 gene in a subject, and for treating a subject having a disorder that would benefit from inhibiting or reducing expression of the PCSK9 gene, e.g., a disorder mediated by expression of PCSK9, e.g., hyperlipidemia, e.g., hypercholesterolemia, with an iRNA composition that effects RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the PCSK9 gene. 【0013】 In one aspect, methods of the invention for inhibiting expression of the PCSK9 gene in a subject, and methods for treating a subject having a disorder that would benefit from inhibiting or reducing expression of the PCSK9 gene, e.g., a disorder mediated by expression of PCSK9, e.g., hyperlipidemia, e.g., hypercholesterolemia, comprise the step of administering to the subject a fixed dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (RNAi) agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:1 by no more than 3 nucleotides, and the antisense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:2 by no more than 3 nucleotides, wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and wherein the sense strand is bound to a ligand attached at its 3' end. 【0014】 In one aspect, the present invention provides a method for inhibiting expression of the PCSK9 gene in a subject, the method comprising administering to the subject a fixed dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (RNAi) agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising a complementary region comprising at least 15 contiguous nucleotides that differ from nucleotides 3544 to 3623 of the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides, thereby inhibiting expression of the PCSK9 gene in the subject. 【0015】 In another aspect, the invention provides a method for reducing low-density lipoprotein (LDLc) levels in a subject, comprising administering to the subject a fixed dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (RNAi) agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, and wherein the antisense strand comprises a complementary region comprising at least 15 contiguous nucleotides that differ from nucleotides 3544 to 3623 of the nucleotide sequence of SEQ ID NO:1 by no more than 3 nucleotides, thereby reducing the level of LDLc in the subject. 【0016】 In another aspect, the present invention provides a method of treating a subject having a disorder that would benefit from a decrease in expression of PCSK9. The method includes administering to the subject a fixed dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (RNAi) agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a duplex region, and the antisense strand comprises a complementary region comprising at least 15 contiguous nucleotides that differ from nucleotides 3544 to 3623 of the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides, thereby treating the subject having a disorder that would benefit from a decrease in expression of PCSK9. 【0017】 In yet another aspect, the present invention provides a method for treating a subject with hyperlipidemia, comprising administering to the subject a fixed dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (RNAi) agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising a complementary region comprising at least 15 contiguous nucleotides that differ from nucleotides 3544 to 3623 of the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides, thereby treating the subject with hypercholesterolemia. 【0018】 The fixed dose may be administered to the subject once weekly, once every two weeks, once monthly, once quarterly, or at semi-annual intervals. 【0019】 In one embodiment, a subject is administered a fixed dose of about 25 mg to about 50 mg once a week. In another embodiment, a subject is administered a fixed dose of about 50 mg to about 100 mg once every two weeks. In another embodiment, a subject is administered a fixed dose of about 100 mg to about 200 mg once a month. In yet another embodiment, a subject is administered a fixed dose of about 300 mg to about 800 mg once quarterly. In another embodiment, a subject is administered a fixed dose of about 300 mg to about 800 mg every six months. 【0020】 The invention also provides methods in which the RNAi agent is administered in a dosing regimen comprising a loading phase and a maintenance phase. 【0021】 Thus, in one aspect, the present invention provides a method for inhibiting expression of the PCSK9 gene in a subject, the method comprising administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen including a loading phase followed by a maintenance phase, wherein the loading phase comprises administering the RNAi agent to the subject at a fixed dose of about 200 mg to about 600 mg, and the maintenance phase comprises administering the RNAi agent to the subject at a fixed dose of about 25 mg to about 100 mg approximately once per month, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, and wherein the antisense strand comprises a complementary region comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from nucleotides 3544 to 3623 of the nucleotide sequence of SEQ ID NO: 1, thereby inhibiting expression of the PCSK9 gene in the subject. 【0022】 In another aspect, the present invention provides a method for reducing low-density lipoprotein (LDLc) levels in a subject, comprising administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen including a loading phase followed by a maintenance phase, wherein the loading phase comprises administering the RNAi agent to the subject at a fixed dose of about 200 mg to about 600 mg, and the maintenance phase comprises administering the RNAi agent to the subject monthly at a fixed dose of about 25 mg to about 100 mg, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a duplex region, and wherein the antisense strand comprises a complementary region comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from nucleotides 3544 to 3623 of the nucleotide sequence of SEQ ID NO: 1, thereby reducing the LDLc level in the subject. 【0023】 In another aspect, the present invention provides a method of treating a subject having a disorder that would benefit from a decrease in expression of PCSK9. The method includes administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen including a loading phase followed by a maintenance phase, wherein the loading phase comprises administering the RNAi agent to the subject at a fixed dose of about 200 mg to about 600 mg, and the maintenance phase comprises administering the RNAi agent to the subject monthly at a fixed dose of about 25 mg to about 100 mg, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a duplex region, and wherein the antisense strand comprises a complementary region comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from nucleotides 3544 to 3623 of the nucleotide sequence of SEQ ID NO: 1, thereby treating the subject having a disorder that would benefit from a decrease in expression of PCSK9. 【0024】 In yet another aspect, the present invention provides a method for treating a subject with hyperlipidemia, comprising administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen including a loading phase followed by a maintenance phase, wherein the loading phase comprises administering the RNAi agent to the subject at a fixed dose of about 200 mg to about 600 mg, and the maintenance phase comprises administering the RNAi agent to the subject once a month at a fixed dose of about 25 mg to about 100 mg, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, and wherein the antisense strand comprises a complementary region comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from nucleotides 3544 to 3623 of the nucleotide sequence of SEQ ID NO: 1, thereby treating the subject with hyperlipidemia. 【0025】 The double-stranded RNAi agent may be administered to the subject subcutaneously, for example, by subcutaneous injection, or intramuscularly. 【0026】 In one embodiment, the antisense strand comprises a nucleotide sequence selected from the group consisting of any one of the unmodified nucleotide sequences presented in Table 1. In one embodiment, the double-stranded RNAi agent targets nucleotides 3601-3623 of SEQ ID NO: 1. In one embodiment, the agent targeting nucleotides 3601-3623 of SEQ ID NO: 1 is AD-60212. 【0027】 In one embodiment, the antisense strand comprises the nucleotide sequence 5'-ACAAAAGCAAAACAGGUCUAGAA-3' (SEQ ID NO: 685). 【0028】 In one embodiment, the sense strand comprises the nucleotide sequence 5'-CUAGACCUGUTUUGCUUUUGU-3' (SEQ ID NO: 686). 【0029】 In one embodiment, the double-stranded ribonucleic acid RNAi agent comprises at least one modified nucleotide. 【0030】 In one embodiment, substantially all of the nucleotides in the sense strand are modified nucleotides. In another embodiment, substantially all of the nucleotides in the antisense strand are modified nucleotides. In yet another embodiment, substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides. 【0031】 In one embodiment, all of the nucleotides in the sense strand are modified nucleotides. In another embodiment, all of the nucleotides in the antisense strand are modified nucleotides. In yet another embodiment, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand are modified nucleotides. 【0032】 In one aspect, the invention provides a method for inhibiting expression of the PCSK9 gene in a subject, the method comprising administering to the subject a fixed dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (RNAi) agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises the nucleotide sequence 5'-ACAAAAGCAAAACAGGUCUAGAA-3' (SEQ ID NO: 685) and the sense strand comprises the nucleotide sequence 5'-CUAGACCUGUTUUGCUUUUGU-3' (SEQ ID NO: 686), and wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, thereby inhibiting expression of the PCSK9 gene in the subject. 【0033】 In another aspect, the present invention provides a method for reducing low-density lipoprotein (LDLc) levels in a subject, the method comprising administering to the subject a fixed dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (RNAi) agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises the nucleotide sequence 5'-ACAAAAGCAAAACAGGUCUAGAA-3' (SEQ ID NO: 685) and the sense strand comprises the nucleotide sequence 5'-CUAGACCUGUTUUGCUUUUGU-3' (SEQ ID NO: 686), and wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, thereby reducing the LDLc level in the subject. 【0034】 In another aspect, the invention provides a method of treating a subject having a disorder that would benefit from a decrease in expression of PCSK9, comprising the step of administering to the subject a fixed dose of from about 25 mg to about 800 mg of a double-stranded ribonucleic acid (RNAi) agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises the nucleotide sequence 5'-ACAAAAGCAAAACAGGUCUAGAA-3' (SEQ ID NO: 685) and the sense strand comprises the nucleotide sequence 5'-CUAGACCUGUTUUGCUUUUGU-3' (SEQ ID NO: 686), and wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, thereby treating a subject having a disorder that would benefit from a decrease in expression of PCSK9. 【0035】 In yet another aspect, the present invention provides a method for treating a subject with hyperlipidemia, comprising administering to the subject a fixed dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (RNAi) agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence 5'-ACAAAAGCAAAACAGGUCUAGAA-3' (SEQ ID NO: 685) and the sense strand comprises the nucleotide sequence 5'-CUAGACCUGUTUUGCUUUUGU-3' (SEQ ID NO: 686), and wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, thereby treating the subject with hyperlipidemia. 【0036】 In one aspect, the invention provides a method for inhibiting expression of the PCSK9 gene in a subject. The method includes administering to the subject a fixed dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (RNAi) agent and a therapeutically effective amount of an anti-PCSK9 antibody or antigen-binding fragment thereof, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises the nucleotide sequence 5'-ACAAAAGCAAAACAGGUCUAGAA-3' (SEQ ID NO: 685) and the sense strand comprises the nucleotide sequence 5'-CUAGACCUGUTUUGCUUUUGU-3' (SEQ ID NO: 686), and wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, thereby inhibiting expression of the PCSK9 gene in the subject. 【0037】 In another aspect, the present invention provides a method for reducing low-density lipoprotein (LDLc) levels in a subject, the method comprising administering to the subject a fixed dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (RNAi) agent and a therapeutically effective amount of an anti-PCSK9 antibody or antigen-binding fragment thereof, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises the nucleotide sequence 5'-ACAAAAGCAAAACAGGUCUAGAA-3' (SEQ ID NO: 685) and the sense strand comprises the nucleotide sequence 5'-CUAGACCUGUTUUGCUUUUGU-3' (SEQ ID NO: 686), and wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, thereby reducing the level of LDLc in the subject. 【0038】 In another aspect, the invention provides a method of treating a subject having a disorder that would benefit from a decrease in expression of PCSK9, comprising the steps of administering to the subject a double-stranded ribonucleic acid (RNAi) agent at a fixed dose of about 25 mg to about 800 mg; and administering to the subject a therapeutically effective amount of an anti-PCSK9 antibody or antigen-binding fragment thereof, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises the nucleotide sequence 5'-ACAAAAGCAAAACAGGUCUAGAA-3' (SEQ ID NO: 685) and the sense strand comprises the nucleotide sequence 5'-CUAGACCUGUTUUGCUUUUGU-3' (SEQ ID NO: 686), and wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, thereby treating the subject having a disorder that would benefit from a decrease in expression of PCSK9. 【0039】 In yet another aspect, the present invention provides a method for treating a subject with hyperlipidemia, comprising administering to the subject a fixed dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (RNAi) agent and a therapeutically effective amount of an anti-PCSK9 antibody or antigen-binding fragment thereof, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence 5'-ACAAAAGCAAAACAGGUCUAGAA-3' (SEQ ID NO: 685) and the sense strand comprises the nucleotide sequence 5'-CUAGACCUGUTUUGCUUUUGU-3' (SEQ ID NO: 686), and wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, thereby treating the subject with hyperlipidemia. 【0040】 The fixed dose may be administered to the subject once weekly, once every two weeks, once monthly, once quarterly, or at semi-annual intervals. 【0041】 In one embodiment, the subject is administered a flat dose of about 25 mg to about 50 mg once weekly. In another embodiment, the subject is administered a flat dose of about 50 mg to about 100 mg once every two weeks. In another embodiment, the subject is administered a flat dose of about 100 mg to about 200 mg once monthly. In yet another embodiment, the subject is administered a flat dose of about 300 mg to about 800 mg once quarterly. In another embodiment, the subject is administered a flat dose of about 300 mg to about 800 mg every six months. 【0042】 In one aspect, the present invention provides a method for inhibiting expression of the PCSK9 gene in a subject. The method comprises administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen including a loading phase followed by a maintenance phase, wherein the loading phase comprises administering the RNAi agent to the subject at a fixed dose of about 200 mg to about 600 mg, and the maintenance phase comprises administering the RNAi agent to the subject once quarterly at a fixed dose of about 25 mg to about 100 mg, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a duplex region, wherein the antisense strand comprises the nucleotide sequence 5'-ACAAAAGCAAAACAGGUCUAGAA-3' (SEQ ID NO: 685) and the sense strand comprises the nucleotide sequence 5'-CUAGACCUGUTUUGCUUUUGU-3' (SEQ ID NO: 686), and wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, thereby inhibiting expression of the PCSK9 gene in the subject. 【0043】 In another aspect, the present invention provides a method for reducing low-density lipoprotein (LDLc) levels in a subject, the method comprising administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen including a loading phase followed by a maintenance phase, wherein the loading phase comprises administering the RNAi agent to the subject at a fixed dose of about 200 mg to about 600 mg, and the maintenance phase comprises administering the RNAi agent to the subject once quarterly at a fixed dose of about 25 mg to about 100 mg, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a duplex region, wherein the antisense strand comprises the nucleotide sequence 5'-ACAAAAGCAAAACAGGUCUAGAA-3' (SEQ ID NO: 685) and the sense strand comprises the nucleotide sequence 5'-CUAGACCUGUTUUGCUUUUGU-3' (SEQ ID NO: 686), wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, thereby reducing the LDLc level in the subject. 【0044】 In another aspect, the invention provides a method of treating a subject having a disorder that would benefit from a decrease in the expression of PCSK9. The method includes administering a double-stranded ribonucleic acid (RNAi) agent to a subject in a dosing regimen including a loading phase followed by a maintenance phase, wherein the loading phase includes administering the RNAi agent to the subject at a fixed dose of about 200 mg to about 600 mg, and the maintenance phase includes administering the RNAi agent to the subject once quarterly at a fixed dose of about 25 mg to about 100 mg, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises the nucleotide sequence 5'-ACAAAAGCAAAACAGGUCUAGAA-3' (SEQ ID NO: 685), and the sense strand comprises the nucleotide sequence 5'-CUAGACCUGUTUUGCUUUUGU-3' (SEQ ID NO: 686), and wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, thereby treating a subject having a disorder that would benefit from a decrease in expression of PCSK9. 【0045】 In yet another aspect, the present invention provides a method of treating a subject with hyperlipidemia, comprising administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen including a loading phase followed by a maintenance phase, wherein the loading phase comprises administering the RNAi agent to the subject at a fixed dose of about 200 mg to about 600 mg, and the maintenance phase comprises administering the RNAi agent to the subject once quarterly at a fixed dose of about 25 mg to about 100 mg, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a duplex region, wherein the antisense strand comprises the nucleotide sequence 5'-ACAAAAGCAAAACAGGUCUAGAA-3' (SEQ ID NO: 685) and the sense strand comprises the nucleotide sequence 5'-CUAGACCUGUTUUGCUUUUGU-3' (SEQ ID NO: 686), wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, thereby treating the subject with hyperlipidemia. 【0046】 In one aspect, the invention provides a method of inhibiting expression of the PCSK9 gene in a subject. The method includes administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen including a loading phase followed by a maintenance phase; and administering to the subject a therapeutically effective amount of an anti-PCSK9 antibody or antigen-binding fragment thereof, wherein the loading phase comprises administering the RNAi agent to the subject at a fixed dose of about 200 mg to about 600 mg, and the maintenance phase comprises administering the RNAi agent to the subject once quarterly at a fixed dose of about 25 mg to about 100 mg, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises the nucleotide sequence 5'-ACAAAAGCAAAACAGGUCUAGAA-3' (SEQ ID NO: 685), and the sense strand comprises the nucleotide sequence 5'-CUAGACCUGUTUUGCUUUUGU-3' (SEQ ID NO: 686), wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, thereby inhibiting expression of the PCSK9 gene in the subject. 【0047】 In another aspect, the present invention provides a method of reducing low density lipoprotein (LDLc) levels in a subject. The method includes administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen including a loading phase followed by a maintenance phase; and administering to the subject a therapeutically effective amount of an anti-PCSK9 antibody or antigen-binding fragment thereof, wherein the loading phase includes administering to the subject a fixed dose of about 200 mg to about 600 mg of the RNAi agent, and the maintenance phase includes administering to the subject once quarterly a fixed dose of about 25 mg to about 100 mg of the RNAi agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises the nucleotide sequence 5'-ACAAAAGCAAAACAGGUCUAGAA-3' (SEQ ID NO: 685), and the sense strand comprises the nucleotide sequence 5'-CUAGACCUGUTUUGCUUUUGU-3' (SEQ ID NO: 686), wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, thereby reducing the level of LDLc in the subject. 【0048】 In another aspect, the present invention provides a method of treating a subject having a disorder that would benefit from a decrease in expression of PCSK9. The method includes administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen including a loading phase followed by a maintenance phase, and administering to the subject a therapeutically effective amount of an anti-PCSK9 antibody or antigen-binding fragment thereof, wherein the loading phase comprises administering to the subject a fixed dose of about 200 mg to about 600 mg of the RNAi agent, and the maintenance phase comprises administering to the subject once quarterly a fixed dose of about 25 mg to about 100 mg of the RNAi agent, wherein the double-stranded RNAi agent comprises a sense strand and a nucleotide sequence that form a double-stranded region. and an antisense strand, wherein the antisense strand comprises the nucleotide sequence 5'-ACAAAAGCAAAACAGGUCUAGAA-3' (SEQ ID NO: 685) and the sense strand comprises the nucleotide sequence 5'-CUAGACCUGUTUUGCUUUUGU-3' (SEQ ID NO: 686), wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, thereby treating a subject having a disorder that would benefit from a decrease in expression of PCSK9. 【0049】 In yet another aspect, the invention provides a method of treating a subject with hyperlipidemia. The method includes administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen including a loading phase followed by a maintenance phase; and administering to the subject a therapeutically effective amount of an anti-PCSK9 antibody or antigen-binding fragment thereof, wherein the loading phase includes administering to the subject a fixed dose of about 200 mg to about 600 mg of the RNAi agent, and the maintenance phase includes administering to the subject once quarterly a fixed dose of about 25 mg to about 100 mg of the RNAi agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises the nucleotide sequence 5'-ACAAAAGCAAAACAGGUCUAGAA-3' (SEQ ID NO: 685), and the sense strand comprises the nucleotide sequence 5'-CUAGACCUGUTUUGCUUUUGU-3' (SEQ ID NO: 686), wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, thereby treating the subject with hyperlipidemia. 【0050】 In one embodiment, the subject is a human. 【0051】 In one embodiment, the disorder that would benefit from a decrease in expression of PCSK9 is hyperlipidemia, eg, hypercholesterolemia. 【0052】 In one embodiment, the hyperlipidemia is hypercholesterolemia. 【0053】 The double-stranded RNAi agent may be administered to the subject subcutaneously, for example, by subcutaneous injection, or intramuscularly. 【0054】 In one embodiment, the sense strand comprises the nucleotide sequence 5'-csusagacCfuGfudTuugcuuuugu-3' (SEQ ID NO: 687), and the antisense strand comprises the nucleotide sequence 5'-asCfsaAfAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 688), where A, G, c, and u are 2'-O-methyl (2'-OMe) A, G, C, or U; Af, Gf, Cf, or Uf is 2'-fluoro A, G, C, or U; dT is 2'-deoxythymidine; and s is a phosphorothioate linkage) (AD-60212). 【0055】 In one embodiment, the double-stranded ribonucleic acid RNAi agent further comprises a ligand. 【0056】 In one embodiment, the ligand is attached to the 3' end of the sense strand of the double-stranded ribonucleic acid RNAi agent. 【0057】 In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative. 【0058】 In one embodiment, the ligand is [ka] is. 【0059】 In one embodiment, the double-stranded ribonucleic acid RNAi agent is shown in the following schematic diagram: [ka] wherein X is O or S. In one embodiment, X is O. 【0060】 In one embodiment, expression of PCSK9 is inhibited by at least about 30%. 【0061】 In one embodiment, the method of the invention further comprises determining the subject's LDLR genotype or phenotype. 【0062】 In one embodiment, administration of the double-stranded RNAi agent results in reduced serum cholesterol and / or reduced PCSK9 protein accumulation in the subject. 【0063】 In one embodiment, the method of the present invention further comprises measuring serum cholesterol levels in the subject. 【0064】 In one embodiment, the method of the invention further comprises administering to the subject an additional therapeutic agent, e.g., a statin and / or an anti-PCSK9 antibody. In one embodiment, the anti-PCSK9 antibody is selected from the group consisting of alirocumab (Praluent), evolocumab (Repatha), and bococizumab. 【0065】 In one embodiment, the RNAi agent is administered as a pharmaceutical composition. 【0066】 RNAi agent can be administered in non-buffered solution, such as saline or water, or can be administered with buffer solution.In one embodiment, buffer solution comprises acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof.In another embodiment, buffer solution is phosphate buffered saline (PBS). 【0067】 In one aspect, the present invention provides a method for inhibiting expression of the PCSK9 gene in a subject, the method comprising the steps of administering to the subject a single fixed dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (RNAi) agent and administering to the subject a therapeutically effective amount of an anti-PCSK9 antibody or antigen-binding fragment thereof, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising the nucleotide sequence 5'-csusagacCfuGfudTuugcuuuugu-3' (SEQ ID NO: 687) and the antisense strand comprising the nucleotide sequence 5'-as CfsaAfAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 688), where A, G, c, and u are 2'-O-methyl (2'-OMe) A, G, C, or U; Af, Gf, Cf, or Uf is 2'-fluoro A, G, C, or U; dT is 2'-deoxythymidine; and s is a phosphorothioate linkage) (AD-60212), thereby inhibiting expression of the PCSK9 gene in a subject. 【0068】 In another aspect, the present invention provides a method for reducing low-density lipoprotein (LDLc) levels in a subject, the method comprising the steps of administering to the subject a double-stranded ribonucleic acid (RNAi) agent at a fixed dose of about 25 mg to about 800 mg, and administering to the subject a therapeutically effective amount of an anti-PCSK9 antibody or antigen-binding fragment thereof, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising the nucleotide sequence 5'-csusagacCfuGfudTuugcuuuugu-3' (SEQ ID NO: 687), and the antisense strand comprising the nucleotide sequence 5'-as CfsaAfAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 688), where A, G, c, and u are 2'-O-methyl (2'-OMe) A, G, C, or U; Af, Gf, Cf, or Uf is 2'-fluoro A, G, C, or U; dT is 2'-deoxythymidine; and s is a phosphorothioate linkage (AD-60212), thereby reducing the level of LDLc in a subject. 【0069】 In another aspect, the present invention provides a method of treating a subject having a disorder that would benefit from a decrease in expression of PCSK9. The method includes administering to the subject a double-stranded ribonucleic acid (RNAi) agent at a fixed dose of about 25 mg to about 800 mg, and administering to the subject a therapeutically effective amount of an anti-PCSK9 antibody or antigen-binding fragment thereof, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises the nucleotide sequence of 5'-csusagacCfuGfudTuugcuuuugu-3' (SEQ ID NO: 687), and the antisense strand comprises the nucleotide sequence of 5'-asCfsaAfAfAf gCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 688), where A, G, c, and u are 2'-O-methyl (2'-OMe) A, G, C, or U; Af, Gf, Cf, or Uf is 2'-fluoro A, G, C, or U; dT is 2'-deoxythymidine; and s is a phosphorothioate linkage (AD-60212), thereby treating a subject having a disorder that would benefit from a decrease in expression of PCSK9. 【0070】 In yet another aspect, the present invention provides a method for treating a subject with hyperlipidemia, comprising the steps of administering to the subject a double-stranded ribonucleic acid (RNAi) agent at a fixed dose of about 25 mg to about 800 mg, and administering to the subject a therapeutically effective amount of an anti-PCSK9 antibody or antigen-binding fragment thereof, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence 5'-csusagacCfuGfudTuugcuuuugu-3' (SEQ ID NO: 687), and the antisense strand comprises the nucleotide sequence 5'- asCfsaAfAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 688), where A, G, c, and u are 2'-O-methyl (2'-OMe) A, G, C, or U; Af, Gf, Cf, or Uf is 2'-fluoro A, G, C, or U; dT is 2'-deoxythymidine; and s is a phosphorothioate linkage (AD-60212), thereby treating a subject with hyperlipidemia. 【0071】 In one embodiment, a subject is administered a flat dose of about 200 mg to about 800 mg once quarterly, hi another embodiment, a subject is administered a flat dose of about 200 mg to about 800 mg every six months. 【0072】 In one aspect, the present invention provides a method for inhibiting expression of the PCSK9 gene in a subject, comprising administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen including a loading phase followed by a maintenance phase, and administering to the subject a therapeutically effective amount of an anti-PCSK9 antibody or antigen-binding fragment thereof, wherein the loading phase comprises administering to the subject a fixed dose of about 200 mg to about 600 mg of the RNAi agent, and the maintenance phase comprises administering to the subject once quarterly a fixed dose of about 25 mg to about 800 mg of the RNAi agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, and the sense strand is 5'-csusagacCfuGfud and the antisense strand comprises the nucleotide sequence 5'-asCfsaAfAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 688), where A, G, c, and u are 2'-O-methyl (2'-OMe) A, G, C, or U; Af, Gf, Cf, or Uf is 2'-fluoro A, G, C, or U; dT is 2'-deoxythymidine; and s is a phosphorothioate linkage) (AD-60212), thereby inhibiting expression of the PCSK9 gene in a subject. 【0073】 In another aspect, the present invention provides a method for reducing low-density lipoprotein (LDLc) levels in a subject, the method comprising administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen including a loading phase followed by a maintenance phase, and administering to the subject a therapeutically effective amount of an anti-PCSK9 antibody or antigen-binding fragment thereof, wherein the loading phase comprises administering to the subject a fixed dose of about 200 mg to about 600 mg of the RNAi agent, and the maintenance phase comprises administering to the subject once quarterly a fixed dose of about 25 mg to about 100 mg of the RNAi agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising the amino acid sequence 5'-csusagacCfuGfu and the antisense strand comprises the nucleotide sequence 5'-asCfsaAfAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 688), where A, G, c, and u are 2'-O-methyl (2'-OMe) A, G, C, or U; Af, Gf, Cf, or Uf is 2'-fluoro A, G, C, or U; dT is 2'-deoxythymidine; and s is a phosphorothioate linkage) (AD-60212), thereby reducing the level of LDLc in a subject. 【0074】 In yet another aspect, the present invention provides a method of treating a subject having a disorder that would benefit from a decrease in expression of PCSK9. The method includes administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen including a loading phase followed by a maintenance phase, and administering to the subject a therapeutically effective amount of an anti-PCSK9 antibody or antigen-binding fragment thereof, wherein the loading phase comprises administering to the subject a fixed dose of about 200 mg to about 600 mg of the RNAi agent, and the maintenance phase comprises administering to the subject once quarterly a fixed dose of about 25 mg to about 100 mg of the RNAi agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, and the sense strand is 5'-csusagacCfuGfudTuugcuuuu and the antisense strand comprises the nucleotide sequence of 5'-asCfsaAfAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 688), where A, G, c, and u are 2'-O-methyl (2'-OMe) A, G, C, or U; Af, Gf, Cf, or Uf is 2'-fluoro A, G, C, or U; dT is 2'-deoxythymidine; and s is a phosphorothioate linkage) (AD-60212), thereby treating a subject having a disorder that would benefit from a decrease in PCSK9 expression. 