Apolipoprotein C3 (APOC3) iRNA compositions and methods of use thereof

Abstract

To provide regulators of APOC3 expression for treating apolipoprotein C3 associated disorders, such as hypertriglyceridemia.SOLUTION: A double stranded RNAi agent for inhibiting expression of apolipoprotein C3 (APOC3) in a cell is provided which comprises a sense strand and an antisense strand that form a double-stranded region. The sense strand comprises at least 15 contiguous nucleotides differing by 3 or less nucleotides from a specific nucleotide sequence, and the antisense strand comprises at least 15 contiguous nucleotides differing by 3 or less nucleotides from another specific nucleotide sequence. Substantially all of the nucleotides of at least one strand are modified nucleotides. The sense strand is conjugated to a ligand attached at the 3'-terminus.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 080,941, filed November 17, 2014, and U.S. Provisional Patent Application No. 62 / 136,159, filed March 20, 2015, the entire contents of each of the foregoing applications being hereby incorporated by reference herein.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety. Said ASCII copy, created on November 17, 2015, is named 121301-02520_SL.txt and is 212,085 bytes in size. [Background technology]

[0003] Apolipoprotein C3 (APOC3) is a very low-density lipoprotein (VLDL) and a key regulator of lipoprotein metabolism. In humans, APOC2 is encoded by the APOC3 gene, which is located in a gene cluster with the APOA1 and APOA4 genes on the long arm of chromosome 11. APOC3 is expressed as a small 99-amino acid protein in the liver and, to a lesser extent, in the intestine. Removal of the 20-amino acid signal peptide in the endoplasmic reticulum results in the formation of the 79-amino acid mature ApoC3 protein, which can exist as either nonglycosylated or glycosylated isoforms.

[0004] The primary role of APOC3 is as a regulator of lipolysis through noncompetitive inhibition of endothelial-bound lipoprotein lipase (LPL). LPL hydrolyzes triacylglycerols in triacylglycerol-rich lipoproteins (TRL), liberating fatty acids into plasma and converting large triacylglycerol-rich particles into small triacylglycerol-depleted remnant lipoproteins. APOC3-deficient individuals have low TRL levels, accompanied by highly efficient lipolysis of triacylglycerols. Furthermore, mice genetically deleted for the APOC3 gene have been shown to have low plasma triacylglycerol levels and efficient TRL catabolism. APOC3 also inhibits hepatic lipase (HL), a lipolytic enzyme with triacylglycerol lipase and phospholipase A1 activity synthesized in the liver. The HL-inhibitory effect of APOC3 further reduces lipolysis and TRL remnant uptake by the liver. APOC3 has also been shown to stimulate the synthesis of very low-density lipoprotein (VLDL). The underlying mechanism associated with this APOC3 effect may involve inhibition of proteasome-mediated APOB degradation, leading to increased APOB synthesis and secretion, as well as increased synthesis of VLDL triacylglycerol. APOC3 may therefore play an important role in regulating hepatic VLDL production.

[0005] Cellular studies have shown that APOC3 can interfere with TRL and remnant binding to hepatic lipoprotein receptors. APOC3 can abolish APOB- and ApoE-mediated lipoprotein binding to the low-density lipoprotein receptor (LDLR) by either shielding APOB and APOE or altering their conformation. Chylomicron and VLDL particle binding to the lipolysis-stimulating receptor (LSR) is also significantly inhibited by APOC3.

[0006] Increased APOC3 levels lead to the development of hypertriglyceridemia, i.e., high (hyper-) triglyceride blood levels (-emia). Elevated triglyceride levels are associated with various diseases, including cardiovascular disease, atherosclerosis, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, polycystic ovary syndrome, kidney disease, obesity, type 2 diabetes mellitus (insulin resistance), hypertension, and skin lesions (xanthomas). Extremely high triglyceride levels also increase the risk of acute pancreatitis. Therefore, modulating APOC3 metabolism may be an important novel therapeutic approach for managing hypertriglyceridemia and related diseases. Summary of the Invention [Problem to be solved by the invention]

[0007] Thus, there is a need in the art for modulators of APOC3 expression to treat apolipoprotein C3-related disorders, such as hypertriglyceridemia. [Means for solving the problem]

[0008] The present invention provides iRNA compositions that inhibit or reduce expression of the APOC3 gene, which can be in a cell, for example, a cell within the body of a subject, such as a human.

[0009] The present invention also provides methods and therapies for treating subjects with disorders that may benefit from inhibiting or reducing expression of the APOC3 gene, such as apolipoprotein C3-related diseases, such as hypertriglyceridemia, using iRNA compositions that inhibit or reduce expression of the APOC3 gene.

[0010] In some embodiments, the present invention provides a double-stranded RNAi agent for inhibiting expression of apolipoprotein C3 (APOC3) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2; Substantially all of the nucleotides of at least one strand are modified nucleotides, and the sense strand is conjugated with a ligand attached to the 3' end.

[0011] In certain embodiments, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.In one embodiment, the sense strand and the antisense strand comprise a complementary region that comprises at least 15 consecutive nucleotides, and differs from any one of the sequences listed in Table 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12 and 13 by 3 nucleotides or less.

[0012] In some embodiments, at least one of the modified nucleotides is a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally fixed nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl (hydroxly) modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide comprising a non-natural base, a tetrahydropyran modified nucleotide, a 1,5- The nucleotide is selected from the group consisting of an anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide containing a 5'-phosphorothioate group, a nucleotide containing a 5'-methylphosphonate group, a nucleotide containing a 5' phosphate or a 5' phosphate mimic, a nucleotide containing a vinyl phosphate, a nucleotide containing an adenosine-glycol nucleic acid (GNA), a nucleotide containing a thymidine-glycol nucleic acid (GNA) S-isomer, a nucleotide containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, a nucleotide containing 2'-deoxythymidine-3' phosphate, a nucleotide containing 2'-deoxyguanosine-3'-phosphate, and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.

[0013] In one embodiment, substantially all of the nucleotides in the sense strand are modified. In another embodiment, substantially all of the nucleotides in the antisense strand are modified. 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. 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.

[0014] In one embodiment, at least one strand comprises a 3' overhang of at least 1 nucleotide, hi another embodiment, at least one strand comprises a 3' overhang of at least 2 nucleotides.

[0015] In some embodiments, the present invention provides a double-stranded RNAi agent capable of inhibiting expression of apolipoprotein C3 (APOC3) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising a region complementary to a portion of an mRNA encoding APOC3, each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent having a structure represented by formula (III): Sense: 5'np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq3' Antisense: 3'np'-Na'-(X'X'X')k-Nb'-Y'Y'Y'-Nb'-(Z'Z'Z')l-Na'-nq'5'(III) [In formula: i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6; each Na and Na' independently represents an oligonucleotide sequence containing 0-25 nucleotides that are either modified or unmodified or a combination thereof, each sequence containing nucleotides of at least two different modifications; each Nb and Nb' independently represents an oligonucleotide sequence comprising 0 to 10 nucleotides that are either modified or unmodified or a combination thereof; each np, np', nq, and nq' (each of which may be present or absent) independently represents an overhanging nucleotide; 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; the modification on Nb is different from the modification on Y, and the modification on Nb' is different from the modification on Y'; and The sense strand is conjugated to at least one ligand.

[0016] In a further embodiment, i is 0; j is 0; i is 1; j is 1; i and j are both 0; or i and j are both 1. In another further embodiment, k is 0; l is 0; k is 1; l is 1; k and l are both 0; or k and l are both 1. In another embodiment, the YYY motif is present at or near the cleavage site of the sense strand. In yet another embodiment, the Y'Y'Y' motif is present at positions 11, 12, and 13 from the 5' end of the antisense strand.

[0017] In one embodiment, Y' is 2'-O-methyl or 2'-fluoro.

[0018] In some embodiments, Formula (III) has Formula (IIIa): Sense: 5'np-Na-YYY-Na-nq3' Antisense: 3'np'-Na'-Y'Y'Y'-Na'-nq'5' (IIIa) is expressed by

[0019] In a further embodiment, the double-stranded region is 15-30 nucleotide pairs in length. In another embodiment, the double-stranded region is 17-23 nucleotide pairs in length. In another embodiment, the double-stranded region is 17-25 nucleotide pairs in length. In yet another embodiment, the double-stranded region is 23-27 nucleotide pairs in length. In a further embodiment, the double-stranded region is 19-21 nucleotide pairs in length. In yet another embodiment, the double-stranded region is 21-23 nucleotide pairs in length.

[0020] In one embodiment, each strand has 15 to 30 nucleotides. In a further embodiment, each strand has 19 to 30 nucleotides.

[0021] In one embodiment, the modification on the nucleotide is selected from the group consisting of modifications as listed in Tables 5, 9, 10, 11B, 12, 13, and combinations thereof.

[0022] In some embodiments, the modifications on the nucleotide are 2'-O-methyl and 2'-fluoro modifications.

[0023] In some embodiments, the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker. In further embodiments, the ligand is [ka] is.

[0024] In some embodiments, the ligand is attached to the 3' end of the sense strand.

[0025] In certain embodiments, the RNAi agent is represented by the following schematic diagram: [ka] wherein X is O or S.

[0026] In some embodiments, the RNAi agent further comprises at least one phosphorothioate or methylphosphonate internucleotide bond.In a further embodiment, the phosphorothioate or methylphosphonate internucleotide bond is at the 3'-end of one strand.In another further embodiment, this strand is an antisense strand.In yet another embodiment, this strand is a sense strand.

[0027] In some embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5'-end of one strand. In a further embodiment, this strand is the antisense strand. In another further embodiment, this strand is the sense strand.

[0028] In certain embodiments, the phosphorothioate or methylphosphonate internucleotide linkages are at both the 5' and 3' ends of one strand, hi one embodiment, this strand is the antisense strand.

[0029] In some embodiments, the RNAi agent comprises 6 to 8 phosphorothioate internucleotide linkages. In further embodiments, 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.

[0030] In some embodiments, the base pair at position 1 of the 5' end of the antisense strand of the duplex is an AU base pair.

[0031] In some embodiments, the Y nucleotide contains a 2'-fluoro modification. In further embodiments, the Y' nucleotide contains a 2'-O-methyl modification.

[0032] In some embodiments, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0033] In some embodiments, the RNAi agent is selected from the group of RNAi agents listed in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, and 12.

[0034] In certain embodiments, the present invention also provides a double-stranded RNAi agent capable of inhibiting expression of apolipoprotein C3 (APOC3) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprises 5'-GCUUAAAAGGGACAGUAUUCU-3' (SEQ ID NO: 13) and the antisense strand comprises 5'-AGAAUACUGUCCCUUUUAAGCAA-3' (SEQ ID NO: 14), substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached to the 3' end, and The ligands are one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

[0035] In another embodiment, the present invention also provides a double-stranded RNAi agent capable of inhibiting expression of apolipoprotein C3 (APOC3) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-GCUUAAAAGGGACAGUAUUCU-3' (SEQ ID NO: 13), and the antisense strand comprises 5'-UGAAUACUGUCCCUUUUAAGCAA-3' (SEQ ID NO: 15), substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached to the 3' end, and The ligands are one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

[0036] In certain embodiments, the present invention also provides a double-stranded RNAi agent capable of inhibiting expression of apolipoprotein C3 (APOC3) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprises 5'-GCUUAAAAGGGACAGUAUUCA-3' (SEQ ID NO: 659) and the antisense strand comprises 5'-UGAAUACUGUCCCUUUUAAGCAA-3' (SEQ ID NO: 670); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached to the 3' end, and The ligands are one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

[0037] In one embodiment, all of the nucleotides in the sense strand are modified nucleotides. In one embodiment, all of the nucleotides in the antisense strand are modified nucleotides. In another embodiment, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand are modified nucleotides.

[0038] In further embodiments, at least one of the modified nucleotides is a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally fixed nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl (hydroxly) modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran modified nucleotide, a 1,5- The nucleotide is selected from the group consisting of an anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide containing a 5'-phosphorothioate group, a nucleotide containing a 5'-methylphosphonate group, a nucleotide containing a 5' phosphate or a 5' phosphate mimic, a nucleotide containing a vinyl phosphate, a nucleotide containing an adenosine-glycol nucleic acid (GNA), a nucleotide containing a thymidine-glycol nucleic acid (GNA) S-isomer, a nucleotide containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, a nucleotide containing 2'-deoxythymidine-3' phosphate, a nucleotide containing 2'-deoxyguanosine-3'-phosphate, and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.

[0039] In one embodiment, the RNAi agent comprises 10 or fewer nucleotides that comprise a 2'-fluoro modification. In another embodiment, the RNAi agent comprises 9 or fewer nucleotides that comprise a 2'-fluoro modification. In another embodiment, the RNAi agent comprises 8 or fewer nucleotides that comprise a 2'-fluoro modification. In another embodiment, the RNAi agent comprises 7 or fewer nucleotides that comprise a 2'-fluoro modification. In another embodiment, the RNAi agent comprises 6 or fewer nucleotides that comprise a 2'-fluoro modification. In another embodiment, the RNAi agent comprises 5 or fewer nucleotides that comprise a 2'-fluoro modification. In yet another embodiment, the sense strand comprises 4 or fewer nucleotides that comprise a 2'-fluoro modification. In another embodiment, the sense strand comprises 4 or fewer nucleotides that comprise a 2'-fluoro modification. In another embodiment, the sense strand comprises 3 or fewer nucleotides that comprise a 2'-fluoro modification. In another embodiment, the sense strand comprises 2 or fewer nucleotides that comprise a 2'-fluoro modification. In another embodiment, the sense strand comprises 2 or fewer nucleotides that comprise a 2'-fluoro modification. In another aspect, the antisense strand comprises 6 or fewer nucleotides that comprise a 2'-fluoro modification. In another embodiment, the antisense strand comprises 5 or fewer nucleotides that comprise a 2'-fluoro modification. In another embodiment, the antisense strand comprises no more than four nucleotides comprising a 2'-fluoro modification. In another embodiment, the antisense strand comprises no more than three nucleotides comprising a 2'-fluoro modification. In yet another aspect, the antisense strand comprises no more than two nucleotides comprising a 2'-fluoro modification.

[0040] In one embodiment, the double-stranded RNAi agent of the present invention further comprises a 5'-phosphate or a 5'-phosphate mimic at the 5' nucleotide of the antisense strand.In another embodiment, the double-stranded RNAi agent further comprises a 5'-phosphate mimic at the 5' nucleotide of the antisense strand.In a specific embodiment, the 5'-phosphate mimic is 5'-vinyl phosphate (5'-VP).

[0041] In certain embodiments, the ligand is [ka] is.

[0042] In some embodiments, the RNAi agent is shown in the following schematic diagram: [ka] wherein X is O or S.

[0043] In some embodiments, the present invention provides a double-stranded RNAi agent comprising an RNAi sequence listed in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13.

[0044] In one embodiment, the RNAi agent has the following sequence: Sense: 5'GfscsUfuAfaAfaGfGfGfaCfaGfuAfuUfcUfL96 3' (SEQ ID NO: 16) Antisense: AD-57553 containing 5'asGfsaAfuAfcUfgUfcccUfuUfuAfaGfcsAfsa 3' (SEQ ID NO: 17).

[0045] In another embodiment, the RNAi agent has the following sequence: Sense: 5'GfscsUfuAfaAfaGfGfGfaCfaGfuAfuUfcUfL96 3' (SEQ ID NO: 18) Antisense: AD-65696 containing 5'VPusGfsaAfuAfcUfgUfcccUfuUfuAfaGfcsasa 3' (SEQ ID NO: 19).

[0046] In yet another embodiment, the RNAi agent has the following sequence: Sense: 5'gscsuuaaAfaGfGfGfacaguauucaL96 3' (SEQ ID NO: 20) Antisense: AD-65703 containing 5'usGfsaauAfcUfGfucccUfuUfuaagcsasa 3' (SEQ ID NO: 21).

[0047] In yet another embodiment, the RNAi agent has the following sequence: Sense: 5'gscsuuaaAfaGfGfGfacaguauucaL96 3' (SEQ ID NO: 22) Antisense: AD-65704 containing 5'usGfsaauacugucccUfuuuaagcsasa 3' (SEQ ID NO: 23).

[0048] In yet another embodiment, the RNAi agent has the following sequence: Sense: 5'cscscaauAfaAfGfCfuggacaagaaL96 3' (SEQ ID NO: 714) Antisense: AD-67221 containing 5'usUfscuuGfuCfCfagcuUfuAfuugggsasg 3' (SEQ ID NO: 718).

[0049] In one embodiment, the RNAi agent has the following sequence: Sense: 5'gscsuuaaaaGfgGfacaguauuca 3' (SEQ ID NO: 738) Antisense: AD-69535 containing 5'sGfsaauacugucCfcUfuuuaagcsasa 3' (SEQ ID NO: 749).

[0050] In another embodiment, the RNAi agent has the following sequence: Sense: 5'gscsuuaaaaGfgGfacagu(Agn)uuca 3' (SEQ ID NO: 744) Antisense: AD-69541 containing 5'usGfsaauacugucCfcUfuuuaagcsasa 3' (SEQ ID NO: 755).

[0051] In certain embodiments, the present invention also provides a composition comprising a modified antisense polynucleotide agent, the agent being capable of inhibiting expression of APOC3 in a cell and comprising a sequence complementary to a sense sequence selected from the group of sequences listed in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13, wherein the polynucleotide is about 14 to about 30 nucleotides in length.

[0052] In some embodiments, the present invention also provides vectors containing the double-stranded RNAi agents described herein. In other embodiments, the present invention also provides cells containing the double-stranded RNAi agents described herein.

[0053] In some embodiments, the invention relates to a pharmaceutical composition comprising a double-stranded RNAi agent, or a composition comprising a modified antisense polynucleotide agent, or a vector as described herein.

[0054] In certain embodiments, the double-stranded RNAi agent is present in a non-buffered solution.In further embodiments, the non-buffered solution is saline or water.In other embodiments, the double-stranded RNAi agent is present in a buffered solution.In further embodiments, the buffered solution comprises acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof.In a specific embodiment, the buffered solution is phosphate buffered saline (PBS).

