Angiopoietin-like 3 (ANGPTL3) iRNA composition and method of use thereof

Abstract

The present invention relates to an RNAi agent, such as a double-stranded RNA (dsRNA) agent that targets the angiopoietin-like 3 (ANGPTL3) gene. The present invention also relates to a method for inhibiting the expression of the ANGPTL3 gene using such an RNAi agent, and to a method for preventing and treating an ANGPTL3-related disorder, such as a lipid metabolism disorder, such as hyperlipidemia or hypertriglyceridemia.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 156,476, filed on 4 March 2021, and to U.S. Provisional Patent Application No. 63 / 308,668, filed on 10 February 2022. The entire contents of each of the aforementioned applications are incorporated herein by reference.

[0002] Sequence List This application includes a sequence listing submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The aforementioned ASCII copy was created on February 28, 2022, named 121301_15120_SL.txt, and has a size of 316,668 bytes. [Background technology]

[0003] Angiopoietin-like 3 (ANGPTL3) is a member of the angiopoietin-like family of secretory factors that regulate lipid metabolism and is mainly expressed in the liver (Koishi, R. et al., (2002) Nat. Genet. 30(2):151-157). ANGPTL3 dually inhibits the catalytic activity of lipoprotein lipase (LPL), which catalyzes the hydrolysis of triglycerides, and endothelial lipase (EL), which hydrolyzes high-density lipoprotein (HDL) phospholipids. In lipid-lowering, still obese KK / Snk mice, reduced ANGPTL3 expression has a protective effect against hyperlipidemia and arteriosclerosis by promoting triglyceride clearance (Ando et al., (2003) J. Lipid Res., 44:1216-1223). Human ANGPTL3 plasma concentrations are positively correlated with plasma HDL cholesterol and HDL phospholipid levels (Shimamura et al., (2007) Arterioscler. Thromb. Vasc. Biol., 27:366-372).

[0004] Disorders of lipid metabolism can result in elevated levels of serum lipids, such as triglycerides and / or cholesterol. Elevated serum lipids are strongly associated with hypertension, cardiovascular disease, diabetes, and other pathological conditions. Hypertriglyceridemia is an example of a lipid metabolism disorder characterized by high blood concentrations of triglycerides. It is associated with atherosclerosis even in the absence of high cholesterol levels (hypercholesterolemia). When triglyceride concentrations are excessive (i.e., greater than 1000 mg / dl or 12 mmol / l), hypertriglyceridemia can also lead to pancreatitis. Hyperlipidemia is another example of a lipid metabolism disorder characterized by elevated levels of any one or all lipids and / or lipoproteins in the blood. Current treatments for lipid metabolism disorders, including diet, exercise, statins, and other medications, are not always effective. Therefore, there is a need in the art for alternative treatments for subjects with lipid metabolism disorders. [Overview of the project]

[0005] The present invention provides an iRNA composition that affects RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the gene encoding angiopoietin-like 3 (ANGPTL3). The ANGPTL3 gene may be present in cells, for example, in cells within a subject such as a human subject. The present invention also provides a method of using the iRNA composition of the present invention to inhibit the expression of the ANGPTL3 gene and / or to treat subjects who would benefit from inhibiting or reducing the expression of the ANGPTL3 gene, for example, subjects suffering from or susceptible to lipid metabolic disorders such as hyperlipidemia or hypertriglyceridemia.

[0006] Accordingly, in one embodiment, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent that inhibits the expression of angiopoietin-like 3 (ANGPTL3) in cells, the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides such as 15, 16, 17, 18, 19, 20, or 21 that differ from the nucleotide sequence of SEQ ID NO: 1 by 0, 1, 2, or 3 nucleotides or less, and the antisense strand comprises at least 15 consecutive nucleotides such as 15, 16, 17, 18, 19, 20, 21, 22, or 23 that differ from the nucleotide sequence of SEQ ID NO: 2 by 1, 2, or 3 nucleotides or less. In one embodiment, the dsRNA agent includes debasing, mismatch with opposing nucleotides in a double helix, and at least one thermally destabilized nucleotide modification, such as debasing sugar modification, 2'-deoxy modification, acyclic nucleotide, unlocked nucleic acid (UNA), or glycerol nucleic acid (GNA), for example, an antisense strand containing at least one thermally destabilized nucleotide modification.

[0007] In one embodiment, the present invention provides a double-stranded ribonucleic acid (dsRNA) that inhibits the expression of angiopoietin-like 3 (ANGPTL3) in cells, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising a region complementary to the mRNA encoding ANGPTL3, the complementary region comprising one of any one antisense nucleotide sequences from Tables 2-3 and 7-8 and at least 15 consecutive nucleotides such as 15, 16, 17, 18, 19, 20, 21, 22, or 23, with 3 or fewer nucleotides differing, for example, 3, 2, 1, or 0 nucleotides.

[0008] In another aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) in cells, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising nucleotides 58-80, 73-102, 73-124, 80-114, 91-113, 92-114, 291-320, 291-342, 307-336, 540-567, 540-589 and 545-5 of SEQ ID NO: 1 The antisense strand comprises at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, or 21, which differ from any one of the 77 nucleotide sequences by three or fewer nucleotides, e.g., 3, 2, 1, or 0 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, which differ from the corresponding nucleotide sequence of SEQ ID NO: 2 by three or fewer nucleotides, e.g., 3, 2, 1, or 0 nucleotides.

[0009] In one embodiment, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) in cells, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising nucleotides 58-80, 80-102, 84-106, 87-109, 91-113, 92-114, 186-208, 307-329, 308-330, 310-332, 314-336, 545-567, 551-573, 553-575, 554-576, 55 The antisense strand comprises at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, or 21, which differ from one of the nucleotide sequences 5-577, 1133-1155, or 1140-1162, by three or fewer nucleotides, e.g., 3, 2, 1, or 0; the antisense strand comprises at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, which differ from the corresponding nucleotide sequence of SEQ ID NO: 2, by three or fewer nucleotides, e.g., 3, 2, 1, or 0.

[0010] In another embodiment, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) in cells, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, or 21, which differ from one of the nucleotide sequences of nucleotides 58-80, 91-113, or 92-114 of SEQ ID NO: 1 by three or fewer nucleotides, e.g., 3, 2, 1, or 0; and the antisense strand comprising at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, which differ from the corresponding nucleotide sequence of SEQ ID NO: 2 by three or fewer nucleotides, e.g., 3, 2, 1, or 0.

[0011] In one embodiment, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) in cells, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, or 21, which differ from the nucleotide sequence of nucleotides 58-80 of SEQ ID NO: 1 by three or fewer, e.g., 3, 2, 1, or 0 nucleotides, and the antisense strand comprising at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, which differ from the corresponding nucleotide sequence of SEQ ID NO: 2 by three or fewer, e.g., 3, 2, 1, or 0 nucleotides.

[0012] In one embodiment, the antisense chains are AD-1331203.1, AD-1331206.1, AD-1331209.1, AD-1331212.1, AD-1331213.1, AD-1331329.1, AD-1331237.1, AD-1331238.1, AD-1331240.1, AD-1331244.1, AD-1331256.1, AD-1331262.1, AD-1331264.1, One of the double-stranded antisense nucleotide sequences selected from the group consisting of AD-1331265.1, AD-1331266.1, AD-1331316.1, AD-1331338.1, and AD-1479372, and at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, with three or fewer nucleotides differing, e.g., 3, 2, 1, or 0 nucleotides.

[0013] In one embodiment, the antisense strand comprises one of the double-stranded antisense strand nucleotide sequences selected from the group consisting of AD-1331203.1, AD-1331206.1, AD-1331209.1, AD-1331212.1, AD-1331213.1, AD-1331329.1, AD-1331240.1, AD-1331262.1, AD-1331264.1, AD-1331265.1, AD-1331266.1, and AD-1479372, and at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, with three or fewer nucleotides differing, e.g., 3, 2, 1, or 0 nucleotides.

[0014] In one embodiment, the antisense strand comprises one of the double-stranded antisense strand nucleotide sequences selected from the group consisting of AD-1331203.1, AD-1331206.1, AD-1331209.1, AD-1331212.1, AD-1331213.1, and AD-1479372, and at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, with three or fewer nucleotides differing, e.g., 3, 2, 1, or 0 nucleotides.

[0015] In one embodiment, the antisense strand comprises one of the double-stranded antisense strand nucleotide sequences selected from the group consisting of AD-1331212.1, AD-1331213.1, and AD-1479372, and at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, with three or fewer nucleotides differing, e.g., 3, 2, 1, or 0 nucleotides.

[0016] In one embodiment, the antisense chain comprises the antisense chain nucleotide sequence of AD-1479372 and at least 15 consecutive nucleotides, for example, 15, 16, 17, 18, 19, 20, 21, 22, or 23, with three or fewer nucleotides different, for example, 3, 2, 1, or 0 nucleotides.

[0017] In one embodiment, the dsRNA agent comprises at least one modified nucleotide.

[0018] In one embodiment, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified, or substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified.

[0019] In one embodiment, all nucleotides in the sense strand are modified, all nucleotides in the antisense strand are modified, or all nucleotides in the sense strand and all nucleotides in the antisense strand are modified.

[0020] In one embodiment, at least one of the modified nucleotides is a deoxy-nucleotide, a 3'-terminal deoxythymidine (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 restricted nucleotide, a restricted ethyl nucleotide, a debasalized nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-hydroxyl-modified nucleotide, a 2'-methoxyethyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, The following are selected from the group consisting of morpholinonucleotides, phosphoramides, unnatural bases containing nucleotides, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimics, thermally destabilized nucleotides, glycol-modified nucleotides (GNAs), nucleotides containing 2'-phosphates, and 2-O-(N-methylacetamide)-modified nucleotides, as well as combinations thereof.

[0021] In one embodiment, the modification to the nucleotide is selected from the group consisting of LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and glycol, and combinations thereof.

[0022] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of deoxy-nucleotides, 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, glycol-modified nucleotides (GNAs), such as Ggn, Cgn, Tgn, or Agn, nucleotides having a 2'-phosphate, such as G2p, C2p, A2p, U2p, and vinyl-phosphonate nucleotides, and combinations thereof.

[0023] In another embodiment, at least one of the modifications to the nucleotide is a thermally destabilizing nucleotide modification.

[0024] In one embodiment, the thermally destabilizing nucleotide modification is selected from the group consisting of debasing modifications, mismatches with opposing nucleotides in the double strand, destabilizing sugar modifications, 2'-deoxy modifications, acyclic nucleotides, unlocked nucleic acids (UNAs), and glycerol nucleic acids (GNAs).

[0025] In some embodiments, the modified nucleotide includes a short sequence of 3'-terminal deoxythymidine nucleotides (dT).

[0026] In some embodiments, the dsRNA agent further comprises at least one phosphorothioate nucleotide linkage. In some embodiments, the dsRNA agent comprises 6 to 8 phosphorothioate nucleotide linkages. In one embodiment, the phosphorothioate or methylphosphonate linkage is located at the 3' end of one strand. Optionally, this strand is an antisense strand. In another embodiment, this strand is a sense strand. In related embodiments, the phosphorothioate or methylphosphonate linkage is located at the 5' end of one strand. Optionally, this strand is an antisense strand. In another embodiment, this strand is a sense strand. In yet another embodiment, the phosphorothioate or methylphosphonate linkage is located at both the 5' and 3' ends of one strand. Optionally, this strand is an antisense strand. In yet another embodiment, this strand is a sense strand.

[0027] The double-stranded region may be 19–30 nucleotide pairs, 19–25 nucleotide pairs, 19–23 nucleotide pairs, 23–27 nucleotide pairs, or 21–23 nucleotide pairs in length.

[0028] In one embodiment, each chain independently has a length of 30 nucleotides or less.

[0029] In one embodiment, the sense strand has a length of 21 nucleotides, and the antisense strand has a length of 23 nucleotides.

[0030] The complementary region may be at least 17 nucleotides long, 19 to 23 nucleotides long, or 19 nucleotides long.

[0031] In one embodiment, at least one strand contains a 3' overhang of at least one nucleotide. In another embodiment, at least one strand contains a 3' overhang of at least two nucleotides.

[0032] In one embodiment, the dsRNA agent further comprises a ligand.

[0033] In one embodiment, the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

[0034] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0035] In one embodiment, the ligand is one or more GalNAc derivatives bound via monovalent, divalent, or trivalent branched linkers.

[0036] In one embodiment, the ligand is as follows: [ka]

[0037] In one embodiment, the dsRNA agent is conjugated to a ligand as shown in the following figure, [ka] In the equation, X is either O or S.

[0038] In one embodiment, X is O.

[0039] In one embodiment, the dsRNA agent further comprises at least one phosphorothioate nucleotide linkage or a methylphosphonate nucleotide linkage.

[0040] In one embodiment, the phosphorothioate nucleotide linkage or methylphosphonate nucleotide linkage is located at the 3' end of one of the strands, for example, the antisense strand or the sense strand.

[0041] In another embodiment, the phosphorothioate nucleotide linkage or methylphosphonate nucleotide linkage is located at the 5' end of one of the strands, for example, the antisense strand or the sense strand.

[0042] In one embodiment, the internucleotide linkages of the phosphorothioate or methylphosphonate are located at both the 5' and 3' ends of one of the strands. In one embodiment, the strand is an antisense strand.

[0043] In one embodiment, the base pair at position 1 of the 5' end of the double-stranded antisense strand is an AU base pair.

[0044] In one embodiment, the nucleotide sequence of the sense strand differs from the nucleotide sequence 5'-asasgcuccuUfCfUfuuuuauuguu-3' (SEQ ID NO: 18) by 4 bases or less, and the nucleotide sequence of the antisense strand differs from the nucleotide sequence 5'-asdAscadAudAaaaadGaAfggagcuusgsg-3' (SEQ ID NO: 19) by 4 bases or less, where a, g, c, and u are 2'-O-methyl(2'-OMe)A, G, C, and U, dA and dG are 2'-deoxyA and G, Cf and Uf are 2'-deoxy-2'-fluoro(2'-F)C and U, and s is a phosphorothioate bond.

[0045] In one embodiment, the nucleotide sequence of the sense strand differs from the nucleotide sequence 5'-asasgcuccuUfCfUfuuuuauuguu-3' (SEQ ID NO: 18) by three bases or less, and the nucleotide sequence of the antisense strand differs from the nucleotide sequence 5'-asdAscadAudAaaaadGaAfggagcuusgsg-3' (SEQ ID NO: 19) by three bases or less, where a, g, c, and u are 2'-O-methyl(2'-OMe)A, G, C, and U, dA and dG are 2'-deoxyA and G, Cf and Uf are 2'-deoxy-2'-fluoro(2'-F)C and U, and s is a phosphorothioate bond.

[0046] In one embodiment, the nucleotide sequence of the sense strand differs from the nucleotide sequence 5'-asasgcuccuUfCfUfuuuuauuguu-3' (SEQ ID NO: 18) by two bases or less, and the nucleotide sequence of the antisense strand differs from the nucleotide sequence 5'-asdAscadAudAaaaadGaAfggagcuusgsg-3' (SEQ ID NO: 19) by two bases or less, where a, g, c, and u are 2'-O-methyl(2'-OMe)A, G, C, and U, dA and dG are 2'-deoxyA and G, Cf and Uf are 2'-deoxy-2'-fluoro(2'-F)C and U, and s is a phosphorothioate bond.

[0047] In one embodiment, the nucleotide sequence of the sense strand differs from the nucleotide sequence 5'-asasgcuccuUfCfUfuuuuauuguu-3' (SEQ ID NO: 18) by one or less bases, the nucleotide sequence of the antisense strand differs from the nucleotide sequence 5'-asdAscadAudAaaaadGaAfggagcuusgsg-3' (SEQ ID NO: 19) by one or less bases, a, g, c, and u are 2'-O-methyl(2'-OMe)A, G, C, and U, dA and dG are 2'-deoxyA and G, Cf and Uf are 2'-deoxy-2'-fluoro(2'-F)C and U, and s is a phosphorothioate bond.

[0048] In one embodiment, the sense strand nucleotide sequence comprises the nucleotide sequence 5'-asasgcuccuUfCfUfuuuuauuguu-3' (SEQ ID NO: 18), the antisense strand nucleotide sequence comprises the nucleotide sequence 5'-asdAscadAudAaaaadGaAfggagcuusgsg-3' (SEQ ID NO: 19), where a, g, c, and u are 2'-O-methyl(2'-OMe)A, G, C, and U, dA and dG are 2'-deoxyA and G, Cf and Uf are 2'-deoxy-2'-fluoro(2'-F)C and U, and s is a phosphorothioate bond.

[0049] In one embodiment, the dsRNA agent further comprises a ligand.

[0050] In one embodiment, the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

[0051] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0052] In one embodiment, the ligand is one or more GalNAc derivatives bound via monovalent, divalent, or trivalent branched linkers.

[0053] In one embodiment, the ligand is as follows: [ka]

[0054] In one embodiment, the sense strand nucleotide sequence comprises the nucleotide sequence 5'-asasgcuccuUfCfUfuuuuauuguuL96-3' (SEQ ID NO: 20), the antisense strand nucleotide sequence comprises the nucleotide sequence 5'-asdAscadAudAaaaadGaAfggagcuusgsg-3' (SEQ ID NO: 19), where a, g, c, and u are 2'-O-methyl(2'-OMe)A, G, C, and U, dA and dG are 2'-deoxy A and G, Cf and Uf are 2'-deoxy-2'-fluoro(2'-F)C and U, s is a phosphorothioate bond, and L96 is N-[tris(GalNAc-alkyl)-amidedecanoyl)]-4-hydroxyprolinol.

[0055] In one embodiment, the sense strand nucleotide sequence comprises the nucleotide sequence 5'-asasgcuccuUfCfUfuuuuauuguu-3' (SEQ ID NO: 18), the antisense strand nucleotide sequence comprises the nucleotide sequence 5'-asdAscadAudAaaaadGaAfggagcuusgsg-3' (SEQ ID NO: 19), where a, g, c, and u are 2'-O-methyl(2'-OMe)A, G, C, and U, dA and dG are 2'-deoxy A and G, Cf and Uf are 2'-deoxy-2'-fluoro(2'-F)C and U, s is a phosphorothioate bond, and the ligand is attached to the 3' end of the sense strand as shown in the figure below. [ka] In the equation, X is O.

[0056] In one embodiment, the nucleotide sequence of the sense strand differs from the nucleotide sequence 5'-asasgcucCfuUfCfUfuuuuauuguu-3' (SEQ ID NO: 21) by 4 bases or less, the nucleotide sequence of the antisense strand differs from the nucleotide sequence 5'-asAfscaaUfaaaaagaAfgGfagcuusasa-3' (SEQ ID NO: 22) by 4 bases or less, or the nucleotide sequence of the sense strand is nucleotide sequence 5'-asgscuccUfuCfUfU fuuuauuguuu-3' (SEQ ID NO: 23) differs from this sequence by four bases or less, and the nucleotide sequence of the antisense strand differs from the nucleotide sequence 5'-asAfsacaAfuaaaaagAfaGfgagcususa-3' (SEQ ID NO: 24) by four bases or less, where a, g, c, and u are 2'-O-methyl(2'-OMe)A, G, C, and U, Cf and Uf are 2'-deoxy-2'-fluoro(2'-F)C and U, and s is a phosphorothioate bond.

[0057] In one embodiment, the nucleotide sequence of the sense strand differs from the nucleotide sequence 5'-asasgcucCfuUfCfUfuuuuauuguu-3' (SEQ ID NO: 21) by 3 bases or less, the nucleotide sequence of the antisense strand differs from the nucleotide sequence 5'-asAfscaaUfaaaaagaAfgGfagcuusasa-3' (SEQ ID NO: 22) by 3 bases or less, or the nucleotide sequence of the sense strand is nucleotide sequence 5'-asgscuccUfuCfUfU fuuuauuguuu-3' (SEQ ID NO: 23) differs from this sequence by three bases or less, and the nucleotide sequence of the antisense strand differs from the nucleotide sequence 5'-asAfsacaAfuaaaaagAfaGfgagcususa-3 (SEQ ID NO: 24) by three bases or less, where a, g, c, and u are 2'-O-methyl(2'-OMe)A, G, C, and U, Cf and Uf are 2'-deoxy-2'-fluoro(2'-F)C and U, and s is a phosphorothioate bond.

[0058] In one embodiment, the nucleotide sequence of the sense strand differs from the nucleotide sequence 5'-asasgcucCfuUfCfUfuuuuauuguu-3' (SEQ ID NO: 21) by two bases or less, the nucleotide sequence of the antisense strand differs from the nucleotide sequence 5'-asAfscaaUfaaaaagaAfgGfagcuusasa-3' (SEQ ID NO: 22) by two bases or less, or the nucleotide sequence of the sense strand is nucleotide sequence 5'-asgscuccUfuCfUfU fuuuauuguuu-3' (SEQ ID NO: 23) differs from this sequence by two bases or less, and the nucleotide sequence of the antisense strand differs from the nucleotide sequence 5'-asAfsacaAfuaaaaagAfaGfgagcususa-3' (SEQ ID NO: 24) by two bases or less, where a, g, c, and u are 2'-O-methyl(2'-OMe)A, G, C, and U, Cf and Uf are 2'-deoxy-2'-fluoro(2'-F)C and U, and s is a phosphorothioate bond.

[0059] In one embodiment, the nucleotide sequence of the sense strand differs from the nucleotide sequence 5'-asasgcucCfuUfCfUfuuuuauuguu-3' (SEQ ID NO: 21) by one or less bases, the nucleotide sequence of the antisense strand differs from the nucleotide sequence 5'-asAfscaaUfaaaaagaAfgGfagcuusasa-3' (SEQ ID NO: 22) by one or less bases, or the nucleotide sequence of the sense strand is nucleotide sequence 5'-asgscuccUfuCfUfU fuuuauuguuu-3' (SEQ ID NO: 23) differs by one base or less, and the nucleotide sequence of the antisense strand differs by one base or less from the nucleotide sequence 5'-asAfsacaAfuaaaaagAfaGfgagcususa-3 (SEQ ID NO: 24), where a, g, c, and u are 2'-O-methyl(2'-OMe)A, G, C, and U, Cf and Uf are 2'-deoxy-2'-fluoro(2'-F)C and U, and s is a phosphorothioate bond.

[0060] In one embodiment, the sense strand nucleotide sequence includes the nucleotide sequence 5'-asasgcucCfuUfCfUfuuuuauuguu-3' (SEQ ID NO: 21), and the antisense strand nucleotide sequence includes the nucleotide sequence 5'-asAfscaaUfaaaaagaAfgGfagcuusasa-3' (SEQ ID NO: 22), or the sense strand nucleotide sequence includes the nucleotide sequence 5'-asgscuccUfuCfUfUfuuuauuguuu-3' (SEQ ID NO: 23), and the antisense strand nucleotide sequence includes the nucleotide sequence 5'-asAfsacaAfuaaaaagAfaGfgagcususa The formula includes -3 (SEQ ID NO: 24), where a, g, c, and u are 2'-O-methyl(2'-OMe)A, G, C, and U, Cf and Uf are 2'-deoxy-2'-fluoro(2'-F)C and U, and s is a phosphorothioate bond.