【0075】 In another aspect, the present invention provides a method for treating a subject with hyperlipidemia, comprising administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen including a loading phase followed by a maintenance phase, and administering to the subject a therapeutically effective amount of an anti-PCSK9 antibody or antigen-binding fragment thereof, wherein the loading phase comprises administering to the subject a fixed dose of about 200 mg to about 600 mg of the RNAi agent, and the maintenance phase comprises administering to the subject a fixed dose of about 25 mg to about 100 mg of the RNAi agent once quarterly, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, and the sense strand is selected from the group consisting of 5'-csusagacCfu GfudTuugcuuuugu-3' (SEQ ID NO: 687), and the antisense strand comprises the nucleotide sequence 5'-asCfsaAfAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 688), where A, G, c, and u are 2'-O-methyl (2'-OMe) A, G, C, or U; Af, Gf, Cf, or Uf is 2'-fluoro A, G, C, or U; dT is 2'-deoxythymidine; and s is a phosphorothioate linkage) (AD-60212), thereby treating a subject with hyperlipidemia. 【0076】 In one embodiment, the subject receives a maintenance dose of about 200 mg to about 800 mg administered once quarterly. In another embodiment, the subject receives a maintenance dose of about 200 mg to about 800 mg administered semi-annually. 【0077】 In one embodiment, the double-stranded ribonucleic acid RNAi agent further comprises a ligand. 【0078】 In one embodiment, the ligand is attached to the 3' end of the sense strand of the double-stranded ribonucleic acid RNAi agent. 【0079】 In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative. 【0080】 In one embodiment, the ligand is [ka] is. 【0081】 In one embodiment, the double-stranded ribonucleic acid RNAi agent is shown in the following schematic diagram: [ka] wherein X is O or S. In one embodiment, X is O. 【0082】 In one embodiment, the anti-PCSK9 antibody, or antigen-binding fragment thereof, is selected from the group consisting of alirocumab (Praluent), evolocumab (Repatha), and bococizumab. 【0083】 In one embodiment, the method further comprises administering to the subject an additional therapeutic agent, for example, a statin. 【0084】 In one aspect, the invention provides a kit for carrying out the method of the invention, the kit comprising an RNAi agent and instructions for use, and optionally, a means for administering the RNAi agent to a subject. 【0085】 The present invention is further illustrated by the following detailed description and drawings. [Brief explanation of the drawings] 【0086】 [Figure 1] 1 is a graph showing knockdown of PCSK9 protein levels, shown as percent mean PCSK9 knockdown relative to baseline, in subjects receiving a single fixed dose of AD-60212. [Figure 2] 1 is a graph showing the reduction in LDL-c levels, expressed as percent mean LDL-C reduction relative to baseline, in subjects receiving a single fixed dose of AD-60212. [Figure 3]1 is a graph showing knockdown of PCSK9 protein levels, shown as percent mean PCSK9 knockdown relative to baseline, in subjects receiving multiple fixed doses of AD-60212. [Figure 4] 1 is a graph showing the reduction in LDL-c levels, expressed as percent mean LDL-C reduction relative to baseline, in subjects receiving multiple fixed doses of AD-60212. DETAILED DESCRIPTION OF THE INVENTION 【0087】 The present invention is based, at least in part, on the surprising discovery that double-stranded RNAi agents containing chemical modifications exhibit exceptional potency and durability in inhibiting expression of PCSK9 at a single dose. Specifically, it is shown herein that RNAi agents targeting the human PCSK9 gene, e.g., nucleotides 3544-3623 of the human PCSK9 gene (nucleotides 3544-3623 of SEQ ID NO:1), e.g., nucleotides 3601-3623 of SEQ ID NO:1 (containing a GalNAc ligand), are exceptionally effective and durable in silencing the activity of the PCSK9 gene at a single fixed dose, e.g., a fixed dose of about 300 mg to about 500 mg. 【0088】 Thus, the present invention provides methods for inhibiting expression of the PCSK9 gene and for treating a subject having a disorder that would benefit from inhibiting or reducing expression of PCSK9, e.g., a disorder mediated by expression of PCSK9, e.g., hyperlipidemia, e.g., hypercholesterolemia, with an iRNA composition that effects RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the PCSK9 gene. 【0089】 The following detailed description discloses methods for making and utilizing compositions containing iRNAs that inhibit PCSK9 gene expression, as well as compositions, uses, and methods for treating subjects with diseases and disorders that would benefit from inhibiting and / or reducing the expression of this gene. 【0090】 I. Definition In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values ​​for a parameter is listed, it is intended that values ​​and ranges intermediate to the listed values ​​are also part of the invention. 【0091】 The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or two or more elements, such as, for example, a plurality of elements. 【0092】 The term "including" is used herein to mean, and is used interchangeably with, the term "including but not limited to." 【0093】 The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly dictates otherwise. For example, "the sense strand or the antisense strand" is understood as "the sense strand or the antisense strand, or the sense strand and the antisense strand." 【0094】 The term "about" is used herein to mean within a typical range accepted in the art. For example, "about" can be understood as about 2 standard deviations from the mean. In certain embodiments, about means +10%. In certain embodiments, about means +5%. When about precedes a series of numbers or a range, it is understood that "about" can modify each of the series of numbers or ranges. 【0095】 The term "at least" preceding a number or a series of numbers is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that can be logically included, as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the specified property. When "at least" is present before a series of numbers or a range, it is understood that "at least" can modify each of the series of numbers or the range. 【0096】 As used herein, "less than" or "less than" is understood as the value adjacent to the term and the logically lower value or integer, up to 0, as the logic of the context dictates. For example, a duplex having an overhang of "two or fewer nucleotides" has 2, 1, or 0 nucleotide overhangs. When "less than" precedes a series of numbers or a range, it is understood that "less than" can modify each of the series of numbers or range. 【0097】 As used herein, "PCSK9" refers to the proprotein convertase subtilisin / kexin type 9 gene or protein. PCSK9 is also known as FH3, HCHOLA3, NARC-1, or NARC1. The term PCSK9 includes human PCSK9 (the amino acid and nucleotide sequences of which may be found, for example, in GenBank Accession No. GI:299523249 (SEQ ID NO:1)); mouse PCSK9 (the amino acid and nucleotide sequences of which may be found, for example, in GenBank Accession No. GI:163644257); and rat PCSK9 (the amino acid and nucleotide sequences of which may be found, for example, in GenBank Accession No. GI:77020249). 【0098】 Further examples of PCSK9 mRNA sequences are readily available using publicly available databases such as GenBank, UniProt, and OMIM. 【0099】 In one embodiment, the subject is a human, e.g., a human being undergoing treatment or evaluation for a disease, disorder, or condition that would benefit from a decrease in expression of PCSK9, as described herein; a human being at risk for a disease, disorder, or condition that would benefit from a decrease in expression of PCSK9; a human being with a disease, disorder, or condition that would benefit from a decrease in expression of PCSK9; and / or a human being undergoing treatment for a disease, disorder, or condition that would benefit from a decrease in expression of PCSK9. 【0100】 As used herein, the term "treat" or "treatment" refers to a beneficial or desired result, including, but not limited to, the alleviation or amelioration of one or more symptoms associated with a disorder that would benefit from a decrease in PCSK9 expression, whether detectable or undetectable, or a slowing or reversal of the progression of such a disorder. For example, in the context of hyperlipidemia, treatment may include lowering serum lipid levels, such as lowering low-density lipoprotein cholesterol (LDLc). "Treatment" may also mean prolonging survival compared to expected survival in the absence of treatment. 【0101】 As used herein, "prevention" or "preventing," when used in reference to a disease, disorder, or condition that would benefit from a decrease in expression of the PCSK9 gene, refers to a reduction in the likelihood that a subject will develop symptoms associated with a disease, disorder, or condition mediated by expression of PCSK9, such as cardiovascular disease, e.g., coronary artery disease (CAD) (also known as coronary heart disease (CHD)), or a transient ischemic attack (TIA) or stroke. The likelihood of developing such symptoms is reduced, for example, when an individual with one or more risk factors for a disease, disorder, or condition mediated by PCSK9 expression, e.g., hypercholesterolemia (e.g., diabetes, a past personal history of CHD or non-coronary atherosclerosis (e.g., abdominal aortic aneurysm, peripheral artery disease, and carotid artery stenosis), a family history of cardiovascular disease, e.g., in a male relative under age 50 or a female relative under age 60, tobacco use, hypertension, and / or obesity (BMI≧30)) either does not develop, for example, coronary artery disease, or develops coronary artery disease that is less severe, e.g., relative to a population with the same risk factors and not receiving treatment as described herein. Failure to develop a disease, disorder, or condition, or a reduction in the onset of symptoms associated with such disease, disorder, or condition (e.g., by at least about 10% on the scale clinically recognized for the disease or disorder), or delayed later symptoms (e.g., by days, weeks, months, or years) is considered effective prevention. Prevention may require the administration of more than one dose. 【0102】 The interchangeably used terms "PCSK9-related disease" and "disorders that would benefit from a decrease in PCSK9 expression" as used herein are intended to include any disease, disorder, or condition associated with the PCSK9 gene or protein. Such diseases may be caused, for example, by overproduction of PCSK9 protein, mutations in the PCSK9 gene, abnormal cleavage of the PCSK9 protein, or abnormal interactions between PCSK9 and other proteins or other endogenous or exogenous substances. Exemplary PCSK9-related diseases include lipidemia, such as hyperlipidemia, and other forms of lipid imbalance, such as hypercholesterolemia, hypertriglyceridemia, and conditions associated with these disorders, such as CHD and atherosclerosis. 【0103】 As used herein, the term "hypercholesterolemia" refers to a form of hyperlipidemia (elevated lipid levels in the blood) in which a subject has high levels of cholesterol in their serum, e.g., at least about 240 mg / dL of total cholesterol. 【0104】 As used herein, the term "cardiovascular disease" refers to diseases that affect the heart or blood vessels, including, for example, arteriosclerosis, coronary artery disease (or narrowing of the arteries), valvular heart disease, arrhythmias, heart failure, hypertension, orthostatic hypotension, shock, endocarditis, diseases of the aorta and its branches, disorders of the peripheral vascular system, heart attack, cardiomyopathies, and congenital heart disease. 【0105】 As used herein, a "therapeutically effective amount" is intended to include an amount of an RNAi agent that, when administered to a patient to treat a PCSK9-related disease, is sufficient to effect treatment of the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of the disease). A "therapeutically effective amount" may vary depending on the RNAi agent, how the agent is administered, the disease and its severity and medical history, age, weight, family history, genetic makeup, the stage of the pathological process mediated by PCSK9 expression, the type of prior or concomitant treatment (if any), and other individual characteristics of the patient to be treated. 【0106】 "Prophylactically effective amount" as used herein is intended to include the amount of RNAi agent that is sufficient to prevent or ameliorate disease or one or more symptoms of disease when administered to a subject who has not yet experienced or exhibited the symptoms of PCSK9-related disease but may be susceptible to the disease.Ameliorating disease includes slowing the course of disease or reducing the severity of later-developing disease.The "prophylactically effective amount" may vary depending on the RNAi agent, how the agent is administered, the degree of disease risk, and medical history, age, weight, family history, genetic makeup, type of previous treatment or concomitant treatment (if any), and other individual characteristics of the patient to be treated. 【0107】 A "therapeutically effective amount" or a "prophylactically effective amount" also includes an amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The RNAi agents used in the methods of the invention may be administered in amounts sufficient to produce a reasonable benefit / risk ratio applicable to such treatment. 【0108】 As used herein, "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the PCSK9 gene, including mRNA, which is an RNA processing product of the primary transcript. In one embodiment, the target portion of the sequence is at least sufficiently long to serve as a substrate for iRNA-directed cleavage at or near the portion of the nucleotide sequence of the mRNA molecule formed during transcription of the PCSK9 gene. 【0109】 The target sequence may be about 9-36 nucleotides in length, such as about 15-30 nucleotides in length. For example, the target sequence may be 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-2 The target sequence may be about 15 to 30 nucleotides in length, such as 6, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In some embodiments, the target sequence is about 19 to about 30 nucleotides in length. In other embodiments, the target sequence is about 19 to about 25 nucleotides in length. In still other embodiments, the target sequence is about 19 to about 23 nucleotides in length. In some embodiments, the target sequence is about 21 to about 23 nucleotides in length. Ranges and lengths intermediate to the recited ranges and lengths are also intended to be part of the invention. 【0110】 As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising the nucleotide strand described by the referenced sequence, using standard nucleotide nomenclature. 【0111】 "G", "C", "A", "T" and "U" generally represent nucleotides containing guanine, cytosine, adenine, thymidine and uracil as bases, respectively.However, it is understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides or alternative replacement moieties, as will be further detailed below (see, for example, Table B).Those skilled in the art are well aware that guanine, cytosine, adenine and uracil can be replaced with other moieties without substantially changing the base pairing properties of the oligonucleotides comprising nucleotides with such replacement moieties.As a non-limiting example, a nucleotide comprising inosine as a base can base pair with a nucleotide containing adenine, cytosine or uracil.Therefore, a nucleotide containing uracil, guanine or adenine can be replaced with a nucleotide containing inosine, for example, in the nucleotide sequence of the dsRNA of the present invention. In another example, adenine and cytosine can be substituted with guanine and uracil, respectively, anywhere in the oligonucleotide to form a GU wobble base pair with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods featured herein. 【0112】 The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interference agent" are used interchangeably herein and refer to an agent that contains RNA, as defined herein, and mediates targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. iRNA induces sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA regulates, e.g., inhibits, PCSK9 expression in a cell, e.g., a cell in a subject, e.g., a mammalian subject. 【0113】 In one embodiment, the RNAi agent of the present invention includes a single-stranded RNA that interacts with a target RNA sequence, such as a PCSK9 target mRNA sequence, to induce cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNA introduced into cells is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes dsRNA into short interfering RNAs of 19 to 23 base pairs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to induce target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding with the appropriate target mRNA, one or more endonucleases in RISC cleave the target and induce silencing (Elbashir, et al., (2001) Genes Dev.15:188).Therefore, in one aspect, the present invention relates to the single-stranded RNA (siRNA) that is produced in cells, promotes RISC complex formation, and leads to the silencing of target gene, i.e., PCSK9 gene.Therefore, the term "siRNA" is also used herein to refer to RNAi as described above. 【0114】 In another embodiment, the RNAi agent may be a "single-stranded siRNA" introduced into a cell or organism to inhibit target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any antisense nucleotide sequence described herein may be used as a single-stranded siRNA as described herein, or chemically modified as described in Lima et al., (2012) Cell 150:883-894. 【0115】 In another embodiment, the "iRNA" used in the compositions, uses, and methods of the present invention is double-stranded RNA, and is herein referred to as a "double-stranded RNAi agent," a "double-stranded RNA (dsRNA) molecule," a "dsRNA agent," or a "dsRNA." The term "dsRNA" refers to a ribonucleic acid molecule complex having a double-stranded structure, comprising two antiparallel and substantially complementary nucleic acid strands, which are referred to as having "sense" and "antisense" orientations with respect to the target RNA, i.e., the PCSK9 gene. In some embodiments of the present invention, double-stranded RNA (dsRNA) causes target RNA degradation, such as mRNA, through a post-transcriptional gene silencing mechanism, herein referred to as RNA interference or RNAi. 【0116】 Generally, the majority of the nucleotides in each strand of dsRNA molecule are ribonucleotides, but as described herein, each strand or both strands can also contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides.Furthermore, as used herein, " RNAi agent " can include ribonucleotides with chemical modification; RNAi agent can contain substantial modifications in multiple nucleotides.Such modification can include any type of modification disclosed herein or known in the art.Any such modification is encompassed by " RNAi agent " in the present specification and claims when used in siRNA type molecules. 【0117】 The double-stranded region may be of any length that allows for specific degradation of the desired target RNA through the RISC pathway, and may range from about 9 to 36 base pairs in length, for example, about 15 to 30 base pairs in length, such as about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, for example, about 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, and 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the recited ranges and lengths are also intended to be part of the invention. 【0118】 The two strands forming the double-stranded structure may be different portions of a larger RNA molecule, or they may be separate RNA molecules. When the two strands are part of a single larger molecule and are thus connected by an uninterrupted stretch of nucleotides between the 3' end of one strand and the 5' end of the other strand, the combined RNA strands are referred to as "hairpin loops." A hairpin loop may comprise at least one unpaired nucleotide; in some embodiments, a hairpin loop may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, or at least 23 or more unpaired nucleotides. In some embodiments, a hairpin loop may be 10 or fewer nucleotides long. In some embodiments, a hairpin loop may be 8 or fewer unpaired nucleotides long. In some embodiments, a hairpin loop may be 4 to 10 unpaired nucleotides long. In some embodiments, a hairpin loop may be 4 to 8 nucleotides long. 【0119】 When the two substantially complementary strands of dsRNA are composed of separate RNA molecules, these molecules can be, but are not necessarily, covalently linked. When the two strands are covalently linked between the 3'-end of one strand forming a double-stranded structure and the 5'-end of the other strand by means other than an uninterrupted nucleotide chain, the linked structure is called a "linker." The RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA minus any overhangs present in the double-stranded structure. In addition to the double-stranded structure, RNAi may also comprise one or more nucleotide overhangs. In one embodiment of an RNAi agent, at least one strand comprises a 3'-overhang of at least one nucleotide. In another embodiment, at least one strand comprises a 3'-overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5' overhang of at least one nucleotide. In certain embodiments, at least one strand comprises a 5' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3' and 5' ends of one strand of the RNAi agent comprise an overhang of at least one nucleotide. 【0120】 In one embodiment, the RNAi agent of the present invention is a dsRNA agent, each strand of which contains 19-23 nucleotides that interact with the target RNA sequence, i.e., the PCSK9 target mRNA sequence. Without wishing to be bound by theory, long double-stranded RNA introduced into cells is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). The RNase III-like enzyme of Dicer processes dsRNA to produce short interfering RNAs of 19-23 base pairs, characterized by a two-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). 【0121】 In another embodiment, the RNAi agent of the present invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, such as a PCSK9 target mRNA sequence, to induce cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNA introduced into cells is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). The RNase III-like enzyme of Dicer processes the dsRNA to produce short interfering RNAs of 19-23 base pairs, characterized by a 2-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). 【0122】 As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of an iRNA, such as a dsRNA. For example, a nucleotide overhang exists when the 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa. A dsRNA can contain an overhang of at least one nucleotide; alternatively, the overhang can contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang can comprise or consist of nucleotide / nucleoside analogs, such as deoxynucleotides / nucleosides. An overhang can be present in the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotide of the overhang can be present at the 5'-end, 3'-end, or both ends of either the antisense or sense strand of a dsRNA. 【0123】 In one embodiment of dsRNA, at least one strand comprises a 3' overhang of at least one nucleotide. In another embodiment, at least one strand comprises a 3' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of RNAi agent comprises a 5' overhang of at least one nucleotide. In certain embodiments, at least one strand comprises a 5' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3' and 5' ends of one strand of RNAi agent comprise an overhang of at least one nucleotide. 【0124】 In certain embodiments, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3'-end and / or 5'-end, e.g., 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3'-end and / or 5'-end, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more of the nucleotides in the overhang are replaced with a nucleoside thiophosphate. 【0125】 In certain embodiments, the overhang on the sense strand or the antisense strand, or both, can comprise an extended length of more than 10 nucleotides, e.g., 1 to 30 nucleotides, 2 to 30 nucleotides, 10 to 30 nucleotides, or 10 to 15 nucleotides. In certain embodiments, the extended overhang is present on the sense strand of the duplex. In certain embodiments, the extended overhang is present on the 3'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is present on the 5'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is present on the antisense strand of the duplex. In certain embodiments, the extended overhang is present on the 3'-end of the antisense strand of the duplex. In certain embodiments, the extended overhang is present on the 5'-end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the overhang are replaced with a nucleoside thiophosphate. In certain embodiments, the overhang comprises a self-complementary portion such that the overhang is capable of forming a hairpin structure that is stable under physiological conditions. 【0126】 " Blunt-ended " or " blunt-ended " means that there is no unpaired nucleotide at the end of double-stranded RNAi agent, i.e., there is no nucleotide overhang. " Blunt-ended " RNAi agent is double-stranded throughout its entire length, i.e., there is no nucleotide overhang at either end of the molecule. The RNAi agent of the present invention includes the RNAi agent that has nucleotide overhang at one end (i.e., the agent that has one overhang and one blunt end) or the RNAi agent that has nucleotide overhang at both ends. 【0127】 The term "antisense strand" or "guide strand" refers to the strand of an iRNA, e.g., a dsRNA, that includes a region that is substantially complementary to a target sequence, e.g., PCSK9 mRNA. As used herein, the term "region of complementarity," as defined herein, refers to a region on the antisense strand that is substantially complementary to a sequence, e.g., a target sequence, e.g., a PCSK9 nucleotide sequence. When the complementary region is not perfectly complementary to the target sequence, mismatches can occur within the internal or terminal regions of the molecule. Generally, mismatches are most tolerated within the terminal regions, e.g., within 5, 4, 3, 2, or 1 nucleotide at the 5' and / or 3' ends of the iRNA. In one embodiment, a double-stranded RNAi agent of the present invention includes a nucleotide mismatch within the antisense strand. In another embodiment, a double-stranded RNAi agent of the present invention includes a nucleotide mismatch within the sense strand. In one embodiment, the nucleotide mismatch is located, e.g., within 5, 4, 3, 2, or 1 nucleotide from the 3' end of the iRNA. In another embodiment, the nucleotide mismatch is located, e.g., within the 3'-terminal nucleotide of the iRNA. 【0128】 The term "sense strand" or "passenger strand," as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand, as those terms are defined herein. 【0129】 As used herein, the term "cleavage region" refers to the region located immediately adjacent to the cleavage site. The cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage region comprises three bases on either side of the cleavage site and immediately adjacent thereto. In some embodiments, the cleavage region comprises two bases on either side of the cleavage site and immediately adjacent thereto. In some embodiments, the cleavage site occurs specifically at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12, and 13. 【0130】 As used herein, unless otherwise specified, the term "complementary," when used to describe a first nucleotide sequence in the context of a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize to an oligonucleotide or polynucleotide comprising the second nucleotide sequence under specified conditions to form a double-stranded structure, as would be understood by one of skill in the art. Such conditions can be, for example, stringent conditions, such as 400 mM NaCl, 40 mM PIPES at pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12-16 hours, followed by washing (see, e.g., "Molecular Cloning: A Laboratory Manual," Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions that may be encountered in an organism, can be applied. One of skill in the art can determine the optimal set of conditions for testing the complementarity of two sequences depending on the end use of the hybridized nucleotides. 【0131】 For example, a complementary sequence in an iRNA, such as a dsRNA described herein, involves base pairing between an oligonucleotide or polynucleotide comprising a first nucleotide sequence and an oligonucleotide or polynucleotide comprising a second nucleotide sequence across the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as "fully complementary" to each other. However, when a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences may be fully complementary, or they may form one or more, but generally no more than 5, 4, 3, or 2 mismatched base pairs upon hybridization of a duplex of up to 30 base pairs, while retaining the ability to hybridize under conditions most appropriate for their end use, such as inhibiting gene expression through the RISC pathway. However, if two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs shall not be considered mismatches in determining complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, where the longer oligonucleotide is perfectly complementary to the shorter oligonucleotide, is still referred to as "perfectly complementary" for purposes described herein. 【0132】 "Complementary" sequences, as used herein, also include or may be formed entirely from non-Watson-Crick base pairs and / or base pairs formed from unnatural and modified nucleotides, so long as the above requirements regarding their hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairs or Hoogsteen base pairs. 【0133】 As used herein, the terms "complementary," "fully complementary," and "substantially complementary" may be used in reference to base matching between the sense and antisense strands of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context in which they are used. 【0134】 As used herein, a polynucleotide that is "substantially complementary to at least a portion" of a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a continuous portion of a subject mRNA (e.g., an mRNA encoding PCSK9). For example, a polynucleotide is complementary to at least a portion of a PCSK9 mRNA if its sequence is substantially complementary to a non-interrupted portion of the mRNA encoding PCSK9. 【0135】 Generally, the majority of the nucleotides in each strand are ribonucleotides, but as described in detail herein, one or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Furthermore, "iRNA" includes ribonucleotides with chemical modifications. Such modifications include all types of modifications disclosed herein or known in the art. For purposes of this specification and claims, any such modifications are encompassed by "iRNA" as used in reference to iRNA molecules. 【0136】 In one embodiment of the present invention, the agent used in the methods and compositions of the present invention is a single-stranded antisense RNA molecule that inhibits target mRNA via an antisense inhibition mechanism. The single-stranded antisense RNA molecule is complementary to a sequence within the target mRNA. Single-stranded antisense oligonucleotides can inhibit translation in a stoichiometric manner by base pairing with the mRNA and physically interfering with the translation machinery; see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense RNA molecule may be about 15 to about 30 nucleotides in length and have a sequence complementary to the target sequence. For example, the single-stranded antisense RNA molecule may comprise a sequence that is at least about 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense sequences described herein. 【0137】 II. The Methods of the Invention The present invention provides methods for inhibiting expression of the proprotein convertase subtilisin / kexin type 9 (PCSK9) gene in a subject. The present invention also provides therapeutic and prophylactic methods for treating or preventing diseases and conditions that can be modulated by downregulating PCSK9 gene expression. For example, the compositions described herein can be used to treat lipidemia, e.g., hyperlipidemia and other forms of lipid imbalance, e.g., hypercholesterolemia, hypertriglyceridemia, and conditions associated with these disorders, e.g., heart disease and circulatory system disease. Other diseases and conditions that can be modulated by downregulating PCSK9 gene expression include, but are not limited to, lysosomal storage diseases, including Niemann-Pick disease, Tay-Sachs disease, lysosomal acid lipase deficiency, and Gaucher disease. The method comprises administering a therapeutically or prophylactically effective amount of an RNAi agent of the invention to a subject. In some embodiments, the method comprises administering an effective amount of a PCSK9 iRNA agent to a patient with a heterozygous LDLR genotype. 