[0055] In one embodiment, the present invention also provides a method of inhibiting apolipoprotein C3 (APOC3) expression in a cell, the method comprising: (a) contacting a cell with a composition, vector, or pharmaceutical composition comprising a double-stranded RNAi agent, or a modified antisense polynucleotide agent, as described herein; (b) maintaining the cells resulting from step (a) for a time sufficient to achieve degradation of the mRNA transcripts of the APOC3 gene, thereby inhibiting expression of the APOC3 gene in the cells.

[0056] In one embodiment, the cell is present in a subject. In a further embodiment, the subject is a human or a rabbit. In one embodiment, the subject is suffering from an APOC3-related disease.

[0057] In some embodiments, APOC3 expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%.

[0058] In some aspects, the present invention provides a method of treating a subject having an apolipoprotein C3 (APOC3)-associated disease, the method comprising administering to the subject a therapeutically effective amount of a composition, or vector, or pharmaceutical composition comprising a double-stranded RNAi agent, or modified antisense polynucleotide agent as described herein, thereby treating the subject.

[0059] In one embodiment, the APOC3-related disease is hypertriglyceridemia. In another embodiment, the APOC3-related disease is selected from the group consisting of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, polycystic ovary syndrome, kidney disease, obesity, type 2 diabetes mellitus (insulin resistance), hypertension, atherosclerosis, and pancreatitis.

[0060] In some embodiments, the double-stranded RNAi agent is administered at a dose of about 0.01 mg / kg to about 10 mg / kg or about 0.5 mg / kg to about 50 mg / kg. In further embodiments, the double-stranded RNAi agent is administered at a dose of about 10 mg / kg to about 30 mg / kg. In another embodiment, the double-stranded RNAi agent is administered at a dose of about 3 mg / kg. In yet another embodiment, the double-stranded RNAi agent is administered at a dose of about 10 mg / kg.

[0061] In one embodiment, the double-stranded RNAi agent is administered subcutaneously. In another embodiment, the double-stranded RNAi agent is administered intravenously. In another embodiment, the double-stranded RNAi agent is administered intramuscularly.

[0062] In some embodiments, the RNAi agent is administered in two or more doses. In further embodiments, the RNAi agent is administered at intervals selected from the group consisting of about once every 12 hours, about once every 24 hours, about once every 48 hours, about once every 72 hours, and about once every 96 hours.

[0063] In certain embodiments, the methods of the present invention further comprise administering to the subject an additional therapeutic agent. In further embodiments, the additional therapeutic agent is selected from the group consisting of an HMG-CoA reductase inhibitor, a fibrate, a bile acid sequestrant, niacin, an antiplatelet agent, an angiotensin-converting enzyme inhibitor, an angiotensin II receptor antagonist, 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 modulator, a bile acid modulator, a peroxisome proliferator-activated receptor (PPAR) agonist, a gene-based therapy, a combined vasoprotectant, a glycoprotein Ilb / IIIa inhibitor, aspirin or an aspirin-like compound, an IBAT inhibitor, a squalene synthase inhibitor, a monocyte chemoattractant protein (MCP)-I inhibitor, or a fish oil. [Brief explanation of the drawings]

[0064] [Figure 1] 1 is a bar graph showing the relative amount of APOC3 mRNA in Hep3B cells after treatment with a single dose of 0.1 nM or 10 mM of the indicated iRNA of the invention. [Figure 2] 1 is a bar graph showing the relative amounts of APOC3 mRNA measured on day 5 in wild-type mice treated with 3, 10, and 30 mg / kg doses of GalNac-conjugated AD-57558. [Figure 3] 1 is a bar graph showing measurements of APOC3 mRNA levels measured in individual APOC3-AAV mice injected with AD-57553, AD-57547, and AD-58924. [Figure 4] 1 is a bar graph showing group means of APOC3 mRNA levels measured in APOC3-AAV mice injected with AD-57553, AD-57547, and AD-58924. [Figure 5]1 is a bar graph showing the relative amount of APOC3 mRNA measured in APOC3-AAV mice pre-injected with 10 11 hAPOC3 AAV genome copies followed by injection of 1.25 mg / kg, 2.5 mg / kg and 5 mg / kg doses of AD-57553. [Figure 6] 1 is a bar graph showing the group means of relative amounts of APOC3 mRNA measured in APOC3-AAV mice pre-injected with 10 hAPOC3 AAV genome copies followed by injection of 1.25 mg / kg, 2.5 mg / kg, and 5 mg / kg doses of AD-57553. [Figure 7A] 1 is a graph showing the 20-day time course of serum APOC3 protein measured in APOC3-AAV mice injected with 10 hAPOC3 AAV genome copies followed by a 3 mg / kg dose of the indicated iRNA of the invention. [Figure 7B] 1 is a graph showing the 30-day time course of serum APOC3 protein measured in APOC3-AAV mice injected with 10 hAPOC3 AAV genome copies followed by a 3 mg / kg dose of the indicated iRNA of the invention. [Figure 8] 1 is a bar graph showing the amount of serum APOC3 protein measured on day 10 in APOC3-AAV mice injected with 10 hAPOC3 AAV genome copies followed by a 3 mg / kg dose of the indicated iRNA of the invention. [Figure 9] 1 is a bar graph showing the amount of serum APOC3 protein measured on day 20 in APOC3-AAV mice injected with 10 hAPOC3 AAV genome copies followed by a 3 mg / kg dose of the indicated iRNA of the invention. [Figure 10] 1 is a time course showing the amount of APOC3 protein measured in APOC3-AAV mice injected with 10 11 hAPOC3 AAV genome copies followed by a 3 mg / kg dose of the indicated iRNA of the invention. [Figure 11]FIG. 1 is a schematic diagram showing the dosing schedule Q2W×4 used in multiple dose studies with AD-57553, AD-65696, AD-65699, AD-65703, and AD-65704. [Figure 12] Figure 12A is a time course showing the amount of APOC3 protein measured in APOC3-AAV mice injected with 10 hAPOC3 AAV genome copies followed by four 0.3 mg / kg doses of the indicated iRNA of the invention administered according to the dosing schedule shown in Figure 11. Figure 12B is a time course showing the amount of APOC3 protein measured in APOC3-AAV mice injected with 10 hAPOC3 AAV genome copies followed by four 1 mg / kg doses of the indicated iRNA of the invention administered according to the dosing schedule shown in Figure 11. Figure 12C is a time course showing the amount of APOC3 protein measured in APOC3-AAV mice injected with 10 hAPOC3 AAV genome copies followed by four 3 mg / kg doses of the indicated iRNA of the invention administered according to the dosing schedule shown in Figure 11. [Figure 13] 1 is a bar graph showing the relative amount of APOC3 protein measured at day 14 in APOC3-AAV mice injected with 10 hAPOC3 AAV genome copies followed by single doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg AD-65704. [Figure 14] 12 is a bar graph showing the relative amount of APOC3 protein measured 20 days after the final dose in APOC3-AAV mice injected with 10 hAPOC3 AAV genome copies followed by multiple doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg AD-65704 administered according to the dosing schedule shown in FIG. [Figure 15] 1 is a time course showing the amount of APOC3 protein measured in APOC3-AAV mice injected with 10 11 hAPOC3 AAV genome copies followed by a 1 mg / kg dose of the indicated iRNA of the invention. [Figure 16]Figure 16A is a bar graph showing the relative amount of APOC3 protein measured on day 10 in APOC3-AAV mice injected with 10 hAPOC3 AAV genome copies followed by a single 1 mg / kg dose of the indicated iRNA of the invention. Figure 16B is a bar graph showing the relative amount of APOC3 protein measured on day 24 in APOC3-AAV mice injected with 10 hAPOC3 AAV genome copies followed by a single 1 mg / kg dose of the indicated iRNA of the invention. [Figure 17] Figure 17A is a bar graph showing the relative amounts of serum APOC3 protein measured on day 14 in APOC3-AAV mice injected with 10 hAPOC3 AAV genome copies followed by a single 1 mg / kg dose of the indicated iRNA. Figure 17B is a graph showing the amounts of serum APOC3 protein measured on days 0, 14, 28, and 42 relative to pre-dose levels in APOC3-AAV mice injected with 10 hAPOC3 AAV genome copies followed by a single 1 mg / kg dose of the indicated iRNA. [Figure 18] Figure 18A is a graph showing the amount of serum APOC3 protein measured on days 1, 8, 11, 15, 22, 29, 36, 43, 57, 64, and 71 in cynomolgus monkeys after a single 1 mg / kg weekly dose of AD-65704 for 8 weeks (QWx8) compared to pre-dose on day -7. Figure 18B is a graph showing the amount of serum APOC3 protein measured on days 1, 8, 11, 15, 22, 29, and 36 in cynomolgus monkeys after a single 1 mg / kg dose of AD-65704 compared to pre-dose on day -7. Figure 18C is a graph showing the amount of liver APOC3 mRNA on day 64 in cynomolgus monkeys after a single 1 mg / kg weekly dose of AD-65704 for 5 weeks (q1wx5) compared to before dosing on day -7, and the amount of liver APOC3 mRNA on day 12 in cynomolgus monkeys after a single 1 mg / kg dose of AD-65704 compared to before dosing on day -7. [Figure 19]Figure 19A is a graph showing serum APOC3 protein levels measured on days 1, 8, 11, 15, 22, 29, and 36 in cynomolgus monkeys after a single 1 mg / kg dose of the indicated iRNA compared to pre-dosing on day -7. Figure 19B is a bar graph showing liver APOC3 mRNA levels measured on day 12 in cynomolgus monkeys after a single 1 mg / kg dose of the indicated iRNA compared to pre-dosing on day -7. [Figure 20] Figure 20A is a graph showing serum APOC3 mRNA levels measured on days 1, 8, 11, 15, 22, 29, 36, 43, 50, 57, 64, and 71 in cynomolgus monkeys after administration of a single 1 mg / kg dose of the indicated iRNA followed by a single subcutaneous 3 mg / kg dose of the same agent on day 36, compared to levels before administration on day -7. Figure 20B is a bar graph showing liver APOC3 mRNA levels measured on day 12 in cynomolgus monkeys after administration of a single 1 mg / kg dose of the indicated iRNA on day 1 followed by a single 3 mg / kg dose of the same iRNA agent on day 36, compared to levels before administration on day -7. DETAILED DESCRIPTION OF THE INVENTION

[0065] The present invention provides iRNA agents, e.g., double-stranded iRNA agents, and compositions that reduce or inhibit expression of the APOC3 gene. This gene can be in a cell, e.g., a cell within the body of a subject, such as a human.

[0066] The present invention also provides methods for treating subjects with disorders that would benefit from inhibiting or reducing the expression of APOC3, such as apolipoprotein C3-related diseases or disorders, such as hypertriglyceridemia, using iRNA compositions that inhibit or reduce expression of the APOC3 gene.

[0067] The iRNA of the present invention may be about 30 nucleotides in length or less, for example, 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 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to The iRNAs of the present invention comprise an RNA strand (antisense strand) having a region of 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 that is substantially complementary to at least a portion of the mRNA transcript of the APOC3 gene. These iRNAs can be used to target and degrade the mRNA of the APOC3 gene in cells. Specifically, extremely low dosages of the iRNAs of the present invention can specifically and efficiently mediate RNA interference (RNAi), resulting in significant inhibition of APOC3 gene expression. The present inventors have demonstrated that iRNA targeting APOC3 gene can mediate RNAi, and can cause APOC3 gene expression to be significantly inhibited and APOC3 protein level to be reduced by using in vitro and in vivo assays.The present inventors have also demonstrated that iRNA targeting APOC3 gene can reduce the symptoms associated with apolipoprotein C3-related disorders, for example, can reduce triglyceride levels.Therefore, the methods and compositions comprising these iRNAs are useful for treating the subjects with apolipoprotein C3-related disorders, such as hypertriglyceridemia.

[0068] The detailed description below discloses how to make and use compositions containing iRNA to inhibit expression of the APOC3 gene, as well as compositions, uses, and methods for treating subjects with diseases and disorders that may benefit from inhibiting and / or reducing the expression of APOC3.

[0069] 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.

[0070] 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.

[0071] The term "including" is used herein to mean, and is used interchangeably with, the term "including but not limited to."

[0072] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly indicates otherwise.

[0073] As used herein, the term "APOC3" refers to the well-known gene encoding apolipoprotein C3 and its protein product, also known in the art as HALP2 or APOCIII.

[0074] The term "APOC3" includes human APOC3 (for its amino acid and complete coding sequence, see, for example, GenBank accession number GI:4557322 (NM_000040.1; SEQ ID NO:1)); Macaca fascicularis APOC3 (for its amino acid and complete coding sequence, see, for example, GenBank accession number GI:544489959 (XM_05579730.1; SEQ ID NO:3)); Macaca mulatta APOC3 (for its amino acid and complete coding sequence, see, for example, GenBank accession number GI:297269260 (XM_001090312.2; SEQ ID NO:5)); mouse (Mus musculus) APOC3 (for its amino acid and complete coding sequence, see, for example, GenBank accession number GI:297269260 (XM_001090312.2; SEQ ID NO:5)); musculus) APOC3 (for its amino acid and complete coding sequence, see, for example, GenBank accession number GI:577019555 (NM_023114.4, SEQ ID NO:7)); rat (Rattus norvegicus) APOC3 (for its amino acid and complete coding sequence, see, for example, GenBank accession number GI:402534545 (NM_012501.2, SEQ ID NO:9)); and rabbit (Oryctolagus cuniculus), GenBank accession number GI:655601498 (XM_002708371.2, SEQ ID NO:11).

[0075] Further examples of APOC3 mRNA sequences are readily available in publicly available databases such as GenBank, UniProt, OMIM, and the Macaca Genome Project website.

[0076] As used herein, the term "APOC3" also refers to the naturally occurring DNA sequence mutation of APOC3 gene, such as the single nucleotide polymorphism (SNP) of APOC3 gene.The exemplary SNP of APOC3 DNA sequence can be found in the dbSNP database available at www.ncbi.nlm.nih.gov / projects / SNP / .Non-limiting examples of sequence mutation in APOC3 gene include, for example, two mutations rs2854116 and rs2854117, described in Petersen, KF et al., (2010), N.Engl.J.Med.362(12):1082-1089 (the entire contents of which are incorporated herein by reference).

[0077] As used herein, "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the APOC3 gene, including mRNA, which is an RNA processing product of a 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 an mRNA molecule formed during transcription of the APOC3 gene.

[0078] 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 length may be about 15-30 nucleotides, 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. Ranges and lengths intermediate to the recited ranges and lengths are also intended to be part of the invention.

[0079] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide that comprises a chain of nucleotides described by a referenced sequence, using standard nucleotide nomenclature.

[0080] "G", "C", "A", "T" and "U" generally represent the nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as base, 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 3).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 oligonucleotide that contains the nucleotide with such replacement moieties.As a non-limiting example, the nucleotide that contains inosine as base can base pair with the nucleotide that contains adenine, cytosine or uracil.Therefore, the nucleotide that contains uracil, guanine or adenine can be replaced with the nucleotide that contains 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 substituted moieties are suitable for the compositions and methods featured herein.

[0081] 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, APOC3 expression in a cell, e.g., a cell in a subject, e.g., a mammalian subject.

[0082] In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence, such as an APOC3 target mRNA sequence, and induces cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNAs introduced into cells are degraded into double-stranded small interfering RNAs (siRNAs) comprising a sense strand and an antisense strand by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). When Dicer, an RNase III-like enzyme, processes these dsRNAs, they become 19-23 base pair small interfering RNAs with unique two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). These siRNAs are then incorporated into RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing complementary antisense strands to guide target recognition (Nykanen, et al., (2001) Cell 107:309).When bound to appropriate target mRNA, one or more endonucleases in RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev.15:188).Therefore, in one aspect, the present invention relates to the single-stranded RNA (ssRNA) (the antisense strand of siRNA duplex) that is produced in cells and promotes the formation of RISC complex, resulting in the silencing of target gene, i.e., APOC3 gene.Therefore, the term "siRNA" is also used herein to refer to RNAi as described above.

[0083] In another embodiment, the RNAi agent may be a single-stranded RNA that is 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 long and chemically modified. The design and testing of single-stranded RNAs 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 hereby incorporated by reference). Any of the antisense nucleotide sequences described herein may be used as single-stranded siRNAs as described herein, or chemically modified by the method described in Lima et al., (2012) Cell 150:883-894.

[0084] 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 "double-stranded RNAi agent," "double-stranded RNA (dsRNA) molecule," "dsRNA agent," "RNAi agent," "RNAi," or "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules with a duplex structure, comprising two antiparallel and substantially complementary nucleic acid strands, which are referred to as having "sense" and "antisense" orientations relative to the target RNA, i.e., APOC3 gene. In some embodiments of the present invention, double-stranded RNA (dsRNA) causes the degradation of target RNA, such as mRNA, via a post-transcriptional gene silencing mechanism, herein referred to as RNA interference or RNAi.

[0085] Generally, the majority of the nucleotides in each strand of dsRNA molecule are ribonucleotides; however, as described in detail herein, each or both strands can also contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides.In addition, as used herein, "RNAi agent" can include ribonucleotides with chemical modifications; RNAi agent can contain substantial modifications in multiple nucleotides.As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide bond, and / or a modified nucleobase.Therefore, the term modified nucleotide includes, for example, the substitution, addition, or removal of functional groups or atoms in internucleoside bond, sugar moiety, or nucleobase.The modifications suitable for use in the agent of the present invention include all kinds of modifications disclosed herein or known in the art.Any such modifications, when used in siRNA-type molecules, are included in the "RNAi agent" for the purpose of this specification and claims.

[0086] 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.

[0087] The two strands that form a double-stranded structure can be different parts of a larger RNA molecule, or they can be separate RNA molecules.When the two strands are parts of one larger molecule, and thus the 3'-end of one strand that forms a double-stranded structure is connected to the 5'-end of the other strand by an uninterrupted nucleotide chain, the connected RNA strands are called "hairpin loops".A hairpin loop can contain at least one unpaired nucleotide; in some embodiments, a hairpin loop can contain 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, at least 23 or more unpaired nucleotides.