[0061] In one embodiment, the dsRNA agent further comprises a ligand.

[0062] In one embodiment, the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

[0063] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0064] In one embodiment, the ligand is one or more GalNAc derivatives bound via monovalent, divalent, or trivalent branched linkers.

[0065] In one embodiment, the ligand is as follows: [ka]

[0066] In one embodiment, the sense strand nucleotide sequence includes the nucleotide sequence 5'-asasgcucCfuUfCfUfuuuuauuguuL96-3' (SEQ ID NO: 25), the antisense strand nucleotide sequence includes the nucleotide sequence 5'-asAfscaaUfaaaaagaAfgGfagcuusasa-3' (SEQ ID NO: 22), or the sense strand nucleotide sequence includes the nucleotide sequence 5'-asgscuccUfuCfUfUfuuuauuguuuL96-3' (SEQ ID NO: 281). The antisense strand nucleotide sequence includes the nucleotide sequence 5'-asAfsacaAfuaaaaagAfaGfgagcususa-3' (SEQ ID NO: 24), where a, g, c, and u are 2'-O-methyl(2'-OMe)A, G, C, and U, Cf and Uf are 2'-deoxy-2'-fluoro(2'-F)C and U, s is a phosphorothioate bond, and L96 is N-[tris(GalNAc-alkyl)-amidedecanoyl)]-4-hydroxyprolinol.

[0067] In one embodiment, the sense strand nucleotide sequence comprises the nucleotide sequence 5'-asasgcucCfuUfCfUfuuuuauuguu-3' (SEQ ID NO: 21), the antisense strand nucleotide sequence comprises the nucleotide sequence 5'-asAfscaaUfaaaaagaAfgGfagcuusasa-3' (SEQ ID NO: 22), where a, g, c, and u are 2'-O-methyl(2'-OMe)A, G, C, and U, Cf and Uf are 2'-deoxy-2'-fluoro(2'-F)C and U, s is a phosphorothioate bond, and the ligand is attached to the 3' end of the sense strand as shown in the following figure. [ka] Furthermore, in the equation, X is O.

[0068] In one embodiment, the sense strand nucleotide sequence comprises the nucleotide sequence 5'-asgscuccUfuCfUfUfuuuauuguuu-3' (SEQ ID NO: 23), the antisense strand nucleotide sequence comprises the nucleotide sequence 5'-asAfsacaAfuaaaaagAfaGfgagcususa-3 (SEQ ID NO: 24), where a, g, c, and u are 2'-O-methyl(2'-OMe)A, G, C, and U, Cf and Uf are 2'-deoxy-2'-fluoro(2'-F)C and U, s is a phosphorothioate bond, and the ligand is attached to the 3' end of the sense strand as shown in the following figure. [ka] Furthermore, in the equation, X is O.

[0069] The present invention also provides cells containing any of the dsRNA agents of the present invention, and pharmaceutical compositions containing any of the dsRNA agents of the present invention.

[0070] The pharmaceutical composition of the present invention may comprise a dsRNA agent in a non-buffered solution, such as physiological saline or water, or it may comprise a dsRNA agent in a buffered solution, such as acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof, or phosphate-buffered saline (PBS).

[0071] In one embodiment, the present invention provides a method for inhibiting the expression of the angiopoietin-like 3 (ANGPTL3) gene in cells. The method comprises contacting cells with any of the dsRNAs of the present invention or any of the pharmaceutical compositions of the present invention, thereby inhibiting the expression of the ANGPTL3 gene in cells.

[0072] In one embodiment, the cells are in a subject, for example, a human subject, or a subject having an angiopoietin-like 3 (ANGPTL3) related disorder, such as a lipid metabolism disorder. In a particular embodiment, the lipid metabolism disorder is hyperlipidemia or hypertriglyceridemia.

[0073] In certain embodiments, ANGPTL3 expression is inhibited by at least approximately 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In one embodiment, inhibiting ANGPTL3 expression reduces the ANGPTL3 protein level in the serum of the subject by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0074] In one embodiment, the present invention provides a method for treating a subject having a disorder that would benefit from reduced angiopoietin-like 3 (ANGPTL3) expression. This method comprises administering a therapeutically effective amount of any of the dsRNAs or pharmaceutical compositions of the present invention to a subject, thereby treating the subject having a disorder that would benefit from reduced ANGPTL3 expression.

[0075] In another embodiment, the present invention provides a method for preventing at least one symptom in a subject with a disorder who would benefit from reduced angiopoietin-like 3 (ANGPTL3) expression. This method comprises administering a prophylactic effective amount of any of the dsRNAs of the present invention or any of the pharmaceutical compositions of the present invention to a subject, thereby preventing at least one symptom in a subject with a disorder who would benefit from reduced ANGPTL3 expression.

[0076] In certain embodiments, the disorder is angiopoietin-like 3 (ANGPTL3)-related disorder, e.g., lipid metabolism disorder. In certain embodiments, the lipid metabolism disorder is hyperlipidemia or hypertriglyceridemia. In certain embodiments, administration of dsRNA to a subject causes a decrease in one or more serum lipids and / or a decrease in ANGPTL3 protein accumulation.

[0077] In a further embodiment, the present invention also provides a method for inhibiting the expression of ANGPTL3 in a subject. The method comprises administering a therapeutically effective amount of one of the dsRNAs provided herein to a subject, thereby inhibiting the expression of ANGPTL3 in the subject.

[0078] In one embodiment, the subject is a human.

[0079] In one embodiment, the dsRNA agent is administered to the subject at a dose ranging from approximately 0.01 mg / kg to approximately 50 mg / kg.

[0080] In one embodiment, the dsRNA agent is administered subcutaneously to the subject.

[0081] In one embodiment, the method of the present invention includes further determining the level of ANGPTL3 in a sample from a subject.

[0082] In one embodiment, the ANGPTL3 level in the target sample is the ANGPTL3 protein level in the blood or serum sample.

[0083] In certain embodiments, the method of the present invention further includes administering an additional therapeutic agent to a target.

[0084] The present invention also provides a kit comprising any of the dsRNAs of the present invention or any of the pharmaceutical compositions of the present invention, and optionally instructions for use. In one embodiment, the present invention provides a kit for carrying out a method for inhibiting the expression of the ANGPTL3 gene in cells by contacting cells with the double-stranded RNAi agent of the present invention in an amount effective to inhibit the expression of ANGPTL3 in cells. The kit comprises the RNAi agent and instructions for use, and optionally means for administering the RNAi agent to a target.

[0085] In one embodiment, the RNAi agent is its pharmaceutically acceptable salt. The “pharmaceutically acceptable salt” of each RNAi agent herein includes, but is not limited to, sodium salts, calcium salts, lithium salts, potassium salts, ammonium salts, magnesium salts, and mixtures thereof. Those skilled in the art will understand that when an RNAi agent is provided as a polycationic salt, it has one cation per free acid group of the possibly modified phosphodiester backbone and / or any other acidic modification (e.g., a phosphonate group at the 5' end). For example, an oligonucleotide of length “n” nucleotides contains n-1 possibly modified phosphodiesters, and as a result, an oligonucleotide of length 21nt may be provided as a salt having up to 20 cations (e.g., 20 sodium cations). Similarly, an RNAi agent having a sense strand of length 21nt and an antisense strand of length 23nt may be provided as a salt having up to 42 cations (e.g., 42 sodium cations). In the above-described examples, if the RNAi agent also includes a phosphate group or a vinylphosphonate group at the 5' terminus, the RNAi agent may be provided as a salt having up to 44 cations (e.g., 44 sodium cations). [Brief explanation of the drawing]

[0086] [Figure 1] Figures 1A and 1B are graphs showing human ANGPTL3 protein levels in serum samples (n=3 per group) from mice subcutaneously administered a single dose of 3 mg / kg of the indicated dsRNA double-stranded protein. Serum samples were collected on day 7 or 14 post-administration. Human ANGPTL3 protein levels were determined by ELISA. Figure 1A shows the group mean with standard deviation. Figure 1B shows individual points with standard deviation. [Figure 2] Figure 2 is a graph showing human ANGPTL3 mRNA levels 14 days post-administration in mice (n=3 per group) that received a single dose of the prescribed dsRNA double-stranded molecule subcutaneously. Human ANGPTL3 mRNA levels are shown relative to control levels detected by PBS treatment. [Figure 3] Figure 3 is a graph showing serum ANGPTL3 protein levels in cynomolgus monkeys (n=3 per group) that received a single subcutaneous dose of 3 mg / kg or 10 mg / kg of AD-1331212, AD-1331213, or AD-1479372. The ANGPTL3 levels are shown as a percentage change relative to day 0 (day of administration). [Modes for carrying out the invention]

[0087] This invention provides iRNA compositions that influence RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the angiopoietin-like 3 (ANGPTL3) gene. This gene may be present within cells, for example, in human cells. The use of these iRNAs enables targeted degradation of the mRNA of the corresponding gene (ANGPTL3) in mammals.

[0088] The iRNAs of the present invention are designed to target the human angiopoietin-like 3 (ANGPTL3) gene, including portions of the gene that are conserved in ANGPTL3 orthologs of other mammalian species. While not intended to be limited to theory, the aforementioned features and specific target sites or specific modification combinations or subcombinations in these iRNAs are thought to enhance the efficacy, stability, potency, durability, and safety of the iRNAs of the present invention.

[0089] Accordingly, the present invention provides a method for treating and preventing angiopoietin-like 3 (ANGPTL3)-related disorders, such as lipid metabolism disorders including hyperlipidemia or hypertriglyceridemia, using an iRNA composition that affects RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the ANGPTL3 gene.

[0090] The iRNA of the present invention comprises an RNA strand (antisense strand) having a region of a length of up to approximately 30 nucleotides or less, for example, a region of a length of 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 nucleotides, wherein the region is substantially complementary to at least a portion of the mRNA transcript of the ANGPTL3 gene.

[0091] In certain embodiments, one or both strands of the double-stranded RNAi agent of the present invention are up to 66 nucleotides in length, for example, 36–66, 26–36, 25–36, 31–60, 22–43, or 27–53 nucleotides in length, and comprise a region of at least 19 consecutive nucleotides that is substantially complementary to at least a portion of the mRNA transcript of ANGPTL3. In some embodiments, such iRNA agents having a longer antisense strand may include, for example, a second RNA strand (sense strand) of 20–60 nucleotides in length, in which case the sense strand and antisense strand form a double strand of 18–30 consecutive nucleotides.

[0092] The use of the iRNAs of the present invention enables the degradation of targeted mRNA of the corresponding gene (ANGPTL3 gene) in mammals. The inventors demonstrated through in vitro assays that iRNAs targeting the ANGPTL3 gene can potently mediate RNAi, resulting in significant inhibition of ANGPTL3 gene expression. Therefore, methods and compositions containing these iRNAs are useful for treating ANGPTL3-related disorders, such as lipid metabolism disorders like hyperlipidemia or hypertriglyceridemia.

[0093] Accordingly, the present invention provides methods and combination therapies for treating subjects with disorders that would benefit from inhibiting or reducing the expression of the ANGPTL3 gene, such as angiopoietin-like 3 (ANGPTL3) related diseases, such as lipid metabolism disorders like hyperlipidemia or hypertriglyceridemia, using iRNA compositions that affect RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the ANGPTL3 gene.

[0094] The present invention also provides a method for preventing at least one symptom in subjects having a lipid metabolism disorder, such as hyperlipidemia or hypertriglyceridemia, which would benefit from inhibiting or reducing the expression of the ANGPTL3 gene.

[0095] The following detailed description of the invention discloses methods for preparing and using compositions containing iRNAs that inhibit the expression of the ANGPTL3 gene, as well as compositions, uses, and methods for treating subjects who would benefit from the inhibition and / or reduction of ANGPTL3 gene expression, for example, subjects who are susceptible to or diagnosed with ANGPTL3-related disorders.

[0096] I. Definition To make the present invention easier to understand, certain terms are defined first. Furthermore, whenever parameter values ​​or ranges of values ​​are listed, it should be noted that intermediate values ​​and ranges of those listed values ​​are also intended to be part of the present invention.

[0097] The articles "a" and "an" are used herein to mean one or more (i.e., at least one) grammatical objects of the articles. For example, "an element" means one or more elements, such as multiple elements.

[0098] The term "including" is used herein to mean "including but not limited to," and is used without distinction from the phrase "including but not limited to."

[0099] The term "or" is used herein to mean the term "and / or" unless otherwise specified in the context, and is used without distinction from that term. For example, "sense strand or antisense strand" is understood as "sense strand or antisense strand, or sense strand and antisense strand."

[0100] The term “approximately” is used herein to mean within a typical tolerance range in the art. For example, “approximately” may be understood as a standard deviation of about 2 from the mean. In certain embodiments, “approximately” means ±10%. In certain embodiments, “approximately” means ±5%. When “approximately” precedes a series of numbers or ranges, it is understood that “approximately” may modify each of those consecutive numbers or ranges.

[0101] The terms “at least,” “greater than,” or “more than,” preceding a number or sequence of numbers are understood, where clear from the context, to include the number adjacent to the term “at least,” and all numbers or integers that logically follow it. For example, the number of nucleotides in a nucleic acid molecule should be an integer. For instance, “at least 19 nucleotides out of a 21-nucleotide nucleic acid molecule” means that 19, 20, or 21 nucleotides have the indicated property. When “at least” precedes a sequence of numbers or a range, it is understood that “at least” can modify each of those sequences of numbers or a range.

[0102] As used herein, “less than or equal to” or “or less than or equal to” is understood to mean a value or integer that is logically smaller than the value adjacent to the phrase and, where logically, up to zero. For example, a double strand with an overhang of “less than or equal to 2 nucleotides” has an overhang of 2, 1, or 0 nucleotides. When “less than or equal to” precedes a sequence of numbers or ranges, it is understood that “less than or equal to” may modify each of those sequence of numbers or ranges. As used herein, a range includes both an upper and lower limit.

[0103] As used herein, a detection method may include determining that the amount of analyte present is below the detection level of the method.

[0104] If the indicated target site is inconsistent with the nucleotide sequence of the sense strand or antisense strand, the indicated sequence takes precedence.

[0105] If a transcript or other sequence does not match the indicated site, the nucleotide sequences listed herein shall prevail.

[0106] As used herein, the term “angiopoietin-like 3,” used interchangeably with the term “ANGPTL3,” refers to a well-known gene encoding a member of a family of secreted proteins that function in angiogenesis. The encoded protein, primarily expressed in the liver, is further processed into an N-terminal coiled-coil domain-containing chain and a C-terminal fibrinogen chain. The N-terminal chain is important for lipid metabolism, while the C-terminal chain may be involved in angiogenesis. Mutations in this gene cause familial hypobetalipoproteinemia type 2.

[0107] The sequences of human ANGPTL3 mRNA can be found, for example, at GenBank deposit number GI:452408443 (NM_014495.3, SEQ ID NO: 1, reverse complement, SEQ ID NO: 2) and GenBank deposit number GI:41327750 (NM_014495.2, SEQ ID NO: 3, reverse complement, SEQ ID NO: 4). The sequences of mouse ANGPTL3 mRNA can be found, for example, at GenBank deposit number GI:142388354 (NM_013913.3, SEQ ID NO: 5, reverse complement, SEQ ID NO: 6). The sequences of rat ANGPTL3 mRNA can be found, for example, at GenBank deposit number GI:68163568 (NM_001025065.1, SEQ ID NO: 7, reverse complement, SEQ ID NO: 8). The sequence of cynomolgus monkey ANGPTL3 mRNA can be found, for example, at GenBank deposit number GI:982227663 (XM_005543185.2, SEQ ID NO: 9, reverse complement, SEQ ID NO: 10). The sequence of rhesus monkey ANGPTL3 mRNA can be found, for example, at GenBank deposit number GI:297278846 (XM_001086114.2, SEQ ID NO: 11, reverse complement, SEQ ID NO: 12).

[0108] Further examples of ANGPTL3 mRNA sequences are readily available through publicly accessible databases such as GenBank, UniProt, OMIM, and the Macaca Genome Project website.

[0109] Further information on ANGPTL3 can be found, for example, at www.ncbi.nlm.nih.gov / gene / ?term=ANGPTL3.

[0110] The entire contents of the aforementioned GenBank accession numbers and gene database numbers are incorporated herein by reference as of the filing date of this application.

[0111] As used herein, the term ANGPTL3 also refers to variations of the ANGPTL3 gene, including variants provided in SNP databases. Numerous sequence variations within the ANGPTL3 gene have been identified and can be found, for example, in NCBI dbSNP and UniProt (see, for example, www.ncbi.nlm.nih.gov / snp / ?term=ANGPTL3, whose entire contents are incorporated herein by reference as of the filing date of this application). Non-limiting examples of SNPs within the ANGPTL3 gene can be found in NCBI dbSNP deposit numbers rs193064039, rs192778191, rs192764027, rs192528948, rs191931953, rs191293319, rs191171206, rs191145608, rs191086880, rs191012841, or rs190255403.

[0112] As used herein, “target sequence” means a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the ANGPTL3 gene, such as mRNA which is the product of RNA processing of the primary transcript. In one embodiment, the target portion of the sequence is of sufficient length to function as a substrate for iRNA-dependent cleavage in or near a portion of the nucleotide sequence of an mRNA molecule formed during transcription of the ANGPTL3 gene.

[0113] The target sequence may be approximately 19 to 36 nucleotides in length, for example, approximately 19 to 30 nucleotides. For example, the target sequence may be approximately 19 to 30 nucleotides in length, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 nucleotides. In certain embodiments, the target sequence is 19–23 nucleotides in length, and optionally 21–23 nucleotides in length. Ranges and lengths existing between the above-listed ranges and lengths are also intended to be part of this disclosure.

[0114] As used herein, the term “sequence-containing chain” refers to an oligonucleotide containing a chain of nucleotides described by a sequence as referred to using the standard nucleotide terminology.

[0115] Generally, "G," "C," "A," "T," and "U" represent nucleotides containing guanine, cytosine, adenine, thymidine, and uracil as bases, respectively. However, it will be understood that the terms "ribonucleotide" or "nucleotide" may also refer to modified nucleotides or alternative substitutions (see, for example, Table 1), as will be described in more detail below. Those skilled in the art will be well aware that guanine, cytosine, adenine, and uracil can be substituted with other parts without substantially altering the base-pairing properties of oligonucleotides containing such substitutions. For example, but not limited to, nucleotides containing inosine as a base can base-pair with nucleotides containing adenine, cytosine, or uracil. Therefore, nucleotides containing uracil, guanine, or adenine can be substituted in the nucleotide sequences of the dsRNAs discussed in this invention with, for example, nucleotides containing inosine. In another embodiment, adenine and cytosine at any location within the oligonucleotide can be substituted with guanine and uracil, respectively, to form GU wobble bases that pair with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods described herein.

[0116] The terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interferant,” used without distinction herein, refer to agents containing RNA as defined herein and mediating the cleavage of RNA transcription targets via the RNA-induced silencing complex (RISC) pathway. iRNAs are responsible for sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNAs regulate, for example, inhibit the expression of the ANGPTL3 gene within cells, such as in mammalian subjects.

[0117] In one embodiment, the RNAi agent of the present invention comprises a target RNA sequence and a single-stranded RNA that interacts with, for example, an ANGPTL3 target mRNA sequence to cleave the target RNA. While we do not wish to be bound by theory, it is thought that long double-stranded RNA introduced into a cell is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). The ribonuclease III-like enzyme Dicer processes this dsRNA into small interfering RNAs of 19-23 base pairs with characteristic 3' overhangs of two bases (Bernstein, et al., (2001) Nature 409:363). Subsequently, the siRNA is incorporated into an RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA double strand, thereby inducing target recognition of the complementary antisense strand (Nykanen, et al., (2001) Cell 107:309). Upon binding to a suitable target mRNA, one or more endonucleases within the RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). In one embodiment, the present invention relates to a single-stranded RNA (siRNA) that is generated in a cell and promotes the formation of the RISC complex, resulting in the silencing of a target gene, namely the ANGPTL3 gene. Accordingly, the term "siRNA" is also used herein to refer to the iRNA described above.

[0118] In certain embodiments, the RNAi agent may be a single-stranded siRNA (ssRNAi) introduced into a cell or organism to inhibit a target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaut 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15–30 nucleotides long and are chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883–894, the entire contents of which are incorporated herein by reference. Any antisense nucleotide sequences described herein may be used as single-stranded siRNAs as described herein, or as single-stranded siRNAs chemically modified by the methods described in Lima et al., (2012) Cell 150:883–894.

[0119] In certain embodiments of the present invention, the “iRNA” used in the compositions, uses, and methods of the present invention is double-stranded RNA, and is referred herein to as “double-stranded RNA agent,” “double-stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA.” The term “dsRNA” refers to a complex of ribonucleic acid molecules having a double-stranded structure containing two antiparallel and substantially complementary nucleic acid strands, referred to as having “sense” or “antisense” orientation with respect to the target RNA, i.e., the ANGPTL3 gene. In some embodiments of the present invention, double-stranded RNA (dsRNA) induces the degradation of the target RNA, for example, mRNA, via a post-transcriptional gene silencing mechanism referred herein to as RNA interference or RNAi.

[0120] Generally, the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides; however, as described in detail herein, each or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. In addition, as used herein, “iRNA” may include chemically modified ribonucleotides, and iRNA may include 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 linkage, or a modified nucleic acid base, or any combination thereof. Thus, the term modified nucleotide includes substitution, addition, or removal of, for example, a functional group or atom, to internucleoside linkages, sugar moieties, or nucleic acid bases. Suitable modifications for use in the agents of the present invention include all types of modifications disclosed herein or known in the art. When used in siRNA-type molecules, any such modification is encompassed by “iRNA” or “RNAi agent” for the purposes of this specification and the claims.

[0121] In certain embodiments of this disclosure, the inclusion of a deoxyribonucleotide can be considered to constitute a modified nucleotide if it is present within the RNAi agent.

[0122] The double-stranded region may be of any length that enables the specific degradation of the desired target RNA via the RISC pathway, such as approximately 19–36 base pairs in length, for example, approximately 19–30 base pairs in length, for example, approximately 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, approximately 19–30, 19 The lengths may range from ~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 certain embodiments, the double-stranded region is 19~21 base pairs long, for example, 21 base pairs long. Ranges and lengths existing between the ranges and lengths listed above are also intended to be part of this disclosure.

[0123] The two strands forming a double-stranded structure may be different parts of one larger RNA molecule, or they may be separate RNA molecules. If the two strands are parts of one larger molecule and are linked by an uninterrupted chain of nucleotides between the 3' end of one strand and the corresponding 5' end of the other, the linked RNA strands are called a “hairpin loop.” A hairpin loop may contain at least one unpaired nucleotide. In some embodiments, a hairpin loop may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 23 or more unpaired nucleotides. In some embodiments, a hairpin loop may contain 10 or fewer nucleotides. In some embodiments, a hairpin loop may contain 8 or fewer unpaired nucleotides. In some embodiments, a hairpin loop may contain 4 to 10 unpaired nucleotides. In some embodiments, a hairpin loop may contain 4 to 8 nucleotides.