【0138】 Because PCSK9 controls the level of LDL receptors and then removes cholesterol-rich LDL particles from plasma, the effect of reducing the expression of PCSK9 gene preferably results in a reduction in the LDLc (low-density lipoprotein cholesterol) level in mammalian blood, more particularly in serum.In some embodiments, the LDLc level is reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the level before treatment.Therefore, the present invention also provides a method for reducing the low-density cholesterol (LDLc) level in the serum of a subject. 【0139】 In certain embodiments of the invention, the double-stranded RNAi agent is administered to the subject as a fixed dose. A "fixed dose" (e.g., a dose in mg) means that a certain dose of the iRNA agent is used for all subjects, regardless of any specific subject-related factors, such as body weight. In other embodiments, the iRNA agent of the invention is administered to the subject as a weight-based dose. A "weight-based dose" (e.g., a dose in mg / kg) is a dose of the iRNA agent that will vary depending on the subject's body weight. 【0140】 In certain embodiments, the RNAi agent may be from about 100 mg to about 700 mg, from about 150 mg to about 700 mg, from about 200 mg to about 700 mg, from about 250 mg to about 700 mg, from about 300 mg to about 700 mg, from about 350 mg to about 700 mg, from about 400 mg to about 700 mg, from about 450 mg to about 700 mg, from about 500 mg to about 700 mg, from about 550 mg to about 700 mg, from about 600 mg to about 700 mg, from about 650 mg to about 700 mg, from about 100 mg to about 650 mg, from about 150 mg to about 650 mg, from about 200 mg to about 650 mg, or from about 250 mg ~650mg, 300mg~650mg, 350mg~650mg, 400mg~650mg, 450mg~650mg, 500mg~650mg, 550~650mg, 600~650mg, 100mg~600 mg, about 150mg to about 600mg, about 200mg to about 600mg, about 250mg to about 600mg, about 300mg to about 600mg, about 350mg to about 600mg, about 400mg to about 600mg, about 450mg to about 600mg, about 500mg to about 600mg, Approximately 550mg to approximately 600mg, approximately 100mg to approximately 550mg, approximately 150mg to approximately 550mg, approximately 200mg to approximately 550mg, approximately 250mg to approximately 550mg, approximately 300mg to approximately 550mg, approximately 350mg to approximately 550mg, approximately 400mg to approximately 550mg, approximately 4 50mg to about 550mg, about 500mg to about 550mg, about 100mg to about 500mg, about 150mg to about 500mg, about 200mg to about 500mg, about 250mg to about 500mg, about 300mg to about 500mg, about 350mg to about 500mg, about 400m The subject may be administered a fixed dose of about 100 mg, about 125 mg, about 150 mg, about 175 mg, 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, or about 700 mg. Values ​​and ranges intermediate to the aforementioned recited values ​​are also contemplated as part of the present invention. 【0141】 Administration may be repeated periodically, for example, a fixed dose may be administered to a subject once a week, once every two weeks, once a month, once a quarter, or every six months for a period of six months or a year or longer, i.e., chronic administration. 【0142】 In one embodiment, a subject is administered a fixed dose of about 25 mg to about 50 mg once a week. In another embodiment, a subject is administered a fixed dose of about 50 mg to about 100 mg once every two weeks. In another embodiment, a subject is administered a fixed dose of about 100 mg to about 200 mg once a month. In yet another embodiment, a subject is administered a fixed dose of about 300 mg to about 600 mg once a quarter. In another embodiment, a subject is administered a fixed dose of about 300 mg to about 600 mg every six months (i.e., twice a year). 【0143】 Thus, in one aspect, the present invention provides a method for inhibiting expression of the PCSK9 gene in a subject. The method includes administering to the subject a double-stranded ribonucleic acid (RNAi) agent of the invention, e.g., a dsRNA (e.g., a pharmaceutical composition comprising a dsRNA of the invention), wherein a total of about 200 mg to about 600 mg of the double-stranded RNAi agent is administered to the subject quarterly or semi-annually, and the double-stranded RNAi agent includes a sense strand and an antisense strand that form a double-stranded region, and the antisense strand includes a complementary region comprising at least 15 contiguous nucleotides that differ from nucleotides 3544 to 3623 of the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides. 【0144】 In another aspect, the present invention provides a method for reducing low-density lipoprotein (LDLc) levels in a subject, the method comprising administering to the subject a double-stranded ribonucleic acid (RNAi) agent, wherein a total of about 200 mg to about 600 mg of the double-stranded RNAi agent is administered to the subject quarterly or semi-annually, and the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising a complementary region comprising at least 15 contiguous nucleotides that differ from nucleotides 3544 to 3623 of the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides, thereby reducing the LDLc level in the subject. 【0145】 In another aspect, the present invention provides a method for treating a subject having a disorder that would benefit from a decrease in PCSK9 expression, e.g., hyperlipidemia, e.g., hypercholesterolemia. The method includes administering to the subject a double-stranded ribonucleic acid (RNAi) agent of the invention, e.g., a dsRNA (e.g., a pharmaceutical composition comprising a dsRNA of the invention), wherein a total of about 200 mg to about 600 mg of the double-stranded RNAi agent is administered to the subject quarterly or semi-annually, and wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, and wherein the antisense strand comprises a complementary region comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from nucleotides 3544 to 3623 of the nucleotide sequence of SEQ ID NO:1. 【0146】 In yet another aspect, the present invention provides a method for treating a subject having hyperlipidemia, e.g., hypercholesterolemia, comprising administering to the subject a double-stranded ribonucleic acid (RNAi) agent of the present invention, e.g., a dsRNA (e.g., a pharmaceutical composition comprising a dsRNA of the present invention), wherein a total of about 200 mg to about 600 mg of the double-stranded RNAi agent is administered to the subject quarterly or semi-annually, and the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, and the antisense strand comprises a complementary region comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from nucleotides 3544 to 3623 of the nucleotide sequence of SEQ ID NO:1. 【0147】 As noted above, administration of an RNAi agent to a subject may be repeated periodically, for example, once weekly, once every two weeks, once monthly, once quarterly, or at semi-annual intervals. 【0148】 Thus, in some embodiments, the RNAi agent is administered in a dosing regimen that includes a "loading phase" of closely spaced administrations, which may be followed by a "maintenance phase" in which the RNAi agent is administered at longer intervals. For example, after weekly or biweekly administration for one month, administration may be repeated once a month for six months or a year or longer, i.e., chronic administration. 【0149】 In one embodiment, the loading phase comprises a single administration of the RNAi agent during the first week. In another embodiment, the loading phase comprises a single administration of the RNAi agent over the first two weeks. In yet another embodiment, the loading phase comprises a single administration of the RNAi agent during the first month. 【0150】 In certain embodiments, the RNAi agent is administered at a concentration of about 100 mg to about 700 mg, about 150 mg to about 700 mg, about 200 mg to about 700 mg, about 250 mg to about 700 mg, about 300 mg to about 700 mg, about 350 mg to about 700 mg, about 400 mg to about 700 mg, about 450 mg to about 700 mg, about 500 mg to about 700 mg, about 550 mg to about 700 mg, about 600 to about 700 mg, about 650 to about 700 mg, about 100 mg to about 650 mg, about 150 mg to about 650 mg, about 200 mg to about 650 mg, about 250mg to about 650mg, about 300mg to about 650mg, about 350mg to about 650mg, about 400mg to about 650mg, about 450mg to about 650mg, about 500mg to about 650mg, about 550mg to about 650mg, about 600 to about 650mg, about 100mg ~600mg, 150mg~600mg, 200mg~600mg, 250mg~600mg, 300mg~600mg, 350mg~600mg, 400mg~600mg, 450mg~600mg, 500mg~60 0mg, about 550mg to about 600mg, about 100mg to about 550mg, about 150mg to about 550mg, about 200mg to about 550mg, about 250mg to about 550mg, about 300mg to about 550mg, about 350mg to about 550mg, about 400mg to about 550mg , approx. 450 mg ~ approx. 550 mg, approx. 500 mg ~ approx. 550 mg, approx. 100 mg ~ approx. 500 mg, approx. 150 mg ~ approx. 500 mg, approx. 200 mg ~ approx. 500 mg, approx. 250 mg ~ approx. The subject may be administered a fixed dose of 0 mg to about 500 mg, or about 450 mg to about 500 mg, for example, about 100 mg, about 125 mg, about 150 mg, about 175 mg, 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, or about 700 mg. Values ​​and ranges intermediate to the aforementioned recited values ​​are also intended to be part of the present invention. 【0151】 In one embodiment, the maintenance phase comprises administering a dose of RNAi agent to the subject once a month, once every two months, once every three months, once every four months, once every five months, or once every six months. In one particular embodiment, the maintenance dose is administered to the subject once a month. 【0152】 The single or multiple maintenance doses may be equal to or less than the initial dose, for example, half of the initial dose. For example, the maintenance dose may be about 25 mg to about 100 mg, for example, about 25 mg to about 75 mg, about 25 mg to about 50 mg, or about 50 mg to about 75 mg, for example, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, or about 100 mg, administered to a subject once a month. Intermediate values ​​and ranges within the above-listed values ​​are also contemplated as being part of the present invention. 【0153】 Any of these schedules can optionally be repeated one or more times.The number of repetitions can depend on the achievement of desired effect, such as suppressing PCSK9 gene, and / or achieving therapeutic or preventive effect, such as reducing serum cholesterol level or alleviating the symptoms of hypercholesterolemia.After treatment, patient can be monitored for changes in his / her condition.The dosage of RNAi agent can be increased if patient does not respond significantly to current dosage level, or dosage can be reduced if the symptom of pathology is alleviated, pathology is eliminated, or undesirable side effects are observed. 【0154】 Thus, in one aspect, the present invention provides a method for inhibiting expression of the PCSK9 gene in a subject, the method comprising administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen including a loading phase followed by a maintenance phase, wherein the loading phase comprises administering the RNAi agent to the subject at a fixed dose of about 200 mg to about 600 mg, and the maintenance phase comprises administering the RNAi agent to the subject at a fixed dose of about 25 mg to about 100 mg approximately once per month, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, and wherein the antisense strand comprises a complementary region comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from nucleotides 3544 to 3623 of the nucleotide sequence of SEQ ID NO: 1, thereby inhibiting expression of the PCSK9 gene in the subject. 【0155】 In another aspect, the present invention provides a method for reducing low-density lipoprotein (LDLc) levels in a subject, comprising administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen including a loading phase followed by a maintenance phase, wherein the loading phase comprises administering the RNAi agent to the subject at a fixed dose of about 200 mg to about 600 mg, and the maintenance phase comprises administering the RNAi agent to the subject monthly at a fixed dose of about 25 mg to about 100 mg, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a duplex region, wherein the antisense strand comprises a complementary region comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from nucleotides 3544 to 3623 of the nucleotide sequence of SEQ ID NO: 1, thereby reducing the LDLc level in the subject. 【0156】 In another aspect, the present invention provides a method of treating a subject having a disorder that would benefit from a decrease in expression of PCSK9. The method includes administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen including a loading phase followed by a maintenance phase, wherein the loading phase comprises administering the RNAi agent to the subject at a fixed dose of about 200 mg to about 600 mg, and the maintenance phase comprises administering the RNAi agent to the subject monthly at a fixed dose of about 25 mg to about 100 mg, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a duplex region, and wherein the antisense strand comprises a complementary region comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from nucleotides 3544 to 3623 of the nucleotide sequence of SEQ ID NO: 1, thereby treating the subject having a disorder that would benefit from a decrease in expression of PCSK9. 【0157】 In yet another aspect, the present invention provides a method for treating a subject with hyperlipidemia, comprising administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen including a loading phase followed by a maintenance phase, wherein the loading phase comprises administering the RNAi agent to the subject at a fixed dose of about 200 mg to about 600 mg, and the maintenance phase comprises administering the RNAi agent to the subject once a month at a fixed dose of about 25 mg to about 100 mg, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a duplex region, the antisense strand comprising a complementary region comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from nucleotides 3544 to 3623 of the nucleotide sequence of SEQ ID NO: 1, thereby treating the subject with hyperlipidemia. 【0158】 In one embodiment, a double-stranded ribonucleic acid (RNAi) agent used in the methods of the invention comprises a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises the nucleotide sequence 5'-ACAAAAGCAAAACAGGUCUAGAA-3' (SEQ ID NO: 685) and the sense strand comprises the nucleotide sequence 5'-CUAGACCUGUTUUGCUUUUGU-3' (SEQ ID NO: 686), and wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides. 【0159】 As used herein, "subject" includes humans or non-human animals, preferably vertebrates, more preferably mammals. A subject may also include a transgenic organism. Most preferably, a subject is a human, for example, a human suffering from or susceptible to a PCSK9-related disease. 【0160】 The methods and uses of the present invention involve administering the compositions described herein to a patient in need of treatment with a compound that inhibits or reduces the expression of the target PCSK9 gene for an extended period of time, for example, about 80 days. , 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, or about 180 days, or longer. 【0161】 The decrease in gene expression can be evaluated by any method known in the art.For example, the decrease in the expression of PCSK9 can be determined by using a method that is routine for those skilled in the art, such as Northern blotting, qRT-PCR to measure the mRNA expression level of PCSK9; by using a method that is routine for those skilled in the art, such as Western blotting, immunological technique to measure the protein level of PCSK9; and / or by measuring the biological activity of PCSK9, such as the effect on one or more serum lipid parameters, such as total cholesterol level, high-density lipoprotein cholesterol (HDL) level, non-HDL level, very-low-density lipoprotein cholesterol (VLDL) level, triglyceride level, Lp(a) level and lipoprotein particle size. 【0162】 The administration of dsRNA according to the method and use of the present invention can cause the severity, signs, symptoms and / or markers of the disease or disorder that will benefit from the reduction in the expression of PCSK9 in patients with the disorder.In this context, " reduction " means a statistically significant reduction in such level.The reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or about 100%. 【0163】 The effectiveness of disease treatment or prevention can be evaluated, for example, by measuring disease progression, disease remission, symptom severity, serum lipid levels (e.g., LDLc levels), quality of life, the dose of drug required to maintain therapeutic effect, disease marker levels, or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. Monitoring the effectiveness of treatment or prevention by measuring any one or any combination of such parameters is well within the capabilities of one skilled in the art. For example, the effectiveness of treatment of hyperlipidemia can be evaluated, for example, by periodically monitoring LDLc levels. Comparison of subsequent readings with initial readings provides a physician with an indication of whether the treatment is effective. Monitoring the effectiveness of treatment or prevention by measuring any one or any combination of such parameters is well within the capabilities of one skilled in the art. 【0164】 Therapeutic or preventive effects are evident when there is a statistically significant improvement in one or more parameters of the disease state, or when symptoms that would not normally be expected do not worsen or develop. For example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50% or more in measurable parameters of the disease can indicate effective treatment. The effectiveness of a given iRNA agent or formulation of the agent can also be determined using an experimental animal model for a given disease that is known in the art. When using an experimental animal model, the effectiveness of treatment is proven when a statistically significant decrease in markers or symptoms is observed. 【0165】 Alternatively, efficacy can be measured by a reduction in the severity of disease, as determined by a person skilled in the art of diagnosis based on a clinically accepted disease severity rating scale. For example, any positive change resulting in a reduction in the severity of disease, as measured using an appropriate scale, indicates appropriate treatment with iRNA or iRNA formulations as described herein. 【0166】 Generally, iRNA agents do not activate the immune system, for example, they do not increase cytokine levels, for example, TNF-α or IFN-α levels.For example, when measured by assays, for example, in vitro PBMC assays, for example, those described herein, the increase in TNF-α or IFN-α levels is less than 30%, 20%, or 10% of control cells treated with control dsRNA, for example, dsRNA that does not target PCSK9. 【0167】 In another embodiment, administration may be administered when low-density lipoprotein cholesterol (LDLc) levels reach or exceed a predetermined minimum level, for example, greater than 70 mg / dL, 130 mg / dL, 150 mg / dL, 200 mg / dL, 300 mg / dL, or 400 mg / dL. 【0168】 The effect of the reduced PCSK9 gene preferably results in a decrease in LDLc (low-density lipoprotein cholesterol) levels in the mammal's blood, more particularly in serum, hi some embodiments, LDLc levels are reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to pre-treatment levels. 【0169】 In some embodiments of the methods of the present invention, PCSK9 expression is reduced for an extended period of time, for example, at least 1 week, 2 weeks, 3 weeks, or 4 weeks or longer. For example, in some cases, PCSK9 gene expression is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administering an iRNA agent described herein. In some embodiments, the PCSK9 gene is suppressed by at least about 60%, 70%, or 80% by administering an iRNA agent. In some embodiments, the PCSK9 gene is suppressed by at least about 85%, 90%, or 95% by administering a double-stranded oligonucleotide. 【0170】 The RNAi agent herein can be administered to the subject by any administration method known in the art, including but not limited to subcutaneous, intravenous, intramuscular, intraocular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, cerebrospinal and any combination thereof.In a preferred embodiment, the agent is administered subcutaneously. 【0171】 In some embodiments, administration is via depot injection.Depot injection can release RNAi agent in a consistent manner for a prolonged period of time.Therefore, depot injection can reduce the frequency of administration required to obtain desired effect, for example, the desired inhibition of PCSK9, or therapeutic or preventive effect.Depot injection can also provide more consistent serum concentration.Depot injection can include subcutaneous injection or intramuscular injection.In a preferred embodiment, depot injection is subcutaneous injection. 【0172】 In some embodiments, the administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is an osmotic pump that is subcutaneously implanted. In other embodiments, the pump is an infusion pump. The infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusion. In a preferred embodiment, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the RNAi agent to the liver. 【0173】 Other modes of administration include epidural, intracerebral, intraventricular, intranasal, intraarterial, intracardiac, intraosseous, intrathecal, and intravitreal, and intrapulmonary. The mode of administration may be selected based on whether local or systemic treatment is desired and on the area to be treated. The route and site of administration may be selected to enhance targeting. 【0174】 The iRNA can be administered by intravenous infusion over a period of time, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or about 25 minutes. Administration can be repeated periodically, for example, weekly or every other week (i.e., every two weeks) for one month, two months, three months, four months, or longer. After an initial treatment regimen, treatment can be administered less frequently. For example, after three months of weekly or every other week administration, administration can be repeated once a month for six months, or one year, or longer. 【0175】 Administration of an iRNA may, for example, increase PCSK9 levels in the patient's cells, tissues, blood, urine, or other compartments by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or , 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more. 【0176】 Prior to administration of the full dose of iRNA, the patient may be administered a smaller dose, e.g., a 5% infusion, and monitored for adverse effects, e.g., allergic reactions. In another example, the patient may be monitored for unwanted immunostimulatory effects, e.g., increased cytokine (e.g., TNF-α or INF-α) levels. 【0177】 Due to the inhibitory effect on the expression of PCSK9, the composition according to the present invention or a pharmaceutical composition prepared therefrom may improve the quality of life. 【0178】 The iRNA of the present invention may be administered in "naked" form or as "free iRNA." Naked iRNA is administered in the absence of a pharmaceutical composition. Naked iRNA may be present in a suitable buffer. The buffer may contain acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer is phosphate-buffered saline (PBS). The pH and osmolality of the buffer containing the iRNA may be adjusted to be suitable for administration to a subject. 【0179】 Alternatively, the iRNA of the present invention may be administered as a pharmaceutical composition, such as a dsRNA liposome formulation. 【0180】 The present invention further provides methods and uses for using iRNAs or pharmaceutical compositions thereof in combination with other pharmaceutical agents and / or other therapeutic methods, such as known pharmaceutical agents and / or known therapeutic methods, such as those currently used to treat these disorders, for example, to treat subjects who would benefit from reduced and / or inhibited PCSK9 expression, e.g., subjects with hyperlipidemia, e.g., hypercholesterolemia. The siRNA and additional therapeutic agent can be administered in combination in the same composition, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or by another method described herein. 【0181】 Examples of additional therapeutic agents include agents known to treat lipid disorders, such as hypercholesterolemia, atherosclerosis, or dyslipidemia. For example, siRNAs featured in the invention can be administered with, for example, an HMG-CoA reductase inhibitor (e.g., statins), a fibrate, a bile acid sequestrant, niacin, an antiplatelet agent, an angiotensin-converting enzyme inhibitor, an angiotensin II receptor antagonist (e.g., losartan potassium, such as Merck & Co's Cozaar®), an acyl-CoA cholesterol acetyltransferase (ACAT) inhibitor, a cholesterol absorption inhibitor, a cholesterol ester transfer protein (CETP) inhibitor, a microsomal triglyceride transfer protein (MTTP) inhibitor, a cholesterol regulator, a bile acid regulator, a peroxisome proliferator-activated receptor (PPAR) agonist, a gene-based therapy, a combination vasoprotectant (e.g., AGI-1067, manufactured by Atherogenics), a glycoprotein IIb / IIIa inhibitor, aspirin or an aspirin-like compound, an IBAT inhibitor (e.g., S-8921, manufactured by Shionogi), a squalene synthase inhibitor, or a monocyte chemoattractant protein (MCP)-I inhibitor.Exemplary HMG-CoA reductase inhibitors include atorvastatin (Lipitor® / Tahor / Sortis / Torvast / Cardyl from Pfizer), pravastatin (Pravachol from Bristol-Myers Squibb, Mevalotin / Sanaprav from Sankyo), simvastatin (Zocor® / Sinvacor from Merck, Lipovas from Denan, Banyu from Boehringer Ingelheim), lovastatin (Mevacor / Mevinacor from Merck, Lovastatina, Cepa from Bexal; Schwarz Pharma's Liposcler), fluvastatin (Novartis's Lescol® / Locol / Lochol, Fujisawa's Cranoc, Solvay's Digaril), cerivastatin (Bayer's Lipobay / GlaxoSmithKline's Baycol), rosuvastatin (AstraZeneca's Crestor®), and picivastatin (itavastatin / risivastatin) (Nissan Chemical, Kowa Kogyo, Sankyo, and Novartis). Exemplary fibrates include, for example, bezafibrate (e.g., Roche's Befizal® / Cedur® / Bezalip®, Kissei's Bezatol), clofibrate (e.g., Wyeth's Atromid-S®), fenofibrate (e.g., Fournier's Lipidil / Lipantil, Abbott's Tricor®, Takeda's Lipantil, generics), gemfibrozil (e.g., Pfizer's Lopid / Lipur), and ciprofibrate (Sanofi-Synthelabo's Modalim®).Exemplary bile acid sequestrants include, for example, cholestyramine (Bristol-Myers Squibb's Questran® and Questran Light™), colestipol (e.g., Pharmacia's Colestid), and coleseveran (Genzyme / Sankyo's WelChol™). Exemplary niacin therapeutics include, for example, immediate-release formulations, such as Aventis' Nicobid, Upsher-Smith's Niacor, Aventis' Nicolar, and Sanwakagaku's Perycit. Exemplary niacin sustained-release formulations include, for example, Kos Pharmaceuticals' Niaspan and Upsher-Smith's SIo-Niacin. Exemplary antiplatelet drugs include, for example, aspirin (e.g., Bayer's Aspirin), clopidogrel (Sanofi-Synthelabo / Bristol-Myers Squibb's Plavix) and ticlopidine (e.g., Sanofi-Synthelabo's Ticlid and Daiichi's Panaldine).Other aspirin-like compounds that are useful in combination with dsRNA targeting PCSK9 include, for example, Asacard (Pharmacia's sustained-release aspirin) and Pamicogrel (Kanebo / Angelini Ricerche / CEPA).Exemplary angiotensin-converting enzyme inhibitors include, for example, ramipril (e.g., Aventis's Altace) and enalapril (e.g., Merck & Co.'s Vasotec). Exemplary acyl-CoA cholesterol acetyltransferase (ACAT) inhibitors include, for example, avasimibe (Pfizer), eflucimibe (BioMerieux Pierre Fabre / Eli Lilly), CS-505 (Sankyo and Kyoto), and SMP-797 (Sumito).Exemplary cholesterol absorption inhibitors include, for example, ezetimibe (Merck / Schering-Plough Pharmaceuticals Zetia®) and pamaqueside (Pfizer).Exemplary CETP inhibitors include, for example, torcetrapib (also known as CP-529414, Pfizer), JTT-705 (Japan Tobacco), and CETi-I (Avant Immunotherapeutics). Exemplary microsomal neutral lipid transfer protein (MTTP) inhibitors include, for example, implitapide (Bayer), R-103757 (Janssen), and CP-346086 (Pfizer). Other exemplary cholesterol regulators include, for example, NO-1886 (Otsuka / TAP Pharmaceutical), CI-1027 (Pfizer), and WAY-135433 (Wyeth-Ayerst). 【0182】 Exemplary bile acid regulators include, for example, HBS-107 (Hisamitsu / Banyu), Btg-511 (British Technology Group), BARI-1453 (Aventis), S-8921 (Shionogi), SD-5613 (Pfizer), and AZD-7806 (AstraZeneca). Exemplary peroxisome proliferator-activated receptor (PPAR) agonists include, for example, tesaglitazar (AZ-242) (AstraZeneca), netoglitazone (MCC-555) (Mitsubishi / Johnson & Johnson), GW-409544 (Ligand Pharmaceuticals / GlaxoSmithKline), GW-501516 (Ligand Pharmaceuticals / GlaxoSmithKline), LY-929 (Ligand Pharmaceuticals and Eli Lilly), LY-465608 (Ligand Pharmaceuticals and Eli Lilly), LY-518674 (Ligand Pharmaceuticals and Eli Lilly), and MK-767 (Merck and Kyorin). Exemplary gene-based therapeutic agents include, for example, AdGWEGF121.10 (GenVec), ApoAl (UCBPharma / Groupe Fournier), EG-004 (Trinam) (Ark Therapeutics), and ATP-binding cassette transporter A1 (ABCA1) (CV Therapeutics / Incyte, Aventis, Xenon). Exemplary glycoprotein Ilb / IIIa inhibitors include, for example, roxifiban (also known as DMP754, Bristol-Myers Squibb), gantofiban (Merck KGaA / Yamanouchi), and chromafiban (Millennium Pharmaceuticals).Exemplary squalene synthase inhibitors include, for example, BMS-1884941 (Bristol-Myers Squibb), CP-210172 (Pfizer), CP-295697 (Pfizer), CP-294838 (Pfizer) and TAK-475 (Takeda).Exemplary MCP-I inhibitors include, for example, RS-504393 (Roche Bioscience).Anti-atherosclerotic agent BO-653 (Chugai Pharmaceuticals) and nicotinic acid derivative Nyclin (Yamanouchi Pharmaceuticals) are also suitable for combined administration with dsRNA of the present invention. Exemplary combination therapies suitable for administration with a dsRNA targeting PCSK9 include, for example, Advicol (niacin / lovastatin from Kos Pharmaceuticals), amlodipine / atorvastatin (Pfizer), and ezetimibe / simvastatin (e.g., Vytorin® 10 / 10 tablets, 10 / 20 tablets, 10 / 40 tablets, and 10 / 80 tablets from Merck / Schering-Plough Pharmaceuticals).Drugs suitable for the treatment of hypercholesterolemia and for administration in combination with dsRNA targeting PCSK9 include, for example, lovastatin, niacin Altoprev® extended-release tablets (AndrxLabs), lovastatin Caduet® tablets (Pfizer), amlodipine besylate, atorvastatin calcium Crestor® tablets (AstraZeneca), rosuvastatin calcium Lescol® capsules (Novartis), fluvastatin sodium Lescol® (Reliant, Novartis), fluvastatin sodium Lipitor® tablets (Parke-Davis), atorvastatin calcium Rofibra® capsules (Gate), Niaspan extended-release tablets (Kos), niacin Pravachol tablets (Bristol-Myers Squibb). Squibb), pravastatin sodium Trichol® tablets (Abbott), fenofibrate Vytorin® 10 / 10 tablets (Merck / Schering-Plough Pharmaceuticals), ezetimibe, simvastatin WelChol™ tablets (Sankyo), colesevelam Zetia® hydrochloride tablets (Schering), ezetimibe Zetia® tablets (Merck / Schering-Plough Pharmaceuticals), and ezetimibe Zocor® tablets (Merck). 【0183】 In one embodiment, the iRNA agent is administered in combination with an ezetimibe / simvastatin combination (eg, Vytorin® (Merck / Schering-Plough Pharmaceuticals)). 【0184】 In another embodiment, the iRNA agent is administered in combination with an anti-PCSK9 antibody. Exemplary anti-PCSK9 antibodies for use in the combination therapies of the invention include, for example, alirocumab (Praluent), evolocumab (Repatha), bococizumab (PF-04950615, RN316, RN-316, L1L3; Pfizer, Rinat), roderucizumab (LFU720, pJG04; Novartis), ralpancizumab (RN317, PF-053358 10; Pfizer, Rinat), RG7652 (MPSK3169A, YW508.20.33b; Genentech), LY3015014 (Lilly), LPD1462 (h1F11; Schering-Plough), AX1 (AX189, 1B20, 1D05; Merck & Co.), ALD306 (Alder); mAb1 (Boehringer), and Ig1-PA4 (Nanjing Normal University). 【0185】 In one embodiment, an iRNA agent is administered to a patient, and then an additional therapeutic agent is administered to the patient (or vice versa). In another embodiment, the iRNA agent and the additional therapeutic agent are administered simultaneously. 【0186】 In another aspect, the invention features a method of instructing an end user, e.g., a caregiver or subject, on how to administer an iRNA agent described herein. The method optionally includes providing the end user with one or more doses of the iRNA agent and instructing the end user to administer the iRNA agent in a dosing regimen described herein, thereby instructing the end user. 【0187】 In one aspect, the present invention provides a method of treating a patient by selecting the patient based on the patient's need for LDL reduction, LDL reduction without HDL reduction, ApoB reduction, or total cholesterol reduction, comprising administering an siRNA to the patient in an amount sufficient to reduce the patient's LDL or ApoB levels, e.g., without substantially reducing HDL levels. 