[0088] When the two substantially complementary strands of dsRNA are constituted by another RNA molecule, these molecules can be covalently linked, but do not necessarily have to be.When the two strands are covalently linked by means other than an uninterrupted nucleotide chain between the 3'-end of one strand that forms a double-stranded structure and the 5'-end of each other strand, the linked structure is called "linker".RNA strands can have the same or different nucleotide numbers.The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA minus any overhangs that exist in the double strand.In addition to the double-stranded structure, RNAi can also include one or more nucleotide overhangs.

[0089] In one embodiment, the RNAi agent of the present invention is a dsRNA, each strand of which contains 20-30 nucleotides that interact with a target RNA sequence, such as an APOC3 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 siRNAs by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). When Dicer, a ribonuclease III-like enzyme, processes dsRNA, it produces 19-23 base pair small interfering RNAs with unique 2-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). These siRNAs are 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 the RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

[0090] 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, or at least five or more nucleotides. A nucleotide overhang can comprise or consist of nucleotide / nucleoside analogs, including deoxyribonucleotides / nucleosides. An 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, the 3'-end, or both ends of either the antisense or sense strand of a dsRNA.

[0091] In one embodiment, the antisense strand of the dsRNA has 1 to 10 nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, overhanging at the 3' and / or 5' end. In one embodiment, the sense strand of the dsRNA has 1 to 10 nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, overhanging at the 3' and / or 5' end. In another embodiment, one or more nucleotides in the overhang are substituted with a thiophosphate nucleoside.

[0092] " Blunt-ended " or " blunt-end " means that there is no unpaired nucleotide at the corresponding 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 has nucleotide overhang at both ends.

[0093] The term "antisense strand" or "guide strand" refers to an iRNA strand, such as a dsRNA, that includes a region that is substantially complementary to a target sequence, such as an APOC3 mRNA. As used herein, the term "region complementary" refers to a region on the antisense strand that is substantially complementary to a sequence, such as a target sequence, such as an APOC3 nucleotide sequence as defined herein. If the complementary region is not completely complementary to the target sequence, mismatches may occur in the internal or terminal regions of the molecule. Generally, mismatches are most tolerated in the terminal regions, such as within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' end of the iRNA.

[0094] 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 defined herein.

[0095] As used herein, the term "cleavage region" refers to the region located immediately adjacent to the cleavage site. The cleavage site is the site where cleavage occurs on the target. In some embodiments, the cleavage region comprises 3 bases on both sides of the cleavage site and immediately adjacent thereto. In some embodiments, the cleavage region comprises 2 bases on both sides of the cleavage site and immediately adjacent thereto. In some embodiments, the cleavage site is specifically located at the site where nucleotides 10 and 11 of the antisense strand bind, and the cleavage region comprises nucleotides 11, 12 and 13.

[0096] 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 encountered in living organisms, 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.

[0097] For example, a complementary sequence in an iRNA, such as a dsRNA described herein, includes 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 double strand 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 comprises a 21 nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, would still be referred to as "fully complementary" for purposes described herein.

[0098] "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.

[0099] 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.

[0100] 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 target mRNA (e.g., an mRNA encoding APOC3). For example, a polynucleotide is complementary to at least a portion of an APOC3 mRNA if its sequence is substantially complementary to a non-interrupted portion of the mRNA encoding APOC3.

[0101] Thus, in some embodiments, the antisense polynucleotides disclosed herein are fully complementary to the target APOC3 sequence. In other embodiments, the sense strand polynucleotides and / or antisense polynucleotides disclosed herein are substantially complementary to the target APOC3 sequence, and include a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary to the equivalent region of the nucleotide sequence of any one of SEQ ID NOs: 1-12, or a fragment of any one of SEQ ID NOs: 1-12, over its entire length.

[0102] In one embodiment, an RNAi agent of the invention comprises a sense strand substantially complementary to an antisense polynucleotide complementary to a target APOC3 sequence, wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary over its entire length to the equivalent region of the nucleotide sequence of any one of SEQ ID NOs: 1-12, or a fragment of any one of SEQ ID NOs: 1-12. In another embodiment, an RNAi agent of the present invention comprises an antisense strand substantially complementary to a target APOC3 sequence, and comprises a contiguous nucleotide sequence that is at least about 80% complementary over its entire length, e.g., about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary to the equivalent region of the nucleotide sequence of any one of SEQ ID NOs: 1-12, or a fragment of any one of SEQ ID NOs: 1-12.

[0103] 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.

[0104] In one embodiment of the present invention, the agent used in the methods and compositions of the present invention is a single-stranded antisense nucleic acid molecule that inhibits target mRNA by an antisense inhibition mechanism. The single-stranded antisense nucleic acid molecule is complementary to a sequence within the target mRNA. Single-stranded antisense oligonucleotides can stoichiometrically inhibit translation 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 nucleic acid molecule may be about 15 to about 30 nucleotides in length and may have a sequence complementary to the target sequence. For example, the single-stranded antisense nucleic acid molecule may contain a sequence that is at least about 15, 16, 17, 18, 19, 20, or more consecutive nucleotides from any one of the antisense sequences described herein.

[0105] As used herein, a "subject" is an animal such as a mammal, including a primate (such as a human or a non-human primate, e.g., a monkey or chimpanzee), a non-primate (such as a cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, horse, and whale), or a bird (e.g., a duck or goose). In one embodiment, the subject is a human, such as a human being treated or evaluated for a disease, disorder, or condition that would benefit from reduced APOC3 expression; a human being at risk for a disease, disorder, or condition that would benefit from reduced APOC3 expression; a human having a disease, disorder, or condition that would benefit from reduced APOC3 expression; and / or a human being treated for a disease, disorder, or condition that would benefit from reduced APOC3 expression as described herein.

[0106] As used herein, the term "treat" or "treatment" refers to beneficial or desired results, including but not limited to, alleviating or improving one or more symptoms associated with undesirable or excessive APOC3 expression, such as hypertriglyceridemia (or high triglyceride levels).Such symptoms can include, for example, skin symptoms (e.g., eruptive xanthomas); eye abnormalities (e.g., lipemia retinalis); hepatosplenomegaly (enlarged liver and spleen); neurological symptoms; or abdominal pain attacks, which can be mild episodes of pancreatitis.Other symptoms associated with undesirable or excessive APOC3 expression can also include any disease, disorder or condition that can be caused by, related to, or result from hypertriglyceridemia, such as non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, polycystic ovarian syndrome, kidney disease, obesity, type 2 diabetes mellitus (insulin resistance), atherosclerosis, cardiovascular disease, or pancreatitis. "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment.

[0107] The term "lower," in the context of a subject's APOC3 levels or disease markers or symptoms, refers to a statistically significant decrease in such levels, which may be, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more, preferably to a level accepted as within the normal range for individuals without such disorder.

[0108] As used herein, " prevention " or " preventing ", when used in connection with a disease, disorder, or its pathology that can be treated or improved by reducing the expression of the APOC3 gene, refers to the reduction in the likelihood that a subject will develop symptoms associated with such disease, disorder, or pathology, such as symptoms of undesirable or excessive APOC3 expression, such as hypertriglyceridemia.For example, the likelihood of developing hypertriglyceridemia is reduced when, for example, an individual with one or more risk factors for hypertriglyceridemia does not develop hypertriglyceridemia, or develops hypertriglyceridemia with less severity than a group of people with the same risk factors and who have not received treatment as described herein.Not developing a disease, disorder, or pathology, or reducing the onset of symptoms associated with such disease, disorder, or pathology (for example, by at least about 10% on the clinically accepted scale for the disease or disorder in question), or showing a delay in symptoms (for example, by days, weeks, months, or years), is considered effective prevention.

[0109] As used herein, the term "apolipoprotein C3-related disease" or "APOC3-related disease" refers to a disease, disorder, or condition caused by or associated with unwanted or excessive APOC3 expression. The term "APOC3-related disease" includes diseases, disorders, or conditions that can be treated or ameliorated by reducing APOC3 expression. The term "APOC3-related disease" includes hypertriglyceridemia, or high triglyceride levels.

[0110] Triglyceride levels in the serum of subjects, e.g., human subjects, that may be symptomatic of hypertriglyceridemia are described in Oh, RC et al., (2007) American Family Physician, 75(9):1366-1371. Specifically, hypertriglyceridemia can be associated with "borderline high serum triglyceride levels" (i.e., 150-199 mg / dL or 1.70-2.25 mmol / L); "high serum triglyceride levels" (i.e., 200-499 mg / dL or 2.26-5.64 mmol / L); or "very high triglyceride levels" (i.e., 500 mg / dL or higher (or 5.65 mmol / L or higher)).

[0111] In one embodiment, the APOC3-related disease is primary hypertriglyceridemia. "Primary triglyceridemia" results from environmental or genetic causes (e.g., the result of an obvious underlying disease). Exemplary diseases characterized as primary hypertriglyceridemia include, but are not limited to, familial chyloproteinemia (hyperlipoproteinemia type 1), primary mixed hyperlipidemia (type 5), familial hypertriglyceridemia (hyperlipoproteinemia type 4), familial combined hyperlipoproteinemia (type 2B), and familial dysbetalipoproteinemia (hyperlipoproteinemia type 3).

[0112] In another embodiment, the APOC3-related disease is secondary hypertriglyceridemia. "Secondary triglyceridemia" is caused by or associated with other underlying disorders and conditions. Such disorders and / or conditions include, for example, obesity, metabolic syndrome, diabetes, fatty liver, alcohol consumption, kidney disease, pregnancy, non-alcoholic fatty liver disease, hypothyroidism, paraproteinemia (such as hypergammaglobulinemia in macroglobulinemia, myeloma, lymphoma and lymphocytic leukemia), autoimmune disorders (such as systemic lupus erythematosus), and medication (such as antiretroviral drugs including ritonavir and lopinavir, and antipsychotic drug therapy including clozapine and olanzapine) (see G. Yuan et al., (2007) Canadian Medical Association Journal, 176(8):1113-1120).

[0113] Any disorder that can cause hypertriglyceridemia (for example, secondary hypertriglyceridemia) or any disorder that can be the result of hypertriglyceridemia (for example, primary or secondary hypertriglyceridemia) is included in the term " APOC3-related disease ".Non-limiting examples of APOC3-related disease include metabolic disorders such as non-alcoholic fatty liver disease, non-alcoholic fatty liver disease, polycystic ovarian syndrome, renal disease, obesity, type 2 diabetes mellitus (insulin resistance); hypertension; cardiovascular disorders such as atherosclerosis; and pancreatitis, for example, acute pancreatitis.

[0114] II. iRNAs of the Invention The present invention provides an iRNA that inhibits the expression of the APOC3 gene. In one embodiment, the iRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of the APOC3 gene in cells, such as cells in the body of a mammal, such as a human, with an APOC3-related disease, such as hypertriglyceridemia. The dsRNA comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed by the expression of the APOC3 gene. The complementary region is about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contact with a cell expressing the APOC3 gene, the iRNA inhibits expression of the APOC3 gene (e.g., a human, primate, non-primate, or avian APOC3 gene) by at least about 10%, as assayed, for example, by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, such as by immunofluorescence analysis, using, for example, Western blotting or flow cytometry.

[0115] 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 the target sequence, and is generally completely complementary.The target sequence can be derived from the sequence of the mRNA formed during the expression of APOC3 gene.The other strand (sense strand) comprises a region complementary to the 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.

[0116] 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.

[0117] 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.

[0118] 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).

[0119] 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 recognized that a double-stranded region, such as a double-stranded region of approximately 9-36 base pairs, such as 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, is the primary functional portion of a dsRNA. 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, within the range that it is processed into, for example, a 15-30 base pair functional duplex that targets a desired RNA for cleavage. Thus, one 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 APOC3 expression is not generated in the target cell by cleavage of a larger dsRNA.

[0120] The dsRNA described herein can further comprise one or more single-stranded nucleotide overhangs, such as 1, 2, 3, or 4 nucleotides.Compared with 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 nucleotide of the overhang can be present on the 5'-end, 3'-end, or both ends of either the antisense or sense strand of dsRNA.

[0121] 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.

[0122] 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 containing 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.

[0123] In one embodiment, dsRNA of the present invention comprises at least two nucleotide sequences, sense sequence and antisense sequence.Sense strand and corresponding antisense strand are each selected from the group of sequences provided in any one of tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12 and 13.In this embodiment, one of these two sequences is complementary to the other of these two sequences, and one of these sequences is substantially complementary to the sequence of the mRNA produced by the expression of APOC3 gene.Therefore, in this embodiment, dsRNA comprises two oligonucleotides, wherein one oligonucleotide is described as sense strand in any one of tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12 and 13, and the second oligonucleotide is described as the corresponding antisense strand of sense strand in any one of tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12 and 13. In one embodiment, the substantially complementary sequences of the dsRNA are contained in separate oligonucleotides, hi another embodiment, the substantially complementary sequences of the dsRNA are contained in a single oligonucleotide.

[0124] Although some of the sequences in Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13 are described as modified and / or conjugated sequences, it will be understood that the RNA of the iRNA of the invention, e.g., the dsRNA of the invention, can include any one of the sequences set forth in Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13, unmodified, unconjugated, and / or modified and / or conjugated in a manner different from that described therein.

[0125] Those skilled in the art are well aware that dsRNAs having a double-stranded structure of approximately 20-23 base pairs, such as 21 base pairs, have been proposed 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 double-stranded 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 embodiments, due to the nature of the oligonucleotide sequences provided in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13, the dsRNAs described herein may 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 any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13, which are missing only a few nucleotides at one or both ends, may 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 consecutive nucleotides derived from one of the sequences in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13, and which differ in their ability to inhibit APOC3 gene expression by about 5, 10, 15, 20, 25, or 30% or less from a dsRNA containing the full-length sequence, are intended to be within the scope of the present invention.

[0126] Furthermore, the RNAs provided in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13 identify sites in the APOC3 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, an iRNA is said to target a specific site in an RNA transcript if it promotes cleavage of the transcript anywhere within that specific site. Such iRNAs generally contain approximately 15 contiguous nucleotides from one of the sequences provided in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13, linked to additional nucleotide sequences from regions adjacent to the selected sequence in the APOC3 gene.

[0127] 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, for example, any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12 and 13 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 the given sequence to identify sequences with equivalent or better inhibitory properties.

[0128] It is contemplated that further optimization of any sequence identified in, for example, any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13 can 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 can lead to further improvements in inhibition efficiency. Still further, such optimized sequences can be adjusted by, for example, introducing modified nucleotides described herein or known in the art, adding or modifying 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.).

[0129] 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 mismatches are 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 are 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 region of the APOC3 gene, 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 APOC3 gene expression. Examining the effectiveness of iRNAs with mismatches in inhibiting APOC3 gene expression is important, especially when a specific complementary region of the APOC3 gene is known to have polymorphic sequence variation within the population.

[0130] III. Modified iRNAs of the Invention In one embodiment, the RNA of an iRNA of the invention, e.g., dsRNA, is native and does not contain chemical modifications and / or linkages, e.g., those known in the art and described herein. In another embodiment, the RNA of an iRNA of the invention, e.g., dsRNA, is chemically modified to enhance stability or other beneficial properties. In certain embodiments of the invention, substantially all of the nucleotides of an iRNA of the invention are modified. In other embodiments of the invention, all of the nucleotides of an iRNA of the invention are modified nucleotides. An iRNA of the invention in which "substantially all of the nucleotides are modified" will have most, if not all, modified nucleotides and may contain no more than 5, 4, 3, 2, or 1 unmodified nucleotide.

[0131] In some aspects of the invention, substantially all of the nucleotides of an iRNA of the invention are modified, and the iRNA agent includes 10 or fewer nucleotides that include a 2'-fluoro modification (e.g., 9 or fewer 2'-fluoro modifications, 8 or fewer 2'-fluoro modifications, 7 or fewer 2'-fluoro modifications, 6 or fewer 2'-fluoro modifications, 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 3 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications). For example, in some embodiments, the sense strand includes 4 or fewer nucleotides that include a 2'-fluoro modification (e.g., 3 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications). In other embodiments, the antisense strand includes 6 or fewer nucleotides that include a 2'-fluoro modification (e.g., 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications). In other aspects of the invention, all of the nucleotides of an iRNA of the invention are modified, and the iRNA agent includes 10 or fewer nucleotides that include 2'-fluoro modifications (e.g., 9 or fewer 2'-fluoro modifications, 8 or fewer 2'-fluoro modifications, 7 or fewer 2'-fluoro modifications, 6 or fewer 2'-fluoro modifications, 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 3 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications).

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] Modified RNAs can also contain one or more substituted sugar moieties. For example, iRNAs, such as dsRNAs featured 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 the alkyl, alkenyl, and alkynyl can be substituted or unsubstituted C1-C10 alkyl, or C2-C10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In another embodiment, the dsRNA comprises one of the following at the 2' position: C1-C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group that improves the pharmacokinetic properties of iRNA, or group that improves the pharmacodynamic properties of iRNA, and other substituents with similar properties. In some embodiments, the modification comprises 2'-methoxyethoxy (2'-O-CH2CHOCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is the 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, i.e., O(CH2)2ON(CH3)2 group, 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.

[0140] 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.

[0141] iRNAs 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.

[0142] 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.

[0143] The RNA of an iRNA may 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, the agent of the present invention may contain one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety contains an additional 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 in a 3'-terminal structural conformation. It has been shown that adding a locked nucleic acid to siRNA increases siRNA stability in serum and reduces 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). Examples of bicyclic nucleosides used in the polynucleotide of the present invention include, without limitation, nucleosides that contain a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agent of the present invention comprises one or more bicyclic nucleosides that contain a 4'-2' bridge.Examples of such 4'-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 "constrained ethyl" or "cEt") and 4'-CH(CHOCH3)-O-2' (and analogs; see, e.g., U.S. Pat. No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and analogs; see, e.g., U.S. Pat. No. 8,278, 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', where 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 herein by reference.

[0144] Additional representative U.S. patents and published U.S. patent applications 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; and U.S. Pat. No. 7,399,845. Nos. 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 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.

[0145] 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).

[0146] The RNA of an iRNA may 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)-0-2' bridge. In one embodiment, the constrained ethyl nucleotide is in the S configuration, referred to herein as "S-cEt."