[0124] If the two substantially complementary strands of dsRNA are composed of separate RNA molecules, these molecules can, though not necessarily, be joined by covalent bonds. If the two strands are covalently linked by means other than the uninterrupted chain of nucleotides between the 3' end of one strand forming the double-stranded structure and the corresponding 5' end of the other strand, the connecting structure is called a "linker." RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA minus any overhangs present in the double-stranded structure. In addition to the double-stranded structure, RNAi may contain one or more nucleotide overhangs. In one embodiment of an RNAi agent, at least one strand contains a 3' overhang of at least one nucleotide. In another embodiment, at least one strand contains a 3' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent includes a 5' overhang of at least one nucleotide. In specific embodiments, at least one strand includes a 5' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet another embodiment, both the 3' and 5' ends of one strand of the RNAi agent include an overhang of at least one nucleotide.

[0125] In certain embodiments, the iRNA agent of the present invention is a dsRNA, each of which contains 19 to 23 nucleotides, and interacts with a target RNA sequence, such as the ANGPTL3 gene, to cleave the target RNA.

[0126] In some embodiments, the iRNA of the present invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, for example, an ANGPTL3 target mRNA sequence, to cleave the target RNA.

[0127] As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of a double-stranded iRNA. 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 may contain an overhang of at least one nucleotide, or the overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang may contain or consist of nucleotide / nucleoside analogs, such as deoxynucleotides / nucleosides. An overhang may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotides of a given overhang may be located on the 5' end, 3' end, or both ends of either the antisense strand or the sense strand of the dsRNA.

[0128] In one embodiment, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at its 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more nucleotides within the overhang are substituted with a nucleoside thiophosphate.

[0129] In certain embodiments, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at its 3' or 5' end, for example, 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides at its 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more nucleotides in the overhang are substituted with a nucleoside thiophosphate.

[0130] In certain embodiments, the antisense strand of dsRNA has an overhang of 1 to 10 nucleotides at its 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In certain embodiments, the overhang on the sense strand or antisense strand, or on both strands, may include an elongated length longer than 10 nucleotides, for example, 1 to 30 nucleotides, 2 to 30 nucleotides, 10 to 30 nucleotides, 10 to 25 nucleotides, 10 to 20 nucleotides, or 10 to 15 nucleotides. In certain embodiments, the elongated overhang is on the double-stranded sense strand. In certain embodiments, the elongated overhang is located at the 3' end of the double-stranded sense strand. In certain embodiments, the elongated overhang is located at the 5' end of the double-stranded sense strand. In certain embodiments, the elongated overhang is located on the double-stranded antisense strand. In certain embodiments, the extended overhang is located on the 3' end of the double-stranded antisense strand. In certain embodiments, the extended overhang is located on the 5' end of the double-stranded antisense strand. In certain embodiments, one or more nucleotides in the extended overhang are substituted with a nucleoside thiophosphate. In certain embodiments, the overhang includes a self-complementary moiety such that the overhang can form a stable hairpin structure under physiological conditions.

[0131] "Blunt" or "blunt end" means that a double-stranded RNA agent has no unpaired nucleotides at its end, i.e., no nucleotide overhangs. A double-stranded RNA agent that is "blunt-ended" is double-stranded along its entire length, i.e., has no nucleotide overhangs at any end of the molecule. The RNAi agents of the present invention include RNAi agents that have no nucleotide overhang at one end (i.e., agents having one overhang and one blunt end), or RNAi agents that have no nucleotide overhangs at either end. In most cases, such molecules will be double-stranded along their entire length.

[0132] The term "antisense strand" or "guide strand" refers to a strand of iRNA, such as a dsRNA, that contains a region substantially complementary to the target sequence, e.g., ANGPTL3 mRNA.

[0133] As used herein, the term “complementary region” refers to a region on the antisense strand that is substantially complementary to a sequence, such as a target sequence, such as the ANGPTL3 nucleotide sequence, as defined herein. If the complementary region is not perfectly complementary to the target sequence, the mismatch may be in an internal or terminal region of the molecule. Generally, the most acceptable mismatches are in terminal regions, for example, within the range of 5, 4, or 3 nucleotides at the 5' or 3' end of the iRNA. In some embodiments, the double-stranded RNA agent of the present invention includes nucleotide mismatches in the antisense strand. In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention includes 4 or fewer mismatches with the target mRNA, for example, the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the target mRNA. In some embodiments, the double-stranded RNA agent of the present invention includes 4 or fewer mismatches with the sense strand, for example, the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the sense strand. In some embodiments, the double-stranded RNA agent of the present invention contains nucleotide mismatches in the sense strand. In some embodiments, the sense strand of the double-stranded RNA agent of the present invention contains 4 or fewer mismatches with the antisense strand, for example, the sense strand contains 4, 3, 2, 1, or 0 mismatches with the antisense strand. In some embodiments, the nucleotide mismatches are, for example, within the range of 5, 4, or 3 nucleotides from the 3' end of the iRNA. In another embodiment, the nucleotide mismatches are, for example, within the 3' terminal nucleotide of the iRNA agent. In some embodiments, the mismatches are not in the seed region.

[0134] Therefore, the RNAi agents described herein may contain one or more mismatches with respect to the target sequence. In one embodiment, the RNAi agents described herein contain three or fewer mismatches (i.e., mismatches of 3, 2, 1, or 0). In one embodiment, the RNAi agents described herein contain two or fewer mismatches. In one embodiment, the RNAi agents described herein contain one or fewer mismatches. In one embodiment, the RNAi agents described herein contain zero mismatches. In certain embodiments, if the antisense strand of the RNAi agent contains a mismatch with respect to the target sequence, such mismatches can optionally be restricted to the last five nucleotides from either the 5' or 3' end of the complementary region. For example, in such embodiments, for a 23-nucleotide RNAi agent, the strand complementary to the ANGPTL3 gene region generally does not contain any mismatches within the central 13-nucleotide range. By using the methods described herein or methods known in the art, it is possible to determine whether an RNAi agent containing a mismatch to the target sequence is effective in inhibiting the expression of the ANGPTL3 gene. In particular, when it is known that specific complementary regions in the ANGPTL3 gene have polymorphic sequence variations within the population, it is important to consider the effectiveness of RNAi agents containing mismatches that inhibit ANGPTL3 expression.

[0135] When used herein, the terms “sense strand” or “passenger strand” refer to a strand of iRNA that contains a region substantially complementary to the antisense strand region as defined herein.

[0136] As used herein, "substantially all nucleotides are modified" means that they are broadly modified but not entirely modified, and may include 5, 4, 3, 2, or 1 or fewer unmodified nucleotides.

[0137] As used herein, the term “cleavage region” refers to a region located directly adjacent to a cleavage site. A cleavage site is a site on the target where a cleavage occurs. In some embodiments, the cleavage region includes three directly adjacent bases at either end of the cleavage site. In some embodiments, the cleavage region includes two directly adjacent bases at either end of the cleavage site. In some embodiments, the cleavage site occurs specifically at a site bound by nucleotides 10 and 11 of the antisense strand, and this cleavage region includes nucleotides 11, 12, and 13.

[0138] As used herein, unless otherwise specified, the term “complementary” means the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence under specific conditions to form a double-stranded structure, as will be understood by those skilled in the art when used to describe a first nucleotide sequence in relation to a second nucleotide sequence. Such conditions may be severe, for example, including 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, and washing after 12–16 hours at 50°C or 70°C (see, e.g., “Molecular Cloning: A Laboratory Manual,” Sambrook, et al. (1989), Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions that may be encountered in living organisms, may be applied. Those skilled in the art can determine the most appropriate set of conditions for testing the complementarity of the two sequences, depending on the final use of the hybridized nucleotides.

[0139] A complementary sequence within an iRNA, for example within a dsRNA as described herein, includes a base pairing of an oligonucleotide or polynucleotide containing the first nucleotide sequence with an oligonucleotide or polynucleotide containing the second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as “fully complementary” to each other. However, where herein a first sequence is “substantially complementary” to a second sequence, the two sequences may be fully complementary, or they may form one or more, but generally five, four, three, or two or fewer, mismatched base pairs during hybridization of a double helix of up to 30 base pairs, while maintaining their ability to hybridize for their final use, for example, in vitro or in vivo, under conditions most relevant to inhibiting gene expression. However, if two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, such overhangs shall not be considered mismatches for the purpose of determining complementarity. For example, a dsRNA comprising one oligonucleotide of length 21 nucleotides and another oligonucleotide of length 23 nucleotides, wherein the longer oligonucleotide contains a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, can still be described as "perfectly complementary" for the purposes described herein.

[0140] When used herein, “complementary” sequences may include, or may be formed entirely from, non-Watson-Crick base pairs or base pairs formed from non-naturally modified nucleotides, provided that they satisfy the above requirements regarding their ability to hybridize. Examples of such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairs or Hoogsteen base pairs.

[0141] The terms “complementary,” “fully complementary,” and “substantially complementary” as used herein may be used in relation to base matching between the sense and antisense strands of a dsRNA, or between two oligonucleotides or polynucleotides, such as the antisense strand of a double-stranded RNA agent and a target sequence, as will be understood from the context of their use.

[0142] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" messenger RNA (mRNA) means a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., the mRNA encoding the ANGPTL3 gene). For example, a polynucleotide is complementary to at least a portion of ANGPTL3 mRNA if its sequence is substantially complementary to an uninterrupted portion of the mRNA encoding the ANGPTL3 gene.

[0143] Accordingly, in some embodiments, the antisense polynucleotides disclosed herein are fully complementary to the target ANGPTL3 sequence. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target ANGPTL3 sequence and comprise a sequence of nucleotides that is at least 80% complementary to the full length of any one nucleotide sequence of SEQ ID NOs: 1, 3, 5, 7, 9, or 11, or any one fragment of SEQ ID NOs: 1, 3, 5, 7, 9, or 11, for example, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

[0144] In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of the target ANGPTL3 sequence and comprise a sequence of nucleotides that is at least 80% complementary to the full length of a fragment of SEQ ID NO: 1 selected from the group of nucleotides 73-102, 73-124, 80-114, 291-320, 291-342, 307-336, 540-567, 540-589, and 545-577, for example, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

[0145] In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target ANGPTL3 sequence fragments, and also to nucleotides 80-102, 84-106, 87-109, 91-113, 92-114, 186-208, 307-329, 308-330, 310-332, 314-336, 545-567, 551-573 of SEQ ID NO: 1. The sequence comprises a contiguous nucleotide sequence that is at least 80% complementary to the full length of the sequence number 1, selected from the group 553-575, 554-576, 555-577, 1133-1155, or 1140-1162, for example, approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99% complementary.

[0146] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target ANGPTL3 sequence and comprise a contiguous nucleotide sequence that is at least about 80% complementary over its entire length to any one sense strand nucleotide sequence in any one of Tables 2-3 and 7-8 or any one fragment of a sense strand nucleotide sequence in any one of Tables 2-3 and 7-8, for example, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary.

[0147] In one embodiment, the RNAi agent of the present disclosure comprises a sense strand substantially complementary to an antisense polynucleotide and thus identical to the target ANGPTL3 sequence, wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary to its full length to the nucleotide sequence equivalent region of SEQ ID NO: 2, 4, 6, 8, 10, or 12, or to any one fragment of SEQ ID NO: 2, 4, 6, 8, 10, or 12, for example, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary.

[0148] In some embodiments, the iRNA of the present invention comprises a sense strand substantially complementary to an antisense polynucleotide similarly complementary to a target ANGPTL3 sequence, wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary over its entire length to any one antisense strand nucleotide sequence in any one of Tables 2-3 and 7-8, or any one fragment of an antisense strand nucleotide sequence in any one of Tables 2-3 and 7-8, for example, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary.

[0149] In one embodiment, the antisense chains are AD-1331203.1, AD-1331206.1, AD-1331209.1, AD-1331212.1, AD-1331213.1, AD-1331329.1, AD-1331237.1, AD-1331238.1, AD-1331240.1, AD-1331244.1, AD-1331256.1, and AD-1331262.1. It comprises at least 15, for example, 15, 16, 17, 18, 19, or 20 consecutive nucleotides that differ by 0, 1, 2, or 3 or fewer nucleotides from any one of the double-stranded antisense nucleotide sequences selected from the group consisting of AD-1331264.1, AD-1331265.1, AD-1331266.1, AD-1331316.1, and AD-1331338.1.

[0150] In one embodiment, the antisense strand comprises at least 15, for example, 15, 16, 17, 18, 19, or 20 consecutive nucleotides that differ by 0, 1, 2, or 3 or fewer nucleotides from any one of the double-stranded antisense strand nucleotide sequences selected from the group consisting of AD-1331203.1, AD-1331206.1, AD-1331209.1, AD-1331212.1, AD-1331213.1, AD-1331329.1, AD-1331240.1, AD-1331262.1, AD-1331264.1, AD-1331265.1, and AD-1331266.1. In one embodiment, the antisense strand comprises at least 15, for example, 15, 16, 17, 18, 19, or 20 consecutive nucleotides that differ by 0, 1, 2, or 3 or fewer nucleotides from any one of the double-stranded antisense strand nucleotide sequences selected from the group consisting of AD-1331203.1, AD-1331206.1, AD-1331209.1, AD-1331212.1, and AD-1331213.1.

[0151] Generally, “iRNA” includes ribonucleotides having chemical modifications. Such modifications may include all types of modifications disclosed herein or known in the art. Any such modifications, when used in a dsRNA molecule, are encompassed by “iRNA” for the purposes of this specification and the claims.

[0152] In certain embodiments of this disclosure, the inclusion of a deoxyribonucleotide can be considered to constitute a modified nucleotide if it is present within the RNAi agent.

[0153] In one aspect of the present invention, the agent for use in the methods and compositions of the present invention is a single-stranded antisense oligonucleotide molecule that inhibits target mRNA via an antisense inhibition mechanism. The single-stranded antisense oligonucleotide molecule is complementary to a sequence in the target mRNA. The single-stranded antisense oligonucleotide can inhibit translation stoichiometrically by base-pairing with the mRNA and physically interfering with the translation mechanism. See Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense oligonucleotide molecule may be about 14 to about 30 nucleotides long and may have a sequence complementary to the target sequence. For example, the single-stranded antisense oligonucleotide molecule may contain a sequence of at least about 14, 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from any of the antisense sequences described herein.

[0154] The phrase "bringing iRNA, such as dsRNA, into contact with a cell," as used herein, encompasses contacting a cell by any possible means. Bringing a cell into contact with iRNA includes bringing a cell into contact with iRNA in vitro or in vivo. Contact can be direct or indirect. For example, iRNA may be brought into physical contact with a cell by performing the method individually, or iRNA may be placed in a situation that allows or results in subsequent contact with a cell.

[0155] Cell contact in vitro can be achieved, for example, by incubating cells with iRNA. Cell contact in vivo can be achieved, for example, by injecting iRNA into or near the tissue in which the cells are present, or by injecting iRNA into another region, for example, into the bloodstream or subcutaneous space, so that the agent subsequently reaches the tissue in which the cells to be contacted are present. For example, iRNA may contain or be bound to a ligand that induces iRNA toward a target site, such as the liver, for example. Combinations of in vitro and in vivo contact methods are also possible. For example, cells can be contacted with iRNA in vitro and then transplanted into a target.

[0156] In certain embodiments, contacting cells with iRNA includes “introducing” or “delivering iRNA into cells” by promoting or causing uptake or absorption into the cells. Absorption or uptake of iRNA may occur via spontaneous diffusion or active intracellular processes, or by auxiliary drugs or devices. Intracellular introduction of iRNA may be in vitro or in vivo. For example, in the case of in vivo introduction, iRNA may be injected into a tissue site or administered systemically. In vitro intracellular introduction methods include those known in the art, such as electroporation and lipofection. Further approaches are described below herein or are known in the art.

[0157] The term “lipid nanoparticle” or “LNP” refers to a vesicle containing a lipid layer that encapsulates a pharmaceutically active molecule, such as a nucleic acid molecule, such as an iRNA or a plasmid from which an iRNA is transcribed. LNPs are described, for example, in U.S. Patents 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0158] As used herein, “Subject” refers to animals such as mammals, including primates (such as humans, monkeys, and chimpanzees), non-primates (such as cattle, pigs, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, or mice), or birds that express the target gene either endogenously or heterologously. In some embodiments, the subject is a human being treated or evaluated for a disease or disorder in which a reduction in ANGPTL3 expression would be beneficial, a human being at risk of a disease or disorder in which a reduction in ANGPTL3 expression would be beneficial, a human being having a disease or disorder in which a reduction in ANGPTL3 expression would be beneficial, or a human being being treated for a disease or disorder in which a reduction in ANGPTL3 expression would be beneficial as described herein. In some embodiments, the subject is a female human being. In other embodiments, the subject is a male human being. In one embodiment, the subject is an adult subject. In another embodiment, the subject is a child subject.

[0159] As used herein, the terms “treat” or “remedy” refer to a beneficial or desirable outcome, such as reducing at least one sign or symptom of ANGPTL3-related disorder in a subject. Treatment also includes a reduction in one or more signs or symptoms associated with undesirable ANGPTL3 expression, a decrease in the degree of undesirable ANGPTL3 activation or stabilization, or improvement or mitigation of undesirable ANGPTL3 activation or stabilization. “Treatment” may also mean extending survival time compared to the survival time expected if no treatment is performed. The term “lower” in the context of ANGPTL3 levels or disease markers or symptoms in a subject refers to a statistically significant reduction in such levels. The reduction may be, for example, at least 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In certain embodiments, the reduction is at least 20%. In certain embodiments, the reduction is at least 50% in disease markers, for example, in protein levels or gene expression levels. "Reducing" in the context of ANGPTL3 levels in a subject preferably means reducing to a level acceptable as being within the normal range in an individual without the disorder. In certain embodiments, "reducing" means reducing the difference between the level of a marker or symptom in a subject suffering from the disease and the level acceptable as being within the normal range for the individual, for example, the level of weight reduction between an obese individual and an individual with a weight acceptable as being within the normal range.

[0160] Where used herein, “prevention” or “prevention” may be treated or improved by reducing the expression of the ANGPTL3 gene, and means a reduction in the likelihood of a subject developing such a disease, disorder, or condition, e.g., undesirable or excessive ANGPTL3 expression and / or activity, e.g., high triglyceride levels or rash xanthoma. The likelihood of developing high triglyceride levels or rash xanthoma is reduced, for example, if an individual with one or more risk factors for high triglyceride levels or rash xanthoma does not develop high triglyceride levels or rash xanthoma, or develops high triglyceride levels or rash xanthoma with less severity compared to a population with the same risk factors but not receiving the treatments described herein. The absence of the onset of a disease, disorder, or condition, or a reduction in the onset of symptoms associated with such disease, disorder, or condition (e.g., a reduction of at least about 10% of a clinically acceptable scale for the disease or disorder), or a delay in the onset of delayed symptoms (e.g., a delay of days, weeks, months, or years) is considered effective prevention.

[0161] As used herein, the term “serum lipids” refers to any major lipid present in the blood. Serum lipids may be present in the blood in free form or as part of protein complexes, such as lipoprotein complexes. Non-limiting examples of serum lipids include triglycerides and cholesterol, such as total cholesterol (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), very low-density lipoprotein cholesterol (VLDL-C), and medium-density lipoprotein cholesterol (IDL-C).

[0162] As used herein, the terms “angiopoietin-like 3-related disease” or “ANGPTL3-related disease” refer to a disease or disorder caused by or associated with ANGPTL3 gene expression or ANGPTL3 protein production. The term “ANGPTL3-related disease” includes any disease, disorder, or condition that benefits from reduced ANGPTL3 gene expression, replication, or protein activity. In some embodiments, ANGPTL3-related disease is a disorder of lipid metabolism.

[0163] As used herein, “disorder of lipid metabolism” means any disorder associated with or caused by a disorder of lipid metabolism. For example, the term includes any disorder, disease or condition that may result in hyperlipidemia, or a condition characterized by an abnormally elevated level of any or all of the lipids and / or lipoproteins in the blood. The term also includes induced or acquired disorders, such as hereditary disorders like familial hypertriglyceridemia, familial partial lipodystrophy type 1 (FPLD1), or disorders induced or acquired as a result of a disease, disorder or condition (e.g., renal failure), diet, or certain medications (e.g., as a result of highly active antiretroviral therapy (HAART) used to treat AIDS or HIV). Examples of lipid metabolism disorders include, but are not limited to, atherosclerosis, dyslipidemia, and hypertriglyceridemia (drug-induced hypertriglyceridemia, diuretic-induced hypertriglyceridemia, alcoholic hypertriglyceridemia, β-adrenergic blocker-induced hypertriglyceridemia, estrogen-induced hypertriglyceridemia, glucocorticoid-induced hypertriglyceridemia, retinoid-induced hypertriglyceridemia, and cimetidine-induced hypertriglyceridemia). This includes conditions such as familial hypertriglyceridemia, acute pancreatitis with hypertriglyceridemia, chylomicron syndrome, familial chylomicronemia, Apo-E deficiency or resistance, LPL deficiency or decreased activity, hyperlipidemia (including familial combined hyperlipidemia), hypercholesterolemia, gout associated with hypercholesterolemia, xanthomatous (subcutaneous cholesterol deposits), hyperlipidemia with heterogeneous LPL deficiency, and hyperlipidemia with hyper-LDL and heterogeneous LPL deficiency.

[0164] Cardiovascular diseases associated with lipid metabolism disorders are also considered “lipid metabolism disorders” as defined herein. These diseases may include coronary artery disease (also known as ischemic heart disease), inflammation and restenosis associated with coronary artery disease, peripheral vascular disease, and stroke.

[0165] Weight-related disorders are also considered lipid metabolism disorders as defined herein. Such disorders may include obesity, metabolic syndromes including their independent components (e.g., central obesity, FBG / prediabetes / diabetes, hypercholesterolemia, hypertriglyceridemia, and hypertension), hypothyroidism, urinary tract disorders, and other conditions associated with weight gain (including rapid weight gain), weight loss, maintenance of weight loss, or risk of weight loss after weight loss.

[0166] Blood glucose disorders are further considered “disorders of lipid metabolism” as defined herein. Such disorders may include diabetes mellitus, hypertension, and polycystic ovary syndrome associated with insulin resistance. Other exemplary disorders of lipid metabolism may include renal transplantation, nephrotic syndrome, Cushing’s syndrome, acromegaly, systemic lupus erythematosus, dysglobulinemia, lipodystrophy, glycogenosis type 1, and Addison’s disease.

[0167] When used herein, “therapeutic dose” is intended to include an amount of RNAi agent sufficient to treat the disease (for example, by reducing, improving, or maintaining one or more symptoms of the existing disease or disease) when administered to a subject with ANGPTL3-related disease. “Therapeutic dose” may vary depending on the RNAi agent, the method of administration of the agent, the disease and its severity and history, age, weight, family history, genetic structure, the type of treatment preceding or concomitant treatment, if any, and other personal characteristics of the subject to be treated.