【0188】 Genetic predisposition plays a role in the development of diseases related to target genes, such as hyperlipidemia.Therefore, patients who need siRNA can be identified by taking family history or by screening for one or more genetic markers or variants.Examples of genes involved in hyperlipidemia include, but are not limited to, LDL receptor (LDLR), apolipoprotein (ApoA1, ApoB, ApoE, etc.), cholesteryl ester transfer protein (CETP), lipoprotein lipase (LPL), hepatic lipase (LIPC), endothelial lipase (EL), and lecithin cholesterol acyltransferase (LCAT). 【0189】 Healthcare provider (for example, doctor, nurse, or family) can take family history before prescribing or administering the iRNA agent of the present invention.In addition, tests can be carried out to determine genotype or phenotype.For example, DNA test can be carried out on a sample from patient, for example, blood sample, to identify the genotype and / or phenotype of PCSK9 before PCSK9 dsRNA is administered to patient.In another embodiment, tests are carried out to identify related genotype and / or phenotype, for example, LDLR genotype. Examples of genetic variants in the LDLR gene can be found in the art, for example in the following publications, which are incorporated by reference: Costanza et al. (2005) Am J Epidemiol. 15; 161(8): 714-24; Yamada et al. (2008) J Med Genet. Jan; 45(1): 22-8, Epub 2007 Aug 31; and Boes et al. (2009) Exp. Gerontol 44: 136-160, Epub 2008 Nov 17. 【0190】 The present invention further provides methods of inhibiting expression of proprotein convertase subtilisin / kexin type 9 (PCSK9) in a cell, eg, a cell within a subject, eg, a human subject. 【0191】 Thus, the present invention provides a method for inhibiting expression of the PCSK9 gene in a cell, the method comprising contacting the cell with an RNAi agent, e.g., a double-stranded RNAi agent, in an amount effective to inhibit expression of the PCSK9 gene in the cell, thereby inhibiting expression of PCSK9 in the cell. 【0192】 The step of contacting cells with double-stranded RNAi agent can be carried out in vitro or in vivo.The step of contacting cells with RNAi agent in vivo includes contacting cells or cell groups in a subject, for example, a human subject, with RNAi agent.The combination of in vitro and in vivo contact methods is also possible.The contacting step can be direct or indirect, as discussed above.In addition, the step of contacting cells can be carried out via a targeting ligand, including any ligand described herein or known in the art.In a preferred embodiment, the targeting ligand is a carbohydrate moiety, for example, a GalNAc3 ligand, or any other ligand that directs RNAi agent to the target site, for example, the liver of a subject. 【0193】 The term "inhibit," as used herein, is used interchangeably with "reduce," "silencing," "downregulate," and other similar terms, and includes any level of inhibition. 【0194】 The phrase "inhibiting the expression of PCSK9" is intended to refer to the inhibition of expression of any PCSK9 gene (such as, for example, the mouse PCSK9 gene, rat PCSK9 gene, monkey PCSK9 gene, or human PCSK9 gene) and variants or mutants of the PCSK9 gene. Thus, the PCSK9 gene may be a wild-type PCSK9 gene, a mutant PCSK9 gene, or a transgenic PCSK9 gene in the context of a genetically engineered cell, cell group, or organism. 【0195】 "Inhibiting the expression of the PCSK9 gene" includes any level of inhibition of the PCSK9 gene, for example, at least partial suppression of the expression of the PCSK9 gene. The expression of the PCSK9 gene may be evaluated based on the level or change in the level of any variable associated with PCSK9 gene expression, for example, PCSK9 mRNA level, PCSK9 protein level, or lipid level. The level may be evaluated in an individual's cell or cell group, including, for example, a sample from a subject. 【0196】 Inhibition may be assessed by a decrease in the absolute or relative level of one or more variables associated with PCSK9 expression compared to a control level, which may be any type of control level utilized in the art, such as a pre-administration baseline level, or a level determined from similar subjects, cells, or samples that are untreated or treated with a control (e.g., a buffer-only control or an inactive agent control, etc.). 【0197】 In some embodiments of the methods of the present invention, expression of the PCSK9 gene is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. 【0198】 Inhibition of expression of the PCSK9 gene may be indicated by a decrease in the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample from a subject) that have been treated (e.g., by contacting one or more cells with an RNAi agent of the invention, or by administering an RNAi agent of the invention to a subject in which the cells are or were present) such that the PCSK9 gene is transcribed and expression of the PCSK9 gene is inhibited, compared to a second cell or group of cells (control cells) that are substantially identical to the first cell or group of cells but have not been so treated. In a preferred embodiment, inhibition is indicated by a decrease in the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample from a subject) that have been treated (e.g., by contacting one or more cells with an RNAi agent of the invention, or by administering an RNAi agent of the invention to a subject in which the cells are or were present) such that the PCSK9 gene is transcribed and expression of the PCSK9 gene is inhibited, compared to a second cell or group of cells (control cells) that are substantially identical to the first cell or group of cells but have not been so treated. 【number】 The level of mRNA in treated cells is assessed by expressing the level of mRNA in treated cells as a percentage of the level of mRNA in control cells using the FTIR function. 【0199】 Alternatively, the inhibition of the expression of PCSK9 gene can be evaluated in terms of the reduction of parameters functionally related to the expression of PCSK9 gene, for example, the expression of PCSK9 protein, such as lipid level, cholesterol level, for example, LDLc level.PCSK9 gene silencing can be determined in any cell that expresses PCSK9, constitutively or by genome engineering, and by any assay known in the art.Liver is the main site of PCSK9 expression.Other prominent expression sites include pancreas, kidney and intestine. 【0200】 Inhibition of PCSK9 protein expression may be indicated by a decrease in the level of PCSK9 protein expressed by a cell or group of cells (e.g., the level of protein expressed in a sample from a subject). As described above for assessing mRNA suppression, inhibition of protein expression levels in a treated cell or group of cells may also be expressed as a percentage of the level of protein in a control cell or group of cells. 【0201】 Control cells or cell groups that can be used to evaluate the inhibition of PCSK9 gene expression include cells or cell groups that have not yet been contacted with the RNAi agent of the present invention.For example, the control cells or cell groups can be derived from an individual subject (e.g., a human or animal subject) prior to treatment with the subject's RNAi agent. 【0202】 The level of PCSK9 mRNA expressed by a cell or a group of cells can be measured using any method known in the art for assessing mRNA expression. In one embodiment, the level of PCSK9 expression in a sample is measured by detecting transcribed polynucleotides, or portions thereof, such as the mRNA of the PCSK9 gene. RNA can be extracted from cells using RNA extraction techniques, such as phenol / guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kit (Qiagen) or PAXgene (PreAnalytix, Switzerland). Typical assay formats that utilize ribonucleic acid hybridization include nuclear run-on assay, RT-PCR, ribonuclease protection assay (Melton et al., Nuc. Acids Res. 12:7035), Northern blotting, in situ hybridization, and microarray analysis. 【0203】 In one embodiment, the expression level of PCSK9 is measured using a nucleic acid probe.The term "probe" as used herein refers to any molecule that can selectively bind to a specific PCSK9.Probes can be synthesized by those skilled in the art or derived from suitable biological preparations.Probes can also be specifically designed to be labeled.Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, protein, antibody, and organic molecules. 【0204】 The isolated mRNA can be used in hybridization or amplification assays, including but not limited to Southern or Northern analysis, polymerase chain reaction (PCR) analysis and probe arrays. One method for measuring mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize with PCSK9 mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with the probe, for example, by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In another embodiment, the probe is immobilized on a solid surface and the mRNA is contacted with the probe, for example, in an Affymetrix gene chip array. Those skilled in the art can easily adapt known mRNA detection methods to applications such as measuring PCSK9 mRNA levels. 【0205】 Alternative methods for measuring the expression level of PCSK9 in a sample include nucleic acid amplification (to prepare cDNA) of, for example, mRNA in the sample and / or reverse transcriptase, such as RT-PCR (Mullis, 1987; experimental embodiment shown in U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcription amplification systems (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta replicase (Lizardi et al. (1988) Bio / Technology 6:1197), rolling circle replication (Lizardi

[0010] The process of detecting the amplified molecules includes PCR (e.g., by PCR amplification using PCR primers (e.g., RT-PCR, e.g., TaqMan™ System) or any other nucleic acid amplification method (e.g., by PCR primers (e.g., RT-PCR, e.g., TaqMan™ System) or any other nucleic acid amplification method), followed by detection of the amplified molecules using techniques well known to those skilled in the art. These detection schemes are particularly useful for detecting nucleic acid molecules when such molecules are present in very low numbers. In certain embodiments of the present invention, the level of PCSK9 expression is measured by quantitative fluorogenic RT-PCR (e.g., TaqMan™ System). 【0206】 The expression level of PCSK9 mRNA can be monitored using membrane blot (for example, used in hybridization analysis such as Northern, Southern, dot, etc.), or microwell, sample tube, gel, bead or fiber (or any solid support that contains bound nucleic acid).See U.S. Patent No. 5,770,722, U.S. Patent No. 5,874,219, U.S. Patent No. 5,744,305, U.S. Patent No. 5,677,195 and U.S. Patent No. 5,445,934 (incorporated herein by reference).Measurement of PCSK9 expression level can also include the use of nucleic acid probe in solution. 【0207】 In a preferred embodiment, the level of mRNA expression is assessed using a branched DNA (bDNA) assay or real-time PCR (qPCR). The use of these methods is described and illustrated in the examples presented herein. 【0208】 The level of PCSK9 protein expression may be measured using any method known in the art for measuring protein levels, including, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitation, absorption spectroscopy, colorimetric assay, spectrophotometric assay, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, electrochemiluminescence assay, and the like. 【0209】 The term "sample," as used herein, refers to a collection of similar bodily fluids, cells, or tissues isolated from a subject, as well as bodily fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serous fluid, plasma, lymph, urine, cerebrospinal fluid, saliva, ocular fluid, and the like. Tissue samples may include samples derived from tissues, organs, or localized regions. For example, samples may be derived from specific organs, parts of organs, or bodily fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g., the whole liver or a specific segment of the liver, or a specific type of cell in the liver, such as hepatocytes). In preferred embodiments, a "sample derived from a subject" refers to blood or plasma taken from a subject. In further embodiments, a "sample derived from a subject" refers to liver tissue from a subject. 【0210】 In some embodiments of the method of the present invention, RNAi agent is administered to a subject so that the RNAi agent is delivered to a specific site in the subject.The inhibition of PCSK9 expression can be evaluated by measuring the level or change in the level of PCSK9 mRNA or PCSK9 protein in the sample derived from body fluid or tissue from a specific site in the subject.In a preferred embodiment, the site is the liver.The site can also be a subsection or subgroup of cells from any one of the above-mentioned sites.The site can also comprise the cells that express a specific type of receptor. 【0211】 III. iRNAs used in the methods of the present invention Described herein are methods for using double-stranded RNAi agents to inhibit expression of the PCSK9 gene in cells, e.g., cells within a subject, e.g., a mammal, e.g., a human, having a PCSK9-related disorder, e.g., hyperlipidemia, e.g., hypercholesterolemia. 【0212】 Thus, the present invention provides double-stranded RNAi agents capable of inhibiting the expression of a target gene (ie, the PCSK9 gene) in vivo for use in the claimed methods. 【0213】 In one embodiment, the RNA, e.g., dsRNA, of an iRNA of the invention is unmodified, e.g., does not contain chemical modifications and / or conjugations known in the art and described herein. In another embodiment, the RNA, e.g., dsRNA, of an iRNA of the invention is chemically modified to enhance stability or other advantageous properties. In certain aspects of the invention, substantially all of the nucleotides of an iRNA of the invention are modified. For example, substantially all of the nucleotides of the sense strand are modified nucleotides, and / or substantially all of the nucleotides of the antisense strand are modified nucleotides, and / or substantially all of the nucleotides of both the sense and antisense strands are modified nucleotides. In other embodiments of the invention, all of the nucleotides of an iRNA of the invention are modified. For example, all of the nucleotides of the sense strand are modified nucleotides, and / or all of the nucleotides of the antisense strand are modified nucleotides, and / or all of the nucleotides of both the sense and antisense strands are modified nucleotides. An iRNA of the invention in which "substantially all of the nucleotides are modified" may contain no more than 5, 4, 3, 2, or 1 unmodified nucleotide, whether or not the majority or entire modifications are present. 【0214】 The dsRNA comprises an antisense strand with a complementary region that is complementary to at least a portion of the mRNA formed during PCSK9 gene expression.The complementary region is about 30 nucleotides or less in length (for example, about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length).When contacting with the cell that expresses PCSK9 gene, the iRNA inhibits the expression of PCSK9 gene (for example, human PCSK9 gene) by at least about 10%, for example, by PCR or branched DNA (bDNA)-based method, or by protein-based method, such as immunofluorescence analysis using Western blot or flow cytometry technology. 【0215】 dsRNA comprises two complementary RNA strands, which hybridize to form a double-stranded structure under the conditions in which dsRNA is used.One strand (antisense strand) of dsRNA comprises a complementary region, which is substantially complementary to target sequence, and is generally completely complementary.Target sequence can be derived from the sequence of mRNA formed during the expression of PCSK9 gene.The other strand (sense strand) comprises a region complementary to antisense strand, so that when combined under appropriate conditions, the two strands hybridize to form a double-stranded structure.As described elsewhere herein and known in the art, the complementary sequence of dsRNA can also be contained as a self-complementary region of a single nucleic acid molecule, as opposed to being on separate oligonucleotides. 【0216】 Generally, the double-stranded structure is, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27 , 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the recited ranges and lengths are also contemplated as part of the invention. 【0217】 Similarly, the complementary region of the target sequence may be, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-21, 19-22, 19-23, 19-24, 19-25, 19-26, 19-29, 19-28, 19-26, 19-27, 19-28, 19-29, 19-30, 19-31, 19-32, 19-33, 19-34, 19-35, 19-36, 19-37, 19-38, 19-39, 19-40, 19-41, 19-42, 19-43, 19-44, 19-45, 19-46, 19-47, 19-48, 19-49, 19-50, 19-51, 19-52, 19-53, 19-54, 19-55, 19-56, 19-57, 19-58, 19-59, 19-60, 19-61 and 15-30 nucleotides in length, such as 7, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the listed ranges and lengths are also contemplated as part of the invention. 【0218】 In some embodiments, the dsRNA is about 15 to about 20 nucleotides in length, or about 25 to about 30 nucleotides in length. Generally, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21 to 23 nucleotides in length can serve as substrates for Dicer. As those skilled in the art will recognize, the target region of an RNA targeted for cleavage is most often a portion of a larger RNA molecule, which is often an mRNA molecule. Where applicable, a "portion" of an mRNA target is a contiguous sequence of the mRNA target that is long enough to be a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway). 【0219】 In certain embodiments, dsRNA agents of the invention may include an RNA strand (antisense strand) that can be longer, e.g., up to 66 nucleotides, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, with a region of at least 19 contiguous nucleotides that is substantially complementary to at least a portion of an mRNA transcript of the PCSK9 gene. These dsRNA agents with longer antisense strand lengths preferably include a second RNA strand (sense strand) of 20-60 nucleotides in length, where the sense strand and antisense strand form a duplex of 18-30 contiguous nucleotides. 【0220】 Those skilled in the art will appreciate that the amount of hydroxybenzoates may be, for example, about 10 to 36, 11 to 36, 12 to 36, 13 to 36, 14 to 36, 15 to 36, 9 to 35, 10 to 35, 11 to 35, 12 to 35, 13 to 35, 14 to 35, 15 to 35, 9 to 34, 10 to 34, 11 to 34, 12 to 34, 13 to 34, 14 to 34, 15 to 34, 9 to 33, 10 to 33, 11 to 33, 12 to 33, 13 to 33, 14 to 3 3, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-1 9, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20 It will also be appreciated that a double-stranded region, such as a double-stranded region of about 9-36 base pairs, such as 28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs, is the primary functional portion of a dsRNA. 【0221】 Thus, in one embodiment, an RNA molecule or RNA molecule complex having a double-stranded region of more than 30 base pairs is a dsRNA, for example, within the range of processing into a functional duplex of 15-30 base pairs that targets a desired RNA for cleavage. Thus, those skilled in the art will recognize that, in one embodiment, an miRNA is a dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful for targeting PCSK9 expression is not generated in a target cell by cleavage of a larger dsRNA. 【0222】 The dsRNA described herein can further comprise one or more single-stranded nucleotide overhangs, such as 1, 2, 3, or 4 nucleotides.Compared to their blunt-end counterparts, dsRNAs with at least one nucleotide overhang can have surprisingly superior inhibitory properties.The nucleotide overhang can comprise or consist of nucleotide / nucleoside analogs, including deoxyribonucleotides / nucleosides.The overhang can be on the sense strand, the antisense strand, or any combination thereof.Furthermore, the overhanging nucleotide can be present on the 5'-end, 3'-end, or both ends of either the antisense or sense strand of dsRNA.In certain embodiments, a longer extended overhang can be present. 【0223】 dsRNA can be synthesized by standard methods known in the art using an automated DNA synthesizer, such as those commercially available from Biosearch, Applied Biosystems, Inc., as discussed further below. 【0224】 The iRNA compounds of the present invention can be prepared using a two-step method. First, the individual strands of the double-stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compounds can be prepared using solution phase or solid phase organic synthesis or both. Organic synthesis offers the advantage that it can easily prepare oligonucleotide strands that comprise unnatural or modified nucleotides. The single-stranded oligonucleotides of the present invention can be prepared using solution phase or solid phase organic synthesis or both. 【0225】 In one aspect, the dsRNA of the present invention comprises at least two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand is selected from the sequences provided in Table 1, and the antisense strand corresponding to the sense strand is selected from the sequences in Table 1. In this aspect, one of the two sequences is complementary to the other of the two sequences, and one of the sequences is substantially complementary to the mRNA sequence generated during PCSK9 gene expression. Thus, in this aspect, the dsRNA comprises two oligonucleotides, one oligonucleotide being described as the sense strand in Table 1, and the second oligonucleotide being described as the antisense strand corresponding to the sense strand in Table 1. In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide. 【0226】 Although some of the sequences in Table 1 are described as modified and / or conjugated sequences, it is understood that the RNA of the iRNA of the invention, e.g., the dsRNA of the invention, may comprise any one of the sequences set forth in Table 1 unmodified, unconjugated, and / or modified and / or conjugated differently than described. 【0227】 Those skilled in the art are well aware that dsRNAs having a duplex structure of approximately 20-23 base pairs, e.g., 21 base pairs, have been advocated as being particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be similarly effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the above-described embodiment, due to the nature of the oligonucleotide sequences provided in Table 1, the dsRNAs described herein can contain at least one strand that is at least 21 nucleotides long. It can be reasonably expected that shorter duplexes having one of the sequences in Table 1, with only a few nucleotides missing from one or both termini, can be similarly effective compared to the dsRNAs described above. Thus, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20 or more contiguous nucleotides derived from one of the sequences in any one of Tables 3, 4, 5, 6, 18, 19, 20, 21 and 23, and which differ in their ability to inhibit PCSK9 gene expression by about 5, 10, 15, 20, 25, or 30% or less from a dsRNA comprising the full-length sequence, are intended to be within the scope of the present invention. 【0228】 Furthermore, the RNAs provided in Table 1 identify sites in the PCSK9 transcript that are highly susceptible to RISC-mediated cleavage. Thus, the present invention further features iRNAs that target within one of these sequences. As used herein, if an iRNA promotes cleavage of the transcript anywhere within that specific site, the iRNA is said to target within that specific site of the RNA transcript. Such iRNAs generally contain about 15 consecutive nucleotides from one of the sequences provided in Table 1 linked to additional nucleotide sequences from regions adjacent to the selected sequence in the PCSK9 gene. 【0229】 Target sequences are generally about 15-30 nucleotides in length, although there is wide variability in the suitability of specific sequences within this range to induce cleavage of any given target RNA. While the various software packages and guidelines presented herein provide guidance for identifying optimal target sequences for any given gene target, an empirical approach can also be taken, in which a "window" or "mask" of a given size (21 nucleotides, as a non-limiting example) is placed, either physically or figuratively (e.g., by computer simulation), on the target RNA sequence to identify sequences within a size range that can serve as target sequences. By successively moving the sequence "window" one nucleotide upstream or downstream of the initial target sequence position, subsequent potential target sequences can be identified until a complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis of the identified sequences and testing (using assays described herein or known in the art) to identify optimally functioning sequences, can identify RNA sequences that mediate the best inhibition of target gene expression when targeted with an iRNA agent. Thus, while the sequences identified in Table 1, for example, represent effective target sequences, it is contemplated that further optimization of inhibitory efficiency may be achieved by successively "window walking" one nucleotide upstream or downstream of a given sequence to identify sequences with equivalent or better inhibitory properties. 【0230】 It is contemplated that further optimization of any sequence, for example, identified in Table 1, may be achieved by systematically adding or removing nucleotides to create longer or shorter sequences, and then testing these created sequences by walking through windows of a size longer or shorter than the target RNA from that position. Again, combining this approach of creating new target candidates with testing the effectiveness of iRNAs based on these target sequences in inhibition assays known in the art and / or described herein may result in further improvements in inhibition efficiency. Still further, such optimized sequences may be adjusted by, for example, introducing modified nucleotides described herein or known in the art, adding or altering overhangs, or other modifications known in the art and / or discussed herein to further optimize the molecule as an expression inhibitor (e.g., increasing serum stability or circulating half-life, increasing thermostability, enhancing transmembrane delivery, targeting specific locations or cell types, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.). 【0231】 The iRNAs described herein may contain one or more mismatches with the target sequence. In one embodiment, the iRNAs described herein contain three or fewer mismatches. When the antisense strand of an iRNA contains mismatches with the target sequence, it is preferable that the range of mismatches is not located in the center of the complementary region. When the antisense strand of an iRNA contains mismatches with the target sequence, it is preferable that the mismatches be limited to the last five nucleotides from either the 5' or 3' end of the complementary region. For example, in a 23-nucleotide iRNA agent strand complementary to a PCSK9 gene region, the RNA strand generally does not contain any mismatches within the central 13 nucleotides. Using the methods described herein or known in the art, it can be determined whether an iRNA containing mismatches with the target sequence is effective in inhibiting PCSK9 gene expression. Examining the effectiveness of iRNAs with mismatches in inhibiting PCSK9 gene expression is important, especially when a specific complementary region of the PCSK9 gene is known to have polymorphic sequence variation within the population. 【0232】 Nucleic acids featured in the present invention can be synthesized and / or modified by methods established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S. Lett. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. For example, modifications include terminal modifications, such as 5'-end modifications (phosphorylation, conjugated linkage, inverted linkage) or 3'-end modifications (conjugated linkage, DNA nucleotide, inverted linkage, etc.); base modifications, such as substitution with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, base removal (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; and backbone modifications, including modification or replacement of phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or RNAs that do not contain natural internucleoside linkages. The RNA with modified backbone particularly includes those that do not have phosphorus atom in backbone.For the purpose of this specification, and as sometimes referred to in the art, the modified RNA that does not have phosphorus atom in their internucleoside backbone is also considered to be oligonucleoside.In some embodiments, modified iRNA has phosphorus atom in its internucleoside backbone. 【0233】 Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and boranophosphates with reverse polarity, in which adjacent nucleoside unit pairs are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.Various salts, mixed salts, and free acid forms are also included. 【0234】 Representative United States patents that teach the preparation of the above phosphorus-containing linkages include U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; and 5,286,717, each of which is hereby incorporated by reference in its entirety. No. 17; U.S. Patent No. 5,321,131; U.S. Patent No. 5,399,676; U.S. Patent No. 5,405,939; U.S. Patent No. 5,453,496; U.S. Patent No. 5,455,233; U.S. Patent No. 5,466,677; U.S. Patent No. 5,476,925; U.S. Patent No. 5,519,126; U.S. Patent No. 5,536,821; U.S. Patent No. 5,541,316; U.S. Patent No. 5,550,111; U.S. Patent No. 5,563,253; U.S. Patent No. 5,57 Nos. 1,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6 ,531,590; U.S. Patent No. 6,534,639; U.S. Patent No. 6,608,035; U.S. Patent No. 6,683,167; U.S. Patent No. 6,858,715; U.S. Patent No. 6,867,294; U.S. Patent No. 6,878,805; U.S. Patent No. 7,015,315; U.S. Patent No. 7,041,816; U.S. Patent No. 7,273,933; U.S. Patent No. 7,321,029; and U.S. Patent No. RE39464, but are not limited thereto. 【0235】 Modified RNA backbones that do not contain phosphorus atoms have backbones formed by short alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, those with amide backbones, and others with mixed N, O, S, and CH2 components. 【0236】 Representative United States patents that teach the preparation of the above oligonucleosides include U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; and 5,470,967, each of which is hereby incorporated by reference in its entirety. Nos.; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439. 【0237】 In another embodiment, suitable RNA mimics are contemplated for use in iRNA, in which both the sugar and internucleoside linkages, i.e., the backbone of the nucleotide units, are replaced with novel groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimic that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly linked to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the contents of each of which are incorporated herein by reference in their entirety. Further suitable PNA compounds for use in the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500. 【0238】 Some embodiments featured in the present invention include RNAs with phosphorothioate backbones, and oligonucleosides with heteroatom backbones that are --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- (known as methylene(methylimino) or MMI backbones), --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- (natural phosphodiester backbones are represented as --O--P--O--CH2--) of the aforementioned U.S. Pat. No. 5,489,677, and amide backbones of the aforementioned U.S. Pat. No. 5,602,240. In some embodiments, the RNA featured herein has a morpholino backbone structure as described in the aforementioned US Pat. No. 5,034,506. 【0239】 Modified RNAs can also contain one or more substituted sugar moieties. For example, iRNAs, such as dsRNAs provided herein, can include one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl, or C2-C 10 It can be alkenyl and alkynyl. Exemplary suitable modifications include O[(CH) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10. In another embodiment, the dsRNA includes one of the following at the 2' position: C1 to C 10

[0033] In some embodiments, the modification is 2'-methoxyethoxy (2'-O-CH2CHOCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 1996). 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is the 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2)2, as described in the Examples herein below. 【0240】 Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an iRNA, specifically at the 3' position of the sugar on the 3'-terminal nucleotide, or in 2'-5'-linked dsRNA, and at the 5' position of the 5'-terminal nucleotide. An iRNA can also have a sugar mimic, such as a cyclobutyl moiety, in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of the above modified sugar structures include, certain of which are commonly owned with the present application: U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; and U.S. Pat.