[0147] The iRNA of the present invention may also contain one or more "conformationally locked nucleotides" ("CRNs"). A CRN is a nucleotide analogue 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 position, increasing hybridization affinity to mRNA. The linker is long enough to place the oxygen in an optimal position for stability and affinity, reducing puckering of the ribose ring.

[0148] Representative literature that teaches the preparation of some of the above-mentioned CRNs includes, but is not limited to, U.S. Patent Application Publication No. 2013 / 0190383; and WO 2013 / 036868, the entire contents of each of which are hereby incorporated by reference herein.

[0149] One or more of the nucleotides of the iRNA of the invention can also comprise a hydroxymethyl-substituted nucleotide. A "hydroxymethyl-substituted nucleotide" is an acyclic 2'-3'-seco-nucleotide, also referred to as an "unlocked nucleic acid" ("UNA") modification.

[0150] Representative U.S. publications that teach the preparation of UNAs include, but are not limited to, U.S. Pat. No. 8,314,227; and U.S. Patent Application Publication No. 2013 / 0096289; U.S. Pat. No. 2013 / 0011922; and U.S. Pat. No. 2011 / 0313020 (the entire contents of each of which are hereby incorporated by reference herein).

[0151] 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.

[0152] A. Modified iRNAs Containing Motifs of the Invention In certain aspects of the present invention, the double-stranded RNAi agent of the present invention includes agents having chemical modifications, for example, as disclosed in International Publication No. 2013 / 075035, filed November 16, 2012 (the entire contents of which are incorporated herein by reference).As shown herein and in International Publication No. 2013 / 075035, excellent 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 completely modified otherwise.The introduction of these motifs, if present, disrupts the modification pattern 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 agent exhibits excellent gene silencing activity.

[0153] More specifically, it has been unexpectedly found that fully modifying the sense and antisense strands of a double-stranded RNAi agent 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 significantly enhanced the gene silencing activity of the RNAi agent.

[0154] Thus, the present invention provides a double-stranded RNAi agent capable of inhibiting the expression of a target gene (i.e., the apolipoprotein C3 (APOC3) gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may be 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.

[0155] The sense and antisense strands typically form a duplex, double-stranded RNA ("dsRNA"), also referred to herein as an "RNAi agent." The duplex region of an RNAi agent may be 12-30 nucleotide pairs in length. For example, the duplex region may be 14-30 nucleotide pairs, 17-30 nucleotide pairs, 27-30 nucleotide pairs, 17-23 nucleotide pairs, 17-21 nucleotide pairs, 17-19 nucleotide pairs, 19-25 nucleotide pairs, 19-23 nucleotide pairs, 19-21 nucleotide pairs, 21-25 nucleotide pairs, or 21-23 nucleotide pairs in length. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

[0156] In one embodiment, an RNAi agent can 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 offset. The overhangs can form mismatches with the target mRNA, or they can be complementary to the targeted gene sequence, or they can be a different sequence. The first and second strands can also be joined by additional bases, e.g., to form a hairpin, or by other non-basic linkers.

[0157] In one embodiment, the nucleotides in the overhang region of the RNAi agent can each independently be modified or unmodified nucleotides, including but not limited to 2'-sugar modifications, such as 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 of either strand.The overhang can form a mismatch with target mRNA, or the overhang can be complementary to the target gene sequence, or can be another sequence.

[0158] The 5'- or 3'-overhang of the sense strand, antisense strand, or both strands of RNAi agent can be phosphorylated.In some embodiments, one or more overhang regions comprise 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, this 3'-overhang is present in the antisense strand.In one embodiment, this 3'-overhang is present in the sense strand.

[0159] RNAi agent may contain only a single overhang, which can enhance the interference activity of RNAi without affecting its overall stability.For example, this single-stranded overhang may be located at the 3'-end of the sense strand or at the 3'-end of the antisense strand.RNAi may also be located at the 5'-end of the antisense strand (or at the 3'-end of the sense strand), or vice versa, have a blunt end.Generally, the antisense strand of RNAi has a nucleotide overhang at the 3'-end, and its 5'-end is blunt.Without wishing to be bound by theory, the asymmetric blunt end of the 5'-end of the antisense strand and the 3'-end overhang of the antisense strand are advantageous for the guide strand to load into RISC process.

[0160] In one embodiment, the RNAi agent is a 19-nucleotide double-ended bluntmer, in which the sense strand contains 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 contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0161] In another embodiment, the RNAi agent is a 20-nucleotide double-ended bluntmer, wherein the sense strand contains 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 contains at least one motif of 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0162] In yet another embodiment, the RNAi agent is a 21-nucleotide double-ended bluntmer, wherein the sense strand contains 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 contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0163] 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 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, wherein one end of the RNAi agent is blunt-ended, and the other end comprises a 2-nucleotide overhang.Preferably, this 2-nucleotide overhang is at the 3' end of the antisense strand.

[0164] When a two-nucleotide overhang is at the 3'-end of the antisense strand, there may be two phosphorothioate internucleotide bonds between the terminal three nucleotides, where two of these 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 has 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.In one embodiment, all nucleotides of the sense strand and antisense strand of the RNAi agent, including the nucleotides that are 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).

[0165] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is 25 to 30 nucleotide residues in length and comprises at least 8 ribonucleotides at positions 1 to 23 of the first strand, starting from the 5'-terminal nucleotide (position 1); the antisense strand is 36 to 66 nucleotide residues in length and comprises at least 8 ribonucleotides at positions 1 to 23 of the sense strand, starting from the 3'-terminal nucleotide, that pair with the sense strand to form a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is unpaired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are unpaired with the sense strand, thereby forming a 3' single-stranded overhang of 1 to 6 nucleotides; and the 5' end of the antisense strand comprises 10 to 30 consecutive nucleotides that are unpaired with the sense strand. The sense strand does not base pair with the antisense strand, thereby forming a 10-30 nucleotide single-stranded 5' overhang; when the sense and antisense strands are aligned for maximum complementarity, at least the 5'- and 3'-terminal nucleotides of the sense strand base pair with nucleotides of the antisense strand, thereby forming a substantially duplex region between the sense and antisense strands; and the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length to reduce target gene expression when the double-stranded nucleic acid is introduced into a mammalian cell; and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, where at least one of these motifs is located at or near the cleavage site. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0166] 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 25 to 29 nucleotides, and a second strand having a length of 30 nucleotides or less, the second strand having 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, the second strand is 1 to 4 nucleotides longer than the first strand at its 3' end, the duplex region 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, such that when the RNAi agent is introduced into a mammalian cell, it reduces target gene expression, and Dicer cleavage of the RNAi agent preferentially produces siRNAs comprising the 3' end of the second strand, thereby reducing target gene expression in the mammal. Optionally, the RNAi agent further comprises a ligand.

[0167] In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications over three consecutive nucleotides, one of which motifs is at the cleavage site of the sense strand.

[0168] In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications over three consecutive nucleotides, where one of these motifs is at or near the cleavage site on the antisense strand.

[0169] For RNAi agents having a duplex region 17-23 nucleotides in length, the cleavage sites of the antisense strand are typically located at approximately positions 10, 11, and 12 from the 5' end. Thus, three identical modification motifs can be located 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 (counting begins from the first nucleotide from the 5' end of the antisense strand, or counting begins from the first paired nucleotide from the 5' end within the duplex region of the antisense strand). The cleavage site of the antisense strand can also vary depending on the length of the duplex region of the RNAi from the 5' end.

[0170] The sense strand of RNAi agent can contain at least one motif of three identical modifications on three consecutive nucleotides at the site of strand breakage; and antisense strand can have at least one motif of three identical modifications on three consecutive nucleotides at or near the site of strand breakage.When sense strand and antisense strand form dsRNA duplex, sense strand and antisense strand can be aligned such 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 or all three nucleotides can overlap.

[0171] 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" as used herein refers to a motif located in another part of the strand that is separated from a motif located at or near the cleavage site of the same strand. The wing modification may be adjacent to the first motif or separated by at least one nucleotide. When the motifs are immediately adjacent to each other, the chemistry of the motifs may be different from each other, and when the motifs are separated by one or more nucleotides, their chemistry may 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 on one side of the first motif located at or near the cleavage site, or on both sides of the lead motif.

[0172] Similar to the sense strand, the antisense strand of the RNAi agent can contain two or more motifs of three identical modifications on three consecutive nucleotides, and at least one of these motifs can be present at or near the site of strand cleavage.This antisense strand can also contain one or more wing modifications, aligned in the same way as the wing modifications that can be present on the sense strand.

[0173] 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.

[0174] 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 in the duplex region at the 3' end, 5' end, or both ends of the strand.

[0175] When the sense and antisense strands of an RNAi agent each contain at least one wing modification, the wing modifications can be located at the same end of the duplex region and have an overlap of 1, 2, or 3 nucleotides.

[0176] When the sense and antisense strands of an RNAi agent each contain at least two wing modifications, the sense and antisense strands may be arranged such that two modifications from each strand are located at one end of the duplex region, with an overlap of 1, 2, or 3 nucleotides; two modifications from each strand are located at the other end of the duplex region, with an overlap of 1, 2, or 3 nucleotides; or two modifications from one strand are located on either side of the lead motif, with an overlap of 1, 2, or 3 nucleotides in the duplex region.

[0177] In one embodiment, all nucleotides of the sense strand and antisense strand of RNAi agent, including the nucleotide that is part of motif, can be modified.Each nucleotide can be modified by the same or different modifications, which can include one or more of one or both of unbound phosphate oxygen and / or bound phosphate oxygen; modification of ribose sugar components, for example, 2' hydroxyl on ribose sugar; large-scale substitution of phosphate moiety with " dephosphorylation " linker; modification or substitution of naturally occurring base; and substitution or modification of ribose phosphate backbone.

[0178] Because nucleic acids are polymers of subunits, many modifications occur at positions that are repeated within nucleic acids, such as modifications of bases, phosphate moieties, or non-linked Os in phosphate moieties. In some cases, modifications can occur at all target positions within a nucleic acid, but in many cases this is not the case. For example, modifications can occur only at the 3' or 5' terminal position, or only in terminal regions, such as at the terminal nucleotide position or the last 2, 3, 4, 5, or 10 nucleotides of the chain. Modifications can occur in double-stranded regions, single-stranded regions, or both. Modifications can occur only in double-stranded regions of RNA, or only in single-stranded regions of RNA. For example, phosphorothioate modifications at non-linked O positions can occur only at one or both ends, or only in terminal regions, such as at the terminal nucleotide position or the last 2, 3, 4, 5, or 10 nucleotides of the chain, or in double-stranded and single-stranded regions, especially at the ends. One or more 5' ends can be phosphorylated.

[0179] For example, to enhance stability, it may be possible to include specific bases in the overhang, or to include modified nucleotides or nucleotide surrogates in the single-stranded overhang, for example, 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 these bases in the 3' or 5' overhang may be modified, for example, by the modifications described herein. Modifications may include modifications known in the art, such as modified deoxyribonucleotides in place of the ribosugar of the nucleobase, modifications at the 2' position of the ribose sugar by using 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl, and modifications at the phosphate group, for example, phosphorothioate modifications. The overhang does not need to be homologous to the target sequence.

[0180] In one embodiment, each residue of sense strand and antisense strand is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl or 2'-fluoro.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.

[0181] There are typically at least two different modifications on the sense and antisense strands, which may be 2'-O-methyl or 2'-fluoro modifications, for example.

[0182] In one embodiment, Na and / or Nb comprise an alternating pattern of modifications. The term "alternating motif," as used herein, refers to a motif having one or more modifications, with each modification occurring on alternating nucleotides on one strand. Alternating nucleotides can refer to every other nucleotide or every third nucleotide, or similar patterns. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif can be "ABABABABABAB...," "AABBAABBAABB...," "AABAABAABAAB...," "AAABAAABAAAB...," "AAABBBAAABBB...," or "ABCABCABCABC...," etc.

[0183] The types of modifications included in the alternating motif can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternating pattern, i.e., the modifications on every other nucleotide, can be the same, except that each of the sense or antisense strands can be selected from several possible modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD..." or "CDCDCD...".

[0184] In one embodiment, the RNAi agent of the present invention comprises an alternating motif on the sense strand whose modification pattern is shifted relative to the alternating motif on the antisense strand. This shift can be such that the modified groups of the nucleotides of the sense strand correspond to differently modified groups of the nucleotides of the antisense strand, and vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, the alternating motif of the sense strand may begin at the 5'-3' end of the strand with "ABABAB," and the alternating motif of the antisense strand may begin at the 5'-3' end of the strand with "BABABA." As another example, the alternating motif of the sense strand may begin at the 5'-3' end of the strand with "AABBAABB," and the alternating motif of the antisense strand may begin at the 5'-3' end of the strand with "BBAABBAA," thereby resulting in a complete or partial shift in the modification pattern between the sense strand and the antisense strand.

[0185] In one embodiment, RNAi agent comprises the alternating motif pattern of 2'-O-methyl modification and 2'-F modification on sense strand, and this initially has a shift with respect to the alternating motif pattern of 2'-O-methyl modification and 2'-F modification on antisense strand, that is, the 2'-O-methyl modified nucleotide on sense strand is base-paired with the 2'-F modified nucleotide on antisense strand, and vice versa.Position 1 of sense strand can start with 2'-F modification, and position 1 of antisense strand can start with 2'-O-methyl modification.

[0186] Introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense strand and / or antisense strand will disrupt the original modification pattern present in the sense strand and / or antisense strand.Unexpectedly, this disruption of the modification pattern of the sense strand and / or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense strand and / or antisense strand will enhance the gene silencing activity against target genes.

[0187] In one embodiment, when a motif of three identical modifications on three consecutive nucleotides is introduced into either of these strands, the modification of the nucleotides adjacent to the motif is a different modification from that of the motif. For example, the portion of the sequence containing the motif is "...NaYYYNb..." (where "Y" represents the modification of the motif of three identical modifications on three consecutive nucleotides, and "Na" and "Nb" represent modifications to the nucleotides adjacent to the motif "YYY" that are different from that of Y, and Na and Nb may be the same or different modifications). Alternatively, when a wing modification is present, Na and / or Nb may or may not be present.

[0188] The RNAi agent may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may be present at any nucleotide at any position on the sense strand, the antisense strand, or both strands. For example, the internucleotide linkage modification may be present at every nucleotide on the sense strand and / or the antisense strand; each internucleotide linkage modification may be present in an alternating pattern on the sense strand and / or the antisense strand; or both the sense strand and the antisense strand may contain internucleotide linkage modifications in an alternating pattern. The alternating pattern of internucleotide linkage modifications on the sense strand may be the same as or different from that of the antisense strand, and the alternating pattern of internucleotide linkage modifications on the sense strand may have a shift relative to the alternating pattern of internucleotide linkage modifications on the antisense strand. In one embodiment, a double-stranded RNAi agent comprises 6 to 8 phosphorothioate internucleotide linkages. In one embodiment, the antisense strand contains two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand contains at least two phosphorothioate internucleotide linkages at either the 5' end or the 3' end.

[0189] In one embodiment, RNAi comprises phosphorothioate or methylphosphonate internucleotide bond modification in overhang region.For example, overhang region can contain two nucleotides with phosphorothioate or methylphosphonate internucleotide bond between them.Internucleotide bond modification can also be made to connect overhang nucleotide with terminal paired nucleotide in double-stranded region.For example, at least 2, 3, 4 or all overhang nucleotides can be linked via phosphorothioate or methylphosphonate internucleotide bond, and optionally there can be additional phosphorothioate or methylphosphonate internucleotide bond that connects overhang nucleotide with paired nucleotide adjacent to overhang nucleotide.For example, there can be at least two phosphorothioate internucleotide bonds between the terminal three nucleotides, two of these three nucleotides are overhang nucleotides, and the third nucleotide is paired nucleotide adjacent to 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.

[0190] In one embodiment, the two-nucleotide overhang is at the 3'-end of the antisense strand, and there are two phosphorothioate internucleotide bonds between the terminal three nucleotides, where two of these three nucleotides are overhanging nucleotides, and the third nucleotide is a paired nucleotide adjacent to the overhanging nucleotide. Optionally, the RNAi agent can 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.

[0191] In one embodiment, the RNAi agent contains mismatches with the target in the duplex, or a combination thereof. Mismatches can be present in the overhang region or the duplex region. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., regarding the free energy of association or dissociation of a particular pairing, the simplest approach is to examine each pair by each base pair, but next-neighbor or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, such as non-canonical or non-canonical pairings (as described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings; and pairings involving universal bases are preferred over canonical pairings.

[0192] In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs from the 5' end within the duplex region 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.

[0193] In one embodiment, the nucleotide at the 1st position from the 5' end in the double-stranded region 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 from the 5' end in the double-stranded region of the antisense strand is an AU base pair.For example, the first base pair from the 5' end in the double-stranded region of the antisense strand is an AU base pair.

[0194] In another embodiment, the 3'-terminal nucleotide of the sense strand is deoxythymine (dT). In another embodiment, the 3'-terminal nucleotide of the antisense strand is deoxythymine (dT). In one embodiment, there is a short sequence of deoxythymine nucleotides, e.g., two dT nucleotides, at the 3'-end of the sense strand and / or antisense strand.

[0195] In one embodiment, the sense strand sequence has formula (I): 5'np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq3'(I) [In formula: i and j are each independently 0 or 1; p and q each independently represent 0 to 6; each Na independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each Nb independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each np and nq independently represents an overhanging nucleotide; wherein Nb and Y do not have the same modification; and wherein 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.

[0196] In one embodiment, Na and / or Nb comprise an alternating pattern of modifications.

[0197] 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 duplex region 17-23 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) ​​(counting begins from the first nucleotide from the 5' end; or optionally, counting begins from the first paired nucleotide from the 5' end within the duplex region).

[0198] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Thus, the sense strand has the following formula: 5'np-Na-YYY-Nb-ZZZ-Na-nq3'(Ib); 5'np-Na-XXX-Nb-YYY-Na-nq3'(Ic); or 5'np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq3'(Id) It can be expressed as:

[0199] When the sense strand is represented by Formula (Ib), Nb represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each Na can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0200] When the sense strand is represented by Formula (Ic), Nb 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. Each Na independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0201] When the sense strand is represented by Formula (Id), each Nb independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Preferably, Nb is 0, 1, 2, 3, 4, 5, or 6. Each Na independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0202] Each of X, Y and Z may be the same as or different from one another.