[0168] "Prophylactic effective dose," as used herein, is intended to include an amount of RNAi agent that, when administered to a subject with ANGPTL3-related disorder, is sufficient to prevent or improve one or more symptoms of the disease. Improvement of the disease includes slowing the course of the disease or reducing the severity of the disease that will develop. "Prophylactic effective dose" may vary depending on the RNAi agent, the method of administration of the agent, the degree of the disease risk, and the patient's medical history, age, weight, family history, genetic makeup, the type of treatment preceding or concomitant in some cases, and other personal characteristics of the patient being treated.

[0169] The “therapeutic effective dose” or “prophylactic effective dose” also includes the amount of RNAi agent that produces several desired effects in a reasonable benefit-to-risk ratio applicable to any treatment. The iRNA used in the method of the present invention may be administered in an amount sufficient to produce a reasonable benefit-to-risk ratio applicable to such treatment.

[0170] The term "pharmaceutically acceptable" is used herein to mean those compounds (including salts), compositions, or dosage forms that are suitable for use in contact with human and animal tissues within the bounds of sound medical judgment, in proportion to a reasonable benefit-to-risk ratio, without excessive toxicity, irritation, allergic reactions, or other problems or complications.

[0171] The term “pharmaceutically acceptable carrier,” as used herein, means a pharmaceutically acceptable material, composition, or vehicle involved in the transport or delivery of a subject compound from one organ or body part to another, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium stearate or zinc stearate, or stearic acid), or solvent encapsulating material. Each carrier must be “acceptable” in the sense that it is compatible with the other raw materials of the formulation and must not be harmful to the subject being treated. Such carriers are known in the art. pharmaceutically acceptable carriers include carriers for administration by injection.

[0172] The term “sample,” as used herein, encompasses similar bodily fluids, cells, or tissues isolated from a subject, and collections of bodily fluids, cells, or tissues present within the subject. Examples of bodily fluids include blood, serum, and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, and saliva. Tissue samples may include samples from tissues, organs, or local areas. For example, a sample may originate from a specific organ, a part of an organ, or bodily fluids or cells within those organs. In certain embodiments, a sample may originate from the liver (e.g., the entire liver or a specific segment of the liver, or a specific type of cell within the liver, e.g., hepatocytes). In some embodiments, “sample derived from subject” refers to urine obtained from the subject. “Sample derived from subject” may also refer to blood or blood-derived serum or plasma from the subject.

[0173] II. The iRNA of the present invention The present invention provides an iRNA that inhibits the expression of the ANGPTL3 gene. In certain embodiments, the iRNA comprises a double-stranded ribonucleic acid (dsRNA) molecule that inhibits the expression of the ANGPTL3 gene in cells, such as cells in a subject, such as a mammal, such as a human, who is prone to developing ANGPTL3-related disorders, such as lipid metabolism disorders, such as hyperlipidemia or hypertriglyceridemia. The dsRNAi agent comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during ANGPTL3 gene expression. The complementary region is approximately 19 to 30 nucleotides in length (e.g., approximately 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides in length).

[0174] When iRNA is brought into contact with cells expressing the ANGPTL3 gene, it inhibits the expression of the ANGPTL3 gene (e.g., human, primate, non-primate, or rat ANGPTL3 gene) by at least approximately 50%, as confirmed by methods such as PCR or branched DNA (bDNA), or by protein-based methods such as immunofluorescence, using techniques such as Western blotting or flow cytometry. In certain embodiments, inhibition of expression is determined by the qPCR method provided in the examples herein, using siRNA at a concentration of, for example, 10 nM, in a suitable biological cell line provided therein. In certain embodiments, inhibition of expression in vivo is determined by knocking down the human gene in rodents expressing the human gene, such as mice expressing a human target gene or AAV-infected mice, when administered, for example, as a single dose at a minimum RNA expression level of 3 mg / kg.

[0175] dsRNA comprises two RNA strands that are complementary and hybridize to form a double-stranded structure under the conditions under which the dsRNA is used. One strand of the dsRNA (the antisense strand) contains a complementary region that is substantially and generally fully complementary to the target sequence. The target sequence can be obtained from the mRNA sequence formed during the expression of the ANGPTL3 gene. The other strand (the sense strand) contains a region complementary to the antisense strand, so that the two strands hybridize to form a double-stranded structure when combined under suitable conditions. As described elsewhere in this specification and as is known in the art, the complementary sequence of the dsRNA can also be included as a self-complementary region of a single nucleic acid molecule, so as to be relative on a separate oligonucleotide.

[0176] Generally, double-stranded structures are 15-30 base pairs long, 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, 19-2 The lengths are 9, 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 certain embodiments, the double-stranded structure is 18–25 base pairs long, for example, 18–25, 18–24, 18–23, 18–22, 18–21, 18–20, 19–25, 19–24, 19–23, 19–22, 19–21, 19–20, 20–25, 20–24, 20–23, 20–22, 20–21, 21–25, 21–24, 21–23, 21–22, 22–25, 22–24, 22–23, 23–25, 23–24, or 24–25 base pairs long, for example, 19–21 base pairs long. Ranges and lengths existing between the ranges and lengths listed above are also intended to be part of this disclosure.

[0177] Similarly, complementary regions to the target sequence are 15-30 nucleotides long, 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, The nucleotide lengths are 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, for example, 19-23 nucleotides or 21-23 nucleotides. The ranges and lengths that exist between the ranges and lengths listed above are also intended to be part of this disclosure.

[0178] In some embodiments, the double-stranded structure is 19–30 base pairs long. Similarly, the region complementary to the target sequence is 19–30 nucleotides long.

[0179] In some embodiments, the dsRNA is about 19–23 nucleotides long, or about 25–30 nucleotides long. Generally, dsRNA is long enough to function as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNA longer than about 21–23 nucleotides can function as a substrate for Dicer. As those skilled in the art will also recognize, the RNA region targeted for cleavage is in most cases a longer RNA molecule, often a portion of an mRNA molecule. Where applicable, the “portion” of the mRNA target is a contiguous sequence of mRNA targets long enough to allow it to serve as a substrate for RNAi-dependent cleavage (i.e., cleavage via the RISC pathway).

[0180] Those skilled in the art will understand that the double-stranded region is the primary functional portion of dsRNA, for example, a double-stranded region of about 19 to about 30 base pairs, for example, about 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs. In other words, in one embodiment, an RNA molecule or complex of RNA molecules having a double-stranded region of more than 30 base pairs is a dsRNA, insofar as it is processed into a functional double-stranded region of, for example, 15-30 base pairs that targets the desired RNA for cleavage. Thus, those skilled in the art will recognize that in one embodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In yet another embodiment, an iRNA agent useful for targeting ANGPTL3 gene expression is not generated in the target cell by cleaving a larger dsRNA.

[0181] The dsRNAs described herein may further include one or more single-stranded nucleotide overhangs, for example, 1-4, 2-4, 1-3, 2-3, 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang may exhibit superior inhibitory properties compared to their blunt-ended counterparts. Nucleotide overhangs may include or consist of nucleotide / nucleoside analogs, such as deoxynucleotides / nucleosides. Overhangs may be on the sense strand, the antisense strand, or a combination thereof. Furthermore, the nucleotides of the overhang may be located on the 5' end, 3' end, or both ends of the antisense or sense strand of the dsRNA.

[0182] dsRNA can be synthesized by standard methods known in the art. The double-stranded RNAi 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 strands of the constituent are annealed. The individual strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis has the advantage that oligonucleotide chains containing non-natural or modified nucleotides can be readily prepared. Similarly, the single-stranded oligonucleotides of the present invention can be prepared using solution-phase or solid-phase organic synthesis or both.

[0183] In one embodiment, the dsRNA of the present invention comprises at least two nucleotide sequences, a sense sequence, and an antisense sequence. The sense strand is selected from the sequence group provided in any one of Tables 2-3 and 7-8, and the corresponding antisense strand of the sense strand is selected from any one of the sequence group provided in Tables 2-3 and 7-8. In this embodiment, one of the two sequences is complementary to the other, and in this case, one of the sequences is substantially complementary to the mRNA sequence produced during the expression of the ANGPTL3 gene. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, one of which is described as the sense strand in any one of Tables 2-3 and 7-8, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in any one of Tables 2-3 and 7-8.

[0184] In certain embodiments, substantially complementary sequences of dsRNA are contained on each oligonucleotide. In other embodiments, substantially complementary sequences of dsRNA are contained on a single oligonucleotide.

[0185] In one embodiment, the antisense chains are AD-1331203.1, AD-1331206.1, AD-1331209.1, AD-1331212.1, AD-1331213.1, AD-1331329.1, AD-1331237.1, AD-1331238.1, AD-1331240.1, AD-1331244.1, AD-1331256.1, and AD-1331262.1. It comprises at least 15, for example, 15, 16, 17, 18, 19, or 20 consecutive nucleotides that differ by 0, 1, 2, or 3 or fewer nucleotides from any one of the double-stranded antisense nucleotide sequences selected from the group consisting of AD-1331264.1, AD-1331265.1, AD-1331266.1, AD-1331316.1, and AD-1331338.1.

[0186] In one embodiment, the antisense strand comprises at least 15, for example, 15, 16, 17, 18, 19, or 20 consecutive nucleotides that differ by 0, 1, 2, or 3 or fewer nucleotides from any one of the double-stranded antisense strand nucleotide sequences selected from the group consisting of AD-1331203.1, AD-1331206.1, AD-1331209.1, AD-1331212.1, AD-1331213.1, AD-1331329.1, AD-1331240.1, AD-1331262.1, AD-1331264.1, AD-1331265.1, and AD-1331266.1.

[0187] In one embodiment, the antisense strand comprises at least 15, for example, 15, 16, 17, 18, 19, or 20 consecutive nucleotides that differ by 0, 1, 2, or 3 or fewer nucleotides from any one of the double-stranded antisense strand nucleotide sequences selected from the group consisting of AD-1331203.1, AD-1331206.1, AD-1331209.1, AD-1331212.1, and AD-1331213.1.

[0188] For example, the sequences in Table 3 are not described as modified or conjugated sequences, but the iRNA of the present invention, for example, the dsRNA of the present invention, may include any one of the sequences specified in any one of Tables 2-3 and 7-8, which are unmodified, unconjugated, or modified or conjugated in a way different from those described therein. In other words, the present invention encompasses the unmodified, unconjugated, modified, or conjugated dsRNAs of Tables 2-3 and 7-8 as described herein.

[0189] Those skilled in the art are well aware that dsRNAs having a double-stranded structure of about 20 to 23 base pairs, for example 21 base pairs, have been welcomed as particularly effective in introducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA double-stranded structures may also be effective (Chu and Rana (2007) RNA 14:1714-1719, Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, due to the properties of the oligonucleotide sequences provided in any one of Tables 2-3 and 7-8, the dsRNAs described herein may include at least one strand of a minimum length of 21 nucleotides. It can be reasonably predicted that a shorter double-stranded molecule having one of the sequences in Tables 2-3 and 7-8, with only a few nucleotides removed from one or both ends, may be equally effective compared to the dsRNAs described above. Therefore, a dsRNA having at least 19, 20, or more consecutive nucleotide sequences, derived from one of the sequences in Tables 2-3 and 7-8, and having a different ability to inhibit ANGPTL3 gene expression by approximately 5, 10, 15, 20, 25, or 30% or less than that of a dsRNA containing the entire sequence, is intended to be within the scope of the present invention.

[0190] Furthermore, the RNAs provided in Tables 2-3 and 7-8 identify sites in the ANGPTL3 transcript that are sensitive to RISC-mediated cleavage. Thus, the present invention further addresses iRNAs that target one of these sites. As used herein, an iRNA is said to target a particular site if the iRNA promotes cleavage of the transcript at any location within that particular site in the RNA transcript. Such an iRNA would generally consist of at least about 19 consecutive nucleotides from any one of the sequences provided in any one of Tables 2-3 and 7-8, bound to an additional nucleotide sequence taken from a region adjacent to a selected sequence in the ANGPTL3 gene.

[0191] III. Modified iRNA of the present invention In certain embodiments, the RNA of the iRNA of the present invention, e.g., dsRNA, is unmodified and does not contain, for example, chemical modifications or conjugations known in the art and described herein. In other embodiments, the RNA of the iRNA of the present invention, e.g., dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the present invention, substantially all of the nucleotides of the iRNA of the present invention are modified. In other embodiments of the present invention, all or substantially all of the nucleotides of the iRNA are modified, i.e., 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer unmodified nucleotides are present in the iRNA chain.

[0192] The nucleic acids discussed in this invention can be synthesized or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. Let al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-terminal modifications (phosphorylation, conjugation, reverse ligation) or 3'-terminal modifications (conjugation, DNA nucleotide, reverse ligation, etc.), base modifications, such as stabilization bases, destabilization bases, substitutions with bases that base-pair with partners in the extended repertoire, base removal (debasing nucleotides), or conjugated bases, sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, or phosphodiester linkage modifications or substitutions, and other skeletal modifications. Specific examples of iRNA compounds useful in the embodiments described herein, but not limited to, include RNAs containing a modified skeleton or unnatural internucleoside linkages. RNAs with modified skeletons include those that do not have a phosphorus atom in their skeleton. For the purposes of this specification, as is sometimes mentioned in the art, modified RNAs that do not have a phosphorus atom in their internucleoside skeleton can also be considered oligonucleosides. In some embodiments, the modified iRNA has a phosphorus atom in its internucleoside skeleton.

[0193] Examples of modified RNA backbones include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotryesters, aminoalkyl phosphotryesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotryesters, as well as boranophosphates having the usual 3'-5' linkage, their 2'-5' linkage analogs, and those having inverted polarity where adjacent pairs of nucleoside units are linked to 3'-5' and 5'-3' or 2'-5' and 5'-2'. Various salt, mixed salt, and free acid forms are also possible. In some embodiments of the present invention, the dsRNA agent of the present invention is in free acid form. In other embodiments of the present invention, the dsRNA agent of the present invention is in salt form. In one embodiment, the dsRNA agent of the present invention is in sodium salt form. In certain embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ions are present in the drug as counterions to substantially all of the phosphodiester and / or phosphorothioate groups present in the drug. Drugs in which substantially all of the phosphodiester and / or phosphorothioate linkages have sodium counterions include 5, 4, 3, 2, or 1 or fewer phosphodiester and / or phosphorothioate linkages that do not have sodium counterions. In some embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ions are present as counterions to all of the phosphodiester and / or phosphorothioate groups present in the drug.

[0194] Representative U.S. patents teaching the preparation of the phosphorus-containing bond described above include, but are not limited to, U.S. Patents 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 5,286,71 No. 7, No. 5,321,131, No. 5,399,676, No. 5,405,939, No. 5,453,496, No. 5,455,233, No. 5,466,677, No. 5,47 No. 6,925, No. 5,519,126, No. 5,536,821, No. 5,541,316, No. 5,550,111, No. 5,563,253, No. 5,571,799, No. 5 , 587,361, 5,625,050, 6,028,188, 6,124,445, 6,160,109, 6,169,170, 6,172,209 No. 6,239,265, No. 6,277,603, No. 6,326,199, No. 6,346,614, No. 6,444,423, No. 6,531,590, No. 6,534, This includes U.S. Patent Nos. 639, 6,608,035, 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029, and U.S. Patent No. RE 39464, the entire contents of each of these are incorporated herein by reference.

[0195] Modified RNA skeletons that do not contain phosphorus atoms in their backbone have skeletons formed by short alkyl or cycloalkyl nucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl nucleoside linkages, or one or more short heteroatoms or heterocyclic nucleoside linkages. These include those with morpholino linkages (partially formed from the sugar portion of the nucleoside), siloxane skeletons, sulfide, sulfoxide and sulfone skeletons, formacetyl and thioformacetyl skeletons, methyleneformacetyl and thioformacetyl skeletons, alkene-containing skeletons, sulfamate skeletons, methyleneimino and methylenehydrazino skeletons, sulfonate and sulfonamide skeletons, amide skeletons, and others having mixed constituent parts of N, O, S and CH2.

[0196] Representative U.S. patents teaching the preparation of the above-mentioned oligonucleotides include, but are not limited to, U.S. Patent 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. 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 are examples, the entire contents of which are incorporated herein by reference.

[0197] RNA mimetic suitable for use in iRNAs provided herein is considered, in which both the sugar and nucleoside links of the nucleotide unit, i.e., its backbone, are substituted with novel groups. This base unit is maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound in which the RNA mimetic is known to have excellent hybridization properties is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is substituted with an amide-containing backbone, particularly with an aminoethylglycine backbone. Nucleic acid bases are retained and directly or indirectly bound to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds, but not limited to, are U.S. Patents 5,539,082, 5,714,331 and 5,719,262, the entire contents of each of which are incorporated herein by reference. Further PNA compounds suitable for use in iRNAs of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0198] Some embodiments of the present invention include RNA having a phosphorothioate backbone and oligonucleosides having a heteroatom backbone, particularly the --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2--[known as the methylene (methylimino) or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2--, and the amide backbone of U.S. Patent No. 5,602,240, as referenced above. In some embodiments, the RNA discussed herein has the morpholino backbone structure of U.S. Patent No. 5,034,506, as referenced above. The natural phosphodiester skeleton can be represented as OP(O)(OH)-OCH2-.

[0199] Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs taken up herein, such as dsRNAs, can contain at one of the 2'-position one of the following: OH, F, O-, S- or N-alkyl, O-, S- or N-alkenyl, O-, S- or N-alkynyl, or O-alkyl-O-alkyl, wherein alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C 10 alkyl or C2-C 10 alkenyl and alkynyl can be. Exemplary suitable modifications include O[(CH2) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON[(CH2) n CH3)]2, wherein n and m are from 1 to about 10. In other embodiments, the dsRNA contains at the 2'-position one of the following: C1-C 10Lower alkyl groups, substituted lower alkyl groups, alkali groups, aralkyl groups, O-alkaryl or O-aralkyl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl groups, heterocycloalkaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleavage groups, reporter groups, interfering substances, groups for improving the pharmacokinetic properties of iRNA or groups for improving the pharmacodynamic properties of iRNA, and other substituents having similar properties. In some embodiments, modifications include 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., alkoxy-alkoxy groups. Other exemplary modifications include the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the following examples herein, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH3)2. Further exemplary modifications include 5'-Me-2'-F nucleotide, 5'-Me-2'-OMe nucleotide, 5'-Me-2'-deoxynucleotide (both R and S isomers within these three families), 2'-alkoxyalkyl, and 2'-NMA (N-methylacetamide).

[0200] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also occur at other positions on the iRNA, particularly on the 3' terminal nucleotide or at the 3' and 5' positions of the sugar in 2'-5' ligated dsRNA. iRNA may also have sugar mimetic molecules such as cyclobutyl moieties instead of pentofuranose sugars. Representative U.S. patents teaching the preparation of such modified sugar structures include U.S. Patent Applications 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 5,567,811. Nos. 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, among others, are included but not limited to, and some of these are shared with this application. The entire contents of each of the aforementioned are incorporated herein by reference.

[0201] iRNA may also include modifications or substitutions of nucleic acid bases (often simply referred to as "bases" in the art). As used herein, "unmodified" or "natural" nucleic acid bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include other synthetic and natural nucleic acid bases, such as deoxythymidine (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, and cytosine. These include thymine, 5-uracil (pseudolacil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 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, as well as 3-deazaguanine and 3-deazaadenine.Further examples of nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808, Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008, The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990, Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and Sanghvi, Y. S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, STand Lebleu, B., Ed., CRC Press, 1993. Some of these nucleic acid bases are particularly useful for increasing the binding affinity of the oligomeric compounds addressed in this invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, such as 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitution has been shown to increase the double-strand stability of nucleic acids by 0.6-1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp.276-278), and more specifically, when combined with 2'-O-methoxyethyl sugar modification, it is an exemplary base substitution.

[0202] Representative U.S. patents teaching the preparation of the above-mentioned modified nucleic acid bases and specific other modified nucleic acid bases include, but are not limited to, U.S. Patents No. 3,687,808, No. 4,845,205, No. 5,130,30, No. 5,134,066, No. 5,175,273, No. 5,367,066, No. 5,432,272, No. 5,457,187, No. 5,459,255, No. 5,484,908, No. 5,502,177, No. 5,525,711, No. 5,552,540, and No. 5,587,469, listed above. Examples include Nos. 5,594,121, 5,596,091, 5,614,617, 5,681,941, 5,750,692, 6,015,886, 6,147,200, 6,166,197, 6,222,025, 6,235,887, 6,380,368, 6,528,640, 6,639,062, 6,617,438, 7,045,610, 7,427,672, and 7,495,088, the entire contents of each of these publications are incorporated herein by reference.

[0203] In some embodiments, the RNAi agents of the present disclosure may also be modified to include one or more bicyclic sugar moieties. A “bicyclic sugar” is a furanosyl ring modified by a ring formed by a bridge between two carbons, whether adjacent or non-adjacent. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety that includes a ring formed by a bridge between two carbon atoms of a sugar ring, whether adjacent or non-adjacent, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring, optionally via a 2'-acyclic oxygen atom. Thus, in some embodiments, the agents of the present invention may include one or more LOK nucleic acids (LNAs). A LOK nucleic acid is a nucleotide having a modified ribose moiety, wherein the ribose moiety includes an additional bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety including a 4'-CH2-O-2' bridge. This structure efficiently "locks" ribose within the 3'-endo conformation. The addition of locking nucleic acids to siRNA is known to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447, Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843, Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the present invention include, but are not limited to, nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agent of the present invention may be one or more bicyclic nucleosides containing a 4'-to-2' bridge.

[0204] A locked nucleoside can be represented by this structure (stereochemistry omitted), [ka] In the formula, B is a nucleic acid base or a modified nucleic acid base, and L is a linking group that connects the 2'-carbon and 4'-carbon of the ribose ring. Examples of such 4'-to-2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH2)-O-2'(LNA), 4'-(CH2)-S-2', 4'-(CH2)2-O-2'(ENA), 4'-CH(CH3)-O-2' (also known as “restricted ethyl” or “cEt”) and 4'-CH(CH2OCH3)-O-2' (and its analogues, see, e.g., U.S. Patent No. 7,399,845), 4'-C(CH3)(CH3)-O-2' (and its analogues, e.g., Examples include U.S. Patent No. 8,278,283, 4'-CH2-N(OCH3)-2' (and its analogues, see, for example, U.S. Patent No. 8,278,425), 4'-CH2-ON(CH3)-2' (see, for example, U.S. Patent Application Publication No. 2004 / 0171570), 4'-CH2-N(R)-O-2', where R is H, C1-C12 alkyl, or nitrogen protecting group (see, for example, U.S. Patent No. 7,427,672), 4'-CH2-C(H)(CH3)-2' (see, for example, Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134), and 4'-CH2-C(=CH2)-2' (and its analogues, see, for example, U.S. Patent No. 8,278,426). The entire contents of each of the aforementioned are incorporated herein by reference.