[0010] Examples of patents that may be used include, but are not limited to, U.S. Patent Nos. 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, the contents of each of which are hereby incorporated by reference in their entirety. 【0241】The RNA of an iRNA may also contain nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include deoxythymine (dT); 5-methylcytosine (5-me-C); 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine, and 2-thiocytosine; 5-halouracil and cytosine; 5-propynyluracil and cytosine; 6-azouracil, cytosine, and thymine; 5-uracil (pseudouracil) ); 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines; 5-halo, specifically 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines; 7-methylguanine and 7-methyladenine; 8-azaguanine and 8-azaadenine; 7-deazaguanine and 7-daazaadenine; and other synthetic and natural nucleobases such as 3-deazaguanine and 3-deazaadenine.Further, the nucleobase can include those disclosed in U.S. Patent No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P.ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990; those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993.Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6 to 1.2°C (Sanghvi, YS, Crooke, ST, and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), making them exemplary base substitutions, especially when combined with 2'-O-methoxyethyl sugar modifications. 【0242】 Representative United States patents that teach the preparation of the above-mentioned specific modified nucleobases, as well as other modified nucleobases, include the above-mentioned U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; and 5,587,469, each of which is hereby incorporated by reference in its entirety. Nos.; U.S. Patent Nos. 5,594,121, 5,596,091; U.S. Patent No. 5,614,617; U.S. Patent No. 5,681,941; U.S. Patent No. 5,750,692; U.S. Patent No. 6,015,886; U.S. Patent No. 6,147,200; U.S. Patent No. 6,166,197; U.S. Patent No. 6,222,025; U.S. Patent No. 6,235,887; U.S. Patent No. 6,380,368; U.S. Patent No. 6,528,640; U.S. Patent No. 6,639,062; U.S. Patent No. 6,617,438; U.S. Patent No. 7,045,610; U.S. Patent No. 7,427,672; and U.S. Patent No. 7,495,088. 【0243】 The RNA of an iRNA can also be modified to contain one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by a two-atom bridge. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety containing a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Thus, in some embodiments, an agent of the present invention may comprise an RNA of an iRNA that can be modified to contain one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide with a modified ribose moiety such that the ribose moiety contains an extra bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety containing a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose into a structural conformation at the 3' end. The addition of immobilized nucleic acids to siRNA has been shown to increase the stability of siRNA in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). 【0244】 Examples of bicyclic nucleosides used in polynucleotides of the invention include, but are not limited to, nucleosides comprising a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agents of the invention comprise one or more bicyclic nucleosides comprising a 4' to 2' bridge. Examples of such 4' to 2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (also referred to as "hindered ethyl" or "cEt") and 4'-CH(CHOCH3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,239,845). ,278,283); 4'-CH2-N(OCH3)-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,425); 4'-CH2-ON(CH3)-2' (see, e.g., U.S. Patent Application Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2' (wherein R is H, C1-C12 alkyl, or a protecting group) (see, e.g., U.S. Pat. No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing are hereby incorporated by reference. 【0245】 Additional representative U.S. patents and U.S. patent application publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. No. 6,268,490; U.S. Pat. No. 6,525,191; U.S. Pat. No. 6,670,461; U.S. Pat. No. 6,770,748; U.S. Pat. No. 6,794,499; U.S. Pat. No. 6,998,484; U.S. Pat. No. 7,053,207; U.S. Pat. No. 7,034,133; U.S. Pat. No. 7,084,125; U.S. Pat. No. 7,399,888; No. 45; U.S. Patent No. 7,427,672; U.S. Patent No. 7,569,686; U.S. Patent No. 7,741,457; U.S. Patent No. 8,022,193; U.S. Patent No. 8,030,467; U.S. Patent No. 8,278,425; U.S. Patent No. 8,278,426; U.S. Patent No. 8,278,283; U.S. Patent Application Publication No. 2008 / 0039618; and U.S. Patent Application Publication No. 2009 / 0012281, the entire contents of each of which are hereby incorporated by reference herein. 【0246】 Any of the foregoing bicyclic nucleosides can be prepared with one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226). 【0247】 The RNA of an iRNA can also be modified to contain one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid containing a bicyclic sugar moiety containing a 4'-CH(CH3)-O-2' bridge. In one embodiment, the constrained ethyl nucleotide is in the S configuration, referred to herein as "S-cEt." 【0248】 The iRNA of the present invention may also contain one or more "conformationally restricted nucleotides" ("CRNs"). A CRN is a nucleotide analog with a linker connecting the C2' and C4' carbons of ribose or the C3 and C5' carbons of ribose. The CRN locks the ribose ring into a stable conformation, increasing hybridization affinity to mRNA. The linker is long enough to position the oxygen optimally for stability and affinity, resulting in reduced puckering of the ribose ring. 【0249】 Representative publications that teach the preparation of certain of the above CRNs include, but are not limited to, U.S. Patent Application Publication No. 2013 / 0190383; and PCT Publication No. WO 2013 / 036868, the entire contents of each of which are hereby incorporated by reference herein. 【0250】 One or more of the nucleotides of the iRNA of the invention may also comprise a hydroxymethyl-substituted nucleotide. A "hydroxymethyl-substituted nucleotide" is an acyclic 2'-3'-seco-nucleotide (also referred to as a "non-locked nucleic acid" ("UNA") modification). 【0251】 Representative U.S. publications that teach the preparation of UNAs include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Patent Application Publication No. 2013 / 0096289; U.S. Patent Application Publication No. 2013 / 0011922; and U.S. Patent Application Publication No. 2011 / 0313020 (the entire contents of each of which are hereby incorporated by reference herein). 【0252】 Other modifications of the nucleotides of the iRNAs of the invention include a 5' phosphate or 5' phosphate mimic, such as a 5' terminal phosphate or phosphate mimic, on the antisense strand of the RNAi agent. Suitable phosphate mimics are disclosed, for example, in U.S. Patent Application Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference. 【0253】 Potential stabilizing modifications to the ends of RNA molecules include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"-phosphate, and inverted base dT (idT). Disclosure of this modification is found in WO 2011 / 005861. 【0254】 A. Modified iRNA containing motifs In certain aspects of the present invention, the double-stranded RNAi agent of the present invention comprises an agent having chemical modifications, such as those disclosed in U.S. Patent Application Publication No. 2014 / 0315835 and PCT Publication No. WO 2013 / 075035 (the entire contents of each of which are incorporated herein by reference).As shown herein and in U.S. Patent Application Publication No. 2014 / 0315835 and PCT Publication No. WO 2013 / 075035, advantageous results can be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense strand and / or antisense strand of the RNAi agent, particularly at or near the cleavage site.In some embodiments, the sense strand and antisense strand of the RNAi agent may be otherwise completely modified.The introduction of these motifs interrupts the modification pattern (if any) of the sense strand and / or antisense strand.The RNAi agent may optionally be conjugated with a GalNAc derivative ligand, for example, on the sense strand. The resulting RNAi agents exhibit superior gene silencing activity. 【0255】 More specifically, it has been surprisingly discovered that when the sense and antisense strands of a double-stranded RNAi agent are fully modified to have one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of the RNAi agent, the gene silencing activity of the RNAi agent is significantly enhanced. 【0256】 Thus, the present invention provides double-stranded RNAi agents capable of inhibiting the expression of a target gene (i.e., the PCSK9 gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may be in the range of 12 to 30 nucleotides in length. For example, each strand may be 14 to 30 nucleotides in length, 17 to 30 nucleotides in length, 25 to 30 nucleotides in length, 27 to 30 nucleotides in length, 17 to 23 nucleotides in length, 17 to 21 nucleotides in length, 17 to 19 nucleotides in length, 19 to 25 nucleotides in length, 19 to 23 nucleotides in length, 19 to 21 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 23 nucleotides in length. 【0257】 The sense and antisense strands typically form a double-stranded RNA ("dsRNA") as a duplex, also referred to herein as an "RNAi agent." The double-stranded region of an RNAi agent may be 12 to 30 nucleotide pairs in length. For example, the double-stranded region may be 14 to 30 nucleotide pairs, 17 to 30 nucleotide pairs, 27 to 30 nucleotide pairs, 17 to 23 nucleotide pairs, 17 to 21 nucleotide pairs, 17 to 19 nucleotide pairs, 19 to 25 nucleotide pairs, 19 to 23 nucleotide pairs, 19 to 21 nucleotide pairs, 21 to 25 nucleotide pairs, or 21 to 23 nucleotide pairs in length. In another example, the double-stranded region may be selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length. 【0258】 In one embodiment, the RNAi agent may include one or more overhang regions and / or capping groups at the 3'-end, 5'-end, or both ends of one or both strands. The overhangs can be 1 to 6 nucleotides in length, e.g., 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhangs can form mismatches with the target mRNA, be complementary to the targeted gene sequence, or be another sequence. The first and second strands can also be linked by additional bases or other non-basic linkers, for example, to form a hairpin. 【0259】 In one embodiment, each nucleotide in the overhang region of RNAi agent can be independently modified or unmodified nucleotide, for example, but not limited to, 2'-sugar modified, for example, 2-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof.For example, TT can be the overhang sequence at either end on either strand.The overhang can form a mismatch with target mRNA, or can be complementary to the gene sequence being targeted, or can be another sequence. 【0260】 The 5'- or 3'-overhang of the sense strand, antisense strand, or both strands of the RNAi agent may be phosphorylated.In some embodiments, the overhang region comprises two nucleotides with phosphorothioate between them, wherein the two nucleotides can be the same or different.In one embodiment, the overhang is present at the 3'-end of the sense strand, antisense strand, or both strands.In one embodiment, the 3'-overhang is present in the antisense strand.In one embodiment, the 3'-overhang is present in the sense strand. 【0261】 RNAi agent may only comprise a single overhang, which can enhance the interference activity of RNAi without affecting its overall stability.For example, the single-stranded overhang may be located at the 3' end of the sense strand, or alternatively at the 3' end of the antisense strand.RNAi may also have a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand) or vice versa.Generally, the antisense strand of RNAi has a nucleotide overhang at the 3' end, and the 5' end is blunt.Without wishing to be bound by theory, it is preferable that the asymmetric blunt end at the 5' end of the antisense strand and the overhang at the 3' end of the antisense strand load the guide strand into RISC process. 【0262】 In one embodiment, the RNAi agent is a 19-nucleotide double-ended bluntmer, wherein the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 7, 8, and 9 from the 5' end, and the antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end. 【0263】 In another embodiment, the RNAi agent is a 20-nucleotide long blunt-ended duplex, wherein the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 from the 5' end, and the antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end. 【0264】 In yet another embodiment, the RNAi agent is a 21-nucleotide long blunt-ended duplex, wherein the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and the antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end. 【0265】 In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand comprises at least one three-nucleotide 2'-F modification motif on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end; and the antisense strand comprises at least one three-nucleotide 2'-O-methyl modification motif on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, wherein one end of the RNAi agent is blunt, while the other end comprises a two-nucleotide overhang.Preferably, the two-nucleotide overhang is present at the 3' end of the antisense strand.When a two-nucleotide overhang is present at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide bonds between the three nucleotide ends, wherein two of the three nucleotides are overhanging nucleotides, and the third nucleotide is a paired nucleotide adjacent to the overhanging nucleotide. In one embodiment, the RNAi agent further comprises two phosphorothioate internucleotide bonds between the three nucleotide ends at both the 5'-end of the sense strand and the 5'-end of the antisense strand.In one embodiment, all nucleotides in the sense strand and antisense strand of the RNAi agent, including the nucleotide that is part of the motif, are modified nucleotides.In one embodiment, each residue is independently modified with 2'-O-methyl or 3'-fluoro, for example, in an alternating motif.Optionally, the RNAi agent further comprises a ligand (preferably GalNAc3). 【0266】 In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length and comprises at least 8 ribonucleotides at positions 1-23 of the first strand, starting from the 5'-most nucleotide (position 1); the antisense strand is 36-66 nucleotide residues in length and comprises at least 8 ribonucleotides at positions that are paired with positions 1-23 of the sense strand, starting from the 3'-most nucleotide of the sense strand, to form a duplex; wherein at least the 3'-most nucleotide of the antisense strand is not paired with the sense strand, and up to 6 consecutive 3'-most nucleotides of the antisense strand are not paired with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides; wherein the 5' end of the antisense strand has 10-30 consecutive ribonucleotides that are not paired with the sense strand. the sense strand comprises at least one ribonucleotide, thereby forming a single-stranded 5' overhang of 10 to 30 nucleotides; wherein at least the 5'- and 3'-terminal nucleotides of the sense strand are base-paired with nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands; and the antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the antisense strand length to reduce expression of the target gene when the double-stranded nucleic acid is introduced into a mammalian cell; and the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides, wherein at least one of the motifs is located at or near the cleavage site. The antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site. 【0267】 In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, wherein the RNAi agent comprises a first strand having a length of at least 25 and at most 29 nucleotides, and a second strand having a length of at most 30 nucleotides, which has at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end; wherein the 3' end of the first strand and the 5' end of the second strand form a blunt end, and the second strand is 1 to 4 nucleotides longer at its 3' end than the first strand, wherein the double-stranded region is a region that is at least 25 nucleotides long, and the second strand is sufficiently complementary to a target mRNA along at least 19 nucleotides of the second strand length to reduce expression of the target gene when the RNAi agent is introduced into a mammalian cell, and wherein Dicer cleavage of the RNAi agent preferentially produces siRNA comprising the 3' end of the second strand, thereby reducing expression of the target gene in a mammal. Optionally, the RNAi agent further comprises a ligand. 【0268】 In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, one of the motifs being at the cleavage site in the sense strand. 【0269】 In one embodiment, the antisense strand of the RNAi agent also contains at least one motif of three identical modifications on three consecutive nucleotides, one of the motifs being at or near the cleavage site in the antisense strand. 【0270】 In RNAi agents having a double-stranded region 17-23 nucleotides long, the cleavage sites of the antisense strand are typically around positions 10, 11, and 12 from the 5' end. Thus, the three identically modified motifs may be present at positions 9, 10, and 11; 10, 11, and 12; 11, 12, and 13; 12, 13, and 14; or 13, 14, and 15 of the antisense strand, with counting starting from the first nucleotide from the 5' end of the antisense strand, or counting starting from the first paired nucleotide within the double-stranded region from the 5' end of the antisense strand. The cleavage site within the antisense strand may also vary depending on the length of the double-stranded region of the RNAi from the 5' end. 【0271】 The sense strand of RNAi agent can have at least one motif of three identical modifications on three consecutive nucleotides at or near the break site of strand; and antisense strand can have at least one motif of three identical modifications on three consecutive nucleotides at or near the break site of strand.When sense strand and antisense strand form dsRNA duplex, sense strand and antisense strand can be aligned so that one motif of three nucleotides on sense strand and one motif of three nucleotides on antisense strand have at least one nucleotide overlap, that is, at least one of the three nucleotides of the motif in sense strand and at least one of the three nucleotides of the motif in antisense strand form base pairs.Alternatively, at least two nucleotides can overlap, or all three nucleotides can overlap. 【0272】 In one embodiment, the sense strand of an RNAi agent may contain two or more motifs of three identical modifications on three consecutive nucleotides. The first motif may be located at or near the cleavage site of the strand, and the other motif may be a wing modification. The term "wing modification" herein refers to a motif located on the other part of the strand, separated from a motif located at or near the cleavage site of the same strand. The wing modification is adjacent to the first motif or separated by at least one or more nucleotides. When the motifs are directly adjacent to each other, the chemistry of the motifs is different from each other, and when the motifs are separated by one or more nucleotides, the chemistry can be the same or different. Two or more wing modifications may be present. For example, when two wing modifications are present, each wing modification may be located at one end of the first motif located at or near the cleavage site, or on either side of the lead motif. 【0273】 Like the sense strand, the antisense strand of an RNAi agent may contain two or more motifs of three identical modifications over three consecutive nucleotides, with at least one of the motifs being at or near the site of strand cleavage. The antisense strand may also contain one or more wing modifications aligned in the same manner as wing modifications that may be present on the sense strand. 【0274】 In one embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand. 【0275】 In another embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two paired nucleotides within the double-stranded region at the 3' end, 5' end, or both ends of the strand. 【0276】 When the sense and antisense strands of an RNAi agent each contain at least one wing modification, the wing modifications may be contained on the same end of the double-stranded region and may have an overlap of 1, 2, or 3 nucleotides. 【0277】 When the sense and antisense strands of an RNAi agent each contain at least two wing modifications, the sense and antisense strands can be arranged such that two modifications from one strand are each contained at one end of a double-stranded region with an overlap of 1, 2, or 3 nucleotides; two modifications from one strand are each contained at the other end of a double-stranded region with an overlap of 1, 2, or 3 nucleotides; and two modifications of one strand are contained on either side of a lead motif with an overlap of 1, 2, or 3 nucleotides within the double-stranded region. 【0278】 In one embodiment, all nucleotides in the sense strand and antisense strand of RNAi agent, including the nucleotide of the motif, may be modified.Each nucleotide may be modified with the same or different modifications, and this modification may include one or more of the non-linked phosphate oxygen and / or one or more linking phosphate oxygen;Modification of ribose sugar components, for example, the 2' hydroxyl of ribose sugar;Modification or substitution of phosphate moiety with " dephosphorylation " linker;Modification or substitution of natural base;And substitution or modification of ribose-phosphate backbone. 【0279】 Because nucleic acids are polymers of subunits, many modifications, such as modifications of bases, phosphate moieties, or non-linked Os in phosphate moieties, occur at positions that are repeated within the nucleic acid. In some cases, modifications will occur at every target position in the nucleic acid, but in many cases, this is not the case. For example, modifications may be limited to the 3' or 5' terminal positions, or may be limited to terminal regions, such as positions on terminal nucleotides or the last 2, 3, 4, 5, or 10 nucleotides of the chain. Modifications may occur in double-stranded regions, single-stranded regions, or both. Modifications may be limited to double-stranded regions of RNA, or may be limited to single-stranded regions of RNA. For example, phosphorothioate modifications at non-linked O positions may be limited to one or both ends, or may be limited to terminal regions, such as positions on terminal nucleotides or the last 2, 3, 4, 5, or 10 nucleotides of the chain, or may occur in double-stranded and single-stranded regions, especially at the ends. The 5' end may be phosphorylated. 【0280】 For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in the single-stranded overhang, for example, in the 5' or 3' overhang, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' or 5' overhang may be modified, for example, with modifications described herein. Modifications may include, for example, the use of modifications known in the art at the 2' position of the ribose sugar, such as deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl modifications, in place of the ribosugar of the nucleobase, and modifications in the phosphate group, such as phosphorothioate modifications. The overhang does not need to be homologous to the target sequence. 【0281】 In one embodiment, each residue of sense strand and antisense strand is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl or 2'-fluoro.These strands can contain two or more modifications.In one embodiment, each residue of sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro. 【0282】 At least two different modifications are typically present on the sense and antisense strands, and the two modifications may be 2'-O-methyl or 2'-fluoro modifications, or others. 【0283】 In one embodiment, N a and / or N b includes an alternating pattern of modifications. The term "alternating motif," as used herein, refers to a motif having one or more modifications, each modification occurring on alternating nucleotides in a strand. The alternating nucleotides may refer to one every other nucleotide or one every third nucleotide, or similar patterns. For example, if A, B, and C each represent one modification type to a nucleotide, the alternating motif could be "ABABABABABAB...," "AABBAABBAABB...," "AABAABAABAAB...," "AAABAAABAAAB...," "AAABBBAAABBB...," or "ABCABCABCABC...," etc. 【0284】 The types of modifications contained within an alternating motif can be the same or different. For example, if A, B, C, and D each represent one modification type to a nucleotide, the alternation pattern, i.e., the modifications to every other nucleotide, can be the same, but each of the sense or antisense strands can be selected from several modification possibilities within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD..." or "CDCDCD...". 【0285】 In one embodiment, the RNAi agents of the present invention include a modification pattern in an alternating motif on the sense strand that is altered relative to the modification pattern in an alternating motif on the antisense strand. The modification groups of nucleotides in the sense strand may correspond to different modification groups of nucleotides in the antisense strand, or vice versa. For example, when the sense strand pairs with the antisense strand in a dsRNA duplex, within the double-stranded region, the alternating motif in the sense strand may begin with "ABABAB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand may begin with "BABABA" from the 5'-3' end of the strand. As another example, within the double-stranded region, the alternating motif in the sense strand may begin with "AABBAABB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand may begin with "BBAABBAA" from the 5'-3' end of the strand, thereby resulting in a complete or partial change in the modification pattern between the sense and antisense strands. 【0286】 In one embodiment, the RNAi agent comprises a pattern of alternating motifs of 2'-O-methyl and 2'-F modifications on the initial sense strand that has a variation relative to the pattern of alternating motifs of 2'-O-methyl and 2'-F modifications on the initial antisense strand, i.e., 2'-O-methyl modified nucleotides on the sense strand base pair with 2'-F modified nucleotides on the antisense strand, and vice versa. Position 1 of the sense strand may start with a 2'-F modification, and position 1 of the antisense strand may start with a 2'-O-methyl modification. 【0287】 By introducing one or more motifs of three identical modifications on three consecutive nucleotides to sense strand and / or antisense strand, the original modification pattern existing in sense strand and / or antisense strand is interrupted.By introducing one or more motifs of three identical modifications on three consecutive nucleotides to sense strand and / or antisense strand, this interruption of the modification pattern of sense strand and / or antisense strand can unexpectedly enhance the gene silencing activity of target gene. 【0288】 In one embodiment, when a motif of three identical modifications on three consecutive nucleotides is introduced into either strand, the modifications of the nucleotides adjacent to the motif are different from the modification of the motif. For example, a portion of a sequence containing a motif may be designated "...N a YYYN b ...", where "Y" represents a modification of a motif of three identical modifications on three consecutive nucleotides, and "N a " and "N b " represents a modification to the nucleotide adjacent to the motif "YYY" that is different from the modification of Y, and a and N b may be the same or different modifications. a and / or N b may be present or absent when wing modifications are present. 【0289】 The RNAi agent may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may be present on any nucleotide at any position in the sense strand, antisense strand, or both strands. For example, the internucleotide linkage modification may be present on every nucleotide on the sense strand and / or antisense strand; each internucleotide linkage modification may be present in an alternating pattern on the sense strand and / or antisense strand; or the sense strand or antisense strand may contain both internucleotide linkage modifications in an alternating pattern. The alternating pattern of internucleotide linkage modifications on the sense strand may be the same or different from that of the antisense strand, and the alternating pattern of internucleotide linkage modifications on the sense strand may have variations relative to the alternating pattern of internucleotide linkage modifications on the antisense strand. In one embodiment, the double-stranded RNAi agent comprises 6 to 8 phosphorothioate internucleotide linkages. In one embodiment, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand comprises at least two phosphorothioate internucleotide linkages at either the 5' end or the 3' end. 【0290】 In one embodiment, the RNAi comprises phosphorothioate or methylphosphonate internucleotide bond modification in the overhang region.For example, the overhang region may comprise two nucleotides with phosphorothioate or methylphosphonate internucleotide bonds between the two nucleotides.Internucleotide bond modification may also be provided to link the overhang nucleotide to the terminal paired nucleotide within the double-stranded region.For example, at least 2, 3, 4, or all of the overhang nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide bonds, and optionally, there may be an additional phosphorothioate or methylphosphonate internucleotide bond that links the overhang nucleotide to the paired nucleotide adjacent to the overhang nucleotide.For example, there may be at least two phosphorothioate internucleotide bonds between the terminal three nucleotides, where two of the three nucleotides are overhang nucleotides and the third is the paired nucleotide adjacent to the overhang nucleotide. These terminal three nucleotides may be at the 3' end of the antisense strand, the 3' end of the sense strand, the 5' end of the antisense strand, and / or the 5' end of the antisense strand. 【0291】 In one embodiment, a two-nucleotide overhang is present at the 3'-end of the antisense strand, and two phosphorothioate internucleotide bonds are present between the terminal three nucleotides, where two of the three nucleotides are overhanging nucleotides, and the third nucleotide is a paired nucleotide adjacent to the overhanging nucleotide. Optionally, the RNAi agent may further have two phosphorothioate internucleotide bonds between the terminal three nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand. 【0292】 In one embodiment, the RNAi agent contains mismatches with the target, within the duplex, or a combination thereof. Mismatches may be present in the overhang region or within the duplex region. Base pairs may be ranked based on their tendency to promote dissociation or melting (for example, the simplest method is to consider the association or dissociation free energy of a particular pairing based on each pairing, but similar or similar analyses can also be used). In terms of promoting dissociation, A:U is preferred to G:C; G:U is preferred to G:C; and I:C is preferred to G:C (I=inosine). Mismatches, such as non-canonical or non-canonical pairings (as described elsewhere herein), are preferred to canonical (A:T, A:U, G:C) pairings; and pairings involving universal bases are preferred to canonical pairings. 