[0203] In other embodiments, i is 0 and j is 0, and the sense strand has the formula: 5'np-Na-YYY-Na-nq3'(Ia) It can be represented by:

[0204] When the sense strand is represented by Formula (Ia), each Na can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0205] In one embodiment, the antisense strand sequence of the RNAi has formula (II): 5'nq'-Na'-(Z'Z'Z')k-Nb'-Y'Y'Y'-Nb'-(X'X'X')l-Na'-np'3'(II) [In formula: k and l are each independently 0 or 1; p' and q' are each independently 0 to 6; each Na' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each Nb' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each np' and nq' independently represents an overhanging nucleotide; wherein Nb' 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.

[0206] In one embodiment, Na' and / or Nb' comprise an alternating pattern of modifications.

[0207] The Y'Y'Y' motif is located 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 located at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand (counting starts from the first nucleotide from the 5' end; or optionally, counting starts from the first paired nucleotide from the 5' end within the double-stranded region). Preferably, the Y'Y'Y' motif is located at positions 11, 12, 13.

[0208] In one embodiment, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0209] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or k and l are both 1.

[0210] Thus, the antisense strand has the following formula: 5'nq'-Na'-Z'Z'Z'-Nb'-Y'Y'Y'-Na'-np'3'(IIb); 5'nq'-Na'-Y'Y'Y'-Nb'-X'X'X'-np'3'(IIc); or 5'nq'-Na'-Z'Z'Z'-Nb'-Y'Y'Y'-Nb'-X'X'X'-Na'-np'3'(IId) It can be represented by:

[0211] When the antisense strand is represented by Formula (IIb), Nb' 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. Each Na' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0212] When the antisense strand is represented by Formula (IIc), Nb' 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. Each Na' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0213] When the antisense strand is represented by formula (IId), each Nb' independently 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. Each Na' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Preferably, Nb is 0, 1, 2, 3, 4, 5, or 6.

[0214] In other embodiments, k is 0 and l is 0, and the antisense strand has the formula: 5'np'-Na'-Y'Y'Y'-Na'-nq'3'(Ia) It can be represented by:

[0215] When the antisense strand is represented as Formula (IIa), each Na' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0216] Each of X', Y' and Z' may be the same as or different from one another.

[0217] Each nucleotide of sense strand and antisense strand can be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl or 2'-fluoro.For example, each nucleotide of sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.Each X, Y, Z, X', Y' and Z' can specifically represent 2'-O-methyl modification or 2'-fluoro modification.

[0218] In one embodiment, the sense strand of the RNAi agent may contain a YYY motif present at positions 9, 10, and 11 of the strand when the duplex region is 21 nt (counting starts from the first nucleotide from the 5' end, or optionally, counting starts from the first paired nucleotide from the 5' end within the duplex region); and Y represents a 2'-F modification. The sense strand may further contain a XXX motif or a ZZZ motif as a wing modification at the opposite end of the duplex region; and XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0219] In one embodiment, the antisense strand may contain 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 first paired nucleotide from the 5' end within the duplex region); and Y' represents a 2'-O-methyl modification. The antisense strand may further contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the duplex region; and X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0220] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with the antisense strand represented by any one of the formulas (IIa), (IIb), (IIc), and (IId), respectively.

[0221] Thus, the RNAi agents used in the methods of the invention can include a sense strand and an antisense strand, each strand having 14-30 nucleotides, and the RNAi duplex can have the formula (III): Sense: 5'np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq3' Antisense: 3'np'-Na'-(X'X'X')k-Nb'-Y'Y'Y'-Nb'-(Z'Z'Z')l-Na'-nq'5' (III) [In formula: i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6; each Na and Na′ independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each Nb and Nb' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; wherein each np', np, nq', and nq (each of which may be present or absent) independently represents 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

[0222] 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.

[0223] Exemplary combinations of sense and antisense strands that form RNAi duplexes include the following formulas: 5'np-Na-YYY-Na-nq3' 3'np'-Na'-Y'Y'Y'-Na'nq'5' (IIIa) 5'np-Na-YYY-Nb-ZZZ-Na-nq3' 3'np'-Na'-Y'Y'Y'-Nb'-Z'Z'Z'-Na'nq'5' (IIIb) 5'np-Na-XXX-Nb-YYY-Na-nq3' 3'np'-Na'-X'X'X'-Nb'-Y'Y'Y'-Na'-nq'5' (IIIc) 5'np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq3' 3'np'-Na'-X'X'X'-Nb'-Y'Y'Y'-Nb'-Z'Z'Z'-Na-nq'5' (IIId)

[0224] When the RNAi agent is represented by Formula (IIIa), each Na independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0225] When an RNAi agent is represented by Formula (IIIb), each Nb independently represents an oligonucleotide sequence containing 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides, and each Na independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0226] When an RNAi agent is represented as Formula (IIIc), each Nb, Nb' independently 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, and each Na independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0227] When an RNAi agent is represented as formula (IIId), each Nb, Nb' independently 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. Each Na, Na' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Each of Na, Na', Nb, and Nb' independently comprises an alternating pattern of modifications.

[0228] Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) may be the same as or different from one another.

[0229] When the RNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides can 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.

[0230] When the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides can 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.

[0231] When the RNAi agent is represented by formula (IIIc) or (IIId), at least one of the X nucleotides can 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.

[0232] 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.

[0233] In one embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is 2'-O-methyl or 2'-fluoro modification.In another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is 2'-O-methyl or 2'-fluoro modification, and np'>0 and at least one np' are linked to adjacent nucleotides via phosphorothioate bonds.In yet another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is 2'-O-methyl or 2'-fluoro modification, np'>0 and at least one np' are linked to adjacent nucleotides via phosphorothioate bonds, and the sense strand is conjugated with one or more GalNAc derivatives (described below) attached via a bivalent or trivalent branched linker. In another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0 and at least one np' is linked to an adjacent nucleotide via a phosphorothioate bond, the sense strand comprises at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives attached via a divalent or trivalent branched linker.

[0234] In one embodiment, when the RNAi agent is represented by formula (IIIa), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0 and at least one np' is linked to an adjacent nucleotide via a phosphorothioate bond, the sense strand comprises at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives attached via a divalent or trivalent branched linker.

[0235] In one embodiment, the RNAi agent is a multimer that contains at least two double strands represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), and the double strands are connected by a linker.The linker can be cleavable or non-cleavable.Optionally, the multimer further comprises a ligand.Each double strand can target the same gene or two different genes; or each double strand can target the same gene at two different target sites.

[0236] In one embodiment, the RNAi agent is a multimer containing 3, 4, 5, 6 or more double strands represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), and 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.

[0237] 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'-end and 3'-end, and are optionally conjugated to a ligand. Each of these agents can target the same gene or two different genes; or each of these agents can target the same gene at two different target sites.

[0238] In certain embodiments, the RNAi agent of the present invention may contain a small number of nucleotides containing 2'-fluoro modifications, for example, 10 or fewer nucleotides containing 2'-fluoro modifications.For example, the RNAi agent may contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 nucleotides containing 2'-fluoro modifications.In a specific embodiment, the RNAi agent of the present invention contains 10 nucleotides containing 2'-fluoro modifications, for example, 4 nucleotides containing 2'-fluoro modifications in the sense strand and 6 nucleotides containing 2'-fluoro modifications in the antisense strand.In another specific embodiment, the RNAi agent of the present invention contains 6 nucleotides containing 2'-fluoro modifications, for example, 4 nucleotides containing 2'-fluoro modifications in the sense strand and 2 nucleotides containing 2'-fluoro modifications in the antisense strand.

[0239] In other embodiments, the RNAi agent of the present invention can contain a very small number of nucleotides that contain 2'-fluoro modification, for example, two or less nucleotides that contain 2'-fluoro modification.For example, the RNAi agent can contain 2, 1 or 0 nucleotides that contain 2'-fluoro modification.In a specific embodiment, the RNAi agent can contain two nucleotides that contain 2'-fluoro modification, for example, 0 nucleotides that contain 2'-fluoro modification in the sense strand and two nucleotides that contain 2'-fluoro modification in the antisense strand.

[0240] Various documents describe the multimeric RNAi agent that can be used in the method of the present invention.Such documents include WO2007 / 091269, US Patent No. 7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520 (each of which is hereby incorporated by reference in its entirety).

[0241] As described in more detail below, an RNAi agent containing one or more carbohydrate moieties conjugated to the RNAi agent can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety can be added 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 to which a carbohydrate ligand is attached. A ribonucleotide subunit in which the ribose sugar of the subunit has been replaced in this manner 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 contain two or more rings, for example, a fused ring. The cyclic carrier can be a fully saturated ring system or contain one or more double bonds.

[0242] Ligands can be attached to polynucleotides via carriers. The carriers contain (i) at least one "backbone attachment point," preferably two, and (ii) at least one "tethering attachment point." As used herein, "backbone attachment point" refers to a functional group, such as a hydroxyl group, or generally to a bond available and suitable for incorporation of the carrier into a backbone, such as the phosphate backbone of a ribonucleic acid, or a modified phosphate backbone, such as a sulfur-containing backbone. A "tethering attachment point" (TAP), in some embodiments, refers to a ring atom, such as 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. This moiety can be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is attached to the cyclic carrier by an intervening tethering. Thus, cyclic carriers often contain a functional group, such as an amino group, or generally provide a bond suitable for incorporation or tethering of another chemical entity, such as a ligand, to the ring.

[0243] The RNAi agent may be conjugated to the ligand via a carrier, which 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.

[0244] 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 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13. These agents may further comprise a ligand.

[0245] IV. Ligand-conjugated iRNA Another modification of the RNA of the iRNA of the invention involves chemically linking to the RNA one or more ligands, moieties, or complexes 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).

[0246] 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 for 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.

[0247] 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.

[0248] The ligand can also include a targeting group such as a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid, or protein, e.g., an antibody that binds to a specific cell type, such as a kidney cell. The targeting group can be thyroid stimulating hormone, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetylglucoseamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptidomimetic.

[0249] 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 ...

[0250] 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.

[0251] 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.

[0252] 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 containing 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 containing 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.

[0253] 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.

[0254] 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.

[0255] 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.

[0256] 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.

[0257] 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.

[0258] 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.

[0259] In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, it binds to 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.

[0260] 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.

[0261] 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).

[0262] 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.

[0263] 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.

[0264] 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, having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 24). An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 25)) can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, capable of transporting numerous polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 26)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 27)) 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.

[0265] 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.

[0266] 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).

[0267] 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 bonded 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 bonded 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 AGT and above (e.g., AGT, C6, C7, or C8); di- and trisaccharides include sugars with two or three monosaccharide units (e.g., AGT, C6, C7, or C8).

[0268] In one embodiment, the carbohydrate conjugate used in the compositions and methods of the present invention is a monosaccharide. In one embodiment, the monosaccharide is [ka] and other N-acetylgalactosamines.

[0269] In another embodiment, the carbohydrate conjugate used in the compositions and methods of the present invention comprises: [ka] [ka] [ka] [ka] [ka] is selected from the group consisting of:

[0270] Other exemplary carbohydrate conjugates for use in the embodiments described herein include, but are not limited to: [ka] (Formula XXIII) is included, where when one of X or Y is an oligonucleotide, the other is hydrogen.

[0271] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, including, but not limited to, a PK modulator and / or a cell-penetrating peptide.

[0272] Additional carbohydrate conjugates suitable for use in the present invention include those described in WO 2014 / 179620 and WO 2014 / 179627, the entire contents of each of which are incorporated herein by reference.

[0273] 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.

[0274] 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, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroaryl Alkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl , alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, etc., in which one or more methylenes can be interrupted or terminated by O, S, S(O), SO, N(R), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle (where R 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.

[0275] 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).

[0276] 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.

[0277] 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.

[0278] 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.

[0279] Linkers containing peptide bonds may be used in targeting peptidase-rich cell types such as hepatocytes and synoviocytes.

[0280] 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 evaluations in cell-free or culture conditions and confirm with further evaluations 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).

[0281] 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.

[0282] ii. Phosphate-based cleavable linkers In another embodiment, the cleavable linker comprises a phosphate-based cleavable linker. The 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.

[0283] 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). In a preferred embodiment, when a carbon is attached to the oxygen of the ester (alkoxy group), the group is 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.

[0284] 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.

[0285] v. Peptide-Based Cleavage Groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linker. Peptide-based cleavable linkers are cleaved intracellularly by enzymes such as peptidases and proteases. Peptide-based cleavable linkers 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 linkers 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.

[0286] In one embodiment, the iRNA of the invention is conjugated to a carbohydrate via a linker. Non-limiting examples of iRNA-carbohydrate conjugates containing linkers of the compositions and methods of the invention include, but are not limited to: [ka] [ka] [ka] [When one of X or Y is an oligonucleotide, the other is hydrogen].

[0287] In certain embodiments of the compositions and methods of the present invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a bivalent or trivalent branched linker.

[0288] In one embodiment, the dsRNA of the present invention has the structure represented by formula (XXXII) to (XXXV): [ka] [In formula: q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C independently represent 0 to 20 for each occurrence, and the repeat units may be the same or different; P2A, P2B, P3A, P3B, P4A, P4B, P5A, P5B, P5C, T2A, T2B, T3A, T3B, T4A, T4B, T4A, T5B, T5C are each independently, at each occurrence, absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CHO; Q2A, Q2B, Q3A, Q3B, Q4A, Q4B, Q5A, Q5B, Q5C, independently at each occurrence, is absent, alkylene, or substituted alkylene, wherein one or more methylenes may be interrupted or terminated by one or more of O, S, S(O), SO2, N(RN), C(R')=C(R"), C≡C, or C(O); R2A, R2B, R3A, R3B, R4A, R4B, R5A, R5B, and R5C each independently represent, for each occurrence, either absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(Ra)C(O), -C(O)-CH(Ra)-NH-, CO, CH=NO, [ka] or heterocyclyl; L2A, L2B, L3A, L3B, L4A, L4B, L5A, L5B, and L5C represent ligands; i.e., each independently at each occurrence represents a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and Ra 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 shown in any of the following: [ka] wherein L5A, L5B and L5C represent monosaccharides such as GalNAc derivatives.

[0289] 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.

[0290] 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.

[0291] 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.

[0292] "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.

[0293] 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.

[0294] V. Delivery of iRNA of the Invention Delivery of an iRNA of the invention to a cell, e.g., a cell in a subject, e.g., a human subject (e.g., a subject in need thereof, e.g., a subject with an APOC3-associated disease), 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 containing 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.

[0295] 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 binding of iRNA molecules (which are negatively charged) and also enhance 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 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.

[0296] A. Vectors Encoding iRNAs of the Invention APOC3 gene-targeting iRNAs can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A., et al., TIG. (1996), 12:5-10; Skillern, A., et al., WO 00 / 22113; Conrad, WO 00 / 22114; and Conrad, U.S. Pat. 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).

[0297] 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.

[0298] 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.

[0299] 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) to transfected cells, such as hygromycin B resistance.

[0300] 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.

[0301] 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.

[0302] 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.

[0303] 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.

[0304] 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.

[0305] 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.

[0306] 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.

[0307] 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.

[0308] 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.

[0309] VI. Pharmaceutical Compositions of the Present Invention The present invention also includes pharmaceutical compositions and formulations containing the iRNA of the present invention. In one embodiment, provided herein is a pharmaceutical composition containing an iRNA as described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing iRNA are useful for treating diseases or disorders associated with APOC3 gene expression or activity. Such pharmaceutical compositions are formulated based on the delivery method. One example is a composition formulated for systemic administration via parenteral delivery, e.g., by subcutaneous (SC) or intravenous (IV) delivery. Another example is a composition formulated for direct delivery to the brain parenchyma, e.g., by injection into the brain, e.g., by continuous pump infusion. Pharmaceutical compositions of the present invention can be administered at a dosage sufficient to inhibit APOC3 gene expression. In general, suitable doses of the iRNA of the present invention can range from about 0.001 to about 200.0 milligrams per kilogram of recipient body weight per day, generally from about 1 to 50 mg per kilogram of body weight per day. For example, the dsRNA can be administered at about 0.01 mg / kg, about 0.05 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, about 5 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, or about 50 mg / kg per single dose.