[0205] Further representative U.S. patents and U.S. patent application publications teaching the preparation of lock nucleic acid nucleotides include, but are not limited to, U.S. Patents 6,268,490, 6,525,191, 6,670,461, 6,770,748, 6,794,499, 6,998,484, 7,053,207, 7,034,133, 7,084,125, and 7,399. Examples include U.S. Patent Publication Nos. 845, 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 these are incorporated herein by reference.

[0206] For example, any of the aforementioned bicyclic nucleosides having one or more stereochemical sugar conformations, including α-L-ribofuranose and β-D-ribofuranose, can be prepared (see International Publication No. 99 / 14226).

[0207] The RNA of the iRNA may also be modified to include one or more constrained ethyl nucleotides. As used herein, “constrained ethyl nucleotide” or “cEt” is a constrained nucleic acid containing a bicyclic sugar moiety including a 4'-CH(CH3)-O-2' bridge (L in the aforementioned structure). In one embodiment, the constrained ethyl nucleotide is in the S conformation and is referred to herein as “S-cEt”.

[0208] The iRNA of the present invention may also comprise one or more “conformally restricted nucleotides” (“CRNs”). A CRN is a nucleotide analog having a linker connecting the C2' and C4' carbons of ribose, or the C3 and -C5' carbons of ribose. The CRN locks the ribose ring into a stable conformation, increasing its hybridization affinity to mRNA. The linker is long enough to position the oxygen in an optimal position for stability and affinity, resulting in reduced puckering of the ribose ring.

[0209] Representative publications teaching the preparation of certain CRNs mentioned above include, but are not limited to, U.S. Patent Application Publication No. 2013 / 0190383 and the PCT publication International Publication No. 2013 / 036868, the entire contents of which are incorporated herein by reference.

[0210] In some embodiments, the iRNA of the present invention comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is an unlocked acyclic nucleic acid in which all of its sugar bonds have been removed to form an unlocked "sugar" residue. In one embodiment, UNA also includes monomers in which the C1'-C4' bond has been removed (i.e., a carbon-oxygen-carbon bond by covalent bonding between the C1' and C4' carbons). In another embodiment, the C2'-C3' bond of the sugar (i.e., a carbon-carbon bond by covalent bonding between the C2' and C3' carbons) has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference).

[0211] Representative U.S. publications teaching the preparation of UNAs include, but are not limited to, U.S. Patent No. 8,314,227, and U.S. Patent Application Publications 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020, the entire contents of each of which are incorporated herein by reference.

[0212] Possible stabilization 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'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyluridine-3'-phosphate, reverse 2'-deoxy-modified ribonucleotides, such as reverse dT (idT) and reverse dA (idA), as well as reverse debasitized 2'-deoxyribonucleotide (iAb) and others. The disclosure of these modifications can be found in International Publication No. 2011 / 005861.

[0213] In one embodiment, the 3' or 5' end of the oligonucleotide is ligated to a reverse 2'-deoxy-modified ribonucleotide, such as a reverse dT (idT), reverse dA (idA), or reverse debased 2'-deoxyribonucleotide (iAb). In one particular embodiment, the reverse 2'-deoxy-modified ribonucleotide is ligated to the 3' end of the oligonucleotide, such as the 3' end of the sense strand described herein, where the ligation is via a 3'-3' phosphodiester linkage or a 3'-3'-phosphorothioate linkage.

[0214] In another embodiment, the 3' end of the sense strand is linked to a reverse debased ribonucleotide (iAb) via a 3'-3'-phosphorothioate linkage. In yet another embodiment, the 3' end of the sense strand is linked to a reverse dA (idA) via a 3'-3'-phosphorothioate linkage.

[0215] In one particular embodiment, a reverse 2'-deoxy-modified ribonucleotide is ligated to the 3' end of an oligonucleotide, such as the 3' end of a sense strand as described herein, where the ligation is via a 3'-3' phosphodiester linkage or a 3'-3'-phosphorothioate linkage.

[0216] In another embodiment, the 3'-terminal nucleotide of the sense strand is reverse-direction dA(idA) and is linked to a preceding nucleotide via a 3'-3' linkage (e.g., a 3'-3'-phosphorothioate linkage).

[0217] Other nucleotide modifications of the iRNA of the present invention include 5' phosphates or 5' phosphate mimetic, such as a 5' terminal phosphate or phosphate mimetic on the antisense strand of the iRNA. Suitable phosphate mimetics are disclosed, for example, in U.S. Patent Application Publication 2012 / 0157511, which is incorporated herein by reference in its entirety.

[0218] A. Modified iRNA containing the motif of the present invention In certain embodiments of the present invention, the double-stranded RNA agent of the present invention includes agents having chemical modifications, such as those disclosed in International Publication No. 2013 / 075035, which is incorporated herein by reference in its entirety. As shown herein and in International Publication No. 2013 / 075035, one or more motifs of three identical modifications on three consecutive nucleotides can be introduced into the sense or antisense strand of the dsRNAi agent, particularly at or near the cleavage site. In some embodiments, the sense and antisense strands of the dsRNAi agent may be otherwise completely modified. The introduction of these motifs interrupts the modification pattern of the sense or antisense strand, if present. The dsRNAi agent may optionally be conjugated with a GalNAc derivative ligand, for example, on the sense strand.

[0219] More specifically, gene silencing activity of the dsRNAi agent was observed when the sense and antisense strands of the double-stranded RNA agent were completely modified to have one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of the dsRNAi agent.

[0220] Accordingly, the present invention provides a double-stranded RNA agent capable of inhibiting the expression of a target gene (i.e., the ANGPTL3 gene) in vivo. This RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may be, for example, 17-30 nucleotides, 25-30 nucleotides, 27-30 nucleotides, 19-25 nucleotides, 19-23 nucleotides, 19-21 nucleotides, 21-25 nucleotides, or 21-23 nucleotides in length.

[0221] The sense strand and antisense strand typically form a double-stranded RNA ("dsRNA"), also referred to herein as the "dsRNAi agent." The double-stranded region of the dsRNAi agent may be, for example, a double-stranded region having a length of 27–30 nucleotide pairs, 19–25 nucleotide pairs, 19–23 nucleotide pairs, 19–21 nucleotide pairs, 21–25 nucleotide pairs, or 21–23 nucleotide pairs. In another embodiment, the double-stranded region is selected from lengths of 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides.

[0222] In certain embodiments, the dsRNAi agent may contain one or more overhang regions or capping groups at the 3' end, 5' end, or both ends of one or both strands. The overhangs may independently be 1 to 6 nucleotides long, 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 certain embodiments, the overhang region may include an extended overhang region as described above. The overhang may result from one strand being longer than the other, or from two strands of equal length being twisted. This overhang may form a mismatch with the target mRNA, or may be complementary to the target gene sequence, or may be a different sequence. The first and second strands may also be linked, for example, by additional bases forming a hairpin, or by other non-base linkers.

[0223] In certain embodiments, the nucleotides within the overhang region of the dsRNAi agent may independently be modified or unmodified nucleotides, including, but not limited to, 2'-sugar-modified, such as 2'-F, 2'-O-methyl thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyladenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof.

[0224] For example, TT could be an overhang sequence for any end on any strand. This overhang could form a mismatch with the target mRNA, or it could be complementary to the target gene sequence, or it could be another sequence altogether.

[0225] The sense strand, antisense strand, or 5'- or 3'- overhangs of both strands of a dsRNAi agent can be phosphorylated. In some embodiments, the overhang region contains two nucleotides having a phosphorothioate between them, which may or may not be the same. In some embodiments, the overhang is located at the 3' end of the sense strand, antisense strand, or both strands. In some embodiments, this 3'-overhang is located within the antisense strand. In some embodiments, this 3'-overhang is located within the sense strand.

[0226] dsRNAi agents may contain only a single overhang that can enhance RNAi interference activity without affecting their overall stability. For example, a single-stranded overhang may be located at the 3' end of the sense strand or the 3' end of the antisense strand. RNAi may also have a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand), or vice versa. Generally, the antisense strand of a dsRNAi agent has a nucleotide overhang at the 3' end and a blunt end at the 5' end. Without wishing to be constrained by theory, such as the blunt end at the 5' end of the antisense strand and the 3' end overhang of the antisense strand are advantageous for guide strand insertion into RISC processes.

[0227] In certain embodiments, the dsRNAi agent is a double-blunt-ended agent with a length of 19 nucleotides, wherein 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. 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.

[0228] In other embodiments, the dsRNAi agent has a double blunt end with a length of 20 nucleotides, and 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. 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.

[0229] In yet another embodiment, the dsRNAi agent has a double blunt end with a length of 21 nucleotides, and 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. 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.

[0230] In certain embodiments, the dsRNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, 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, with one end of the RNAi agent being blunt and the other end containing a 2-nucleotide overhang. In one embodiment, the 2-nucleotide overhang is at the 3' end of the antisense strand.

[0231] If a two-nucleotide overhang is located at the 3' end of the antisense strand, there may be two phosphorothioate nucleotide links between the three terminal nucleotides, two of which are the overhang nucleotides and the third nucleotide is a paired nucleotide adjacent to the overhang nucleotide. In one embodiment, the RNAi agent further has two phosphorothioate nucleotide links between the three terminal nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand. In a particular embodiment, all nucleotides in the sense and antisense strands of the dsRNAi agent, including nucleotides that are part of a motif, are modified nucleotides. In a particular embodiment, each residue is independently modified with 2'-O-methyl or 3'-fluoro, for example, in an alternating motif. Optionally, the dsRNAi agent further comprises a ligand (e.g., GalNAc3).

[0232] In certain embodiments, the dsRNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is 25-30 nucleotides long, starting from the 5' terminal nucleotide (position 1), and positions 1-23 of the first strand contain at least 8 ribonucleotides; the antisense strand is 36-66 nucleotides long, starting from the 3' terminal nucleotide, and forming a double helix with at least 8 ribonucleotides at positions 1-23 of the sense strand, wherein at least the 3' terminal nucleotide of the antisense strand does not pair with the sense strand, and up to 6 consecutive 3' terminal nucleotides do not pair with the sense strand, thereby forming a 3' single-strand overhang of 1-6 nucleotides, and the 5' end of the antisense strand contains 10-30 nucleotides that do not pair with the sense strand. The sense strand contains a sequence of nucleotides, thereby forming a single-stranded 5' overhang of 10-30 nucleotides, wherein at least the 5' and 3' terminal nucleotides of the sense strand base-pair with the nucleotides of the antisense strand when the sense and antisense strands are aligned in maximum complementarity, thereby forming a substantially double-stranded 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, reducing target gene expression when the double-stranded nucleic acid is introduced into mammalian cells, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, at least one of which occurs 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.

[0233] In certain embodiments, the dsRNAi agent comprises a sense strand and an antisense strand, wherein the dsRNAi agent comprises a first strand having a length of at least 25 nucleotides and at most 29 nucleotides, and a second strand having a length of at most 30 nucleotides 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, the 3' end of the first strand and the 5' end of the second strand form blunt ends, and the second strand is 1 to 4 nucleotides longer than the first strand at its 3' end, the double-stranded region having a length of at least 25 nucleotides, and the second strand is sufficiently complementary to the target mRNA along at least 19 nucleotides of the length of the second strand, and the RNAi agent reduces target gene expression when introduced into mammalian cells, and Dicer cleavage of the dsRNAi agent results in siRNA including the 3' end of the second strand, thereby reducing target gene expression in mammals. Optionally, the dsRNAi agent further comprises a ligand.

[0234] In certain embodiments, the sense strand of the dsRNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, one of which occurs at a cleavage site in the sense strand.

[0235] In certain embodiments, the antisense strand of the dsRNAi agent may also contain at least one motif of three identical modifications on three consecutive nucleotides, one of which occurs at or near a cleavage site within the antisense strand.

[0236] For dsRNAi agents with a double-stranded region of 19–23 nucleotides in length, the cleavage sites on the antisense strand are typically located at positions 10, 11, and 12 from the 5' end. Therefore, the three identical modification motifs can occur at positions 9, 10, 11, 10, 11, 12, 11, 12, 13, 12, 13, 12, 13, 14, or 13, 14, 15 of the antisense strand, with these numbers starting from the first nucleotide from the 5' end of the antisense strand, or starting from the first paired nucleotide within the double-stranded region from the 5' end of the antisense strand. The cleavage sites within the antisense strand can also vary depending on the length of the double-stranded region of the dsRNAi agent from the 5' end.

[0237] The sense strand of a dsRNAi agent may contain at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site, and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site. When the sense strand and antisense strand form a dsRNA double helix, the sense strand and antisense strand may be aligned such that one motif of the three nucleotides on the sense strand and one motif of the three nucleotides on the antisense strand have at least one nucleotide overlap, that is, at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may be duplicated, or all three nucleotides may be duplicated.

[0238] In some embodiments, the sense strand of a dsRNAi agent may contain two or more motifs of three identical modifications on three consecutive nucleotides. The first motif may occur at or near a cleavage site on the strand, and the other motifs may be wing modifications. In this specification, the term “wing modification” refers to a motif occurring in a different part of the strand separated from a motif at or near a cleavage site on the same strand. Wing modifications are adjacent to the first motif or separated by at least one or more nucleotides. If the motifs are immediately adjacent to each other, their chemical properties are distinct from each other; if the motifs are separated by one or more nucleotides, their chemical properties may be identical or different. There may be two or more wing modifications. For example, if there are two wing modifications, each wing modification may occur at one end relative to the first motif at or near a cleavage site, or on either side of the lead motif.

[0239] Similar to the sense strand, the antisense strand of a dsRNAi agent may contain two or more motifs of three identical modifications on three consecutive nucleotides, at least one of which occurs at or near a cleavage site on the strand. This antisense strand may also contain one or more wing modifications within an alignment similar to that of the wing modifications that may be present on the sense strand.

[0240] In some embodiments, the wing modifications on the sense or antisense strand of the dsRNAi agent typically do not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0241] In other embodiments, the wing modifications on the sense or antisense strand of the dsRNAi agent typically do not include the first one or two paired nucleotides in the double-stranded region at the 3' end, 5' end, or both ends of the strand.

[0242] If the sense strand and antisense strand of the dsRNAi agent each contain at least one wing modification, the wing modification may be located at the same end of the double-stranded region and may have an overlap of one, two, or three nucleotides.

[0243] If the sense strand or antisense strand of a dsRNAi agent each contains at least two wing modifications, the sense strand and antisense strand may be aligned such that two modifications from each single strand enter one end of a double-stranded region having one, two, or three nucleotide overlaps, and two modifications from each single strand enter the other end of the double-stranded region having one, two, or three nucleotide overlaps, and two modifications from the single strand enter on both sides of a read motif having one, two, or three nucleotide overlaps within the double-stranded region.

[0244] In some embodiments, any nucleotide in the sense and antisense strands of a dsRNAi agent containing a nucleotide that is part of a motif may be modified. Each nucleotide may be modified with the same or different modifications, which may include changes to one or both of the unbound phosphate oxygens, or one or more of the bound phosphate oxygens, changes to components of the ribose sugar, such as the 2'-hydroxyl on the ribose sugar, large-scale substitution of the phosphate moiety with a "dephosphorylated" linker, modification or substitution of naturally occurring bases, and substitution or modification of the ribose phosphate backbone.

[0245] Since nucleic acids are polymers of subunits, most modifications occur at repeating positions within the nucleic acid, such as modifications of base or phosphate moieties, or unbound oxygen atoms in phosphate moieties. In some cases, modifications may occur at all target positions in the nucleic acid, but often they do not. For example, modifications may occur only at the 3' or 5' end, only in terminal regions, for example, at the terminal nucleotides of a strand, or at the last 2, 3, 4, 5, or 10 nucleotides of a strand. Modifications may occur in double-stranded regions, single-stranded regions, or both. Modifications may occur only within the double-stranded regions of RNA, or only within the single-stranded regions of RNA. For example, phosphorothioate modifications at unbound oxygen atoms may occur only at one or both ends, only in terminal regions, for example, at the terminal nucleotides of a strand, or at the last 2, 3, 4, 5, or 10 nucleotides of a strand, or both within double-stranded and single-stranded regions, especially at the terminals. It is possible to phosphorylate the 5' end.

[0246] This may allow for, for example, enhanced stability, inclusion of specific bases within the overhang, or inclusion of modified nucleotides or nucleotide substitutes within single-stranded overhangs, e.g., within the 5' or 3' overhang, or within both overhangs. For example, inclusion of purine nucleotides within the overhang may be desirable. In some embodiments, all or some of the bases within the 3' or 5' overhang may be modified with modifications, for example, those described herein. Modifications may include, for example, the use of modifications at the 2' position of the ribose sugar in modifications known in the art, e.g., the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro(2'-F), or 2'-O-methyl modified in place of the ribosaccharide of the nucleic acid base, and modifications at the phosphate group, e.g., phosphorothioate modifications. The overhang does not need to be homologous to the target sequence.

[0247] In some embodiments, each residue in the sense and antisense chains 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. The chains may contain two or more modifications. In one embodiment, each residue in the sense and antisense chains is independently modified with 2'-O-methyl or 2'-fluoro.

[0248] At least two distinct modifications are typically present on the sense and antisense chains. These two modifications may include 2'-O-methyl or 2'-fluoro modifications.

[0249] In a particular embodiment, N a or N b This includes alternating pattern modifications. The term “alternating motif,” as used herein, refers to a motif having one or more modifications, where each modification occurs on a single-stranded alternating nucleotide. An alternating nucleotide can refer to a pattern of one or similar modifications for every other nucleotide, or one or similar patterns for every three nucleotides. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif could be “ABABABABABAB···”, “AABBAABBAABB···”, “AABAABAABAAB···”, “AAABAAABAAAB···”, “AAABBBAAABBB···”, or “ABCABCABCABC···”.

[0250] The modifications of this type contained within the alternating motif may be identical 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, may be identical, but each of the sense strand or antisense strand may be selected from several possible modifications within the range of the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...".

[0251] In some embodiments, the dsRNAi agent of the present invention includes a modification pattern of alternating motifs on the sense strand that is shifted relative to the modification pattern of alternating motifs on the antisense strand. This shift may be made such that the modifying groups of the nucleotides on the sense strand correspond to differently modified groups of the nucleotides on the antisense strand, and vice versa. For example, when the sense strand pairs with the antisense strand within the dsRNA double helix, the alternating motifs in the sense strand may begin with "ABABAB" from 5' to 3' of the strand, and the alternating motifs in the antisense strand may begin with "BABABA" from 5' to 3' of the strand within the double helix region. As another example, the alternating motifs in the sense strand may begin with "AABBAABB" from 5' to 3' of the strand, and the alternating motifs in the antisense strand may begin with "BBAABBAA" from 5' to 3' of the strand within the double helix region, resulting in a complete or partial shift of the modification patterns between the sense and antisense strands.

[0252] In one particular example, the alternating motif within the sense strand is "ABABAB" from the 5'–3' of the strand, where each A is an unmodified ribonucleotide and each B is a 2'-O methyl-modified nucleotide.

[0253] In one particular example, the alternating motif within the sense strand is "ABABAB" from the 5'–3' of the strand, where each A is a 2'-deoxy-2'-fluoro-modified nucleotide and each B is a 2'-O-methyl-modified nucleotide.

[0254] In another specific embodiment, the alternating motif within the antisense chain is "BABABA" from 3'-5' of the chain, where each A is a 2'-deoxy-2'-fluoro modified nucleotide and each B is a 2'-O-methyl modified nucleotide.

[0255] In one particular example, the alternating motif in the sense strand is "ABABAB" from 5'–3' of the strand, and the alternating motif in the antisense strand is "BABABA" from 3'–5 of the strand, where each A is an unmodified ribonucleotide and each B is a 2'-O methyl-modified nucleotide.

[0256] In one particular example, the alternating motif in the sense strand is "ABABAB" from 5'–3' of the strand, and the alternating motif in the antisense strand is "BABABA" from 3'–5 of the strand, where each A is a 2'-deoxy-2'-fluoro-modified nucleotide and each B is a 2'-O-methyl-modified nucleotide.

[0257] In some embodiments, the dsRNAi agent comprises a pattern of alternating 2'-O-methyl and 2'-F modifications on the sense strand, initially having a relative shift to the pattern of alternating 2'-O-methyl and 2'-F modifications on the antisense strand, i.e., comprising 2'-O-methyl modified nucleotides on the sense strand base pair and 2'-F modified nucleotides on the antisense strand, and vice versa. Position 1 of the sense strand may begin with a 2'-F modification, and position 1 of the antisense strand may begin with a 2'-O-methyl modification.

[0258] Introducing one or more motifs of three identical modifications on three consecutive nucleotides into a sense or antisense strand disrupts the initial modification pattern present in the sense or antisense strand. Disruption of the modification pattern of the sense or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense or antisense strand may enhance gene silencing activity against the target gene.

[0259] In some embodiments, when three identical modification motifs on three consecutive nucleotides are introduced into any one of the chains, the modifications of nucleotides adjacent to the motif are different from the modifications of the motif. For example, the portion of the sequence containing the motif is "···Na YYYN b ..." where "Y" represents the modification of three identical modification motifs on three consecutive nucleotides, and "N a " and "N b " represents a modification to a nucleotide adjacent to the motif "YYY", which is different from the modification of Y, and here N a and N b These can be the same or different modifications. Alternatively, if a wing modification exists, N a or N b It may or may not exist.

[0260] iRNA may further contain at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur at any position on the strand, on the sense strand, the antisense strand, or on any nucleotide on both strands. For example, the internucleotide linkage modification may occur on any nucleotide on the sense strand or the antisense strand, each internucleotide linkage modification may occur in an alternating pattern on the sense strand or the antisense strand, or the sense strand or antisense strand may contain both internucleotide linkage modifications in an alternating pattern. The alternating pattern of internucleotide linkage modifications on the sense strand may be the same as or different from that on the antisense strand, and the alternating pattern of internucleotide linkage modifications on the sense strand may have a relative shift to the alternating pattern of internucleotide linkage modifications on the antisense strand. In one embodiment, the double-stranded RNAi agent contains 6-8 phosphorothioate internucleotide linkages. In some embodiments, the antisense strand includes two phosphorothioate nucleotide linkages at its 5' end and two phosphorothioate nucleotide linkages at its 3' end, and the sense strand includes at least two phosphorothioate nucleotide linkages at either its 5' or 3' end.

[0261] In some embodiments, the dsRNAi agent includes a phosphorothioate or methylphosphonate internucleotide linkage modification within the overhang region. For example, the overhang region may contain two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between them. The internucleotide linkage modification may also be made to link the overhang nucleotide to a terminal paired nucleotide within the double-stranded region. For example, at least 2, 3, 4 or all of the overhang nucleotides may be linked by a phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may be further phosphorothioate or methylphosphonate internucleotide links linking the overhang nucleotide to an adjacent paired nucleotide. For example, there may be at least two phosphorothioate internucleotide links between three terminal nucleotides, where two of the three nucleotides are overhang nucleotides and the third is an adjacent paired nucleotide to the overhang nucleotide. These three terminal nucleotides can be located at the 3' end of the antisense strand, the 3' end of the sense strand, the 5' end of the antisense strand, or the 5' end of the antisense strand.