【0293】 In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the double-stranded region from the 5' end of the antisense strand independently selected from the group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical or non-canonical pairings or pairings containing universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex. 【0294】 In one embodiment, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U and dT.Alternatively, at least one of the first 1, 2 or 3 base pairs in the double-stranded region from the 5' end of the antisense strand is an AU base pair.For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair. 【0295】 In another embodiment, the nucleotide at the 3' end of the sense strand is deoxythymine (dT). In another embodiment, the nucleotide at the 3' end of the antisense strand is deoxythymine (dT). In one embodiment, there is a short sequence of deoxythymine nucleotides, e.g., two dT nucleotides, on the 3' end of the sense strand and / or antisense strand. 【0296】 In one embodiment, the sense strand sequence has formula (I): 5'n p -N a -(XXX)iN b -YYY-N b -(ZZZ) j -N a -n q 3'(I) (In the formula, i and j are each independently 0 or 1; p and q each independently represent 0 to 6; each N a represents oligonucleotide sequences containing independently 0 to 25 modified nucleotides, each sequence containing at least two different modified nucleotides; each N b represents an oligonucleotide sequence containing independently 0 to 10 modified nucleotides; each n p and n q independently represent overhanging nucleotides; N b and Y do not have the same modification; and XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides) Preferably, YYY are all 2'-F modified nucleotides. 【0297】 In one embodiment, N a and / or N b includes alternating pattern modifications. 【0298】 In one embodiment, the YYY motif is located at or near the cleavage site of the sense strand. For example, when the RNAi agent has a double-stranded region of 17 to 23 nucleotides in length, the YYY motif can be located at or near the cleavage site of the sense strand (e.g., at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13), where counting begins at the first nucleotide from the 5' end, or optionally, counting begins at the first paired nucleotide within the double-stranded region from the 5' end. 【0299】 In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or i and j are both 1. Thus, the sense strand has the following formula: 5'n p -N a -YYY-N b -ZZZ-N a -n q 3'(Ib); 5'n p -N a -XXX-N b -YYY-N a -n q 3'(Ic); or 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3'(Id) It can be represented by: 【0300】 When the sense strand is represented by formula (Ib), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. 【0301】 When the sense strand is represented by formula (Ic), N brepresents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. 【0302】 When the sense strand is represented by formula (Id), each N b represents an oligonucleotide sequence containing independently 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6. Each N a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Each of X, Y, and Z can be the same or different from each other. 【0303】 In other embodiments, i is 0 and j is 0, and the sense strand has the formula: 5'n p -N a -YYY-N a -n q 3'(Ia) It may be represented by: 【0304】 When the sense strand is represented by formula (Ia), each N a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. 【0305】 In one embodiment, the antisense strand sequence of the RNAi has the formula (II): 5'n q '-N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p '3' (II) (In the formula, k and l are each independently 0 or 1; p' and q' are each independently 0 to 6; each N a ' represents oligonucleotide sequences containing independently 0 to 25 modified nucleotides, each sequence containing at least two different modified nucleotides; each N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p ' and n q ' independently represents an overhanging nucleotide; where N b ' and Y' do not have the same modification; and X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides. It may be represented by: 【0306】 In one embodiment, N a ' and / or N b ' includes alternating pattern modifications. 【0307】 The Y'Y'Y' motif is present at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a double-stranded region of 17 to 23 nucleotides in length, the Y'Y'Y' motif can be present at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, where counting begins with the first nucleotide from the 5' end, or optionally, counting begins with the first paired nucleotide within the double-stranded region from the 5' end. Preferably, the Y'Y'Y' motif is present at positions 11, 12, or 13. 【0308】 In one embodiment, the Y'Y'Y' motif is all 2'-OMe modified nucleotides. 【0309】 In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1. 【0310】 Thus, the antisense strand has the following formula: 5'n q '-N a '-Z'Z'Z'-N b '-Y'Y'Y'-N a '-n p '3'(IIb); 5'n q '-N a '-Y'Y'Y'-N b '-X'X'X'-n p '3' (IIc); or 5'n q '-N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'-N a '-n p '3' (IId) It can be represented by: 【0311】 When the antisense strand is represented by formula (IIb), N b ' represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' represents an oligonucleotide sequence containing, independently, 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. 【0312】 When the antisense strand is represented by formula (IIc), N b ' represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' represents an oligonucleotide sequence containing, independently, 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. 【0313】 When the antisense strand is represented by formula (IId), each N bEach N' represents an oligonucleotide sequence that independently contains 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6. 【0314】 In other embodiments, k is 0 and l is 0, and the antisense strand has the formula: 5'n p '-N a '-Y'Y'Y'-N a '-n q '3'(Ia) It may be represented by: 【0315】 When the antisense strand is represented by formula (IIa), each N a X', Y', and Z' independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Each of X', Y', and Z' may be the same or different from one another. Each nucleotide in the sense and antisense strands may be independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide in the sense and antisense strands is independently modified with 2'-O-methyl or 2'-fluoro. Each of X, Y, Z, X', Y', and Z' may specifically represent a 2'-O-methyl modification or a 2'-fluoro modification. 【0316】 In one embodiment, the sense strand of the RNAi agent may include a YYY motif occurring at positions 9, 10, and 11 of the strand when the double-stranded region is 21 nt, counting starting at the first nucleotide from the 5' end, or optionally counting starting at the first paired nucleotide within the double-stranded region from the 5' end, and Y representing a 2'-F modification. The sense strand may further include a XXX motif or a ZZZ motif as a wing modification at its opposite end of the double-stranded region; XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification. 【0317】 In one embodiment, the antisense strand may include a Y'Y'Y' motif at positions 11, 12, and 13 of the strand, counting starting from the first nucleotide from the 5' end, or optionally counting starting from the 5' end with the first paired nucleotide within the double-stranded region, and Y' representing a 2'-O-methyl modification. The antisense strand may further include an X'X'X' motif or a Z'Z'Z' motif as a wing modification at its opposite end of the double-stranded region; X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification. 【0318】 Each of the sense strands represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with an antisense strand represented by any one of the above formulas (IIa), (IIb), (IIc), and (IId). 【0319】 Thus, the RNAi agent used in the methods of the invention may comprise a sense strand and an antisense strand, each strand having 14-30 nucleotides, and the RNAi duplex may have the formula (III): Sense:5'n p -N a -(XXX)iN b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p '-Na '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III) (In the formula, i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6; each N a and N a ' represents oligonucleotide sequences containing independently 0 to 25 modified nucleotides, each sequence containing at least two different modified nucleotides; each N b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; where each n p ',n p , n q ', and n q each may be present or absent and independently represent an overhanging nucleotide; and XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides. is expressed by 【0320】 In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or i and j are both 0; or i and j are both 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; or k is 0 and l is 1; or k and l are both 0; or k and l are both 1. 【0321】 An exemplary combination of sense and antisense strands that form an RNAi duplex is of the following formula: 5'n p -N a -YYY-N a -nq 3' 3'n p '-N a '-Y'Y'Y'-N a 'n q '5' (IIIa) 5'n p -N a -YYY-N b -ZZZ-N a -n q 3' 3'n p '-N a '-Y'Y'Y'-N b '-Z'Z'Z'-N a 'n q '5' (IIIb) 5'n p -N a -XXX-N b -YYY-N a -n q 3' 3'n p '-N a '-X'X'X'-N b '-Y'Y'Y'-N a '-n q '5' (IIIc) 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' 3'n p '-N a '-X'X'X'-N b '-Y'Y'Y'-N b '-Z'Z'Z'-N a -n q '5' (IIId) Includes: 【0322】 When the RNAi agent is represented by formula (IIIa), each N a represents an oligonucleotide sequence that independently contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. 【0323】 When the RNAi agent is represented by formula (IIIb), each N b represents an oligonucleotide sequence that independently contains 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. a represents an oligonucleotide sequence that independently contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. 【0324】 When the RNAi agent is represented by formula (IIIc), each N b , N b Each N' represents an oligonucleotide sequence that independently contains 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a represents an oligonucleotide sequence that independently contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. 【0325】 When the RNAi agent is represented by formula (IIId), each N b , N b ' represents an oligonucleotide sequence that independently contains 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a , N a N' represents an oligonucleotide sequence containing, independently, 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. a , N a ', N b and N b ' each independently includes an alternating pattern of modifications. 【0326】 Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) may be the same as or different from one another. 【0327】 When an RNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides may be base-paired with one of the Y' nucleotides, alternatively, at least two of the Y nucleotides are base-paired with the corresponding Y' nucleotide; or all three of the Y nucleotides are base-paired with the corresponding Y' nucleotide. 【0328】 When the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides may be base-paired with one of the Z' nucleotides. Alternatively, at least two of the Z nucleotides are base-paired with the corresponding Z' nucleotide; or all three of the Z nucleotides are base-paired with the corresponding Z' nucleotide. 【0329】 When an RNAi agent is represented as formula (IIIc) or (IIId), at least one of the X nucleotides may be base-paired with one of the X' nucleotides, alternatively, at least two of the X nucleotides are base-paired with the corresponding X' nucleotide; or all three of the X nucleotides are base-paired with the corresponding X' nucleotide. 【0330】 In one embodiment, the modification on the Y nucleotide is different from the modification on the Y' nucleotide, the modification on the Z nucleotide is different from the modification on the Z' nucleotide, and / or the modification on the X nucleotide is different from the modification on the X' nucleotide. 【0331】 In one embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification. In another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, and n p '>0 and at least one n pIn yet another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, and n p '>0 and at least one n p In another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, and n is linked to the adjacent nucleotide via a phosphorothioate bond, and the sense strand is linked to one or more GalNAc derivatives linked via a bivalent or trivalent branched linker (described below). p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate bonds, and the sense strand contains at least one phosphorothioate bond, and the sense strand is linked to one or more GalNAc derivatives linked via a bivalent or trivalent branched linker. 【0332】 In one embodiment, when the RNAi agent is represented by Formula (IIIa), the Na modification is a 2'-O-methyl or a 2'-fluoro modification, and n p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate bonds, and the sense strand contains at least one phosphorothioate bond, and the sense strand is linked to one or more GalNAc derivatives linked via a bivalent or trivalent branched linker. 【0333】 In one embodiment, the RNAi agent is a multimer comprising at least two double strands represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), wherein the double strands are connected by a linker.The linker can be cleavable or non-cleavable.Optionally, the multimer further comprises a ligand.Each of the double strands can target the same gene or two different genes; or each of the double strands can target the same gene at two different target sites. 【0334】 In one embodiment, the RNAi agent is a multimer comprising 3, 4, 5, 6 or more double strands represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), wherein the double strands are connected by a linker.The linker can be cleavable or non-cleavable.Optionally, the multimer further comprises a ligand.Each of the double strands can target the same gene or two different genes; or each of the double strands can target the same gene at two different target sites. 【0335】 In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at one or both of the 5' and 3' ends, and optionally are linked to a ligand. Each of the agents can target the same gene or two different genes; or each of the agents can target the same gene at two different target sites. 【0336】 Various publications describe multimeric RNAi agents that can be used in the methods of the present invention, including WO 2007 / 091269, U.S. Patent No. 7,858,769, WO 2010 / 141511, WO 2007 / 117686, WO 2009 / 014887, and WO 2011 / 031520 (the entire contents of each of which are hereby incorporated by reference). 【0337】 As described in further detail below, an RNAi agent comprising one or more carbohydrate moieties attached thereto can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, such as a non-carbohydrate (preferably cyclic) carrier attached with a carbohydrate ligand. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose-replacement modified subunit (RRMS). The cyclic carrier can be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms can be a heteroatom, such as nitrogen, oxygen, or sulfur. The cyclic carrier can be a monocyclic ring system or can contain two or more rings, such as fused rings. The cyclic carrier can be a fully saturated ring system or can contain one or more double bonds. 【0338】 The ligand may be attached to the polynucleotide by a carrier. The carrier comprises (i) at least one "backbone attachment point," preferably two "backbone attachment points," and (ii) at least one "tether attachment point." As used herein, "backbone attachment point" refers to a functional group, e.g., a hydroxyl group, or generally to a bond available and suitable for incorporation of the carrier into the backbone of a ribonucleic acid, e.g., a phosphate backbone, or, e.g., a sulfur-containing modified phosphate backbone. In some embodiments, a "tether attachment point" (TAP) refers to a ring atom, e.g., a carbon atom or heteroatom (different from the atom providing the backbone attachment point), of the cyclic carrier to which the selected moiety is attached. The moiety may be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is connected to the cyclic carrier by an intervening tether. Thus, the cyclic carrier will often contain a functional group, such as an amino group, or generally provide a bond suitable for incorporation or tethering another chemical entity, such as a ligand, to the constituent ring. 【0339】 The RNAi agent may be attached to the ligand via a carrier, where the carrier can be a cyclic or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is selected from a serinol backbone or a diethanolamine backbone. 【0340】 In certain specific embodiments, the RNAi agent used in the methods of the invention is an agent selected from the group of agents listed in any one of Tables 3, 4, 5, 6, 18, 19, 20, 21, and 23. These agents may further comprise a ligand. 【0341】 IV. Ligand-conjugated iRNA Another modification of the RNA of an iRNA suitable for use in the methods of the invention involves chemically linking to the RNA one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, or cellular uptake of the iRNA. Such moieties include lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:6553-6556); cholic acid (Manoharan et al., Bior. Med. Chem. Let., 1994, 4:1053-1060); thioethers such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Bior. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538); dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54); phospholipids, for example, di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783); polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973); or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654); palmityl moiety (Mishra et al., Biochim. Biophys.Acta, 1995, 1264:229-237); or octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937). 【0342】 In one embodiment, the ligand changes the distribution, targeting or life span of the iRNA agent into which it is incorporated. In a preferred embodiment, the ligand provides improved affinity to a selected target, such as a molecule, a cell or cell type, a compartment, such as a subcellular or organ compartment, a tissue or organ or region of the body, for example, compared to a chemical species in the absence of such a ligand. Preferred ligands do not participate in double-stranded pairing in duplexed nucleic acid. 【0343】 Ligands can include natural substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines are polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, polyamine quaternary salts, or alpha helical peptides. 【0344】 The ligand may also include a targeting group, such as a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid, or protein, for example, an antibody that binds to a specific cell type, such as a kidney cell. The targeting group may be thyroid-stimulating hormone, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, monovalent or polyvalent galactose, N-acetyl-galactosamine, N-acetylglucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acid, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimic. In certain embodiments, the ligand includes monovalent or polyvalent galactose. In certain embodiments, the ligand includes cholesterol. 【0345】 Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithophosphate, and the like. Examples of suitable cleavage inhibitors include acetylcholinesterases (e.g., acetylcholinesterase ... 【0346】 Ligands can be proteins, such as glycoproteins; peptides, such as molecules with specific affinity for co-ligands; or antibodies, such as antibodies that bind to specific cell types, such as liver cells. Ligands can also include hormones and hormone receptors. They can also include lipids, lectins, carbohydrates, vitamins, cofactors, and non-peptide species, such as multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. Ligands can be, for example, lipopolysaccharides, p38 MAP kinase activators, or NF-κB activators. 【0347】 The ligand can be a substance, such as a drug, that can increase uptake of an iRNA agent into a cell, e.g., by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments, e.g., by disrupting the cell's cytoskeleton. The drug can be, e.g., taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin. 【0348】 In some embodiments, the ligand attached to the iRNA described herein refers to a pharmacokinetic modulator (PK modulator). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides comprising several phosphorothioate linkages are also known to bind to serum proteins. Therefore, short oligonucleotides, such as, for example, about 5-, 10-, 15-, or 20-base oligonucleotides comprising multiple phosphorothioate linkages in the backbone, are also suitable as ligands (e.g., as PK-modulating ligands) for the present invention. In addition, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein. 【0349】 Ligand-conjugated oligonucleotides of the invention may be synthesized by using oligonucleotides bearing pendant reactive functional groups, such as those derived from the addition of a binding molecule onto an oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially available ligands, synthesized ligands bearing any of a variety of protecting groups, or ligands bearing an attached binding moiety. 【0350】 The oligonucleotides used in the conjugates of the present invention may be conveniently and routinely produced through well-known solid-phase synthesis techniques. Equipment for such synthesis is sold by several suppliers, including Applied Biosystems (Foster City, Calif.). Additionally or alternatively, any other means for such synthesis known in the art may be used. It is also known to use similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives. 【0351】 In the ligand-conjugated oligonucleotides and sequence-specific linked nucleosides bearing ligand molecules of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer using standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors already bearing a linking moiety, ligand-nucleotide or nucleoside-conjugate precursors already bearing a ligand molecule, or building blocks bearing a non-nucleoside ligand. 【0352】 When using a nucleotide conjugate precursor that already has a binding moiety, synthesis of the sequence-specific linked nucleoside is typically completed, and then a ligand molecule is reacted with the binding moiety to produce the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the invention are synthesized by automated synthesizers using phosphoramidites derived from ligand-nucleoside conjugates, in addition to standard and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis. 【0353】 A. Lipid Complex In one embodiment, the ligand or complex is a lipid or lipid-based molecule.Such lipid or lipid-based molecule preferably binds to serum protein, for example, human serum albumin (HSA).HSA-binding ligand allows the distribution of complex to target tissue, for example, non-renal target tissue of the body.For example, the target tissue can be the liver, including the parenchymal cells of the liver.Other molecules that can bind to HSA can also be used as ligand.For example, naproxen or aspirin can be used.Lipid or lipid-based ligand can (a) increase the degradation resistance of complex, (b) increase the targeting or transport to target cell or cell membrane, and / or (c) be used to regulate the binding of serum protein, for example, HSA. 【0354】 For example, lipid-based ligand can be used for inhibition, such as controlling the binding of complex to target tissue.For example, the lipid or lipid-based ligand that binds more strongly to HSA is less likely to be targeted to the kidney, and therefore less likely to be removed from the body.The lipid or lipid-based ligand that binds weaker to HSA can be used to target complex to the kidney. 【0355】 In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, it binds HSA with sufficient affinity so that the conjugate preferably distributes to non-renal tissues. However, the affinity is preferably not so strong that HSA ligand binding cannot be reversed. 【0356】 In another preferred embodiment, the lipid-based ligand binds weakly or not at all to HSA, such that the conjugate preferably distributes to the kidney. Other moieties that target kidney cells may also be used in place of or in addition to the lipid-based ligand. 【0357】 In another embodiment, the ligand is a moiety, such as a vitamin, that is taken up by target cells, e.g., proliferating cells. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., malignant or non-malignant types, e.g., cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, e.g., folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by target cells, e.g., liver cells. Also included are HSA and low-density lipoprotein (LDL). 【0358】 B. Cell-penetrating agents In another embodiment, the ligand is a cell-penetrating agent, preferably a helical cell-penetrating agent. Preferably, the cell-penetrating agent is amphipathic. An exemplary cell-penetrating agent is a peptide such as tat or antennopedia. When the cell-penetrating agent is a peptide, it can be modified, including peptidylmimetic, invertomer, non-peptide or pseudo-peptide bond, and D-amino acid use. The helical agent is preferably an α-helical agent with a lipophilic and lipophobic phase. 【0359】 The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules that can fold into defined three-dimensional structures similar to natural peptides. The addition of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic distribution of iRNAs, such as by facilitating cellular recognition and uptake. The peptide or peptidomimetic moiety can be about 5-50 amino acids in length, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. 【0360】 The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 3). An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 4)) can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, which can transport a number of polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 5)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 6)) have been shown to function as delivery peptides. Peptides or peptidomimetics can be encoded by random sequences of DNA, such as peptides identified from phage-display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). For cell targeting purposes, an example of a peptide or peptidomimetic tethered to a dsRNA agent through an incorporated monomer unit is an arginine-glycine-aspartic acid (RGD)-peptide or RGD mimic. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications to increase stability or induce conformational properties. Any of the structural modifications described below can be used. 【0361】 The RGD peptide used in the compositions and methods of the present invention can be linear or cyclic, and can be modified, for example, by glycosylation or methylation, to facilitate targeting to specific tissues.RGD-containing peptides and peptidomimetics include D-amino acids and synthetic RGD mimics.In addition to RGD, other moieties that target integrin ligands can be used.Preferred complexes of this ligand target PECAM-1 or VEGF. 【0362】 A "cell-penetrating peptide" can penetrate cells, such as microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or cecropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two key amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain a nuclear localization signal (NLS). For example, cell-penetrating peptides can be bisected amphipathic peptides, such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003). 【0363】 C. Carbohydrate Complex In some embodiments of the compositions and methods of the present invention, the iRNA oligonucleotide further comprises a carbohydrate. Carbohydrate-conjugated iRNAs are advantageous for in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic applications, as described herein. As used herein, "carbohydrate" refers to either a carbohydrate itself, composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), with an oxygen, nitrogen, or sulfur atom attached to each carbon atom; or a compound having a carbohydrate moiety as part of its structure, composed of one or more monosaccharide units, each having at least six carbon atoms (which may be linear, branched, or cyclic), with an oxygen, nitrogen, or sulfur atom attached to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Particular monosaccharides include sugars of C5 and above (e.g., C5, C6, C7, or C8); di- and trisaccharides include sugars with two or three monosaccharide units (e.g., C5, C6, C7, or C8). 【0364】 In one embodiment, the carbohydrate complexes used in the compositions and methods of the present invention are monosaccharides. In another embodiment, the carbohydrate complexes used in the compositions and methods of the present invention are [ka] [ka] [ka] [ka] is selected from the group consisting of: 【0365】 In one embodiment, the monosaccharide is N-acetylgalactosamine, e.g. [ka] is. 【0366】 Other exemplary carbohydrate complexes for use in the embodiments described herein include, but are not limited to: [ka] (When one of X or Y is an oligonucleotide, the other is hydrogen). 【0367】 In certain embodiments of the invention, GalNAc or GalNAc derivatives are attached to iRNA agents of the invention via a monovalent linker. In some embodiments, GalNAc or GalNAc derivatives are attached to iRNA agents of the invention via a bivalent linker. In yet other embodiments of the invention, GalNAc or GalNAc derivatives are attached to iRNA agents of the invention via a trivalent linker. 【0368】 In one embodiment, a double-stranded RNAi agent of the invention comprises one GalNAc or GalNAc derivative attached to the iRNA agent. In another embodiment, a double-stranded RNAi agent of the invention comprises multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently attached to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers. 【0369】 In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of a larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of each strand of the other, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide within the hairpin loop may comprise a GalNAc or GalNAc derivative independently attached via a monovalent linker. Hairpin loops may also be formed by extended overhangs on one strand of the duplex. 【0370】 In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and / or a cell-penetrating peptide. 【0371】 Additional carbohydrate conjugates suitable for use in the present invention include those described in PCT Publications WO 2014 / 179620 and WO 2014 / 179627, the entire contents of each of which are incorporated herein by reference. 【0372】 D. Linker In some embodiments, the conjugates or ligands described herein may be attached to the iRNA oligonucleotide by various linkers, which may be cleavable or non-cleavable. 