[0310] For example, the dsRNA may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, The compound may be administered at a dose of 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0311] In another embodiment, the dsRNA is about 0.1 to about 50 mg / kg, about 0.25 to about 50 mg / kg, about 0.5 to about 50 mg / kg, about 0.75 to about 50 mg / kg, about 1 to about 50 mg / kg, about 1.5 to about 50 mg / kg, about 2 to about 50 mg / kg, about 2.5 to about 50 mg / kg, about 3 to about 50 mg / kg, about 3.5 to about 50 mg / kg, about 4 to about 50 mg / kg, about 4.5 to about 50 mg / kg, about 5 to about 50 mg / kg, about 7.5 to about 50 mg / kg, about 10 to about 50 mg / kg, about 15 to about 50 mg / kg, about 20 to about 50 mg / kg, or about 20 to about 50 mg / kg, about 25 to about 50 mg / kg, about 25 to about 50 mg / kg, about 30 to about 50 mg / kg, about 35 to about 50 mg / kg, about 40 to about 50 mg / kg, about 45 to about 50 mg / kg, about 0.1 to about 45 mg / kg, about 0.25 to about 45 mg / kg, about 0. 5 to about 45 mg / kg, about 0.75 to about 45 mg / kg, about 1 to about 45 mg / kg, about 1.5 to about 45 mg / kg, about 2 to about 45 mg / kg, about 2.5 to about 45 mg / kg, about 3 to about 45 mg / kg, about 3.5 to about 45 mg / kg, about 4 to about 45 mg / kg, about 4.5 to about 45 mg / kg, about 5 to about 45 mg / kg, about 7.5 to about 45 mg / kg, about 10 to about 45 mg / kg, about 15 to about 45 mg / kg, about 20 to about 45 mg / kg, about 20 to about 45 mg / kg, about 25 to about 45 mg / kg, about 25 to about 45 mg / kg, about 30 to about 45 mg / kg kg, about 35 to about 45 mg / kg, about 40 to about 45 mg / kg, about 0.1 to about 40 mg / kg, about 0.25 to about 40 mg / kg, about 0.5 to about 40 mg / kg, about 0.75 to about 40 mg / kg, about 1 to about 40 mg / kg, about 1.5 to about 40 mg / kg, about 2 to about 40 mg / kg kg, about 2.5 to about 40 mg / kg, about 3 to about 40 mg / kg, about 3.5 to about 40 mg / kg, about 4 to about 40 mg / kg, about 4.5 to about 40 mg / kg, about 5 to about 40 mg / kg, about 7.5 to about 40 mg / kg, about 10 to about 40 mg / kg, about 15 to about 40 mg / kg, about 20 to about 40 mg / kg, about 20 to about 40 mg / kg, about 25 to about 40 mg / kg, about 25 to about 40 mg / kg, about 30 to about 40 mg / kg, about 35 to about 40 mg / kg, about 0.1 to about 30 mg / kg, about 0.25 to about 30 mg / kg, about 0.5 to about 30 mg / kg, about 0.75 to about 30 mg / kg, about 1 to about 30 mg / kg, about 1.5 to about 30 mg / kg, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg, about 4 to about 30 mg / kg, about 4.5 to about 30mg / kg, about 5 to about 30mg / kg, about 7.5 to about 30mg / kg, about 10 to about 30mg / kg, about 15 to about 30mg / kg, about 20 to about 30mg / kg, about 20 to about 30mg / kg, about 25 to about 30mg / kg, about 0.1 to about 20m g / kg, about 0.25 to about 20 mg / kg, about 0.5 to about 20 mg / kg, about 0.75 to about 20 mg / kg, about 1 to about 20 mg / kg, about 1.5 to about 20 mg / kg, about 2 to about 20 mg / kg, about 2.5 to about 20 mg / kg, about 3 to about 20 mg / kg, about 3.5 to about 20 mg / kg, about 4 to about 20 mg / kg, about 4.5 to about 20 mg / kg, about 5 to about 20 mg / kg, about 7.5 to about 20 mg / kg, about 10 to about 20 mg / kg, or about 15 to about 20 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of the invention.

[0312] For example, the dsRNA may have a concentration of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5 , 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0313] In another embodiment, the dsRNA is about 0.5 to about 50 mg / kg, about 0.75 to about 50 mg / kg, about 1 to about 50 mg / kg, about 1.5 to about 50 mg / kg, about 2 to about 50 mg / kg, about 2.5 to about 50 mg / kg, about 3 to about 50 mg / kg, about 3.5 to about 50 mg / kg, about 4 to about 50 mg / kg, about 4.5 to about 50 mg / kg, about 5 to about 50 mg / kg, about 7.5 to about 50 mg / kg, about 10 to about 50 mg / kg, about 15 to about 50 mg / kg, about 20 to about 50 mg / kg, about 20 to about 50 mg / kg, about 25 to about 50 mg / kg, or about 25 to about 50 mg / kg. g / kg, about 30 to about 50 mg / kg, about 35 to about 50 mg / kg, about 40 to about 50 mg / kg, about 45 to about 50 mg / kg, about 0.5 to about 45 mg / kg, about 0.75 to about 45 mg / kg, about 1 to about 45 mg / kg, about 1.5 to about 45 mg / kg, about 2 to about 45 mg / kg, About 2.5 to about 45 mg / kg, about 3 to about 45 mg / kg, about 3.5 to about 45 mg / kg, about 4 to about 45 mg / kg, about 4.5 to about 45 mg / kg, about 5 to about 45 mg / kg, about 7.5 to about 45 mg / kg, about 10 to about 45 mg / kg, about 15 to about 45 mg / kg, about 20 to about 4 5mg / kg, about 20 to about 45mg / kg, about 25 to about 45mg / kg, about 25 to about 45mg / kg, about 30 to about 45mg / kg, about 35 to about 45mg / kg, about 40 to about 45mg / kg, about 0.5 to about 40mg / kg, about 0.75 to about 40mg / kg, about 1 to about 40mg / k g, about 1.5 to about 40 mg / kg, about 2 to about 40 mg / kg, about 2.5 to about 40 mg / kg, about 3 to about 40 mg / kg, about 3.5 to about 40 mg / kg, about 4 to about 40 mg / kg, about 4.5 to about 40 mg / kg, about 5 to about 40 mg / kg, about 7.5 to about 40 mg / kg, about 10 to about Approximately 40 mg / kg, approximately 15 to approximately 40 mg / kg, approximately 20 to approximately 40 mg / kg, approximately 20 to approximately 40 mg / kg, approximately 25 to approximately 40 mg / kg, approximately 25 to approximately 40 mg / kg, approximately 30 to approximately 40 mg / kg, approximately 35 to approximately 40 mg / kg, approximately 0.5 to approximately 30 mg / kg, approximately 0.75 to approximately 30 m g / kg, about 1 to about 30 mg / kg, about 1.5 to about 30 mg / kg, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg, about 4 to about 30 mg / kg, about 4.5 to about 30 mg / kg, about 5 to about 30 mg / kg, about 7.It is administered at a dose of 5 to about 30 mg / kg, about 10 to about 30 mg / kg, about 15 to about 30 mg / kg, about 20 to about 30 mg / kg, about 20 to about 30 mg / kg, about 25 to about 30 mg / kg, about 0.5 to about 20 mg / kg, about 0.75 to about 20 mg / kg, about 1 to about 20 mg / kg, about 1.5 to about 20 mg / kg, about 2 to about 20 mg / kg, about 2.5 to about 20 mg / kg, about 3 to about 20 mg / kg, about 3.5 to about 20 mg / kg, about 4 to about 20 mg / kg, about 4.5 to about 20 mg / kg, about 5 to about 20 mg / kg, about 7.5 to about 20 mg / kg, about 10 to about 20 mg / kg, or about 15 to about 20 mg / kg. In one embodiment, the dsRNA is administered at a dose of about 10 mg / kg to about 30 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of the invention.

[0314] For example, targets may include approximately 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 75, 76, 77, 78, 79, 80, 81 .1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6 .4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.6, 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, 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, A single therapeutic dose of iRNA may be administered, for example, subcutaneously or intravenously, such as 8, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of the invention.

[0315] In some embodiments, the subject is administered a dose of about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0. 975,1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9,4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9,5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9,6, 6.1, 6.2, 6 .3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, Multiple doses of therapeutic amounts of iRNA are administered, for example, subcutaneously or intravenously, such as 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg / kg. A multiple dose regimen can include daily administration of a therapeutic amount of iRNA, such as over 2, 3, 4, 5, 6, 7, or more days.

[0316] In other embodiments, the subject is administered a dose of about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0. 975,1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9,4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9,5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9,6, 6.1, 6.2, 6 .3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, Repeated doses of therapeutic iRNA, such as 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg / kg, are administered, for example, subcutaneously or intravenously. Repeated dose regimens can include periodic administration of therapeutic iRNA, such as every other day, every three days, every four days, twice a week, once a week, every other week, or once a month.

[0317] In certain embodiments, for example, when the compositions of the present invention comprise a dsRNA and a lipid as described herein, the subject is administered a dose of about 0.01 mg / kg to about 5 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.05 mg / kg to about 5 mg / kg, about 0.05 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 10 mg / kg, or about 0.2 mg / kg to about 5 mg / kg, about 0.2mg / kg to about 10mg / kg, about 0.3mg / kg to about 5mg / kg, about 0.3mg / kg to about 10mg / kg, about 0.4mg / kg to about 5mg / kg, about 0.4mg / kg to about 10mg / kg, about 0.5mg / kg to about 5mg / kg, about 0.5mg / kg to about 10mg / kg, about 1mg / kg to about 5mg / kg, about 1mg / kg to about 10mg / kg, about 1.5mg / kg to about 5mg / kg, about 1 .5mg / kg~about 10mg / kg, about 2mg / kg~about 2.5mg / kg, about 2mg / kg~about 10mg / kg, about 3mg / kg~about 5mg / kg, about 3mg / kg~about 10mg / kg, about 3.5mg / k g~about 5mg / kg, about 4mg / kg~about 5mg / kg, about 4.5mg / kg~about 5mg / kg, about 4mg / kg~about 10mg / kg, about 4.5mg / kg~about 10mg / kg, about 5mg / kg~about 10mg / Therapeutic amounts of iRNA may be administered, such as about 5.5 mg / kg to about 10 mg / kg, about 6 mg / kg to about 10 mg / kg, about 6.5 mg / kg to about 10 mg / kg, about 7 mg / kg to about 10 mg / kg, about 7.5 mg / kg to about 10 mg / kg, about 8 mg / kg to about 10 mg / kg, about 8.5 mg / kg to about 10 mg / kg, about 9 mg / kg to about 10 mg / kg, or about 9.5 mg / kg to about 10 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of the invention.

[0318] For example, the dsRNA may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.2, 10.3, 1 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of the invention.

[0319] In certain embodiments of the invention, for example, when a double-stranded RNAi agent includes modifications (e.g., one or more motifs of three identical modifications on three consecutive nucleotides), e.g., one such motif at or near the cleavage site of the agent, six phosphorothioate linkages, and a ligand, such an agent may be administered at a dose of about 0.01 to about 0.5 mg / kg, about 0.01 to about 0.4 mg / kg, about 0.01 to about 0.3 mg / kg, about 0.01 to about 0.2 mg / kg, about 0.01 to about 0.1 mg / kg, about 0.01 mg / kg to about 0.09 mg / kg, about 0.01 mg / kg to about 0.1 ... about 0.08mg / kg, about 0.01mg / kg to about 0.07mg / kg, about 0.01mg / kg to about 0.06mg / kg, about 0.01mg / kg to about 0.05mg / kg, about 0.02 to about 0.5mg / kg, about 0.02 to about 0.4mg / kg, about 0.02 to about 0.3mg / kg, about 0.02 to about 0.2mg / kg, about 0.02 to about 0.1mg / kg, about 0.02mg / kg to about 0.09mg / kg, about 0.02mg / kg to about 0.08mg / kg, about 0.02mg / kg to about 0.07mg / kg, about 0.02mg / kg to about 0.06mg / kg, about 0 0.02mg / kg to about 0.05mg / kg, about 0.03 to about 0.5mg / kg, about 0.03 to about 0.4mg / kg, about 0.03 to about 0.3mg / kg, about 0.03 to about 0.2mg / kg, about 0.03 to about 0.1mg / kg, about 0.03mg / kg to about 0.09mg / kg, about 0.03mg / kg to about 0.08mg / kg, about 0.03mg / kg to about 0.07mg / kg, about 0.03mg / kg to about 0.06mg / kg, about 0.03mg / kg to about 0.05mg / kg, about 0.04 to about 0.5mg / kg, about 0.04 to about 0.4mg / kg, about 0.04 ~0.3mg / kg, 0.04~0.2mg / kg, 0.04~0.1mg / kg, 0.04mg / kg~0.09mg / kg, 0.04mg / kg~0.08mg / kg, 0.04mg / kg~0.07mg / kg, 0.04mg / kg~0. 0.06 mg / kg, about 0.05 to about 0.5 mg / kg, about 0.05 to about 0.4 mg / kg, about 0.05 to about 0.3 mg / kg, about 0.05 to about 0.2 mg / kg, about 0.05 to about 0.1 mg / kg, about 0.05 mg / kg to about 0.09 mg / kg, about 0.05 mg / kg to about 0.The RNAi agent may be administered at a dose of about 0.08 mg / kg, or about 0.05 mg / kg to about 0.07 mg / kg. Values ​​and ranges intermediate to the above-listed values ​​are also contemplated as part of the invention; for example, the RNAi agent may be administered to a subject at a dose of about 0.015 mg / kg to about 0.45 mg / kg.

[0320] For example, the RNAi agent, e.g., the RNAi agent in the pharmaceutical composition, may be about 0.01 mg / kg, 0.0125 mg / kg, 0.015 mg / kg, 0.0175 mg / kg, 0.02 mg / kg, 0.0225 mg / kg, 0.025 mg / kg, 0.0275 mg / kg, 0.03 mg / kg, 0.0325 mg / kg, 0.035 mg / kg, 0.0 375mg / kg, 0.04mg / kg, 0.0425mg / kg, 0.045mg / kg, 0.0475mg / kg, 0.05mg / kg, 0.0525mg / kg, 0 .055mg / kg, 0.0575mg / kg, 0.06mg / kg, 0.0625mg / kg, 0.065mg / kg, 0.0675mg / kg, 0.07mg / kg, 0 .0725mg / kg, 0.075mg / kg, 0.0775mg / kg, 0.08mg / kg, 0.0825mg / kg, 0.085mg / kg, 0.0875mg / k g, 0.09mg / kg, 0.0925mg / kg, 0.095mg / kg, 0.0975mg / kg, 0.1mg / kg, 0.125mg / kg, 0.15mg / kg, 0 The compound may be administered at a dose of 0.175 mg / kg, 0.2 mg / kg, 0.225 mg / kg, 0.25 mg / kg, 0.275 mg / kg, 0.3 mg / kg, 0.325 mg / kg, 0.35 mg / kg, 0.375 mg / kg, 0.4 mg / kg, 0.425 mg / kg, 0.45 mg / kg, 0.475 mg / kg, or about 0.5 mg / kg. Values ​​intermediate to the foregoing recited values ​​are also contemplated as part of this invention.

[0321] The pharmaceutical composition can be administered by intravenous infusion for a predetermined period of time, such as 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, biweekly (i.e., every two weeks), for one, two, three, four, or more months. After the 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 or one year or more.

[0322] The pharmaceutical composition can be administered once daily, or the iRNA can be administered as two, three, or more subdoses at appropriate intervals throughout the day, or even via continuous infusion or controlled-release delivery. In this case, the amount of iRNA contained in each subdose must be correspondingly smaller to achieve the total daily dose. The dosage unit can also be formulated for delivery over several days, for example, using a conventional sustained-release formulation that provides sustained release of the iRNA over several days. Sustained-release formulations are well known in the art and are particularly useful for site-specific agent delivery, such as those used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

[0323] In another embodiment, a single dose of the pharmaceutical composition can be administered over an extended period of time, such that subsequent doses are administered no more than 3, 4, or 5 days apart, or no more than 1, 2, 3, or 4 weeks apart. In some embodiments of the present invention, a single dose of the pharmaceutical composition of the present invention is administered once a week. In another embodiment of the present invention, a single dose of the pharmaceutical composition of the present invention is administered twice a month.

[0324] 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.

[0325] 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.

[0326] The iRNA can be delivered in a manner that targets a specific tissue, such as the liver (e.g., liver parenchymal cells).

[0327] 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, distearoylphosphatidylcholine), anionic (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The iRNAs featured in the present invention can be encapsulated in or complexed with liposomes, particularly cationic liposomes. Alternatively, the iRNAs can be complexed with lipids, particularly cationic lipids. Suitable fatty acids and esters include, but are not limited to, 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, acylcarnitines, acylcholines, or C1-20 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, incorporated herein by reference.

[0328] A. iRNA formulations containing membrane-like molecular assemblies The iRNAs used in the compositions and methods of the present invention may be formulated for delivery within membrane-like molecular assemblies, such as liposomes or micelles. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one or more bilayers. Liposomes include unilamellar or multilamellar vesicles, with a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material separates the aqueous interior from the aqueous exterior, which typically does not contain the iRNA composition, but may in some cases. Liposomes are useful for transporting and delivering active ingredients to their site of action. Because the liposome membrane is structurally similar to biological membranes, upon application of the liposome to a tissue, the liposomal bilayer fuses with the bilayer of the cell membrane. As fusion of the liposome with the cell proceeds, the internal aqueous contents, including the iRNA, are delivered into the cell, where the iRNA can specifically bind to the target RNA and mediate iRNA activity. In some cases, liposomes are also specifically targeted, for example, to direct iRNA to a particular cell type.

[0329] Liposomes containing iRNA agents can be prepared by a variety of methods. In one example, the lipid components of the liposomes are dissolved in a detergent so that micelles form without the lipid components. For example, the lipid components can be amphipathic cationic lipids or lipid complexes. The detergent can have a high critical micelle concentration and can be non-ionic. Exemplary detergents include cholic acid, CHAPS, octylglucoside, deoxycholic acid, and lauroyl sarcosine. The iRNA agent preparation is then added to the micelles containing the lipid components. The cationic groups on the lipids interact with the iRNA agent, condensing around the iRNA agent to form liposomes. After condensation, the detergent is removed, for example, by dialysis, to yield a liposome preparation of the iRNA agent.

[0330] If necessary, a carrier compound, e.g., to aid in condensation, can be added during the condensation reaction by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). The pH can also be adjusted to aid in condensation.

[0331] Methods for generating stable polynucleotide delivery vehicles that incorporate polynucleotide / cationic lipid complexes as structural components of the delivery vehicle are further described, for example, in WO 96 / 37194, the entire contents of which are incorporated herein by reference. Liposome formation is described by Felgner, PLet al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678; Bangham, et al. al.Biochim.Biophys.Acta 557:9,1979;Szoka,et al.Proc.Natl.Acad.Sci.75:4194,1978;Mayhew,et al.Biochim.Biophys.Acta 775:169,1984;Kim,et al.Biochim.Biophys.Acta 728:339,1983; and Fukunaga, et al. The present invention may also include one or more embodiments of the exemplary method described in [Endocrinol. 115:757, 1984]. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and a combination of freeze-thawing and extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). If consistently small (50-200 nm) and relatively uniform aggregates are desired, microfluidization can be used (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are easily adapted to the packaging of RNAi agent preparations within liposomes.