[0262] In some embodiments, a 2-nucleotide overhang is located at the 3' end of the antisense strand, with two phosphorothioate nucleotide links between the three terminal nucleotides, two of which are the overhang nucleotides, and the third nucleotide being a paired nucleotide adjacent to the overhang nucleotide. Optionally, the dsRNAi agent may further have two phosphorothioate nucleotide links between the three terminal nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand.

[0263] In one embodiment, the dsRNAi agent includes a double-stranded mismatch or combination thereof with a target. The mismatch may occur within an overhang region or within a double-stranded region. Base pairs may be graded based on their tendency to promote dissociation or dissolution (e.g., based on the free energy of association or dissociation of a particular pairing, the simplest approach being to examine each individual pair, although adjacent or similar analyses may 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-standard or non-standard pairings (described elsewhere in this specification), are preferred over standard pairings (A:T, A:U, G:C), and pairings containing universal bases are preferred over standard pairings.

[0264] In certain embodiments, the dsRNAi agent comprises at least one of the following: the first 1, 2, 3, 4, or 5 base pairs in the double-stranded region from the 5' end of the antisense strand, independently selected from the groups A:U, G:U, and I:C; and a mismatch pair, which is, for example, a non-standard pairing, a non-standard pairing, or a pairing containing a universal base, thereby promoting the dissociation of the antisense strand at the 5' end of the double-stranded region.

[0265] In certain embodiments, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand is selected from A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pairs in the double-stranded region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0266] In other embodiments, the nucleotide at the 3' end of the sense strand is deoxythymidine (dT), or the nucleotide at the 3' end of the antisense strand is deoxythymidine (dT). For example, there may be a short sequence of deoxythymidine nucleotides, e.g., two dT nucleotides on the 3' ends of the sense strand, antisense strand, or both strands.

[0267] In a particular embodiment, the sense strand sequence is given by formula (I): 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3'(I) It can be expressed as, During the ceremony, i and j are independently either 0 or 1. p and q are each independently between 0 and 6. each N a Each independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two differently modified nucleotides. each N b Each independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides. each n p and n q These independently represent overhang nucleotides, In the formula, Nb and Y do not have the same modification, and XXX, YYY, and ZZZ each independently represent a single motif of three identical modifications on three consecutive nucleotides. In one embodiment, all YYY are 2'-F modified nucleotides.

[0268] In some embodiments, N a or N b This includes alternating modification patterns.

[0269] In some embodiments, the YYY motif occurs at or near a cleavage site on the sense strand. For example, if the dsRNAi agent has a double-stranded region of 17–23 nucleotides in length, the YYY motif may occur at or near a cleavage site on 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), and this digit may start from the 5' end with the first nucleotide, or optionally, from the 5' end with the first paired nucleotide in the double-stranded region.

[0270] 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. Therefore, the sense chain is given by the following formula: 5'n p -N a -YYY-N b -ZZZ-N a -n q 3'(Ib), 5'n p -N a -XXX-N b -YYY-N a -n q 3'(Ic), or 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3'(Id) It can be represented as follows.

[0271] If the sense chain is represented by formula (Ib), then N b This represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-5, 0-4, 0-2, or 0. a These can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0272] If the sense chain is represented by equation (Ic), then Nb This represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a These can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0273] If the sense chain is represented by the expression (Id), then each N b Independently, each represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-5, 0-4, 0-2, or 0. In one embodiment, N b is 0, 1, 2, 3, 4, 5, or 6. a This can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0274] Each of X, Y, and Z may be the same as or different from one another.

[0275] In other embodiments, i is 0 and j is 0, and the sense chain is given by the formula 5'n p -N a -YYY-N a -n q 3'(Ia) It can be represented as follows.

[0276] If the sense chain is represented by equation (Ia), then each N a These can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0277] In one embodiment, the antisense strand sequence of RNAi is given by formula (II): 5'n q’ -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p '3'(II) It can be expressed as, During the ceremony, k and l are independently either 0 or 1. p' and q' are each independently between 0 and 6. each N a Each of these independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two differently modified nucleotides. each N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides. each n p 'and n q ' independently represents an overhang nucleotide, In the formula, N b 'and Y' do not have the same modifier, and X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif of three identical modifications on three consecutive nucleotides.

[0278] In some embodiments, N a 'or N b ' includes alternating modification patterns.

[0279] The Y'Y'Y' motif occurs at or near the cleavage site of the antisense strand. For example, if a dsRNAi agent has a double-stranded region of 17-23 nucleotides in length, the Y'Y'Y' motif may occur at positions 9, 10, 11, 10, 11, 12, 11, 12, 13, 12, 13, 12, 13, 14, or 13, 14, 15 of the antisense strand, with the digits starting from the first nucleotide from the 5' end, or optionally, starting from the first paired nucleotide within the double-stranded region from the 5' end. In one embodiment, the Y'Y'Y' motif occurs at positions 11, 12, and 13.

[0280] In certain embodiments, all Y'Y'Y' motifs are 2'-OMe modified nucleotides.

[0281] In certain embodiments, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

[0282] Thus, the antisense strand has the following formula: 5’n q’ -N a ’-Z’Z’Z’-N b ’-Y’Y’Y’-N a ’-n p’ 3’ (IIb), 5’n q’ -N a ’-Y’Y’Y’-N b ’-X’X’X’-n p’ 3’ (IIc), or 5’n q’ -N a ’-Z’Z’Z’-N b ’-Y’Y’Y’-N b ’-X’X’X’-N a ’-n p’ 3’ (IId) and can be represented by.

[0283] When the antisense strand is represented by formula (IIb), N b ’ represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N a ’ independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0284] When the antisense strand is represented by formula (IIc), N b ’ represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N a ’ independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0285] When the antisense strand is represented by formula (IId), each Nb ' independently represents oligonucleotide sequences containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a ' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. In one embodiment, N b It is 0, 1, 2, 3, 4, 5, or 6.

[0286] In other embodiments, k is 0 and l is 0, and the antisense chain is given by the formula: 5'n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 3'(Ia) It can be represented as follows.

[0287] If the antisense chain is represented by equation (IIa), then each N a ' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0288] Each of X', Y', and Z' may be the same as or different from one another.

[0289] Each nucleotide in the sense and antisense strands 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 in the sense and antisense strands can be independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y', and Z' may, in particular, represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0290] In some embodiments, the sense strand of the dsRNAi agent may contain a YYY motif occurring at positions 9, 10, and 11 of the strand when the double-stranded region is 21nt (nucleotides), where the digits start from the first nucleotide from the 5' end, or optionally, from the first paired nucleotide in the double-stranded region from the 5' end, and Y represents a 2'-F modification. The sense strand may further contain an XXX motif or a ZZZ motif as a wing modification at the opposite ends of the double-stranded region, where XXX and ZZZ independently represent a 2'-OMe modification or a 2'-F modification.

[0291] In some embodiments, the antisense strand may contain a Y'Y'Y' motif occurring at positions 11, 12, and 13 of the strand, where the digits start from the first nucleotide from the 5' end, or optionally, from the first paired nucleotide in the double-stranded region from the 5' end, 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 ends of the double-stranded region, where X'X'X' and Z'Z'Z' independently represent a 2'-OMe modification or a 2'-F modification.

[0292] Each sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a double helix with an antisense strand represented by any one of the above formulas (IIa), (IIb), (IIc), and (IId).

[0293] Therefore, the dsRNAi agent used in the method of the present invention may include a sense strand and an antisense strand, each having 14 to 30 nucleotides, and the iRNA double strand is given by the following formula (III): Sense: 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p ’ -N a ’ -(X'X'X') k -N b ’ -Y'Y'Y'-N b ’ -(Z'Z'Z') l -N a ’ -n q ’ 5' (III) It is represented as, During the ceremony, i, j, k, and l are each independently either 0 or 1. p, p', q, and q' are each independently between 0 and 6. each N a and N a ’ Each independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two distinctly modified nucleotides. each N b and N b ’ Each independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides. In the formula, each n p ',n p , n q 'and n q Each of these may or may not exist, and independently represents an overhang nucleotide. XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif of three identical modifications on three consecutive nucleotides.

[0294] 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 both i and j are 0, or both i and j are 1. In another embodiment, k is 0 and l is 0, or k is 1 and l is 0, k is 0 and l is 1, or both k and l are 0, or both k and l are 1.

[0295] The following formula is an example of a combination of sense strands and antisense strands that form an iRNA double helix. 5'n p -N a -YYY-N a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y'-N a ’ n q ’ 5' (IIIa) 5'n p -N a -YYY-N b -ZZZ-N a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a ’ n q ’ 5' (IIIb) 5'n p -N a -XXX-N b -YYY-N a -n q 3' 3'n p ’ -N a ’ -X'X'X'-N b’ -Y'Y'Y'-N a ’ -n q ’ 5' (IIIc) 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' 3'n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a -n q ’ 5' (IIId)

[0296] When a dsRNAi agent is represented by formula (IIIa), each N a Each of these independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0297] When a dsRNAi agent is represented by formula (IIIb), each N b Each N independently represents an oligonucleotide sequence containing 1-10, 1-7, 1-5, or 1-4 modified nucleotides. a Each of these independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0298] When a dsRNAi agent is represented by formula (IIIc), each N b , N b ' independently represents oligonucleotide sequences containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a Each of these independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0299] When a dsRNAi agent is represented by formula (IIId), each N b , N b ' independently represents oligonucleotide sequences containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a , N a ’ Each of these independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. a , N a ', N b , and N b ’ Each of these independently includes alternating modification patterns.

[0300] In equations (III), (IIIa), (IIIb), (IIIc), and (IIId), X, Y, and Z may be the same as or different from each other.

[0301] If a dsRNAi agent is represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), then at least one of the Y nucleotides may form a base pair with one of the Y' nucleotides. Alternatively, at least two Y nucleotides may form a base pair with the corresponding Y' nucleotide, or all three Y nucleotides may form a base pair with the corresponding Y' nucleotide.

[0302] If a dsRNAi agent is represented by formula (IIIb) or (IIId), then at least one of the Z nucleotides may form a base pair with one of the Z' nucleotides. Alternatively, at least two Z nucleotides may form base pairs with the corresponding Z' nucleotides, or all three Z nucleotides may form base pairs with the corresponding Z' nucleotides.

[0303] If a dsRNAi agent is represented by formula (IIIc) or (IIId), then at least one of the X nucleotides may form a base pair with one of the X' nucleotides. Alternatively, at least two of the X nucleotides may form a base pair with the corresponding X' nucleotide, or all three of the X nucleotides may form a base pair with the corresponding X' nucleotide.

[0304] In certain embodiments, modifications on the Y nucleotide are different from modifications on the Y' nucleotide, modifications on the Z nucleotide are different from modifications on the Z' nucleotide, or modifications on the X nucleotide are different from modifications on the X' nucleotide.

[0305] In certain embodiments, if the dsRNAi agent is represented by formula (IIId), N a The modification is a 2'-O-methyl or 2'-fluoro modification. In other embodiments, if the RNAi agent is represented by formula (IIId), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, and also n p '>0 and at least one n p ' is linked to an adjacent nucleotide via phosphorothioate linkage. In yet another embodiment, if the RNAi agent is represented by formula (IIId), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p ' is linked to an adjacent nucleotide via a phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker (described below). In other embodiments, if the RNAi agent is represented by formula (IIId), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n pThe sense strand is linked to an adjacent nucleotide via a phosphorothioate linkage, and the sense strand contains at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0306] In some embodiments, if the dsRNAi agent is represented by formula (IIIa), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p The sense strand is linked to an adjacent nucleotide via a phosphorothioate linkage, and the sense strand contains at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0307] In some embodiments, the dsRNAi agent is a multimer containing at least two double-stranded molecules represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), the double-stranded molecules being linked by a linker. The linker may or may not be cleavable. Optionally, the multimer further contains ligands. Each of the double-stranded molecules may target the same gene or two different genes, or each of the double-stranded molecules may target the same gene at two different target sites.

[0308] In some embodiments, the dsRNAi agent is a multimer containing three, four, five, six, or more double strands represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), the double strands being linked by a linker. The linker may or may not be cleavable. Optionally, the multimer further contains ligands. Each double strand may target the same gene or two different genes, or each double strand may target the same gene at two different target sites.

[0309] In one embodiment, two dsRNAi agents represented by at least one of formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) are ligated together at their 5' ends and at one or both of their 3' ends, and optionally conjugated to a ligand. Each agent may target the same gene or two different genes, or each agent may target the same gene at two different target sites.

[0310] In certain embodiments, the RNAi agent of the present invention may contain a small number of nucleotides having 2'-fluoro modifications, for example, 10 or fewer nucleotides having 2'-fluoro modifications. For example, the RNAi agent may contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 nucleotides having 2'-fluoro modifications. In certain embodiments, the RNAi agent of the present invention contains 10 nucleotides having 2'-fluoro modifications, for example, 4 nucleotides having 2'-fluoro modifications in the sense strand and 6 nucleotides having 2'-fluoro modifications in the antisense strand. In another particular embodiment, the RNAi agent of the present invention contains 6 nucleotides having 2'-fluoro modifications, for example, 4 nucleotides having 2'-fluoro modifications in the sense strand and 2 nucleotides having 2'-fluoro modifications in the antisense strand.

[0311] In other embodiments, the RNAi agent of the present invention may contain a very small number of nucleotides containing 2'-fluoro modifications, for example, two or fewer nucleotides containing 2'-fluoro modifications. For example, the RNAi agent may contain 2, 1, or 0 nucleotides having 2'-fluoro modifications. In a particular embodiment, the RNAi agent may contain 2 nucleotides having 2'-fluoro modifications, for example, 0 nucleotides having 2'-fluoro modifications in the sense strand and 2 nucleotides having 2'-fluoro modifications in the antisense strand.

[0312] Various published patent applications describe multimeric iRNAs that can be used in the methods of the present invention. These applications include International Publication No. 2007 / 091269, U.S. Patent No. 7,858,769, International Publication No. 2010 / 141511, International Publication No. 2007 / 117686, International Publication No. 2009 / 014887, and International Publication No. 2011 / 031520, the entire contents of each of these publications which are incorporated herein by reference.

[0313] In certain embodiments, the compositions and methods of the Disclosure include vinyl phosphonate (VP) modification of an RNAi agent as described herein. In exemplary embodiments, the 5'-vinyl phosphonate-modified nucleotide of the Disclosure has the following structure: [ka] In the formula, X is either O or S. R is hydrogen, hydroxyl, fluoro, or C 1~20 It is an alkoxy (e.g., methoxy or n-hexadecyloxy), R 5’ It is =C(H)-P(O)(OH)2, and also C5' carbon and R 5’ The double bond between them is in either the E or Z conformation (for example, the E conformation), and B is a nucleic acid base or a modified nucleic acid base, and optionally, B is adenine, guanine, cytosine, thymine, or uracil.

[0314] The vinyl phosphonates of the Disclosure may bind to either the antisense or sense strand of the dsRNA of the Disclosure. In certain embodiments, the vinyl phosphonates of the Disclosure may optionally bind to the antisense strand of the dsRNA, at its 5' end.

[0315] Vinyl phosphate modifications are also intended for the compositions and methods of the present disclosure. Exemplary vinyl phosphate structures include the structures described above, where R5' is = C(H)-OP(O)(OH)2, and the double bond between the C5' carbon and R5' is in either an E or Z conformation (e.g., E conformation).

[0316] As will be described in more detail below, iRNAs containing the conjugation of one or more carbohydrate moieties can optimize one or more properties of the iRNA. Often, the carbohydrate moieties bind to a modified subunit of the iRNA. For example, the ribose sugar of one or more ribonucleotide subunits of the iRNA may be replaced with another moiety, such as a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is bound. A ribonucleotide subunit in which the ribose sugar of the subunit is thus replaced is referred to herein as a ribose-substituted modified subunit (RRMS). The cyclic carrier may be a cyclic carbon system, i.e., all ring atoms are carbon atoms, or it may be a heterocyclic system, i.e., one or more ring atoms may be heteroatoms such as nitrogen, oxygen, and sulfur. The cyclic carrier may be a monocyclic ring system, or it may contain two or more rings, e.g., a fused ring. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.

[0317] A ligand can be bound to a polynucleotide via a carrier. The carrier comprises (i) at least one “skeletal linkage site,” for example, two “skeletal linkage sites,” and (ii) at least one “tethering linkage site.” “Skeleton linkage site,” as used herein, refers to a functional group, for example, a hydroxyl group, or generally, a linkage that is available and suitable for incorporating the carrier into the skeleton of a ribonucleic acid, for example, a phosphate, or a modified phosphate, for example, one containing sulfur. In some embodiments, a “tethering linkage site” (TAP) refers to a ring atom of the cyclic carrier connecting a selected portion, for example, a carbon atom or a heteroatom (different from the atom providing the skeletal linkage sites). This portion may be, for example, a carbohydrate, and may be, for example, a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected portion is connected to the cyclic carrier by an intervening anchorage. Therefore, cyclic carriers often contain a functional group, such as an amino group, or are generally bonds, providing a bond suitable for the incorporation or anchoring of another chemical entity, such as a ligand, to the constituting ring.

[0318] iRNAs can be conjugated to ligands via a carrier, which may be a cyclic or acyclic group. In one embodiment, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuryl, and decalin. In one embodiment, the acyclic group is a selinol skeleton or a diethanolamine skeleton.

[0319] i. Thermal destabilization modification In certain embodiments, a dsRNA molecule can be optimized for RNA interference by incorporating a thermal destabilization modification into the seed region of the antisense strand. As used herein, “seed region” refers to positions 2–9 of the 5' end of the referenced strand. For example, incorporating a thermal destabilization modification into the seed region of the antisense strand can reduce or inhibit off-target gene silencing.

[0320] The term "thermal destabilization modification" refers to the melting temperature (T) of dsRNA that does not have such modifications. m The overall melting temperature (T) is lower than ) m This includes modifications that result in dsRNA having ) . For example, thermal destabilization modifications result in dsRNA having T m This can be reduced by 1 to 4°C, for example, by 1, 2, 3, or 4 degrees Celsius. Furthermore, the term "thermally destabilized nucleotide" refers to a nucleotide containing one or more thermally destabilizing modifications.

[0321] We have found that dsRNAs having an antisense strand containing at least one double-stranded thermal destabilization modification located within the first nine nucleotides from the 5' end of the antisense strand exhibit reduced off-target gene silencing activity. Therefore, in some embodiments, the antisense strand contains at least one (e.g., 1, 2, 3, 4, 5, or more) double-stranded thermal destabilization modification within the first nine nucleotides from the 5' region of the antisense strand. In some embodiments, one or more double-stranded thermal destabilization modifications are located at positions 2–9 from the 5' end of the antisense strand, for example, positions 4–8. In some further embodiments, the double-stranded thermal destabilization modifications are located at positions 6, 7, or 8 from the 5' end of the antisense strand. In some even further embodiments, the double-stranded thermal destabilization modifications are located at position 7 from the 5' end of the antisense strand. In some embodiments, the double-stranded thermal destabilization modifications are located at positions 2, 3, 4, 5, or 9 from the 5' end of the antisense strand.

[0322] An iRNA agent contains a sense strand and an antisense strand, each having 14 to 40 nucleotides. The RNAi agent is represented by the following formula (L): [ka] (L) can be represented as (L).

[0323] In formula (L), B1, B2, B3, B1', B2', B3', and B4' are each independently nucleotides containing a modification selected from the group consisting of 2'-O-alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halo, ENA, and BNA / LNA. In one embodiment, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe modification. In one embodiment, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe modification or a 2'-F modification. In one embodiment, at least one of B1, B2, B3, B1', B2', B3', and B4' contains a 2'-ON-methylacetamide (2'-O-NMA, 2'-O-CH2C(O)N(Me)H) modification.

[0324] C1 is a thermally destabilized nucleotide located at a site opposite the seed region of the antisense strand (i.e., at positions 2-8 of the 5' end of the antisense strand). For example, C1 is located on the sense strand opposite a nucleotide at positions 2-8 of the 5' end of the antisense strand. In one embodiment, C1 is located at position 15 from the 5' end of the sense strand. The C1 nucleotide may harbor thermally destabilized modifications, including debasing, mismatch with an opposing nucleotide in a double helix, such as 2'-deoxyribonucleotide modifications or acyclic nucleotides, such as unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA), or 2'-5' linked ribonucleotides (3'-RNA). In one embodiment, C1 may have i) a mismatch with an opposing nucleotide in the antisense strand, or ii) a debasing modification selected from the group consisting of: [ka] , iii) sugar modification, [ka] , [ka] , [ka] , [ka] , [ka] , and [ka] Selected from the group consisting of, where B is a modified or unmodified nucleic acid base, R 1 and R 2 C1 independently has a thermal destabilization modification selected from the group consisting of sugar modifications, where R3 is H, alkyl, OR3, or alkyl, and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. In one embodiment, the thermal destabilization modification at C1 is a mismatch selected from the group consisting of G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, and U:T, where optionally, at least one nucleic acid base in the mismatch pair is a 2'-deoxy nucleic acid base. In one example, the thermal destabilization modification at C1 is GNA or [ka] That is the case.

[0325] T1, T1', T2', and T3' each independently represent a nucleotide containing a modification that provides the nucleotide with a steric bulk equal to or less than the steric bulk of the 2'-OMe modification. Steric bulk refers to the sum of the steric effects of the modifications. Methods for determining the steric effects of nucleotide modifications are known to those skilled in the art. The modifications may be at the 2' position of the ribose sugar of the nucleotide, or modifications to the nucleotide backbone that are similar to or equivalent to the 2' position of the ribose sugar of a non-ribose nucleotide, an acyclic nucleotide, or a nucleotide, and provide the nucleotide with a steric bulk less than or equal to the steric bulk of the 2'-OMe modification. For example, T1, T1', T2', and T3' each independently are selected from DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl. In one embodiment, T1 is DNA. In one embodiment, T1' is DNA, RNA, or LNA. In one embodiment, T2' is DNA or RNA. In another embodiment, T3' is DNA or RNA.

[0326] n 1 , n 3 , and q 1 Each of these is independently 4 to 15 nucleotides long.

[0327] n 5 , q 3 , and q 7 Each of these is independently 1 to 6 nucleotides long.

[0328] n 4 , q 2 , and q 6 Each is independently 1 to 3 nucleotides long, or n 4 This represents a length of 0 nucleotides.

[0329] q 5 Each of these is independently between 0 and 10 nucleotides in length.

[0330] n 2 , and q 4 Each of these is independently 0 to 3 nucleotides long.

[0331] Or, n 4 Its length is between 0 and 3 nucleotides.

[0332] In one embodiment, n 4 n can be 0. In one embodiment, n 4 is 0, and q 2 and q 6 In another embodiment, n 4 is 0, and q 2 and q 6 This is 1, and these are accompanied by two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23.

[0333] In one embodiment, n 4 , q 2 , and q 6 Each of these values ​​is 1.