【0373】 The term "linker" or "linking group" means an organic moiety that joins two parts of a compound, for example, by covalently bonding the two parts of the compound. Linkers are typically a direct bond, or an atom such as oxygen or sulfur, a unit such as NR, C(O), C(O)NH, SO, SO, SONH, or a group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, aryl and alkylaryl, alkenylaryl, alkynylaryl, alkylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, wherein one or more methylenes are selected from O, S, S(O), SO, N(R 8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle (wherein R 8 is hydrogen, acyl, aliphatic, or substituted aliphatic). In one embodiment, the linker is about 1-24 atoms, 2-24 atoms, 3-24 atoms, 4-24 atoms, 5-24 atoms, 6-24 atoms, 6-18 atoms, 7-18 atoms, 7-17 atoms, 8-17 atoms, 6-16 atoms, 7-16 atoms, or 8-16 atoms. 【0374】 A cleavable tether is one that is sufficiently stable outside the cell but is cleaved upon entry into a target cell to release the two moieties tethered by the linker. In preferred embodiments, the cleavable tether is cleaved at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or more, or at least about 100-fold more rapidly in the target cell, or under first standard conditions (e.g., which may be selected to mimic or correspond to intracellular conditions), than in the subject's blood, or under second standard conditions (e.g., which may be selected to mimic or correspond to conditions found in blood or serum). 【0375】 Cleavable linking groups are susceptible to cleavage agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleavage agents are more common in cells than in serum or blood, or are found at higher levels or activity. Examples of such degradable agents include oxidizing or reducing enzymes or reducing agents such as mercaptans present in cells, which can degrade redox-cleavable linking groups by reduction, and are selective for specific substrates or do not have substrate specificity; esterases; agents that can create an acidic environment, such as endosomes or those that produce a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which can be substrate specific), and phosphatases. 【0376】 Cleavable linking groups, such as disulfide bonds, can be highly sensitive to pH. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers have cleavable linking groups that are cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand in the cell or to a desired compartment of the cell. 【0377】 Linker can contain cleavable linking group that can be cleaved by specific enzyme.The type of cleavable linking group incorporated into linker can depend on the cell to be targeted.For example, the ligand for targeting liver can be linked to cationic lipid through a linker that contains ester group.Hepatocytes are rich in esterase, therefore linker is more efficiently cleaved in hepatocytes than in cell types that are not rich in esterase.Other cell types that are rich in esterase include lung, renal cortex and testicular cells. 【0378】 Linkers containing peptide bonds may be used in targeting peptidase-rich cell types such as hepatocytes and synoviocytes. 【0379】 In general, the suitability of a candidate cleavable linker can be evaluated by testing the ability of a degradable agent (condition) to cleave the candidate linker. It may also be desirable to test candidate cleavable linkers for their ability to resist cleavage in blood or upon contact with other non-target tissues. Thus, the relative susceptibility to cleavage between first and second conditions can be determined, with the first condition selected to indicate cleavage in target cells and the second condition selected to indicate cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in a whole animal. It may be useful to perform initial evaluation in a cell-free or culture condition and confirm with further evaluation in a whole animal. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times more rapidly in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions). 【0380】 i. Redox-cleavable linker In one embodiment, the cleavable linker is a redox-cleavable linker that is cleaved upon reduction or oxidation. One example of a reductively cleavable linker is a disulfide linker (-SS-). To determine whether a candidate cleavable linker is a suitable "reductively cleavable linker" or suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, one can rely on the methods described herein. For example, candidates can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate observed in cells, such as target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. A candidate compound is cleaved at a maximum of about 10% in blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times more rapidly in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of a candidate compound may be determined using standard enzyme kinetic assays under conditions selected to mimic intracellular media compared to conditions selected to mimic extracellular media. 【0381】 ii. Phosphate-based cleavable linkers In another embodiment, the cleavable linker comprises a phosphate-based cleavable linker. A phosphate-based cleavable linker can be cleaved by an agent that degrades or hydrolyzes the phosphate group. An example of an agent that cleaves a phosphate group in a cell is an enzyme such as an intracellular phosphatase. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O, -SP(S)(H)-O-, -SP(O)(H)-S-, -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above. 【0382】 iii. Acid-cleavable linking group In another embodiment, the cleavable linker comprises an acid-cleavable linker. An acid-cleavable linker is a linker that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linker is cleaved in an acidic environment of about pH 6.5 or below (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0 or below) or by an agent such as an enzyme that can act as a general acid. Within a cell, certain low-pH organelles, such as endosomes and lysosomes, may provide a cleavage environment for the acid-cleavable linker. Examples of acid-cleavable linkers include, but are not limited to, hydrazones, esters, and amino acid esters. Acid-cleavable groups may have the general formula -C=NN-, C(O)O, or -OC(O). A preferred embodiment is when the carbon is attached to the oxygen of the ester (alkoxy group), an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates may be evaluated using methods similar to those described above. 【0383】 iv. Ester-based linking groups In another embodiment, the cleavable linker comprises an ester-based cleavable linker. Ester-based cleavable linkers are cleaved intracellularly by enzymes such as esterases and amidases. Examples of ester-based cleavable linkers include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linkers have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above. 【0384】 v. Peptide-Based Cleavage Groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable tether. Peptide-based cleavable tethers are cleaved intracellularly by enzymes such as peptidases and proteases. Peptide-based cleavable tethers are peptide bonds formed between amino acids to give rise to oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (—C(O)NH—). Amide groups can be formed between any alkylene, alkenylene, or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to give rise to peptides and proteins. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to give rise to peptides and proteins, but do not include the entire amide functionality. Peptide-based cleavable tethers have the general formula —NHCHRAC(O)NHCHRBC(O)—, where R and R are the R groups of two adjacent amino acids. These candidates may be evaluated using methods similar to those described above. 【0385】 In one embodiment, the iRNA of the present invention is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrates conjugated to linkers in the compositions and methods of the present invention include: [ka] [ka] (In the formula, wherein one of X or Y is an oligonucleotide and the other is hydrogen). 【0386】 In certain embodiments of the compositions and methods of the present invention, the ligand is one or more GalNAc (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker. 【0387】 In one embodiment, the dsRNA of the invention is Formulas (XXXII) to (XXXV), [ka] (In the formula, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C independently represent each occurrence from 0 to 20, and the repeat units may be identical or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C is, each independently for each occurrence, absent, CO, NH, O, S, OC(O), NHC(O), CH, CHNH, or CHO; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C is independently for each occurrence absent, alkylene, or substituted alkylene, and one or more methylenes are selected from O, S, S(O), SO, N(R N ), C(R')=C(R''), C≡C or C(O); R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5Cis independently for each occurrence absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocyclyl; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represents a ligand; i.e., independently for each occurrence, a monosaccharide (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; R a is H or an amino acid side chain). The trivalent conjugated GalNAc derivative is conjugated to a bivalent or trivalent branched linker selected from the group of structures represented by: Formula (XXXVI), [ka] (In the formula, L 5A , L 5B and L 5C It is particularly useful for use with RNAi agents to inhibit the expression of target genes such as ribonucleotides (wherein represents a monosaccharide, such as a GalNAc derivative). 【0388】 Examples of suitable divalent and trivalent branched linker groups for conjugation to GalNAc derivatives include, but are not limited to, the structures listed above as Formulas II, VII, XI, X, and XIII. 【0389】 Representative United States patents that teach the preparation of RNA complexes include U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; and 5,591,584, the contents of each of which are hereby incorporated by reference in their entirety. Details; U.S. Patent Nos. 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737 Nos.; U.S. Patent Nos. 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469 ;U.S. Patent No. 5,258,506;U.S. Patent No. 5,262,536;U.S. Patent No. 5,272,250;U.S. Patent No. 5,292,873;U.S. Patent No. 5,317,098;U.S. Patent No. 5,371,241, U.S. Patent No. 5,391,723;U.S. Patent No. 5,416,203, U.S. Patent No. 5,451,463;U.S. Patent No. 5,510,475;U.S. Patent No. 5,512,667;U.S. Patent No. 5,514,785;U.S. Patent No. 5,565,552;Examples of such patents include, but are not limited to, U.S. Patent Nos. 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; and 8,106,022. 【0390】 It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the foregoing modifications may be incorporated in a single compound, or even in a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds. 【0391】 "Chimeric" iRNA compounds or "chimeras," in the context of the present invention, are iRNA compounds, preferably dsRNA, that contain two or more chemically distinct regions, each composed of at least one monomer unit, i.e., nucleotides in the case of dsRNA compounds. These iRNAs typically contain at least one region in which the RNA has been modified to confer on the iRNA increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity for the target nucleic acid. Additional regions of the iRNA may serve as substrates for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. As an example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H therefore results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. As a result, comparable results are often obtained with shorter iRNAs when chimeric dsRNAs are used compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art. 【0392】 In some cases, the RNA of an iRNA may be modified by a non-ligand group. To enhance the activity, cellular distribution, or intracellular uptake of an iRNA, several non-ligand molecules have been conjugated to the iRNA, and procedures for performing such conjugation are available in the scientific literature.Such non-ligand moieties include lipid moieties such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923).Representative U.S. patents that teach the preparation of such RNA complexes are listed above. A typical conjugation protocol involves the synthesis of RNA with an amino linker at one or more positions in the sequence. The amino group is then reacted with the molecule to be conjugated using an appropriate coupling or activation reagent. The conjugation reaction can be carried out in solution phase while the RNA is still bound to the solid support, or following RNA cleavage. Purification of the RNA complex by HPLC typically yields a pure complex. 【0393】 IV. Delivery of iRNA of the Invention Delivery of an iRNA of the invention to a cell, such as a cell in a subject, e.g., a human subject (e.g., a subject in need thereof, e.g., a subject with a disorder that would benefit from reduced PCSK9 expression), can be achieved in several different ways. For example, delivery may be performed by contacting a cell with an iRNA of the invention, either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an iRNA, e.g., dsRNA, to a subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and induce expression of the iRNA. These alternatives are discussed further below. 【0394】 Generally, any method for delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the iRNAs of the present invention (see, for example, Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and International Publication No. WO 94 / 02595, the entire contents of which are incorporated herein by reference). For in vivo delivery, factors to consider for delivering iRNA molecules include, for example, the biological stability of the delivered molecule, prevention of nonspecific effects, and accumulation of the delivered molecule in the target tissue. Nonspecific effects of iRNA can be minimized by local administration, such as direct injection or implantation into tissue or local administration of the formulation. Local administration at the treatment site maximizes the local concentration of the agent, limits exposure to the agent in systemic tissues that may otherwise be harmed by or degrade the agent, and allows for administration of a lower total dose of the iRNA molecule. Several studies have demonstrated successful gene product knockdown when iRNA is administered locally. For example, intraocular delivery of VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, MJ., et al (2004) Retina 24:132-138) and by subretinal injection in mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) has been shown to prevent neovascularization in experimental models of age-related macular degeneration. In addition, direct intratumoral injection of dsRNA in mice reduced tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and prolonged the survival of tumor-bearing mice (Kim, WJ., et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523).RNA interference can be delivered to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, PH., et al. (2005) Gene Ther. 12:59-66; Makimura, H., et al. (2002) BMC Neurosci. 3:18; Shishkina, GT., et al. (2004) Neuroscience 129:521-528; Thakker, ER., et al. (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594-602) and to the lung by intranasal administration (Howard, KA., et (2006) Mol. Ther. 14:476-484; Zhang, X., et al. (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al. (2005) Nat. Med. 11:50-55) have demonstrated successful localized delivery. To administer iRNA systemically to treat disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent rapid degradation of dsRNA by endogenous endo- and exonucleases. Modification of the RNA or pharmaceutical carrier can also enable targeting of iRNA compositions to target tissues, avoiding undesirable nonspecific effects. iRNA molecules can be modified by chemical attachment of lipophilic groups, such as cholesterol, to enhance cellular uptake and prevent degradation. For example, systemic injection of iRNAs directed against ApoB conjugated to lipophilic cholesterol moieties into mice resulted in apoB mRNA knockdown in both the liver and jejunum (Soutschek, J., et al. (2004) Nature 432:173-178). Conjugation of iRNAs to aptamers has been shown to suppress tumor growth and mediate tumor regression in mouse models of prostate cancer (McNamara, J.O., et al. (2006) Nat. Biotechnol. 24:1005-1015).In alternative embodiments, iRNAs can be delivered using drug delivery systems such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems facilitate the binding of iRNA molecules (which are negatively charged) and also enhance their interaction with the negatively charged cell membrane, allowing for efficient uptake of iRNA by cells. Cationic lipids, dendrimers, or polymers can be bound to iRNAs or induced to form vesicles or micelles that encapsulate iRNAs (see, for example, Kim SH., et al. (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents degradation of iRNAs upon systemic administration. Methods for making and administering cationic iRNA complexes are well within the capabilities of one of ordinary skill in the art (see, e.g., Sorensen, D.R., et al. (2003) J. Mol. Biol 327:761-766; Verma, U.N., et al. (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A.S. et al. (2007) J. Hypertens. 25:197-205, the contents of which are incorporated herein by reference in their entirety).Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA include DOTAP (Sorensen, D.R., et al. (2003), supra; Verma, U.N., et al. (2003), supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, T.S., et al. (2006) Nature 441:111-114), cardiolipin (Chien, P.Y., et al. (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al. (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M.E., et al. (2005) Int J. Oncol. 26:1087-1091), and PEG-1 (Polymerase Chain Receptor Blockers). al (2008) Pharm. Res. August 16, advance online publication; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamine (Tomalia, D. A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, for systemic administration, the iRNA is complexed with cyclodextrin. Methods and pharmaceutical compositions for administering iRNA and cyclodextrin are described in U.S. Patent No. 7,427,605, the entire contents of which are incorporated herein by reference. 【0395】 A. Vectors Encoding iRNAs of the Invention PCSK9 gene-targeting iRNAs can be expressed from transcription units inserted into DNA or RNA vectors (see, for example, Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International Publication No. WO 00 / 22113; Conrad, International Publication No. WO 00 / 22114; and Conrad, U.S. Patent No. 6,054,299). Expression can be transient (hours to weeks) or persistent (weeks to months or longer), depending on the specific construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrative or non-integrative vectors. Transgenes can also be constructed to allow them to be inherited as extrachromosomal plasmids (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292). 【0396】 Each iRNA strand or strands can be transcribed from the promoter on the expression vector.When expressing two separate strands to produce, for example, dsRNA, two separate expression vectors can be simultaneously introduced into target cells (for example, by transfection or infection).Alternatively, each of the promoters can be transcribed from the individual strands of dsRNA by being located on the same expression plasmid.In one embodiment, dsRNA is expressed as an inverted repeat polynucleotide that is linked by a linker polynucleotide sequence, so that dsRNA has a stem-loop structure. 【0397】 iRNA expression vectors are generally DNA plasmids or viral vectors. Recombinant constructs for expressing iRNAs described herein can be produced using expression vectors compatible with eukaryotic cells, preferably vertebrate cells. Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction enzyme recognition sites for inserting desired nucleic acid fragments. Delivery of iRNA expression vectors can be by systemic administration, such as intravenous or intramuscular administration, administration to target cells explanted from a patient and then reintroduced into the patient, or any other means that allows for introduction into desired target cells. 【0398】 iRNA expression plasmids can be transfected into target cells as complexes with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdown, targeting different regions of a target RNA over a period of one week or more, are also contemplated by the present invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be indicated by a reporter, such as a fluorescent marker like green fluorescent protein (GFP). Stable transfection of cells in vitro can be ensured using markers that confer resistance to specific environmental factors (e.g., antibiotics and drugs) on transfected cells, such as hygromycin B resistance. 【0399】 Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to, (a) adenoviral vectors; (b) retroviral vectors, including but not limited to lentiviral vectors, Moloney murine leukemia virus, and the like; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) poxvirus vectors, such as orthopox, e.g., vaccinia virus vectors, or avipox, e.g., canarypox or fowlpox; and (j) helper-dependent or gutless adenoviruses. Replication-defective viruses may also be advantageous. Different vectors may or may not integrate into the cellular genome. The constructs may contain viral sequences for transfection, if desired. Alternatively, the constructs may be incorporated into vectors capable of episomal replication, such as EPV and EBV vectors. Constructs for recombinant expression of iRNA generally require regulatory elements, such as promoters, enhancers, etc., to ensure iRNA expression in target cells. Other contemplated aspects of vectors and constructs are described in more detail below. 【0400】 Vectors useful for delivering iRNA contain sufficient regulatory elements (promoters, enhancers, etc.) for expression of the iRNA in the desired target cells or tissues. Regulatory elements can be selected to provide for either constitutive or regulated / inducible expression. 【0401】 The expression of iRNA can be precisely regulated using inducible regulatory sequences that are sensitive to specific physiological regulators, such as circulating glucose levels or hormones (Docherty et al., 1994, FASEB J. 8:20-24).Such inducible expression systems suitable for controlling dsRNA expression in cells or mammals include, for example, regulation by ecdysone, estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-β-D1-thiogalactopyranoside (IPTG).Those skilled in the art can select appropriate regulatory / promoter sequences based on the intended use of the iRNA transgene. 【0402】 Viral vectors containing nucleic acid sequences encoding iRNAs can be used. For example, retroviral vectors can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for correct packaging of the viral genome and integration into host cell DNA. The nucleic acid sequences encoding iRNAs are cloned into one or more vectors, which facilitates delivery of the nucleic acid to patients. More details regarding retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells, for example, to generate stem cells that are more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy include Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors that may be used include, for example, HIV-based vectors described in U.S. Patent No. 6,143,520; U.S. Patent No. 5,665,557; and U.S. Patent No. 5,981,276, which are incorporated herein by reference. 【0403】 Adenoviruses are also contemplated for use in delivering iRNAs of the present invention. Adenoviruses are particularly attractive vehicles for delivering genes to, for example, respiratory epithelia. Adenoviruses naturally infect respiratory epithelia, causing a mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993), present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994), demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other examples of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); WO 94 / 12649; and Wang et al., Gene Therapy 2:775-783 (1995). Suitable AV vectors for expressing iRNAs featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described in Xia H et al. (2002), Nat. Biotech. 20:1006-1010. 【0404】 Adeno-associated virus (AAV) vectors can also be used to deliver the iRNAs of the invention (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, the iRNAs can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector, e.g., with either the U6 or H1 RNA promoter, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described in Samulski R et al. (1987), J.Virol. 61:3096-3101; Fisher KJ et al. (1996), J.Virol, 70:520-532; Samulski R et al. (1989), J.Virol. 63:3822-3826; U.S. Patent No. 5,252,479; U.S. Patent No. 5,139,941; WO 94 / 13788; and WO 93 / 24641, the entire disclosures of which are incorporated herein by reference. 【0405】 Another viral vector suitable for delivering the iRNA of the invention is a vaccinia virus, e.g., an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, or a poxvirus, e.g., an avipox, e.g., fowlpox or canarypox. 【0406】 The tropism of viral vectors can be modified, if necessary, by pseudotyping the vector with envelope proteins or other surface antigens from other viruses, or by substituting capsid proteins from different viruses. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, etc. AAV vectors can be engineered to target different cells by expressing different capsid protein serotypes; see, for example, Rabinowitz JE et al. (2002), J Virol 76:791-801, the entire disclosure of which is incorporated herein by reference. 【0407】 The vector pharmaceutical preparation can include the vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system. 【0408】 V. PHARMACEUTICAL COMPOSITIONS OF THE INVENTION The present invention also includes pharmaceutical compositions and formulations that include the iRNAs of the present invention. In one embodiment, the present invention provides a pharmaceutical composition containing an iRNA described herein and a pharmaceutically acceptable carrier. 【0409】 The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are within the scope of sound medical judgment and suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, consistent with a reasonable benefit / risk ratio. 【0410】 The phrase "pharmaceutically acceptable carrier," as used herein, means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium, or zinc stearate, or stearic acid), or solvent encapsulating material, that is involved in carrying or transporting a compound of interest from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject being treated. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffers; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; and (24) other non-toxic affinity materials used in pharmaceutical formulations. 【0411】 Pharmaceutical compositions containing iRNA are useful for treating diseases or disorders associated with PCSK9 expression or activity, such as diseases or disorders that would benefit from a decrease in PCSK9 expression. Such pharmaceutical compositions are formulated based on the delivery method. One example is a composition formulated for systemic administration via parenteral delivery, for example, subcutaneous (SC), intramuscular (IM), or intravenous (IV) delivery. Another example is a composition formulated for direct delivery to brain parenchyma, for example, by injection into the brain, for example, by continuous pump infusion. The pharmaceutical compositions of the present invention may be administered at a dose sufficient to inhibit the expression of PCSK9 gene. 【0412】 Preferably, in the methods of the invention, the iRNA agent is administered to the subject as a fixed dose. In one particular embodiment, the fixed dose of the iRNA agent of the invention is based on a predetermined weight or age. 【0413】 In some embodiments, the RNAi agent is between about 200 mg and about 850 mg, between about 200 mg and about 500 mg, between about 200 mg and about 400 mg, between about 200 mg and about 300 mg, between about 100 mg and about 800 mg, between about 100 mg and about 750 mg, between about 100 mg and about 700 mg, between about 100 mg and about 650 mg, between about 100 mg and about 600 mg, between about 100 mg and about 550 mg, between about 100 mg and about 500 mg, between about 200 mg and about 850 mg, between about 200 mg and about 800 mg, between about 200 mg and about 750 mg, between about 200 mg and about 700 mg, between about 200 mg and about 650 mg, between about 200 mg and about 600 mg, It is administered as a fixed dose of between about 200 mg and about 500 mg, between about 300 mg and about 850 mg, between about 300 mg and about 800 mg, between about 300 mg and about 750 mg, between about 300 mg and about 700 mg, between about 300 mg and about 650 mg, between about 300 mg and about 600 mg, between about 300 mg and about 550 mg, between about 300 mg and about 500 mg, between about 400 mg and about 850 mg, between about 400 mg and about 800 mg, between about 400 mg and about 750 mg, between about 400 mg and about 700 mg, between about 400 mg and about 650 mg, between about 400 mg and about 600 mg, between about 400 mg and about 550 mg, or between about 400 mg and about 500 mg. 【0414】 In some embodiments, the RNAi agent is administered as a fixed dose of about 100 mg, about 125 mg, about 150 mg, about 175 mg, 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, or about 850 mg. 【0415】 In some embodiments, a therapeutic amount of iRNA is administered to a subject in multiple doses, for example, subcutaneously or intramuscularly. 【0416】 The iRNA may be formulated in a pharmaceutical composition at a suitable concentration such that a suitable volume of the composition is administered to a subject, such as about 1.0 ml, 1.1 ml, 1.2 ml, 1.3 ml, 1.4 ml, 1.5 ml, 1.6 ml, 1.7 ml, 1.8 ml, 1.9 ml, or about 2.0 ml of the pharmaceutical composition. For example, in one embodiment, an iRNA agent of the invention is formulated in a suitable pharmaceutical formulation at about 200 mg / ml such that administration of about 1.5 ml of the formulation to a subject provides a fixed dose of 300 mg of the agent. 【0417】 As described herein, a single dose of an iRNA agent or pharmaceutical composition containing such an agent can be long-lasting, such that subsequent doses are administered at weekly, biweekly, monthly, two-monthly, three-monthly, four-monthly, five-monthly, or six-monthly intervals or less. 【0418】 In some embodiments, a therapeutic amount of iRNA is administered to a subject in repeated doses, for example, subcutaneously or intramuscularly. Repeated administration regimens may include regular administration of a therapeutic amount of iRNA, for example, once a month, once every two months, once a quarter, once every four months, once every five months, or every six months. In some embodiments of the present invention, the pharmaceutical composition of the present invention is administered in a single dose once every quarter (qQ). In other embodiments of the present invention, the pharmaceutical composition of the present invention is administered in a single dose every six months (i.e., every six months). Administration can be repeated, for example, once a quarter for six months, one year, two years, or longer, for example, chronic administration. 