[0332] Liposomes are divided into two broad classes: cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes; the positively charged nucleic acid / liposome complexes bind to the negatively charged cell surface and are internalized inside endosomes; the acidic pH within the endosomes causes the liposomes to rupture, releasing their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0333] pH-sensitive or negatively charged liposomes do not complex with nucleic acids but rather encapsulate them. Because both nucleic acids and lipids have similar charges, repulsion occurs rather than complexation. Nevertheless, some nucleic acid is encapsulated within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected within the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0334] One major type of liposome composition contains phospholipids in addition to naturally occurring phosphatidylcholine. For example, neutral liposome compositions can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are primarily formed from dioleoylphosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0335] Other examples of methods for introducing liposomes into cells in vitro and in vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94 / 00569; WO 93 / 24640; WO 91 / 16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

[0336] Nonionic liposomal systems, particularly those containing nonionic surfactants and cholesterol, have been studied to determine their efficacy in delivering drugs to the skin. Cyclosporine A was delivered into the dermis of mouse skin using nonionic liposomal formulations containing Novasome™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether). The results suggested that such nonionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al. STP Pharma. Sci., 1994, 4, 6, 466).

[0337] Liposomes also include "sterically stabilized" liposomes, which, as used herein, refer to liposomes containing one or more specialized lipids that, when incorporated into the liposome, result in improved circulation life compared to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which a portion of the vesicle-forming lipid portion of the liposome (A) contains one or more glycolipids, such as monosialoganglioside GM1, or (B) is derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. While not wishing to be bound by any particular theory, it is believed in the art that the improved circulation half-life of these sterically stabilized liposomes, at least for those containing gangliosides, sphingomyelin, or PEG-derivatized lipids, is due to reduced uptake into reticuloendothelial system (RES) cells (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0338] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., 1987, 507, 64) reported that monosialoganglioside GM1, galactocerebroside sulfate and phosphatidylinositol can improve the blood half-life of liposomes. These findings were elaborated by Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). U.S. Patent No. 4,837,028 and International Publication No. 88 / 04924, both to Allen et al., disclose liposomes containing (1) sphingomyelin and (2) ganglioside GM1 or galactocerebroside sulfate. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97 / 13499 (Lim et al.).

[0339] In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse with the cell membrane. Non-cationic liposomes cannot fuse efficiently with the plasma membrane, but can be taken up by macrophages in vivo and used to deliver iRNA agents to macrophages.

[0340] Additional advantages of liposomes include: Liposomes derived from natural phospholipids are biocompatible and biodegradable; Liposomes can incorporate a wide range of water- and lipid-soluble drugs; and Liposomes can protect iRNA agents encapsulated in their internal compartments from metabolism and degradation (Rosoff, "Pharmaceutical Dosage Forms," ​​Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in preparing liposome formulations are the lipid surface charge, vesicle size, and aqueous volume of the liposomes.

[0341] The positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), can be used to form small liposomes that spontaneously interact with nucleic acids to form lipid-nucleic acid complexes that can fuse with the negatively charged lipids of the plasma membrane of tissue culture cells, resulting in iRNA agent delivery (see, e.g., Felgner, PL et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987, and U.S. Pat. No. 4,897,355, for a description of DOTMA and its use in combination with DNA).

[0342] The ADOTMA analog 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with phospholipids to form DNA-complexed vesicles. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivering highly anionic nucleic acids to living tissue culture cells. It contains positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. If sufficiently positively charged liposomes are used, the net charge on the resulting complexes is also positive. The positively charged complexes thus prepared spontaneously adhere to negatively charged cell surfaces and fuse with the plasma membrane, efficiently delivering functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Indiana), differs from DOTMA in that the oleoyl moiety is attached by an ester rather than an ether bond.

[0343] Other reported cationic lipid compounds include those conjugated to a variety of moieties, including carboxyspermine conjugated to one of two lipid types, such as compounds such as 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (Transfectam™, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide ("DPPES") (see, e.g., U.S. Pat. No. 5,171,678).

[0344] Another cationic lipid complex involves derivatizing lipids with cholesterol ("DC-Chol") in combination with DOPE and formulated into liposomes (see Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, produced by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). In certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more efficient transfection than DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California), and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for delivery of oligonucleotides are described in WO 98 / 39359 and WO 96 / 37194.

[0345] Liposomal formulations are particularly suitable for topical administration, and liposomes offer several advantages over other formulations. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer the iRNA agent intradermally. In some implementations, liposomes are used to deliver iRNA agents to epidermal cells and to enhance the penetration of iRNA agents into skin tissues, such as within the skin. For example, liposomes can be applied topically. Topical delivery of therapeutic agents formulated as liposomes to the skin has been demonstrated (e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, RJ and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276, 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R M and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C Y and See Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

[0346] Nonionic liposomal systems, particularly those containing nonionic surfactants and cholesterol, have been studied to determine their utility in delivering drugs to the skin. Nonionic liposomal formulations containing Novasome I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) have been used to deliver drugs into the dermis of mouse skin. Such formulations containing iRNA agents are useful for treating skin diseases.

[0347] Liposomes containing iRNA can be highly deformable. Such deformability can allow the liposome to penetrate pores smaller than the average diameter of the liposome. For example, transferosomes are a type of deformable liposome. Transferosomes can be created by adding a surface edge activator, usually a surfactant, to a standard liposome composition. Transfersomes containing iRNA agents can be delivered subcutaneously, for example, by infection, to deliver the iRNA agent to keratinocytes within the skin. To cross intact mammalian skin, lipid vesicles must pass through a series of pores, each less than 50 nm in diameter, under the influence of an appropriate transdermal gradient. Furthermore, due to their lipid properties, these transferosomes can self-optimize (e.g., adapt to the shape of skin pores), self-repair, frequently reach their targets without fragmentation, and are often self-loading.

[0348] Other formulations to which the present invention may be applied are described in U.S. Provisional Patent Application Nos. 61 / 018,616, filed January 2, 2008; 61 / 018,611, filed January 2, 2008; 61 / 039,748, filed March 26, 2008; 61 / 047,087, filed April 22, 2008; and 61 / 051,528, filed May 8, 2008. PCT application PCT / US2007 / 080331, filed October 3, 2007, also describes formulations to which the present invention may be applied.

[0349] Transfersomes are yet another type of liposome, highly deformable lipid aggregates that are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets that are so highly deformable that they can easily penetrate pores smaller than droplets. Transfersomes can adapt to the environment in which they are used; for example, they self-optimize (adapt to the shape of skin pores), self-repair, often reach their targets without fragmentation, and are often self-loading. To create transfersomes, a surface edge activator, usually a surfactant, can be added to a standard liposome composition. Transfersomes have been used to deliver serum albumin to the skin. Transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0350] Surfactants have a wide range of applications in formulations such as emulsions (including microemulsions) and liposomes. The most common method of classifying and ranking the properties of the many different surfactant types, both natural and synthetic, is by using the hydrophile / lipophile balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means of classifying different surfactants used in formulations (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0351] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants have a wide range of applications in pharmaceutical and cosmetic products and can be used over a wide pH range. Their HLB values ​​generally range from 2 to approximately 18, depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers, are also included in this class. Polyoxyethylene surfactants are the most common members of the nonionic surfactant class.

[0352] If surfactant molecule carries negative charge when dissolved or dispersed in water, surfactant is classified as anionic.Anionic surfactants include carboxylates such as soap, acyl lactylate, acyl amide of amino acid, sulfate esters such as alkyl sulfate and ethoxylated alkyl sulfate, sulfonates such as alkyl benzene sulfonate, acyl isethionate, acyl taurate and acyl sulfosuccinate, and acyl phosphate.The most important members of anionic surfactant class are alkyl sulfate and soap.

[0353] If the surfactant molecule carries a positive charge when dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used members of this class.

[0354] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phospholipids.

[0355] The use of surfactants in pharmaceutical preparations and emulsions has been reviewed (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0356] The iRNA used in the methods of the present invention can also be provided as a micellar formulation. A "micelle" is defined herein as a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure so that the hydrophobic portions of the molecules all face inward and the hydrophilic portions remain in contact with the surrounding aqueous phase. If the environment is hydrophobic, the opposite arrangement will exist.

[0357] Mixed micelle formulations suitable for transdermal delivery may be prepared by mixing an siRNA composition, an alkali metal C8-C22 alkyl sulfate, and an aqueous solution of a micelle-forming compound. Exemplary micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleate, monolaurate, borage oil, evening primrose oil, menthol, trihydroxyoxocholanylglycine and its pharmaceutically acceptable salts, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and their analogs, polidocanol alkyl ethers and their analogs, chenodeoxycholic acid, deoxycholic acid, and mixtures thereof. The micelle-forming compound may be added simultaneously with or after the addition of the alkali metal alkyl sulfate. Mixed micelles form with virtually any mixing of the components, but are mixed vigorously to provide smaller micelles.

[0358] In one method, a first micelle composition is prepared, containing an siRNA composition and at least an alkali metal alkyl sulfate.The first micelle composition is then mixed with at least three micelle-forming compounds to form a mixed micelle composition.In another method, a micelle composition is prepared by mixing an siRNA composition, an alkali metal alkyl sulfate, and at least one micelle-forming compound, followed by adding the remaining micelle-forming compounds with vigorous mixing.

[0359] Phenol and / or m-cresol may be added to the mixed micelle composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and / or m-cresol may be added along with the micelle-forming components. An isotonicity agent, such as glycerin, may also be added after the mixed micelle composition is formed.

[0360] To deliver a micelle formulation as a spray, the formulation can be placed in an aerosol dispensing device, which can then be loaded with the propellant. The propellant, under pressure, is in liquid form within the dispensing device. The ratio of the components is adjusted so that the aqueous phase and the propellant phase are one, i.e., one phase. If there are two phases, the dispensing device must be shaken before dispensing a portion of the contents, for example, through a metered valve. The dispensed dose of the pharmaceutical product is expelled from the metered valve in a fine spray.

[0361] Propellants include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether, and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2-tetrafluoroethane) may be used.

[0362] The specific concentrations of the essential ingredients can be determined by relatively simple experimentation. For absorption through the oral cavity, it is often desirable to increase the dose, e.g., at least two or three times, that for administration through injection or through the gastrointestinal tract.

[0363] B. Lipid particles The iRNA, e.g., dsRNA, of the invention may be fully encapsulated in a lipid formulation, e.g., an LNP, or other nucleic acid-lipid particle.

[0364] As used herein, the term "LNP" refers to a stable nucleic acid-lipid particle. LNPs typically include cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). LNPs exhibit a long circulatory life after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically separated from the administration site), making them extremely useful for systemic application. Examples of LNPs include "pSPLPs" containing encapsulated condensing agent-nucleic acid complexes, as described in International Publication No. WO 00 / 03683. The particles of the present invention typically have an average diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially nontoxic. Additionally, when present in the nucleic acid-lipid particles of the present invention, the nucleic acid is resistant to nuclease degradation in aqueous solution. Nucleic acid-lipid particles, and methods for preparing them, are disclosed, for example, in U.S. Pat. No. 5,976,567; U.S. Pat. No. 5,981,501; U.S. Pat. No. 6,534,484; U.S. Pat. No. 6,586,410; U.S. Pat. No. 6,815,432; U.S. Patent Application Publication No. 2010 / 0324120; and WO 96 / 40964.

[0365] In one embodiment, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to dsRNA ratio) ranges from about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above-cited ranges are also considered part of the invention.

[0366] Cationic lipids include, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLenDMA), and 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLenDMA). 1,2-Dilinoleyl-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP).Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanedio(propanedio) (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), or or its analogs, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoic acid (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or mixtures thereof. The cationic lipid may comprise from about 20 mol% to about 50 mol% or about 40 mol% of the total lipid present in the particle.

[0367] In another embodiment, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles.The synthesis of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. Provisional Patent Application No. 61 / 107,998, filed October 23, 2008, which is incorporated herein by reference.

[0368] In one embodiment, the lipid-siRNA particles comprise 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane:10% DSPC:40% cholesterol:10% PEG-C-DOMG (molar percentages), with a particle size of 63.0±20 nm and an siRNA / lipid ratio of 0.027.

[0369] Ionic / non-cationic lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine-4-(N-maleimidomethyl)-2-methylpropional (DOPE), and dioleoyl-phosphatidylethanolamine-4-(N-maleimidomethyl)-2-methylpropional (DOPE). The lipid may be an anionic or neutral lipid, including, but not limited to, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), cholesterol, or mixtures thereof. When cholesterol is included, the non-cationic lipid may be about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipid present in the particle.

[0370] The conjugated lipid that inhibits particle aggregation can be, for example, without limitation, a polyethylene glycol (PEG)-lipid, including PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (C]8). The conjugated lipid that inhibits particle aggregation can be 0 mol% to about 20 mol% or about 2 mol% of the total lipid present in the particles.

[0371] In some embodiments, the nucleic acid-lipid particles further comprise cholesterol, for example, from about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

[0372] In one embodiment, lipid-dsRNA nanoparticles (i.e., LNP01 particles) can be created using lipidoid ND98·4HCl (MW 1487) (see U.S. Patent Application No. 12 / 056,230, filed March 26, 2008, the contents of which are incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar Lipids). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml; PEG-ceramide C16, 100 mg / ml. The ND98, cholesterol, and PEG-ceramide C16 stock solutions can then be combined in a molar ratio of, for example, 42:48:10. The combined lipid solution can then be mixed with aqueous dsRNA (e.g., in sodium acetate at pH 5) to achieve a final ethanol concentration of approximately 35-45% and a final sodium acetate concentration of approximately 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously when mixed.Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cutoff) using a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc.).In some cases, the extrusion step can be omitted.Ethanol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration.The buffer can be exchanged with phosphate buffered saline (PBS) at about pH 7, for example, about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4. [ka]

[0373] LNP01 formulations are described, for example, in WO 2008 / 042973, which is hereby incorporated by reference.

[0374] Further exemplary lipid-dsRNA formulations are listed in Table 1.

[0375] [Table 1]

[0376] [Table 2]

[0377] [Table 3]

[0378] Formulations containing SNALP (1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) are described in WO 2009 / 127060, filed April 15, 2009, which is incorporated herein by reference.

[0379] Formulations containing XTC are described, for example, in U.S. Provisional Application Nos. 61 / 148,366, filed January 29, 2009; 61 / 156,851, filed March 2, 2009; 61 / 156,851, filed June 10, 2009; 61 / 228,373, filed July 24, 2009; 61 / 239,686, filed September 3, 2009; and International Application No. PCT / US2010 / 022614, filed January 29, 2010, which are incorporated herein by reference.

[0380] Formulations containing MC3 are described, for example, in US Patent Application Publication No. 2010 / 0324120, filed June 10, 2010, the entire contents of which are incorporated herein by reference.

[0381] ALNY-100-containing formulations are described, for example, in International Application PCT / US09 / 63933, filed November 10, 2009, which is incorporated herein by reference.

[0382] C12-200-containing formulations are described in U.S. Provisional Patent Application No. 61 / 175,770, filed May 5, 2009, and International Application No. PCT / US10 / 33777, filed May 5, 2010, which are incorporated herein by reference.

[0383] 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.

[0384] 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.

[0385] 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.

[0386] 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.

[0387] 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.

[0388] 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 containing 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 containing 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.

[0389] 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).

[0390] 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 parts. 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).

[0391] 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.

[0392] 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).

[0393] Hydrophilic colloids, or hydrocolloids, include natural gums and synthetic polymers such as polysaccharides (e.g., acacia, agar, alginate, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethyl cellulose and carboxypropyl cellulose), and synthetic polymers (e.g., carbomer, cellulose ethers, and carboxyvinyl polymers), which disperse in or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around dispersed phase droplets and by increasing the viscosity of the external phase.

[0394] Emulsions often contain several components, such as carbohydrates, proteins, sterols, and phospholipids, which can easily support the growth of microorganisms, so preservatives are often incorporated into these preparations.The commonly used preservatives contained in emulsion preparations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid.Antioxidants are also generally added to emulsion preparations to prevent the preparation from deteriorating.The antioxidants used can be free radical scavengers such as tocopherol, alkyl gallate, butylated hydroxyanisole, butylated hydroxytoluene; or reducing agents such as ascorbic acid and sodium metabisulfite; and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0395] The application of emulsion formulations via dermal, oral, and parenteral routes and methods for preparing them have been reviewed in the 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; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Emulsion formulations for oral delivery are very widely used due to their ease of preparation and efficiency in terms of absorption and bioavailability (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; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Mineral oil-based laxatives, oil-soluble vitamins, and high-fat nutrients are among the materials commonly administered orally as o / w emulsions.

[0396] ii. Microemulsions In one embodiment of the present invention, the iRNA and nucleic acid compositions are formulated as microemulsions. A microemulsion can be defined as a system of water, oil, and an amphiphile that is a single optically isotropic and thermodynamically stable solution (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; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245). Typically, microemulsions are prepared by first dispersing oil in an aqueous surfactant solution, and then adding a sufficient amount of a fourth component, typically a medium-chain alcohol, to form a transparent system. Thus, microemulsions are described as thermodynamically stable, isotropically transparent dispersions of two immiscible liquids stabilized by an interfacial film of surface-active molecules (Leung and Shah, Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions are typically prepared through the combination of three to five components, including oil, water, surfactant, cosurfactant, and electrolyte. Whether a microemulsion is water-in-oil (w / o) or oil-in-water (o / w) depends on the properties of the oil and surfactant used and the structure and geometric packing of the polar head and hydrocarbon tail of the surfactant molecule (Schott, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0397] The phenomenological approach using phase diagrams has been extensively studied, providing those skilled in the art with comprehensive knowledge of microemulsion formulation (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; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245; Block, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs into a formulation of spontaneously formed, thermodynamically stable droplets.

[0398] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, nonionic surfactants, Brij 96, polyoxyethylene oleyl ether, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), and decaglycerol decaoleate (DAO750), either alone or in combination with cosurfactants. Cosurfactants, which are typically short-chain alcohols such as ethanol, 1-propanol, and 1-butanol, help increase interfacial fluidity by penetrating the surfactant film, resulting in irregular coatings due to the gaps between surfactant molecules. However, microemulsions can be prepared without the use of cosurfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, aqueous pharmaceutical solutions, glycerol, PEG 300, PEG 400, polyglycerol, propylene glycol, and ethylene glycol derivatives. The oil phase can include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium-chain (C8-C12) mono-, di-, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oils.