[0334] In one embodiment, n 2 , n 4 , q 2 , q 4 , and q 6 Each of these values ​​is 1.

[0335] In one embodiment, C1 has a sense strand with a length of 19 to 22 nucleotides, and n 4 When is 1, it is located at positions 14-17 of the 5' end of the sense strand. In one embodiment, C1 is located at position 15 of the 5' end of the sense strand.

[0336] In one embodiment, T3' starts at position 2 from the 5' end of the antisense chain. In one embodiment, T3' is at position 2 from the 5' end of the antisense chain, and q 6 It is equal to 1.

[0337] In one embodiment, T1' starts at position 14 from the 5' end of the antisense chain. In one embodiment, T1' is located at position 14 from the 5' end of the antisense chain, and q 2 It is equal to 1.

[0338] In an exemplary embodiment, T3' starts at position 2 from the 5' end of the antisense chain, and T1' starts at position 14 from the 5' end of the antisense chain. In one example, T3' starts at position 2 from the 5' end of the antisense chain, and q 6 is equal to 1, and T1' starts from position 14 from the 5' end of the antisense chain, and q 2 It is equal to 1.

[0339] In one embodiment, T1' and T3' are separated by a length of 11 nucleotides (i.e., T1' and T3' nucleotides are not counted).

[0340] In one embodiment, T1' is located at position 14 from the 5' end of the antisense chain. In one embodiment, T1' is located at position 14 from the 5' end of the antisense chain, and q 2 When this is equal to 1, and the acyclic or 2'-position modification in the skeleton of non-ribose has less steric bulk than 2'-OMe-ribose.

[0341] In one embodiment, T3' is located at position 2 from the 5' end of the antisense chain. In one embodiment, T3' is located at position 2 from the 5' end of the antisense chain, and q 6 If the value is equal to 1, and the modification at the 2' position in the acyclic or skeletal structure of non-ribose is less than or equal to that of 2'-OMe-ribose, then the steric bulk is less than or equal to that of 2'-OMe-ribose.

[0342] In one embodiment, T1 is located at the cleavage site of the sense strand. In one embodiment, T1 is located when the sense strand is 19 to 22 nucleotides long and n 2When is 1, it is located at position 11 from the 5' end of the sense strand. In an exemplary embodiment, T1 is such that the sense strand is 19-22 nucleotides long and n 2 When it is 1, it is located at the sense strand cleavage site at position 11 from the 5' end of the sense strand.

[0343] In one embodiment, T2' starts at position 6 from the 5' end of the antisense chain. In one embodiment, T2' is located at positions 6-10 from the 5' end of the antisense chain, and q 4 The value is 1.

[0344] In exemplary embodiments, T1 is located at the cleavage site of the sense strand, for example, if the sense strand is 19-22 nucleotides long and n 2 When is 1, it is located at position 11 from the 5' end of the sense strand, and T1' is located at position 14 from the 5' end of the antisense strand, and also q 2 is equal to 1, and the modification to T1' is at the 2' position of the ribose sugar, or at an acyclic or skeletal position of non-ribose with less steric bulk than 2'-OMe-ribose, T2' is at the 6-10 position from the 5' end of the antisense chain, and q 4 is 1, and T3' is at position 2 from the 5' end of the antisense strand, and q 6 The value is equal to 1, and the modification to T3' is either at the 2' position or in an acyclic or skeletal position of non-ribose with less steric bulk than 2'-OMe ribose.

[0345] In one embodiment, T2' starts at position 8 from the 5' end of the antisense chain. In one embodiment, T2' is located at position 8 from the 5' end of the antisense chain, and q 4 The answer is 2.

[0346] In one embodiment, T2' starts at position 9 from the 5' end of the antisense chain. In one embodiment, T2' is located at position 9 from the 5' end of the antisense chain, and q 4 The value is 1.

[0347] In one embodiment, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 1, and B3' is 2'-OMe or 2'-F, then q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 This configuration is 1, and these are accompanied by two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23.

[0348] In one embodiment, n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 1, and B3' is 2'-OMe or 2'-F, then q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 This configuration is 1, and these are accompanied by two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23.

[0349] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1.

[0350] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7This configuration is 1, and these are accompanied by two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23.

[0351] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 6, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 7, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1.

[0352] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 6, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 7, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 This configuration is 1, and these are accompanied by two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23.

[0353] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 1, and B3' is 2'-OMe or 2'-F, then q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1.

[0354] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 1, and B3' is 2'-OMe or 2'-F, then q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 This configuration is 1, and these are accompanied by two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23.

[0355] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 5, T2' is 2'-F, and q 4 If is 1, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 1 is present, and optionally, these are accompanied by at least 2 further TTs at the 3' end of the antisense chain.

[0356] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 5, T2' is 2'-F, and q 4 If is 1, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The function is 1, and optionally, these are accompanied by at least 2 further TTs at the 3' end of the antisense strand, which are accompanied by two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23.

[0357] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7The value is 1.

[0358] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 This configuration is 1, and these are accompanied by two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 (counting from the 5' end) of the sense strand, two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23.

[0359] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1.

[0360] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 This configuration is 1, and these are accompanied by two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23.

[0361] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1.

[0362] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 This configuration is 1, and these are accompanied by two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23.

[0363] RNAi agents may contain a phosphorus-containing group at the 5' end of the sense or antisense strand. The 5'-terminal phosphorus-containing group may be a 5'-terminal phosphate (5'-P), a 5'-terminal phosphorothioate (5'-PS), a 5'-terminal phosphorodithioate (5'-PS2), a 5'-terminal vinylphosphonate (5'-VP), a 5'-terminal methylphosphonate (MePhos), or 5'-deoxy-5'-C-malonyl [ka] It is possible. If the 5'-terminal phosphorus-containing group is a 5'-terminal vinyl phosphonate (5'-VP), then 5'-VP is a 5'-E-VP isomer (i.e., trans-vinyl phosphonate). [ka] ), 5'-Z-VP isomer (i.e., cis-vinyl phosphonate, [ka] ) It may be any one of them, or a mixture thereof.

[0364] In one embodiment, the RNAi agent contains a phosphorus-containing group at the 5' end of the sense strand. In another embodiment, the RNAi agent contains a phosphorus-containing group at the 5' end of the antisense strand.

[0365] In one embodiment, the RNAi agent contains 5'-P. In another embodiment, the RNAi agent contains 5'-P within the antisense strand.

[0366] In one embodiment, the RNAi agent contains 5'-PS. In one embodiment, the RNAi agent contains 5'-PS within the antisense strand.

[0367] In one embodiment, the RNAi agent contains 5'-VP. In one embodiment, the RNAi agent contains 5'-VP in the antisense strand. In one embodiment, the RNAi agent contains 5'-E-VP in the antisense strand. In one embodiment, the RNAi agent contains 5'-Z-VP in the antisense strand.

[0368] In one embodiment, the RNAi agent contains 5'-PS2. In one embodiment, the RNAi agent contains 5'-PS2 in the antisense strand.

[0369] In one embodiment, the RNAi agent contains 5'-PS2. In another embodiment, the RNAi agent contains 5'-deoxy-5'-C-malonyl in the antisense strand.

[0370] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also include 5'-PS.

[0371] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q7 The value is 1. RNAi agents also contain 5'-P.

[0372] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also include 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0373] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q7 The value is 1. RNAi agents also contain 5'-PS2.

[0374] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0375] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7This is 1, and these involve two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23. RNAi agents also contain 5'-P.

[0376] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 These are 1, and they involve two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23. RNAi agents also contain 5'-PS.

[0377] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 This is characterized by 1, and these involve two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23. RNAi agents also contain 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0378] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7This is 1, and these are accompanied by two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23. RNAi agents also contain 5'-PS2.

[0379] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The compound is 1, and these are accompanied by two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23. The RNAi agent also contains 5'-deoxy-5'-C-malonyl.

[0380] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also contain 5'-P.

[0381] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. dsRNA agents also include 5'-PS.

[0382] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also include 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0383] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also contain 5'-PS2.

[0384] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0385] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 This is represented by 1, and these involve two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 (counting from the 5' end) of the sense strand, two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23. RNAi agents also contain 5'-P.

[0386] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 This is 1, and these are accompanied by two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 (counting from the 5' end) of the sense strand, two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23. RNAi agents also contain 5'-PS.

[0387] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7This is characterized by 1, and these involve two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 (counting from the 5' end) of the sense strand, two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23. RNAi agents also contain 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0388] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 This is represented by 1, and these involve two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 (counting from the 5' end) of the sense strand, two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23. The RNAi agent also contains 5'-PS2.

[0389] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The compound is 1, and these are accompanied by two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 (counting from the 5' end) of the sense strand, two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23. The RNAi agent also contains 5'-deoxy-5'-C-malonyl.

[0390] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also contain 5'-P.

[0391] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also include 5'-PS.

[0392] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also include 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0393] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. dsRNAi RNA agents also contain 5'-PS2.

[0394] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0395] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 This is 1, and these involve two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23. RNAi agents also contain 5'-P.

[0396] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 These are 1, and they involve two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23. RNAi agents also contain 5'-PS.

[0397] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 This is characterized by 1, and these involve two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23. RNAi agents also contain 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0398] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 This is 1, and these are accompanied by two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23. RNAi agents also contain 5'-PS2.

[0399] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The compound is 1, and these are accompanied by two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23. The RNAi agent also contains 5'-deoxy-5'-C-malonyl.

[0400] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also contain 5'-P.

[0401] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also include 5'-PS.

[0402] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also include 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0403] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also contain 5'-PS2.

[0404] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0405] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q4 is 0, B3’ is 2’-OMe or 2’-F, and q 5 is 7, T3’ is 2’-F, and q 6 is 1, B4’ is 2’-F, and also q 7 is 1, and these are associated with internucleotide linkage modifications between two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5’ end of the sense strand) of the sense strand, internucleotide linkage modifications between two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand, and internucleotide linkage modifications between two phosphorothioate nucleotides within the range of positions 18 to 23. The RNAi agent also contains 5’-P.

[0406] In one embodiment, B1 is 2’-OMe or 2’-F, and n 1 is 8, T1 is 2’F, and n 2 is 3, B2 is 2’-OMe, and n 3 is 7, and n 4 is 0, B3 is 2’-OMe, and n 5 is 3, B1’ is 2’-OMe or 2’-F, and q 1 is 9, T1’ is 2’-F, and q 2 is 1, B2’ is 2’-OMe or 2’-F, and q 3 is 4, and q 4 is 0, B3’ is 2’-OMe or 2’-F, and q 5 is 7, T3’ is 2’-F, and q 6 is 1, B4’ is 2’-F, and also q 7 is 1, and these are associated with internucleotide linkage modifications between two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5’ end of the sense strand) of the sense strand, internucleotide linkage modifications between two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand, and internucleotide linkage modifications between two phosphorothioate nucleotides within the range of positions 18 to 23. The RNAi agent also contains 5’-PS.

[0407] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 This is characterized by 1, and these involve two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23. RNAi agents also contain 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0408] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 This is 1, and these are accompanied by two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23. RNAi agents also contain 5'-PS2.

[0409] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The compound is 1, and these are accompanied by two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23. The RNAi agent also contains 5'-deoxy-5'-C-malonyl.

[0410] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The nucleotides are 1, and these include two phosphorothioate nucleotide ligation modifications located within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications located within the range of positions 18-23. The RNAi agent also includes 5'-P and a targeting ligand. In one embodiment, 5'-P is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0411] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, and q 7 is 1, and these are two phosphorothioate nucleotide internucleotide linkage modifications within the range of positions 1 to 5 (counting from the 5’ end of the sense strand) of the sense strand, two phosphorothioate nucleotide internucleotide linkage modifications at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand, and two phosphorothioate nucleotide internucleotide linkage modifications within the range of positions 18 to 23. The RNAi agent also includes 5’-PS and a targeting ligand. In one embodiment, 5’-PS is at the 5’ end of the antisense strand, and the targeting ligand is at the 3’ end of the sense strand.

[0412] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 [[ID=2,6]]is 4, T2’ is 2’-F, q<o000976>is 2, B3’ is 2’-OMe or 2’-F, q 5 is, 5 T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, and q 7 It should be noted that there seems to be a typo in the original text where "<o000976>" should probably be " 4 ". This has been retained as is in the translation for the purpose of following the instruction to preserve all 7 - digit tags exactly.The relationship is 1, and these involve two phosphorothioate nucleotide ligation modifications within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications within the range of positions 18-23. The RNAi agent also includes 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand.

[0413] In one embodiment, the 5'-VP is located at the 5' end of the antisense chain, and the targeting ligand is located at the 3' end of the sense chain.

[0414] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7The relationship is 1, and these involve two phosphorothioate nucleotide ligation modifications located within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications located within the range of positions 18-23. The RNAi agent also comprises 5'-PS2 and a targeting ligand. In one embodiment, 5'-PS2 is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0415] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7The relationship is 1, and these involve two phosphorothioate nucleotide ligation modifications located within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications located within the range of positions 18-23. The RNAi agent also comprises 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, 5'-deoxy-5'-C-malonyl is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0416] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The nucleotides are 1, and these include two phosphorothioate nucleotide ligation modifications located in the range of positions 1-5 (counting from the 5' end) of the sense strand, two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end), and two phosphorothioate nucleotide ligation modifications located in the range of positions 18-23. The RNAi agent also includes 5'-P and a targeting ligand. In one embodiment, 5'-P is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0417] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The nucleotides are 1, and these include two phosphorothioate nucleotide ligation modifications located in the range of positions 1-5 (counting from the 5' end) of the sense strand, two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end), and two phosphorothioate nucleotide ligation modifications located in the range of positions 18-23. The RNAi agent also includes 5'-PS and a targeting ligand. In one embodiment, 5'-PS is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0418] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 34 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The RNAi agent is 1, and these consist of two phosphorothioate nucleotide ligation modifications located within the range of positions 1-5 (counting from the 5' end) of the sense strand, and two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end), as well as two phosphorothioate nucleotide ligation modifications located within the range of positions 18-23. The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0419] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7The relationship is 1, and these involve two phosphorothioate nucleotide ligation modifications located in the range of positions 1-5 (counting from the 5' end) of the sense strand, two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end), and two phosphorothioate nucleotide ligation modifications located in the range of positions 18-23. The RNAi agent also comprises 5'-PS2 and a targeting ligand. In one embodiment, 5'-PS2 is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0420] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The relationship is 1, and these involve two phosphorothioate nucleotide ligation modifications located within the range of positions 1-5 (counting from the 5' end) of the sense strand, two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end), and two phosphorothioate nucleotide ligation modifications located within the range of positions 18-23. The RNAi agent also comprises 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, 5'-deoxy-5'-C-malonyl is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0421] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The nucleotides are 1, and these include two phosphorothioate nucleotide ligation modifications located within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications located within the range of positions 18-23. The RNAi agent also includes 5'-P and a targeting ligand. In one embodiment, 5'-P is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0422] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The relationship is 1, and these involve two phosphorothioate nucleotide ligation modifications located within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications located within the range of positions 18-23. The RNAi agent also comprises 5'-PS and a targeting ligand. In one embodiment, 5'-PS is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0423] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7The RNAi agent is 1, and these are accompanied by two phosphorothioate nucleotide ligation modifications located in the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), as well as two phosphorothioate nucleotide ligation modifications located in the range of positions 18-23. The RNAi agent also comprises a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0424] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7The relationship is 1, and these involve two phosphorothioate nucleotide ligation modifications located within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications located within the range of positions 18-23. The RNAi agent also comprises 5'-PS2 and a targeting ligand. In one embodiment, 5'-PS2 is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0425] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 is 4, T2' is 2'-F, and q 4 If is 2, and B3' is 2'-OMe or 2'-F, then q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7The relationship is 1, and these involve two phosphorothioate nucleotide ligation modifications located within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications located within the range of positions 18-23. The RNAi agent also comprises 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, 5'-deoxy-5'-C-malonyl is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0426] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The nucleotides are 1, and these include two phosphorothioate nucleotide ligation modifications located within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications located within the range of positions 18-23. The RNAi agent also includes 5'-P and a targeting ligand. In one embodiment, 5'-P is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0427] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The relationship is 1, and these involve two phosphorothioate nucleotide ligation modifications located within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications located within the range of positions 18-23. The RNAi agent also comprises 5'-PS and a targeting ligand. In one embodiment, 5'-PS is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0428] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The RNAi agent is 1, and these are accompanied by two phosphorothioate nucleotide ligation modifications located in the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), as well as two phosphorothioate nucleotide ligation modifications located in the range of positions 18-23. The RNAi agent also comprises a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0429] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7The relationship is 1, and these involve two phosphorothioate nucleotide ligation modifications located within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications located within the range of positions 18-23. The RNAi agent also comprises 5'-PS2 and a targeting ligand. In one embodiment, 5'-PS2 is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0430] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 If is 1, and B2' is 2'-OMe or 2'-F, then q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The relationship is 1, and these involve two phosphorothioate nucleotide ligation modifications located within the range of positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide ligation modifications located within the range of positions 18-23. The RNAi agent also comprises 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, 5'-deoxy-5'-C-malonyl is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0431] In certain embodiments, the RNAi agent of the present invention is (a) sense chain having the following: (i) Length of 21 nucleotides, (ii) An ASGPR ligand bound to the 3' terminus, comprising three GalNAc derivatives bound via a trivalent branched linker, and (iii) 2'-F modifications at positions 1, 3, 5, 7, 9 through 11, 13, 17, 19, and 21, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14 through 16, 18, and 20 (counting from the 5' end), Furthermore (b) Antisense chain having the following: (i) Length of 23 nucleotides, (ii) 2'-OMe modifications at positions 1, 3, 5, 9, 11 through 13, 15, 17, 19, 21 and 23, and 2'-F modifications (counting from the 5' end) at positions 2, 4, 6 through 8, 10, 14, 16, 18, 20 and 22, and (iii) Including phosphorothioate nucleotide ligations between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end), The dsRNA agent has a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0432] In another specific embodiment, the RNAi agent of the present invention is (a) sense chain having the following: (i) Length of 21 nucleotides, (ii) An ASGPR ligand bound to the 3' terminus, comprising three GalNAc derivatives bound via a trivalent branched linker, (iii) 2'-F modifications at positions 1, 3, 5, 7, 9 through 11, 13, 15, 17, 19, and 21, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 (counting from the 5' end), (iv) Phosphothioate internucleotide ligations between positions 1 and 2 of a nucleotide, and between positions 2 and 3 of a nucleotide (counting from the 5' end), Furthermore (b) Antisense chain having the following: (i) Length of 23 nucleotides, (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11 through 13, 15, 17, 19, and 21 through 23, and 2'F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5' end), (iii) Phosphothioate internucleotide ligations between positions 1 and 2 of a nucleotide, between positions 2 and 3 of a nucleotide, between positions 21 and 22 of a nucleotide, and between positions 22 and 23 of a nucleotide (counting from the 5' end), The RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0433] In another specific embodiment, the RNAi agent of the present invention is (a) sense chain having the following: (i) Length of 21 nucleotides, (ii) An ASGPR ligand bound to the 3' terminus, comprising three GalNAc derivatives bound via a trivalent branched linker, (iii) 2'-OMe modifications at positions 1 through 6, 8, 10, and 12 through 21, 2'-F modifications at positions 7 and 9, and a deoxyribonucleotide (e.g., dT) at position 11 (counting from the 5' end), (iv) Phosphothioate internucleotide ligations between positions 1 and 2 of a nucleotide, and between positions 2 and 3 of a nucleotide (counting from the 5' end), Furthermore (b) Antisense chain having the following: (i) Length of 23 nucleotides, (ii) 2'-OMe modifications at positions 1, 3, 7, 9, 11, 13, 15, 17, and 19 through 23, and 2'-F modifications at positions 2, 4 through 6, 8, 10, 12, 14, 16, and 18 (counting from the 5' end), (iii) Phosphothioate internucleotide ligations between positions 1 and 2 of a nucleotide, between positions 2 and 3 of a nucleotide, between positions 21 and 22 of a nucleotide, and between positions 22 and 23 of a nucleotide (counting from the 5' end), The RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0434] In another specific embodiment, the RNAi agent of the present invention is (a) sense chain having the following: (i) Length of 21 nucleotides, (ii) An ASGPR ligand bound to the 3' terminus, comprising three GalNAc derivatives bound via a trivalent branched linker, (iii) 2'-OMe modifications in positions 1 through 6, 8, 10, 12, 14, and 16 through 21, and 2'-F modifications in positions 7, 9, 11, 13, and 15, and (iv) Phosphothioate internucleotide ligations between positions 1 and 2 of a nucleotide, and between positions 2 and 3 of a nucleotide (counting from the 5' end), Furthermore (b) Antisense chain having the following: (i) Length of 23 nucleotides, (ii) 2'-OMe modifications at positions 1, 5, 7, 9, 11, 13, 15, 17, 19, and 21 through 23, and 2'-F modifications (counting from the 5' end) at positions 2 through 4, 6, 8, 10, 12, 14, 16, 18, and 20, and (iii) Phosphothioate internucleotide ligations between positions 1 and 2 of a nucleotide, between positions 2 and 3 of a nucleotide, between positions 21 and 22 of a nucleotide, and between positions 22 and 23 of a nucleotide (counting from the 5' end), The RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0435] In another specific embodiment, the RNAi agent of the present invention is (a) sense chain having the following: (i) Length of 21 nucleotides, (ii) An ASGPR ligand bound to the 3' terminus, comprising three GalNAc derivatives bound via a trivalent branched linker, (iii) 2'-OMe modifications in positions 1 through 9 and 12 through 21, and 2'-F modifications in positions 10 and 11, (iv) Phosphothioate internucleotide ligations between positions 1 and 2 of a nucleotide, and between positions 2 and 3 of a nucleotide (counting from the 5' end), Furthermore (b) Antisense chain having the following: (i) Length of 23 nucleotides, (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11 through 13, 15, 17, 19, and 21 through 23, and 2'-F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5' end), (iii) Phosphothioate internucleotide ligations between positions 1 and 2 of a nucleotide, between positions 2 and 3 of a nucleotide, between positions 21 and 22 of a nucleotide, and between positions 22 and 23 of a nucleotide (counting from the 5' end), The RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0436] In another specific embodiment, the RNAi agent of the present invention is (a) sense chain having the following: (i) Length of 21 nucleotides, (ii) An ASGPR ligand bound to the 3' terminus, comprising three GalNAc derivatives bound via a trivalent branched linker, (iii) 2'-F modifications in positions 1, 3, 5, 7, 9 through 11, and 13, and 2'-OMe modifications in positions 2, 4, 6, 8, 12, and 14 through 21, (iv) Phosphothioate internucleotide ligations between positions 1 and 2 of a nucleotide, and between positions 2 and 3 of a nucleotide (counting from the 5' end), Furthermore (b) Antisense chain having the following: (i) Length of 23 nucleotides, (ii) 2'-OMe modifications at positions 1, 3, 5 through 7, 9, 11 through 13, 15, 17 through 19, and 21 through 23, and 2'-F modifications at positions 2, 4, 8, 10, 14, 16, and 20 (counting from the 5' end), (iii) Phosphothioate internucleotide ligations between positions 1 and 2 of a nucleotide, between positions 2 and 3 of a nucleotide, between positions 21 and 22 of a nucleotide, and between positions 22 and 23 of a nucleotide (counting from the 5' end), The RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0437] In another specific embodiment, the RNAi agent of the present invention is (a) sense chain having the following: (i) Length of 21 nucleotides, (ii) An ASGPR ligand bound to the 3' terminus, comprising three GalNAc derivatives bound via a trivalent branched linker, (iii) 2'-OMe modifications in positions 1, 2, 4, 6, 8, 12, 14, 15, 17, and 19 through 21, and 2'-F modifications in positions 3, 5, 7, 9 through 11, 13, 16, and 18, and (iv) Phosphothioate internucleotide ligations between positions 1 and 2 of a nucleotide, and between positions 2 and 3 of a nucleotide (counting from the 5' end), Furthermore (b) Antisense chain having the following: (i) Length of 25 nucleotides, (ii) 2'-OMe modifications at positions 1, 4, 6, 7, 9, 11 to 13, 15, 17, and 19 to 23, 2'-F modifications at positions 2, 3, 5, 8, 10, 14, 16, and 18, and deoxyribonucleotides (e.g., dT) at positions 24 and 25 (counting from the 5' end), and (iii) Phosphothioate internucleotide ligations between positions 1 and 2 of a nucleotide, between positions 2 and 3 of a nucleotide, between positions 21 and 22 of a nucleotide, and between positions 22 and 23 of a nucleotide (counting from the 5' end), The RNAi agent has a four-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0438] In another specific embodiment, the RNAi agent of the present invention is (a) sense chain having the following: (i) Length of 21 nucleotides, (ii) An ASGPR ligand bound to the 3' terminus, comprising three GalNAc derivatives bound via a trivalent branched linker, (iii) 2'-OMe modifications in positions 1 through 6, 8, and 12 through 21, and 2'-F modifications in position 7 and 9 through 11, (iv) Phosphothioate internucleotide ligations between positions 1 and 2 of a nucleotide, and between positions 2 and 3 of a nucleotide (counting from the 5' end), Furthermore (b) Antisense chain having the following: (i) Length of 23 nucleotides, (ii) 2'-OMe modifications at positions 1, 3 through 5, 7, 8, 10 through 13, 15, and 17 through 23, and 2'-F modifications at positions 2, 6, 9, 14, and 16 (counting from the 5' end), (iii) Phosphothioate internucleotide ligations between positions 1 and 2 of a nucleotide, between positions 2 and 3 of a nucleotide, between positions 21 and 22 of a nucleotide, and between positions 22 and 23 of a nucleotide (counting from the 5' end), The RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0439] In another specific embodiment, the RNAi agent of the present invention is (a) sense chain having the following: (i) Length of 21 nucleotides, (ii) An ASGPR ligand bound to the 3' terminus, comprising three GalNAc derivatives bound via a trivalent branched linker, (iii) 2'-OMe modifications in positions 1 through 6, 8, and 12 through 21, and 2'-F modifications in position 7 and 9 through 11, (iv) Phosphothioate internucleotide ligations between positions 1 and 2 of a nucleotide, and between positions 2 and 3 of a nucleotide (counting from the 5' end), Furthermore (b) Antisense chain having the following: (i) Length of 23 nucleotides, (ii) 2'-OMe modifications at positions 1, 3 through 5, 7, 10 through 13, 15, and 17 through 23, and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end), (iii) Phosphothioate internucleotide ligations between positions 1 and 2 of a nucleotide, between positions 2 and 3 of a nucleotide, between positions 21 and 22 of a nucleotide, and between positions 22 and 23 of a nucleotide (counting from the 5' end), The RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0440] In another specific embodiment, the RNAi agent of the present invention is (a) sense chain having the following: (i) Length of 19 nucleotides, (ii) An ASGPR ligand bound to the 3' terminus, comprising three GalNAc derivatives bound via a trivalent branched linker, (iii) 2'-OMe modifications in positions 1 through 4, 6, and 10 through 19, and 2'-F modifications in positions 5 and 7 through 9, (iv) Phosphothioate internucleotide ligations between positions 1 and 2 of a nucleotide, and between positions 2 and 3 of a nucleotide (counting from the 5' end), Furthermore (b) Antisense chain having the following: (i) Length of 21 nucleotides, (ii) 2'-OMe modifications at positions 1, 3 through 5, 7, 10 through 13, 15, and 17 through 21, and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end), (iii) Phosphothioate internucleotide ligatures between positions 1 and 2 of a nucleotide, between positions 2 and 3 of a nucleotide, between positions 19 and 20 of a nucleotide, and between positions 20 and 21 of a nucleotide (counting from the 5' end), The RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0441] In certain embodiments, the iRNA used in the method of the present invention is selected from any one of the agents selected from Tables 2-3 and 7-8. These agents may further contain ligands.