【0419】 In some embodiments, the RNAi agent is administered in a dosing regimen that includes a loading phase followed by a maintenance phase. 【0420】 The loading stage may be, for example, about 100 mg to about 700 mg, about 150 mg to about 700 mg, about 200 mg to about 700 mg, about 250 mg to about 700 mg, about 300 mg to about 700 mg, about 350 mg to about 700 mg, about 400 mg to about 700 mg, about 450 mg to about 700 mg, about 500 mg to about 700 mg, about 550 mg to about 700 mg, about 600 to about 700 mg, about 650 to about 700 mg, about 100 mg to about 650 mg, about 150 mg to about 650 mg, about 200 mg to about 650 mg, about 250 mg to about 650 mg, about 300 mg to about 650 mg, approx. 350 mg ~ approx. 650 mg, approx. 400 mg ~ approx. 650 mg, approx. 450 mg ~ approx. 650 mg, approx. 500 mg ~ approx. 650 mg, approx. 550 mg ~ approx. 650 mg, approx. 600mg, about 250mg to about 600mg, about 300mg to about 600mg, about 350mg to about 600mg, about 400mg to about 600mg, about 450mg to about 600mg, about 500mg to about 600mg, about 550mg to about 600mg, about 100mg to about 550mg, about 15 A fixed dose of about 0 mg to about 550 mg, about 200 mg to about 550 mg, about 250 mg to about 550 mg, about 300 mg to about 550 mg, about 350 mg to about 550 mg, about 400 mg to about 550 mg, about 450 mg to about 550 mg, about 500 mg to about 550 mg, about 100 mg to about 500 mg, about 150 mg to about 500 mg, about 200 mg to about 500 mg, about 250 mg to about 500 mg, about 300 mg to about 500 mg, about 350 mg to about 500 mg, about 400 mg to about 500 mg, or about 450 mg to about 500 mg, for example, about 100 mg, about It may comprise a single administration of the RNAi agent during the first week, a single administration of the RNAi agent over the first two weeks, or a single administration of the RNAi agent during the first month at a fixed dose of 125 mg, about 150 mg, about 175 mg, 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, or about 700 mg.Values ​​and ranges intermediate to the above-listed values ​​are also intended to be part of this invention. 【0421】 The maintenance phase may involve administering a dose of an RNAi agent to a subject once a month, once every two months, once every three months, once every four months, once every five months, or once every six months. In one particular embodiment, the maintenance dose is administered to a subject once a month. 【0422】 The one or more maintenance doses may be the same or smaller than the initial dose, for example, half the initial dose. For example, the maintenance dose may be about 25 mg to about 100 mg, for example, about 25 mg to about 75 mg, about 25 mg to about 50 mg, or about 50 mg to about 75 mg, for example, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, or about 100 mg, administered to a subject monthly. Intermediate values ​​and ranges within the above-listed values ​​are also contemplated as part of the present invention. 【0423】 The pharmaceutical composition can be administered by intravenous infusion over a long period of time, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, or about 25 minutes. Administration can be repeated periodically, for example, weekly or every other week (i.e., every two weeks), for one, two, three, four months, or longer. Following an initial treatment regimen, treatment can be administered less frequently. For example, after three months of weekly or biweekly administration, administration can be repeated monthly for six months, one year, or longer. 【0424】 Those skilled in the art will understand that certain factors, including but not limited to, the severity of the disease or disorder, previous treatments, the subject's overall health and / or age, and other diseases present, may influence the dosage and timing required to effectively treat a subject. Moreover, treatment of a subject with a therapeutically effective amount of a composition may include a single treatment or a series of treatments. The effective dosage and in vivo half-life of the individual iRNAs encompassed by the present invention may be estimated using conventional procedures or based on in vivo studies using appropriate animal models, as described elsewhere herein. 【0425】 The pharmaceutical compositions of the present invention can be administered in several ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration can be topical (e.g., via a transdermal patch), pulmonary, for example, by inhalation or insufflation of powders or aerosols, including nebulizers; intratracheal, intranasal, transepidermal, transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subdermal administration, for example, via an implanted device; or intracranial administration, for example, intracerebral parenchyma, intrathecal, or intraventricular. 【0426】 The iRNA can be delivered in a manner that targets a specific tissue, such as the liver (e.g., liver parenchymal cells). 【0427】 Pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may be necessary or desirable. Coated condoms, gloves, and the like may also be useful. Suitable topical formulations include those in which the iRNA featured in the present invention is in admixture with a topical delivery agent, such as a lipid, liposome, fatty acid, fatty acid ester, steroid, chelating agent, or surfactant. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearolyphosphatidylcholine), anionic (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The iRNA featured in the present invention can be encapsulated in or complexed with liposomes, particularly cationic liposomes. Alternatively, the iRNA can be complexed with lipids, particularly cationic lipids. Suitable fatty acids and esters include arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C 1~20 Topical formulations include, but are not limited to, alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Topical formulations are described in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference. 【0428】 Compositions and preparations for oral administration include powder or granule, microparticle, nanoparticle, suspension or solution in water or non-aqueous medium, capsule, gel capsule, sachet, tablet or mini-tablet.Thickener, flavoring agent, diluent, emulsifier, dispersing aid or binder may be required.In some embodiments, oral preparations are those in which the DsRNA of the present invention is administered in combination with one or more penetration-promoting surfactants and chelating agents.Suitable surfactants include fatty acid and / or ester or their salt, bile acid and / or their salt. Suitable bile acids / salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glycolic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or monoglyceride, diglyceride, or pharmaceutically acceptable salts thereof (e.g., sodium). In some embodiments, a combination of penetration enhancers is used, such as fatty acid / salts combined with bile acids / salts. One exemplary combination is the sodium salt of lauric acid, capric acid, and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. The DsRNA featured in the present invention can be orally delivered in granular form, including spray-dried particles, or can be complexed to form micro- or nanoparticles.DsRNA complexing agents include polyamino acids, polyimines, polyacrylates, polyalkyl acrylates, polyoxetanes, polyalkylcyanoacrylates, cationized gelatin, albumin, starch, acrylates, polyethylene glycol (PEG) and starch, polyalkylcyanoacrylates, DEAE-derivatized polyimines, pullulans, cellulose, and starch.Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate. acrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid) (PLGA), alginate, and polyethylene glycol (PEG). Oral formulations of dsRNA and their preparation are described in detail in U.S. Pat. No. 6,887,906, U.S. Patent Application Publication No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference. 【0429】 Compositions and formulations for parenteral, intraparenchymal (intracerebral), intrathecal, intraventricular, or intrahepatic administration can include sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives, including, but not limited to, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients. 【0430】 Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. When treating liver disorders, such as liver cancer, liver-targeted formulations are particularly preferred. 【0431】 The pharmaceutical preparation of the present invention, which can be conveniently presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include combining active ingredients with pharmaceutical carriers or excipients. Generally, the preparation is prepared by uniformly and intimately combining active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. 【0432】 The compositions of the present invention can be formulated into any of a number of possible dosage forms, including, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions can further contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension can also contain stabilizers. 【0433】 C. Additional Formulations i. Emulsion The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another liquid in the form of droplets, usually greater than 0.1 μm in diameter (e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 199; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and (See Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 2, p. 335; Higuchi et al., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed within one another. In general, emulsions can be either water-in-oil (w / o) or oil-in-water (o / w). When the aqueous phase is finely dispersed and dispersed as minute droplets within the bulk oily phase, the resulting composition is referred to as a water-in-oil (w / o) emulsion. Alternatively, when the oily phase is finely dispersed and dispersed as minute droplets within the bulk aqueous phase, the resulting composition is referred to as an oil-in-water (o / w) emulsion.In addition to the dispersed phase and the active agent, which may be present as a solution in either the aqueous or oily phase or as a separate phase, emulsions may contain additional components. Pharmaceutical excipients, such as emulsifiers, stabilizers, dyes, and antioxidants, may also be present in the emulsion as needed. Pharmaceutical emulsions may also be multiple emulsions comprising more than two phases, such as oil-in-water-in-oil (o / w / o) and water-in-oil-in-water (w / o / w) emulsions. Such complex formulations often offer certain advantages not offered by simple binary emulsions. Multiple emulsions in which individual oil droplets of an o / w emulsion surround small water droplets constitute w / o / w emulsions. Similarly, oil droplet systems encapsulated in globules of water and stabilized within an oily continuous phase provide o / w / o emulsions. 【0434】 Emulsions are characterized by little or no thermodynamic stability. Frequently, the dispersed or discontinuous phase of an emulsion is well dispersed within the external or continuous phase and is maintained in this form through the use of emulsifiers or formulation viscosity. Either of the emulsion phases can be semi-solid or solid, as in the case of emulsion-type ointment bases and creams. Another means of stabilizing emulsions involves the use of emulsifiers, which can be incorporated into either of the emulsion phases. Emulsifiers can be broadly classified into four categories: synthetic surfactants, natural emulsifiers, absorption bases, and finely dispersed solids (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). 【0435】 Synthetic surfactants, also known as surface active agents, have a wide range of uses in emulsion formulations and have been reviewed in literature (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.285; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, NY, 1988, volume 1, p.199).Surfactants are typically amphiphilic and comprise hydrophilic and hydrophobic moieties. The ratio of hydrophilicity to hydrophobicity is called the hydrophilic / lipophilic balance (HLB) of surfactant, and is a useful tool for classifying and selecting surfactant in the preparation of formulation.Surfactant can be classified into different classes based on the nature of hydrophilic group: nonionic, anionic, cationic and amphoteric (see for example Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY Rieger, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.285). 【0436】 Natural emulsifiers used in emulsion formulations include lanolin, beeswax, phospholipids, lecithin, and acacia. Absorbent bases with hydrophilic properties, such as anhydrous lanolin and hydrophilic petrolatum, can absorb water to form water-in-oil emulsions while still maintaining their semi-solid consistency. Finely dispersed solids are also used as excellent emulsifiers in viscous preparations, especially in combination with surfactants. These include polar inorganic solids such as heavy metal hydroxides, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments, and non-polar solids such as carbon or glyceryl tristearate. 【0437】 A wide variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of the emulsion, including fats, oils, waxes, fatty acids, fatty alcohols, fatty acid esters, humectants, hydrophilic colloids, preservatives, and antioxidants (Block, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 335; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199...

Claims

[Claim 1] A pharmaceutical composition for subcutaneous administration for use in a method of treating hypercholesterolemia in human subjects, wherein the composition comprises a constant dose of 275 mg to 325 mg of a double-stranded ribonucleic acid (RNAi) agent or a salt thereof. Here, the administration of an initial fixed dose of 275 mg-325 mg of the double-stranded RNAi agent or a salt thereof resulted in at least a 30% reduction in the level of low-density lipoprotein cholesterol (LDLc) from baseline in the human subjects 84 days after the initial fixed dose administration. Here, the double-stranded RNAi agent or a salt thereof comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising the nucleotide sequence 5'-csusagacCfuGfudTuugcuuuugu-3' (SEQ ID NO: 687), and the antisense strand comprising the nucleotide sequence 5'-asCfsaAfAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 688), Here, a, g, c, and u are 2'-O-methyl(2'-OMe)A, G, C, and U, respectively; Af, Gf, Cf, and Uf are 2'-fluoroA, G, C, and U, respectively; dT is 2'-deoxythymidine; and s is a phosphorothioate bond, and Here, the N-acetylgalactosamine (GalNAc) 3 ligand is shown in the following schematic diagram. 【Chemistry 1】 A pharmaceutical composition in which a bond is attached to the 3' end of the sense chain as shown in the formula, where X is O. [Claim 2] The pharmaceutical composition for use according to claim 1, wherein administration of the double-stranded RNAi agent or a salt thereof in an initial fixed dose of 275 mg to 325 mg results in at least a 40% reduction in the level of LDLc in the human subject from baseline 84 days after the initial fixed dose administration. [Claim 3] The pharmaceutical composition for use according to claim 1, wherein administration of the double-stranded RNAi agent or a salt thereof in an initial fixed dose of 275 mg to 325 mg results in at least a 50% reduction in the level of LDLc in the human subject from baseline 84 days after the initial fixed dose administration. [Claim 4] The pharmaceutical composition for use according to claim 1, wherein, 84 days after administration of the initial fixed dose of the double-stranded RNAi agent or a salt thereof in 275 mg to 325 mg, the LDLc level in the human subject is reduced by at least 30% from baseline, and the administration of the fixed dose of the double-stranded RNAi agent or a salt thereof in 275 mg to 325 mg is repeated. [Claim 5] The pharmaceutical composition for use according to claim 4, wherein a fixed dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof is administered repeatedly three months after the initial administration. [Claim 6] The pharmaceutical composition for use according to claim 4, wherein the administration of a fixed dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof is repeated every six months. [Claim 7] The aforementioned method, (a) A step of evaluating the level of LDLc in the human subject after administering the initial fixed dose of the double-stranded RNAi agent or a salt thereof, ranging from 275 mg to 325 mg; (b) a step of comparing the level of LDLc evaluated in step (a) with the baseline LDLc level of the human subject; and (c) A step of determining that the level of LDLc in the human subject has decreased by at least 30%. A pharmaceutical composition for use according to claim 1, further comprising: [Claim 8] The aforementioned method, (d) The step of repeating the administration of a constant dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof after step (c). A pharmaceutical composition for use according to claim 7, further comprising: [Claim 9] The pharmaceutical composition for use according to claim 8, wherein the administration of a fixed dose of the double-stranded RNAi agent or a salt thereof in a constant dose of 275 mg to 325 mg is repeated three months after the initial fixed dose administration. [Claim 10] The pharmaceutical composition for use according to claim 8, wherein the administration of a fixed dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof is repeated every six months. [Claim 11] The aforementioned method, (a) A step of evaluating the level of LDLc in the human subject after administering the initial fixed dose of the double-stranded RNAi agent or a salt thereof, ranging from 275 mg to 325 mg; (b) a step of comparing the level of LDLc evaluated in step (a) with the baseline LDLc level of the human subject; and (c) A step of determining that the level of LDLc in the human subject has decreased by at least 40%. A pharmaceutical composition for use according to claim 1, further comprising: [Claim 12] The aforementioned method, (d) The step of repeating the administration of a constant dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof after step (c). A pharmaceutical composition for use according to claim 11, further comprising: [Claim 13] The pharmaceutical composition for use according to claim 12, wherein the administration of a fixed dose of the double-stranded RNAi agent or a salt thereof in a constant dose of 275 mg to 325 mg is repeated three months after the initial fixed dose administration. [Claim 14] The pharmaceutical composition for use according to claim 12, wherein the administration of a fixed dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof is repeated every six months. [Claim 15] The aforementioned method, (a) A step of evaluating the level of LDLc in the human subject after administering the initial fixed dose of the double-stranded RNAi agent or a salt thereof, ranging from 275 mg to 325 mg; (b) a step of comparing the level of LDLc evaluated in step (a) with the baseline LDLc level of the human subject; and (c) A step of determining that the level of LDLc in the human subject has decreased by at least 50%. A pharmaceutical composition for use according to claim 1, further comprising: [Claim 16] The aforementioned method, (d) The step of repeating the administration of a constant dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof after step (c). A pharmaceutical composition for use according to claim 15, further comprising: [Claim 17] The pharmaceutical composition for use according to claim 16, wherein the administration of a fixed dose of the double-stranded RNAi agent or a salt thereof in a constant dose of 275 mg to 325 mg is repeated three months after the initial fixed dose administration. [Claim 18] The pharmaceutical composition for use according to claim 16, wherein the administration of a fixed dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof is repeated every six months. [Claim 19] The aforementioned method, (a) A step of evaluating the level of LDLc in the human subject after repeated administration of a constant dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof; (b) a step of comparing the level of LDLc evaluated in step (a) with the baseline LDLc level of the human subject; and (c) A step of determining that the level of LDLc in the human subject has decreased by at least 30%. A pharmaceutical composition for use according to claim 4, further comprising: [Claim 20] The aforementioned method, (d) The step of repeating the administration of a constant dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof after step (c). A pharmaceutical composition for use according to claim 19, further comprising: [Claim 21] The pharmaceutical composition for use according to claim 20, wherein in step (d), the administration of a fixed dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof is repeated 6 months after the repeated administration described in step (a). [Claim 22] The aforementioned method, (a) A step of evaluating the level of LDLc in the human subject after repeated administration of a constant dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof; (b) a step of comparing the level of LDLc evaluated in step (a) with the baseline LDLc level of the human subject; and (c) A step of determining that the level of LDLc in the human subject has decreased by at least 40%. A pharmaceutical composition for use according to claim 4, further comprising: [Claim 23] The aforementioned method, (d) The step of repeating the administration of a constant dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof after step (c). A pharmaceutical composition for use according to claim 22, further comprising: [Claim 24] The pharmaceutical composition for use according to claim 23, wherein in step (d), the administration of a fixed dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof is repeated 6 months after the repeated administration described in step (a). [Claim 25] The aforementioned method, (a) A step of evaluating the level of LDLc in the human subject after repeated administration of a constant dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof; (b) a step of comparing the level of LDLc evaluated in step (a) with the baseline LDLc level of the human subject; and (c) A step of determining that the level of LDLc in the human subject has decreased by at least 50%. A pharmaceutical composition for use according to claim 4, further comprising: [Claim 26] The aforementioned method, (d) The step of repeating the administration of a constant dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof after step (c). A pharmaceutical composition for use according to claim 25, further comprising: [Claim 27] The pharmaceutical composition for use according to claim 26, wherein in step (d), the administration of a constant dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof is repeated 6 months after the repeated administration described in step (a). [Claim 28] The pharmaceutical composition for use according to claim 2, wherein, 84 days after administration of the initial fixed dose of the double-stranded RNAi agent or a salt thereof in 275 mg to 325 mg, the LDLc level in the human subject is reduced by at least 40% from baseline, and the administration of the fixed dose of the double-stranded RNAi agent or a salt thereof in 275 mg to 325 mg is repeated. [Claim 29] The pharmaceutical composition for use according to claim 28, wherein a fixed dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof is administered repeatedly three months after the initial administration. [Claim 30] The pharmaceutical composition for use according to claim 28, wherein the administration of a fixed dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof is repeated every six months. [Claim 31] The aforementioned method, (a) A step of evaluating the level of LDLc in the human subject after repeated administration of a constant dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof; (b) a step of comparing the level of LDLc evaluated in step (a) with the baseline LDLc level of the human subject; and (c) A step of determining that the level of LDLc in the human subject has decreased by at least 40%. A pharmaceutical composition for use according to claim 28, further comprising: [Claim 32] The aforementioned method, (d) The step of repeating the administration of a constant dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof after step (c). A pharmaceutical composition for use according to claim 31, further comprising: [Claim 33] The pharmaceutical composition for use according to claim 32, wherein in step (d), the administration of a constant dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof is repeated 6 months after the repeated administration described in step (a). [Claim 34] The pharmaceutical composition for use according to claim 3, wherein, 84 days after administration of the initial fixed dose of the double-stranded RNAi agent or a salt thereof in 275 mg to 325 mg, the LDLc level in the human subject is reduced by at least 50% from baseline, and the administration of the fixed dose of the double-stranded RNAi agent or a salt thereof in 275 mg to 325 mg is repeated. [Claim 35] The pharmaceutical composition for use according to claim 34, wherein a fixed dose of the double-stranded RNAi agent or a salt thereof, ranging from 275 mg to 325 mg, is administered repeatedly three months after the initial administration. [Claim 36] The pharmaceutical composition for use according to claim 34, wherein the administration of a fixed dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof is repeated every six months. [Claim 37] The aforementioned method, (a) A step of evaluating the level of LDLc in the human subject after repeated administration of a constant dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof; (b) a step of comparing the level of LDLc evaluated in step (a) with the baseline LDLc level of the human subject; and (c) A step of determining that the level of LDLc in the human subject has decreased by at least 50%. A pharmaceutical composition for use according to claim 34, further comprising: [Claim 38] The aforementioned method, (d) The step of repeating the administration of a constant dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof after step (c). A pharmaceutical composition for use according to claim 37, further comprising: [Claim 39] The pharmaceutical composition for use according to claim 37, wherein in step (d), the administration of a constant dose of 275 mg to 325 mg of the double-stranded RNAi agent or a salt thereof is repeated six months after the repeated administration described in step (a). [Claim 40] The pharmaceutical composition for use according to claim 1, wherein the fixed dose administered to the human subject is 300 mg. [Claim 41] The pharmaceutical composition for use according to claim 40, wherein administration of 300 mg of the initial fixed dose of the double-stranded RNAi agent or a salt thereof results in at least a 40% reduction in the level of LDLc in the human subject from baseline 84 days after the initial fixed dose administration. [Claim 42] The pharmaceutical composition for use according to claim 40, wherein administration of 300 mg of the initial fixed dose of the double-stranded RNAi agent or a salt thereof results in at least a 50% reduction in the level of LDLc in the human subject from baseline 84 days after the initial fixed dose administration. [Claim 43] The aforementioned method, (a) A step of evaluating the level of LDLc in the human subject after administering an initial fixed dose of 300 mg of the double-stranded RNAi agent or a salt thereof; (b) a step of comparing the level of LDLc evaluated in step (a) with the baseline LDLc level of the human subject; and (c) A step of determining that the level of LDLc in the human subject has decreased by at least 30%. A pharmaceutical composition for use according to claim 40, further comprising: [Claim 44] The aforementioned method, (d) The step of repeating the administration of a fixed dose of 300 mg of the double-stranded RNAi agent or a salt thereof after step (c). A pharmaceutical composition for use according to claim 43, further comprising: [Claim 45] The pharmaceutical composition for use according to claim 44, wherein the administration of a fixed dose of 300 mg of the double-stranded RNAi agent or a salt thereof is repeated three months after the initial fixed dose administration. [Claim 46] The pharmaceutical composition for use according to claim 44, wherein the administration of a fixed dose of 300 mg of the double-stranded RNAi agent or a salt thereof is repeated every six months. [Claim 47] The aforementioned method, (a) A step of evaluating the level of LDLc in the human subject after administering an initial fixed dose of 300 mg of the double-stranded RNAi agent or a salt thereof; (b) a step of comparing the level of LDLc evaluated in step (a) with the baseline LDLc level of the human subject; and (c) A step of determining that the level of LDLc in the human subject has decreased by at least 40%. A pharmaceutical composition for use according to claim 40, further comprising: [Claim 48] The aforementioned method, (d) The step of repeating the administration of a fixed dose of 300 mg of the double-stranded RNAi agent or a salt thereof after step (c). A pharmaceutical composition for use according to claim 47, further comprising: [Claim 49] The pharmaceutical composition for use according to claim 48, wherein the administration of a fixed dose of 300 mg of the double-stranded RNAi agent or a salt thereof is repeated three months after the initial fixed dose administration. [Claim 50] The pharmaceutical composition for use according to claim 48, wherein the administration of a fixed dose of 300 mg of the double-stranded RNAi agent or a salt thereof is repeated every six months. [Claim 51] The aforementioned method, (a) A step of evaluating the level of LDLc in the human subject after administering an initial fixed dose of 300 mg of the double-stranded RNAi agent or a salt thereof; (b) a step of comparing the level of LDLc evaluated in step (a) with the baseline LDLc level of the human subject; and (c) A step of determining that the level of LDLc in the human subject has decreased by at least 50%. A pharmaceutical composition for use according to claim 40, further comprising: [Claim 52] The aforementioned method, (d) The step of repeating the administration of a fixed dose of 300 mg of the double-stranded RNAi agent or a salt thereof after step (c). A pharmaceutical composition for use according to claim 51, further comprising: [Claim 53] The pharmaceutical composition for use according to claim 52, wherein the administration of a fixed dose of 300 mg of the double-stranded RNAi agent or a salt thereof is repeated three months after the initial fixed dose administration. [Claim 54] The pharmaceutical composition for use according to claim 52, wherein the administration of a fixed dose of 300 mg of the double-stranded RNAi agent or a salt thereof is repeated every six months. [Claim 55] The pharmaceutical composition according to claim 1, further comprising the step of administering an additional therapeutic agent to the human subject. [Claim 56] The pharmaceutical composition according to claim 55, wherein the additional therapeutic agent is a statin.

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