[0399] Microemulsions are of particular interest from the standpoint of drug solubilization and improved drug absorption. Lipid-based microemulsions (both o / w and w / o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see, e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions offer the advantages of improved drug solubilization, drug protection from enzymatic hydrolysis, potential drug absorption enhancement due to surfactant-induced changes in membrane fluidity and permeability, ease of preparation, ease of oral administration compared to solid dosage forms, improved clinical efficacy, and reduced toxicity (see, e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Microemulsions can often form spontaneously when their components are combined at ambient temperature. This can be particularly advantageous when formulating thermolabile drugs, peptides, or iRNAs. Microemulsions have been effective in transdermal delivery of active ingredients for both cosmetic and pharmaceutical applications. The microemulsion compositions and formulations of the present invention are expected to facilitate increased systemic absorption of iRNA and nucleic acids from the gastrointestinal tract and improve local cellular uptake of iRNA and nucleic acids.

[0400] The microemulsions of the present invention may also contain additional ingredients and additives, such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers, to improve the properties of the formulation and enhance the absorption of the iRNA and nucleic acids of the present invention. The penetration enhancers used in the microemulsions of the present invention can be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

[0401] iii. Particulates The iRNA agents of the invention may be incorporated into particles, such as, for example, microparticles. Microparticles can be produced by spray drying, but they can also be produced by other methods, including freeze drying, evaporation, fluidized bed drying, vacuum drying, or a combination of these techniques.

[0402] iv. Penetration enhancers In one embodiment, the present invention uses various penetration enhancers to achieve efficient delivery of nucleic acids, particularly iRNA, to animal skin. Most drugs exist in solution in both ionized and non-ionized forms. However, usually, only lipid-soluble or lipophilic drugs can easily pass through cell membranes. It has been discovered that even non-lipophilic drugs can pass through cell membranes if the membrane they pass through is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also increase the permeability of lipophilic drugs.

[0403] Penetration enhancers can be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the aforementioned classes of penetration enhancers is described in more detail below.

[0404] Surfactants (or "surface-active agents") are chemicals that, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, resulting in improved iRNA absorption through mucosal membranes. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, and polyoxyethylene-20-cetyl ether (see, e.g., Malmsten, M., Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0405] Various fatty acids and their derivatives that act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, their C1-20 alkyl esters (e.g., methyl, isopropyl, and t-butyl), and their mono- and di-glycerides (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.). (See, e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0406] The physiological role of bile includes promoting the dispersion and absorption of lipids and fat-soluble vitamins (see, for example, Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Brunton, Chapter 38, Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts and their synthetic derivatives act as penetration enhancers. Thus, the term "bile salt" includes any of the natural components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glycolic acid (sodium glycolate), glycolic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydrofusidate (STDHF), sodium glycodihydrofusidate, and polyoxyethylene-9-lauryl ether (POE).(See, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39, Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0407] Chelating agents used in the context of the present invention can be defined as compounds that complex with metal ions, removing them from solution and resulting in improved iRNA absorption through mucous membranes. Regarding their use as penetration enhancers in the present invention, chelating agents have the added advantage of also acting as deoxyribonuclease inhibitors, since most DNA nucleases require divalent metal ions for catalysis and are inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylic acid, and homovanilate), N-acyl derivatives of collagen, laureth-9, and N-aminoacyl derivatives of β-diketones (enamines). (See, e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0408] As used herein, a non-chelating, non-surfactant penetration enhancer may be defined as a compound that demonstrates insignificant activity as a chelating agent or as a surfactant, but still enhances the absorption of iRNA through the gastrointestinal mucosa (see, e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancer includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and nonsteroidal anti-inflammatory agents such as diclofenac sodium, indomethacin, and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0409] Agents that enhance cellular level iRNA uptake can also be added to the pharmaceutical and other compositions of the present invention.For example, cationic lipids such as lipofectin (U.S. Patent No. 5,705,188 to Junichi et al.), cationic glycerol derivatives, and polycationic molecules such as polylysine (WO 97 / 30731 to Lollo et al.) are also known to enhance cellular uptake of dsRNA. Examples of commercially available transfection reagents include, for example, Lipofectamine™ (Invitrogen; Carlsbad, CA), Lipofectamine 2000™ (Invitrogen; Carlsbad, CA), 293fectin™ (Invitrogen; Carlsbad, CA), Cellfectin™ (Invitrogen; Carlsbad, CA), DMRIE-C™ (Invitrogen; Carlsbad, CA), FreeStyle™ MAX (Invitrogen; Carlsbad, CA), Lipofectamine™ 2000, among others. CD (Invitrogen; Carlsbad, CA), Lipofectamine(TM) (Invitrogen; Carlsbad, CA), iRNAMAX(Invitrogen; Carlsbad, CA), Oligofectamine(TM) (Invitrogen; Carlsbad, CA), Optifect(TM) (Invitrogen; Carlsbad, CA), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;(Madison, WI), TransFast (trademark) Transfection Reagent (Promega; Madison, WI), Tfx (trademark)-20 Reagent (Promega; Madison, WI), Tfx (trademark)-50 Reagent (Promega; Madison, WI), DreamFect (trademark) (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPassa D1 Transfection Reagent (New England Biolabs; Ipswich, MA, USA), LyoVec (trademark) / LipoGen (trademark) (Invitrogen; San Diego, CA, USA), PerFectin Transfection Reagent (Genlantis; San Diego, CA, USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), GenePORTER Transfection Reagent (Genlantis; San Diego, CA, USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, CA, USA), Cytofectin Transfection Reagent (Genlantis; San Diego, CA, USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), TroganPORTER (trademark) transfection Reagent (Genlantis; San Diego, CA, USA), RiboFect (Bioline; Taunton, MA, USA), PlasFect (Bioline; Taunton, MA, USA), UniFECTOR (B-Bridge International; Mountain View, CA, USA), SureFECTOR (B-Bridge International;Mountain View, CA, USA), or HiFect™ (B-Bridge International, Mountain View, CA, USA);

[0410] Other agents can be utilized to enhance the penetration of the administered nucleic acid, including glycols, such as ethylene glycol and propylene glycol; pyrroles, such as 2-pyrrole; azone; and terpenes, such as limonene and menthone.

[0411] v. Carrier Certain compositions of the present invention also incorporate a carrier compound into their formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid, or an analog thereof, that is inert (i.e., has no biological activity itself) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of biologically active nucleic acids, for example, by degrading the biologically active nucleic acid or facilitating its removal from the circulation. Co-administration of nucleic acids and carrier compounds, typically in excess of the latter substance, can result in a substantial reduction in the amount of nucleic acid recovered in the liver, kidneys, or other extracirculatory reservoirs, likely due to competition between the carrier compound and the nucleic acid for their normal receptors. For example, recovery of partial phosphorothioate dsRNA in liver tissue can be reduced when it is co-administered with polyinosinic acid, dextran sulfate, polycytidic, or 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0412] vi. excipients In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. Excipients can be liquid or solid and are selected with the intended mode of administration in mind to provide the desired bulk, consistency, etc., when combined with the nucleic acids and other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binders (such as pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (such as lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, or calcium hydrogen phosphate); lubricants (such as magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (such as starch, sodium starch glycolate, etc.); and wetting agents (such as sodium lauryl sulfate, etc.).

[0413] The composition of the present invention can be prepared using pharmaceutically acceptable organic or inorganic excipients that do not adversely react with nucleic acid and are suitable for oral administration.Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solution, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone etc.

[0414] The preparation for topical administration of nucleic acid can include sterile and non-sterile aqueous solution, non-aqueous solution in common solvent such as alcohol, or nucleic acid solution in liquid or solid oil base.The solution can also contain buffer, diluent and other suitable additives.Pharmaceutically acceptable organic or inorganic excipients suitable for oral administration that do not adversely react with nucleic acid can be used.

[0415] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

[0416] vii. Other ingredients The compositions of the present invention may further contain other auxiliary ingredients conventionally found in pharmaceutical compositions at their technically established usage levels.Thus, for example, the compositions may contain additional compatible pharmacologically active ingredients, such as antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful for physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickeners, and stabilizers.However, when added, such materials should not excessively interfere with the biological activity of the components of the compositions of the present invention.The formulations may be sterilized and, if desired, mixed with auxiliary agents that do not adversely interact with the nucleic acid of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, colorants, flavorings and / or aromatic substances, etc.

[0417] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers.

[0418] In some embodiments, pharmaceutical compositions featured herein include (a) one or more iRNA compounds and (b) one or more agents that function via non-iRNA mechanisms and are useful for treating APOC-related disorders. Examples of such agents include, but are not limited to, anti-inflammatory agents, anti-lipid agents, antiviral agents, and / or anti-fibrotic agents. In addition, other substances commonly used to protect the liver, such as silymarin, may also be used in combination with the iRNAs described herein. Other agents useful for treating liver disease include telbivudine, entecavir, telaprevir, and protease inhibitors, such as those disclosed in U.S. Patent Application Publication Nos. 2005 / 0148548, 2004 / 0167116, and 2003 / 0144217 to Tung et al.; and U.S. Patent Application Publication No. 2004 / 0127488 to Hale et al.

[0419] The toxicity and therapeutic effect of such compounds can be determined by standard pharmaceutical procedures, for example, in cell cultures or experimental animals to determine LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Compounds that exhibit high therapeutic indices are preferred.

[0420] Data obtained from cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage of the compositions featured herein generally lies within a range of circulating concentrations, including the ED50, with little or no toxicity. Dosages can vary within this range depending on the dosage form employed and the route of administration utilized. For any compound used in the methods featured herein, a therapeutically effective dose can be estimated initially from cell culture assays. A dose can also be formulated in animal models to achieve a circulating plasma concentration range (e.g., achieve a reduction in polypeptide concentrations) of the compound, or, if appropriate, of the polypeptide product of the target sequence, including the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms), as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0421] In addition to the administrations discussed above, the iRNAs described herein can be administered in combination with other known agents that are effective in treating pathological processes mediated by APOC3 expression. In any case, the treating physician can adjust the amount and timing of iRNA administration based on the results observed using standard measures of effectiveness known in the art or described herein.

[0422] VII. Methods of the Invention The invention provides methods of treatment and prevention comprising administering to a subject having or susceptible to developing an APOC3-related disease, disorder, and / or condition (e.g., hypertriglyceridemia) a pharmaceutical composition comprising an iRNA agent of the invention, or a vector comprising an iRNA.

[0423] In one aspect, the present invention provides methods of treating a subject having a disorder that would benefit from reduced APOC3 expression, e.g., hypertriglyceridemia and other APOC-3-related diseases, e.g., non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, polycystic ovary syndrome, kidney disease, obesity, type 2 diabetes mellitus (insulin resistance); hypertension; cardiovascular disorders, e.g., atherosclerosis; and pancreatitis, e.g., acute pancreatitis.

[0424] Therapeutic methods (and uses) of the invention include administering to a subject, e.g., a human, a therapeutically effective amount of an iRNA agent that targets the APOC3 gene or a pharmaceutical composition that includes an iRNA agent that targets the APOC3 gene, thereby treating the subject having a disorder that would benefit from reduced APOC3 expression.

[0425] In one aspect, the present invention provides a method for preventing at least one symptom of the subject with disorders that can benefit from reducing APOC3 expression, such as APOC3-related diseases, such as hypertriglyceridemia, and other diseases that can be caused by, associated with, or result from hypertriglyceridemia.The latter diseases include but are not limited to non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, polycystic ovarian syndrome, kidney disease, obesity, type 2 diabetes mellitus (insulin resistance), atherosclerosis, cardiovascular disease, or pancreatitis.This method includes administering a therapeutically effective amount of iRNA agent, such as dsRNA, or vector of the present invention to the subject, thereby preventing at least one symptom of the subject with disorders that can benefit from reducing APOC3 expression.

[0426] In another aspect, the invention provides the use of a therapeutically effective amount of an iRNA agent of the invention to treat a subject, eg, a subject that may benefit from reducing and / or inhibiting APOC3 expression.

[0427] In a further aspect, the present invention provides the use of an iRNA agent, e.g., a dsRNA of the present invention, that targets the APOC3 gene, or a pharmaceutical composition comprising an iRNA agent that targets the APOC3 gene, in th...

Claims

1. 1. A salt of a double-stranded RNAi agent for inhibiting expression of apolipoprotein C3 (APOC3) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises at least 20 contiguous nucleotides from the nucleotide sequence 5'-GCUUAAAAGGGACAGUAUUCU-3' (SEQ ID NO: 81), and the antisense strand comprises at least 21 contiguous nucleotides from the nucleotide sequence 5'-AGAAUACUGUCCCUUUUAAGCAA-3' (SEQ ID NO: 184); all of the nucleotides of the sense strand comprise a nucleotide modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification, and all of the nucleotides of the antisense strand comprise a nucleotide modification selected from the group consisting of a 2'-O-methyl modification, a 2'-fluoro modification, and a 2'-deoxy modification; the sense strand and the antisense strand independently further comprise at least two phosphorothioate internucleotide linkages; and At least one chain is conjugated to a ligand; salt.

2. 2. The salt of claim 1, wherein the sense strand comprises at least two 2'-fluoro nucleotide modifications, at least seven 2'-O-methyl nucleotide modifications, and at least two phosphorothioate internucleotide linkages.

3. The salt of claim 1 or 2, wherein the antisense strand comprises at least nine 2'-O-methyl nucleotide modifications and at least four phosphorothioate internucleotide linkages.

4. 4. The salt of claim 2 or 3, wherein at least two of the 2'-fluoro nucleotide modifications on the sense strand are 2'-fluoro modified guanosine nucleotides at positions 10 and 11 of the sense strand from the 5' end.

5. The salt of any one of claims 2 to 4, wherein at least one of the 2'-O-methyl nucleotide modifications on the sense strand is a 2'-O-methyl modified adenosine nucleotide at position 6 of the sense strand from the 5' end.

6. 6. The salt of any one of claims 2 to 5, wherein at least one of the 2'-O-methyl nucleotide modifications on the sense strand is a 2'-O-methyl modified uridine nucleotide at position 18 of the sense strand from the 5' end.

7. 7. The salt of any one of claims 3 to 6, wherein at least one of the 2'-O-methyl nucleotide modifications on the antisense strand is a 2'-O-methyl modified adenosine nucleotide at position 19 of the antisense strand from the 5' end.

8. 8. The salt of any one of claims 3 to 7, wherein at least one of the 2'-O-methyl nucleotide modifications on the antisense strand is a 2'-O-methyl modified uridine nucleotide at position 17 of the antisense strand from the 5' end.

9. 9. The salt of any one of claims 3 to 8, wherein at least two of the 2'-O-methyl nucleotide modifications on the antisense strand are 2'-O-methyl modified cytosine nucleotides at positions 12 and 13 of the antisense strand from the 5' end.

10. 10. The salt of any one of claims 3 to 9, wherein 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.

11. The salt of any one of claims 1 to 10, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

12. The salt of any one of claims 1 to 11, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

13. The salt of any one of claims 1 to 11, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides.

14. The salt according to any one of claims 1 to 13, wherein the ligand is one or more GalNAc derivatives.

15. 15. The salt of claim 14, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

16. The ligand is 【Chemistry 1】 16. The salt of claim 15, wherein

17. The salt according to any one of claims 1 to 16, wherein the ligand is attached to the 3' end of the sense strand.

18. 10. The double-stranded RNAi agent according to claim 1, wherein the double-stranded RNAi agent is 【Chemistry 2】 18. The salt of any one of claims 1 to 17, conjugated to a ligand as shown in the formula: wherein X is O or S.

19. 19. The salt of claim 18, wherein X is O.

20. An isolated cell containing the salt of any one of claims 1 to 19.

21. A pharmaceutical composition comprising the salt of any one of claims 1 to 19.

22. 22. The pharmaceutical composition of claim 21, wherein the salt of the double-stranded RNAi agent is in a non-buffered solution.

23. 23. The pharmaceutical composition of claim 22, wherein the non-buffered solution is saline or water.

24. 22. The pharmaceutical composition of claim 21, wherein the salt of the double-stranded RNAi agent is present in a buffer.

25. 25. The pharmaceutical composition of claim 24, wherein the buffer comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.

26. 25. The pharmaceutical composition of claim 24, wherein the buffer is phosphate buffered saline (PBS).

27. 1. An in vitro method for inhibiting apolipoprotein C3 (APOC3) expression in a cell, comprising: (a) contacting the cells with a salt according to any one of claims 1 to 19 or a pharmaceutical composition according to any one of claims 21 to 26; and (b) maintaining the cells resulting from step (a) for a time sufficient to achieve degradation of mRNA transcripts of the APOC3 gene, thereby inhibiting expression of the APOC3 gene in the cells. An in vitro method comprising:

28. 27. The salt of any one of claims 1 to 19 or the pharmaceutical composition of any one of claims 21 to 26 for use in a method for treating a subject having an apolipoprotein C3 (APOC3)-associated disease.

29. 29. The salt or pharmaceutical composition of claim 28, wherein the APOC3-related disorder is hypertriglyceridemia.

30. 29. The salt or pharmaceutical composition of claim 28, wherein the APOC3-associated disease is selected from the group consisting of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, polycystic ovary syndrome, kidney disease, obesity, type 2 diabetes mellitus, hypertension, atherosclerosis, and pancreatitis.

31. The salt or pharmaceutical composition according to any one of claims 28 to 30, wherein the salt or pharmaceutical composition is for subcutaneous administration.

32. 32. The salt or pharmaceutical composition of any one of claims 28 to 31, wherein the salt or pharmaceutical composition is for administration in combination with an additional therapeutic agent.

33. 33. The salt or pharmaceutical composition of claim 32, wherein the additional therapeutic agent is selected from the group consisting of an HMG-CoA reductase inhibitor, a fibrate, a bile acid sequestrant, niacin, an antiplatelet agent, an angiotensin-converting enzyme inhibitor, an angiotensin II receptor antagonist, 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 modulator, a bile acid modulator, a peroxisome proliferator-activated receptor (PPAR) agonist, a gene-based therapy, a combination vasoprotectant, a glycoprotein Ilb / IIIa inhibitor, aspirin or an aspirin-like compound, an IBAT inhibitor, a squalene synthase inhibitor, a monocyte chemoattractant protein (MCP)-I inhibitor, or a fish oil.

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