[0442] III. iRNA conjugated to a ligand Another modification of the iRNA of the present invention involves chemically linking the iRNA with one or more ligands, moieties, or conjugates that enhance the activity, cell distribution, or, for example, cellular uptake into cells of the iRNA. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:6553-6556).In other embodiments, the ligand may be cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, such as beryl-S-tritylthiol (Manoharan et al., Ann. NYAcad. Sci., 1992, 660:306-309, Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), or fatty acid chains, such as dodecanediol residues 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, e.g., dihexadecyl-rac-glycerol or triethylammonium 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), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetate (Manoharan et al., Tetrahedron These are the palmityl moiety (Lett., 1995, 36:3651-3654), the palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or the octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0443] In certain embodiments, ligands alter the distribution, targeting, or lifespan of the iRNA agent into which they are incorporated. In some embodiments, ligands result in enhanced affinity for selected targets, such as molecules, cells, or cell types, compartments such as compartments of cells or organs, tissues, organs, or regions of the body, compared to species in which such ligands are absent. In some embodiments, ligands do not participate in double-strand pairing in double-stranded nucleic acids.

[0444] Ligands can be naturally occurring substances, such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid), or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers or 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-ethylacryllic acid), N-isopropylacrylamide polymer, or polyphosphatidine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptide-mimicking polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helix peptides.

[0445] The ligand may also contain a targeting group, such as a cell or tissue targeting agent, such as a lectin, such as a glycoprotein, lipid or protein that binds to a specific cell type, such as kidney cells, such as an antibody. The targeting group may be thyroid-stimulating hormone, melanocyte-stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, vitamin A, biotin, or RGD peptide or RGD peptide mimetic. In certain embodiments, the ligand is polyvalent galactose, such as N-acetyl-galactosamine.

[0446] Other examples of ligands include dyes, inserts (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol, cholic acid, adamantane acetate, 1-pyrenebutanoic acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl) lithoglycerol Examples include lic acid, O3-(oleoyl)colenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., Antennapedia peptide, Tat peptide), alkylating agents, phosphates, amino acids, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyls, substituted alkyls, radiolabeling markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of the tetraazamacrocycle), dinitrophenyl, HRP, or AP.

[0447] Ligands can be proteins, such as glycoproteins, or peptides, such as molecules that have a specific affinity for a co-ligand, or antibodies, such as antibodies that bind to a specific cell type, such as hepatocytes. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, or polyvalent fucose. Ligands can be, for example, lipopolysaccharides, p38 MAP kinase activators, or NF-κB activators.

[0448] A ligand can be a substance, such as a drug, that can increase the uptake of an iRNA agent into a cell, for example, by disrupting the cytoskeleton of a cell, for example, by disrupting the microtubules, microfibrils, or intermediate fibers of a cell. Drugs may include, for example, taxol, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latruncrine A, phalloidin, swinholide A, indanocine, or myoservin.

[0449] In some embodiments, the ligands that bind to iRNAs described herein act as pharmacokinetic modulators (PK modulators). Examples of PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, and vitamins. Exemplary PK modulators, but not limited to, include cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, and biotin. Oligonucleotides containing several phosphorothioate linkages are also known to bind to serum proteins; therefore, short oligonucleotides, such as oligonucleotides of about 5, 10, 15, or 20 bases containing multiple phosphorothioate linkages in their backbone, are also suitable as ligands (e.g., as PK-modulating ligands) in the present invention. Furthermore, aptamers that bind serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0450] The iRNA conjugated with the ligand of the present invention can be synthesized using an oligonucleotide having a reactive functional pendant side chain, for example, the pendant side chain is derived from the binding of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be directly reacted with a commercially available ligand, a synthesized ligand having any of the various protecting groups, or a ligand having a linking portion that binds thereto.

[0451] The oligonucleotides used in the conjugate of the present invention can be conveniently and commonly prepared by well-known solid-phase synthesis techniques. Equipment for such synthesis is available from several vendors, including, for example, Applied Biosystems® (Foster City, California). Any other methods for such synthesis known in the art may be used additionally or as an alternative. Similar techniques for preparing other oligonucleotides, such as phosphorothioates and alkylated derivatives, are also known.

[0452] In the sequence-specific linked nucleosides possessing the ligand-conjugated iRNA and ligand molecule of the present invention, the oligonucleotide and oligonucleoside can be assembled on a suitable DNA synthesizer utilizing a standard nucleotide or nucleoside precursor, or on a conjugate precursor of a nucleotide or nucleoside already possessing a linking portion, or on a ligand nucleotide or ligand nucleoside conjugate precursor already possessing a ligand molecule, or on a non-nucleoside ligand-possessing structural unit.

[0453] When using nucleotide-conjugate precursors that already possess a linking site, typically, the synthesis of a sequence-specifically linked nucleoside is completed, and then the ligand molecule reacts with the linking site to form a ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using commercially available standard and non-standard phosphoramidites commonly used in oligonucleotide synthesis, as well as phosphoramidites derived from ligand-nucleoside conjugates.

[0454] A. Lipid conjugates In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule. In one embodiment, such a lipid or lipid-based molecule binds to a serum protein, such as human serum albumin (HSA). The HSA-binding ligand enables the distribution of the conjugate to a target tissue in the body, for example, a target tissue other than the kidney. For example, the target tissue could be the liver, including the parenchymal cells of the liver. Other molecules capable of binding to HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipid or lipid-based ligands can be used to (a) increase the resistance of the conjugate to degradation, (b) increase targeting or transport into target cells or cell membranes, or (c) modulate binding to serum proteins, such as HSA.

[0455] Lipid ligands can be used to inhibit, for example, control the binding of conjugates to target tissues. For instance, lipids or lipid ligands that bind more strongly to HSA are less likely to target the kidneys and therefore less likely to be eliminated from the body. Lipids or lipid ligands that do not bind too strongly to HSA can be used so that the conjugate targets the kidneys.

[0456] In certain embodiments, a lipid ligand binds to HSA. In one embodiment, it binds to HSA with sufficient affinity so that the conjugate is distributed to non-renal tissue. However, it is preferable that this affinity is not so strong as to prevent the HSA-ligand binding from being reversed.

[0457] In other embodiments, the lipid ligand either weakly binds to the HSA or does not bind at all. In one embodiment, the conjugate is distributed to the kidney. Other parts that target kidney cells can be used instead of, or in addition to, the lipid ligand.

[0458] In another embodiment, the ligand is a portion, such as a vitamin, that is taken up by target cells, such as proliferating cells. These are particularly useful in treating disorders characterized by unwanted cell proliferation, such as cancer cells, of malignant or non-malignant species. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include vitamin B, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by target cells such as hepatocytes. HSA and low-density lipoprotein (LDL) are also examples.

[0459] B. Cell permeability agents In another embodiment, the ligand is a cell permeabilizer, such as a helix cell permeabilizer. In one embodiment, the cell permeabilizer is amphiphilic. Exemplary cell permeabilizers include peptides such as tat or antennopedia. If the cell permeabilizer is a peptide, it is modifiable, including the use of peptidyl mimes, invertomers, linkage of non-peptides or pseudopeptides, and D-amino acids. In one embodiment, the helix agent is an alpha-helix agent having a lipophilic phase and an oleophobic phase.

[0460] Ligands can be peptides or peptide mimes. Peptide mimes (also referred to herein as oligopeptide mimes) are molecules that can fold into defined three-dimensional structures similar to natural peptides. Binding of peptides and peptide mimes to iRNA agents can affect the pharmacokinetic distribution of the iRNA, for example, by enhancing cell recognition and absorption. The peptide or peptide mime portion can be about 5 to 50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0461] Peptides or peptide mimes may be, for example, cell-permeable peptides, cationic peptides, amphiphilic peptides, or hydrophobic peptides (e.g., mainly composed of Tyr, Trp, or Phe). The peptide moiety may be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. Alternatively, the peptide moiety may contain a hydrophobic membrane transport sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 14). RFGF analogs containing hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 15) can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, which can carry large polar molecules including peptides, oligonucleotides, and transmembrane proteins. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 16)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 17)) have been found to have the ability to function as delivery peptides. Peptides or peptide mimetic compounds can be encoded by random sequences of DNA, e.g., phage display libraries or one-bead-one compound (OBOC) combinatorial libraries (Lam et al.). These include peptides identified in (al., Nature, 354:82-84, 1991). Examples of peptides or peptide mimes anchored to dsRNA agents via monomeric units incorporated for cell targeting purposes include arginine-glycine-aspartic acid (RGD) peptides or RGD mimes. The peptide portion may range in length from approximately 5 to 40 amino acids. The peptide portion may have structural modifications, such as increasing stability or governing conformational properties. Any of the structural modifications described below are available.

[0462] The RGD peptides for use in the compositions and methods of the present invention may be linear or cyclic, and may be modified, for example, glycosylated or methylated, to facilitate targeting to specific tissues. RGD-containing peptides and peptidiomimemtics may comprise D-amino acids and synthetic RGD mimics. In addition to RGD, other moieties that target integrin ligands, such as PECAM-1 or VEGF, may be used.

[0463] A "cell-permeable peptide" is capable of permeating cells, such as microbial cells (e.g., bacterial or fungal cells) or mammalian cells (e.g., human cells). Microbial cell-permeable peptides may be, for example, α-helix linear peptides (e.g., LL-37 or ceropin P1), disulfide bond-containing peptides (e.g., α-defensin, β-defensin, or bactenecin), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidine). Cell-permeable peptides may also contain nuclear localization signals (NLS). For example, a cell-permeable peptide may be a bipartite amphiphilic peptide, such as MPG, derived from the fusion peptide domain of the NLS of HIV-1 gp41 and SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0464] C. Carbohydrate Conjugate In some embodiments of the compositions and methods of the present invention, the iRNA further comprises a carbohydrate. The carbohydrate-conjugated iRNA is a composition that is advantageous for in vivo delivery of nucleic acids and suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” means a compound that is either a carbohydrate itself (which may be linear, branched, or cyclic) composed of one or more monosaccharide units having at least six carbon atoms, each with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom, or a compound that has as part a carbohydrate portion (which may be linear, branched, or cyclic) composed of one or more monosaccharide units, each having at least six carbon atoms, each with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Typical carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides consisting of about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide resins. Specific monosaccharides include C5 and the above-mentioned (e.g., C5, C6, C7, or C8) sugars, while disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0465] In certain embodiments, the carbohydrate conjugate used in the compositions and methods of the present invention is a monosaccharide.

[0466] In certain embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). GalNAc conjugates comprising one or more N-acetylgalactosamine (GalNAc) derivatives are described, for example, in U.S. Patent No. 8,106,022, the entire contents of which are incorporated herein by reference. In some embodiments, the GalNAc conjugate functions as a ligand for iRNA to target specific cells. In some embodiments, the GalNAc conjugate functions as a ligand for asialoglycoprotein receptors in liver cells (e.g., hepatocytes), thereby allowing iRNA to target liver cells.

[0467] In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives can be conjugated via a linker, for example, via a divalent or trivalent branched linker. In some embodiments, the GalNAc conjugate is conjugated to the 3' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 3' end of the sense strand) via a linker, for example, via a linker as described herein. In some embodiments, the GalNAc conjugate is conjugated to the 5' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 5' end of the sense strand) via a linker, for example, via a linker as described herein.

[0468] In certain embodiments of the present invention, GalNAc or a GalNAc derivative binds to the iRNA agent of the present invention via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative binds to the iRNA agent of the present invention via a divalent linker. In yet another embodiment of the present invention, GalNAc or a GalNAc derivative binds to the iRNA agent of the present invention via a trivalent linker. In yet another embodiment of the present invention, GalNAc or a GalNAc derivative binds to the iRNA agent of the present invention via a tetravalent linker.

[0469] In certain embodiments, the double-stranded RNAi agent of the present invention comprises one GalNAc or GalNAc derivative conjugated to an iRNA agent. In certain embodiments, the double-stranded RNAi agent of the present invention comprises a plurality of (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently conjugated to a plurality of nucleotides of the double-stranded RNAi agent via a plurality of monovalent linkers.

[0470] In some embodiments, for example, if the two strands of the iRNA agent of the present invention are part of a larger molecule linked by an uninterrupted chain of nucleotides between the 3' end of one strand and the corresponding 5' end of the other strand, forming a hairpin loop containing multiple unpaired nucleotides, each of the unpaired nucleotides in the hairpin loop may independently contain GalNAc or a GalNAc derivative linked via a monovalent linker. This hairpin loop may also be formed by an extended overhang within one of the two strands.

[0471] In some embodiments, for example, if the two strands of the iRNA agent of the present invention are part of a larger molecule linked by an uninterrupted chain of nucleotides between the 3' end of one strand and the corresponding 5' end of the other strand, forming a hairpin loop containing multiple unpaired nucleotides, each of the unpaired nucleotides in the hairpin loop may independently contain GalNAc or a GalNAc derivative linked via a monovalent linker. This hairpin loop may also be formed by an extended overhang within one of the two strands.

[0472] In one embodiment, the carbohydrate conjugate for use in the compositions and methods of the present invention is selected from the group consisting of: [ka] Formula II, [ka] Formula III, [ka] Formula IV, [ka] Formula V, [ka] Formula VI

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[0473] In another embodiment, the carbohydrate conjugate used in the compositions and methods of the present invention is a monosaccharide. In one embodiment, the monosaccharide is N-acetylgalactosamine, for example. [ka] Formula II, And so on.

[0474] In some embodiments, the RNAi agent is bound to a carbohydrate conjugate via a linker as shown in the schematic diagram below, where X is O or S. [ka]

[0475] In some embodiments, the RNAi agent is defined in Table 1 and is conjugated to L96 as shown below. [ka]

[0476] Other typical carbohydrate conjugates for use in the embodiments described herein include, but are not limited to, [ka] (Formula XXXVI) is an example, where if one of X or Y is an oligonucleotide, the other is hydrogen.

[0477] In some embodiments, a suitable ligand is the ligand disclosed in International Publication No. 2019 / 055633, the entirety of which is incorporated herein by reference. In one embodiment, the ligand includes the following structure: [ka]

[0478] In certain embodiments of the present invention, GalNAc or a GalNAc derivative binds to the iRNA agent of the present invention via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative binds to the iRNA agent of the present invention via a divalent linker. In yet another embodiment of the present invention, GalNAc or a GalNAc derivative binds to the iRNA agent of the present invention via a trivalent linker.

[0479] In one embodiment, the double-stranded RNAi agent of the present invention comprises one or more GalNAc or GalNAc derivatives bound to an iRNA agent. GalNAc may be bound to any nucleotide via a linker on the sense strand or antisense strand. GalNAc may be bound to the 5' end of the sense strand, the 3' end of the sense strand, the 5' end of the antisense strand, or the 3' end of the antisense strand. In one embodiment, GalNAc is bound to the 3' end of the sense strand, for example, via a trivalent linker.

[0480] In other embodiments, the double-stranded RNAi agent of the present invention comprises a plurality of (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently binding to a plurality of nucleotides of the double-stranded RNAi agent via a plurality of linkers, such as monovalent linkers.

[0481] In some embodiments, for example, if the two strands of the iRNA agent of the present invention are part of a larger molecule linked by an uninterrupted chain of nucleotides between the 3' end of one strand and the corresponding 5' end of the other strand, forming a hairpin loop containing a plurality of unpaired nucleotides, each of the unpaired nucleotides in the hairpin loop may independently contain GalNAc or a GalNAc derivative linked via a monovalent linker.

[0482] In some embodiments, the carbohydrate conjugate further comprises one or more further ligands, such as but not limited to PK modulators or cell-permeable peptides.

[0483] Further carbohydrate conjugates and linkers suitable for use in the present invention are those described in the PCT publications International Publication No. 2014 / 179620 and International Publication No. 2014 / 179627, the entire contents of which are incorporated herein by reference.

[0484] D. Linker In some embodiments, the conjugates or ligands described herein can be conjugated to iRNA oligonucleotides using various linkers, some of which may be cleavable and others may not.

[0485] The term "linker" or "linking group" refers to an organic part that connects two parts of a compound, for example, by a covalent bond.Typically, the linker is a direct bond, or a unit such as an atom like oxygen or sulfur, NR8, C(O), C(O)NH, SO, SO2, SO2NH, or a chain of atoms, but is not limited to, for example, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkyl Alkyl alkenyl, alkenyl heteroaryl alkinyl, alkynyl heteroaryl alkyl, alkynyl heteroaryl alkenyl, alkynyl heteroaryl alkinyl, alkyl heterocyclyl alkyl, alkyl heterocyclyl alkenyl, alkyl hererocyclyl alkynyl, alkenyl heterocyclyl alkyl, alkenyl heterocyclyl alkenyl, alkenyl heterocyclyl alkynyl, alkynyl heterocyclyl alkyl, alkynyl heterocyclyl alkenyl The formula includes alkynyl heterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, and alkynylhereroaryl, in which one or more methylene groups may be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycles, where R8 is hydrogen, acyl, aliphatic, or substituted aliphatic.In one embodiment, the linker is composed of approximately 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18, 7-17, 8-17, 6-16, 7-17, or 8-16 atoms.

[0486] A cleavable linking group is a group that is sufficiently stable outside the cell but, upon entering the target cell, is cleaved, releasing the two parts held together by the linker. In one exemplary embodiment, the cleavable linking group is cleaved at a rate at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or more, or at least 100 times faster in the target cell or under a first reference condition (which may be selected to mimic or represent intracellular conditions) than in the target blood or under a second reference condition (which may be selected to mimic or represent conditions found in blood or serum).

[0487] Cleavable linking groups are susceptible to the effects of cleavage agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleavage agents are more widespread or found at higher levels or activity inside cells than in serum or blood. Examples of such degrading agents include redox agents selected for specific substrates or those without substrate specificity, such as oxidases or reductases or reducing agents present in cells, such as mercaptans, which can degrade redox-cleavable linking groups by reduction, as well as esterases, reagents that can create endosomes or acidic environments, such as reagents that produce a pH of 5 or less, and enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0488] Cleavable linking groups, such as disulfide bonds, can be susceptible to pH changes. While human serum has a pH of 7.4, the average intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes have a more acidic pH in the range of 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers possess cleavable linking groups that, at a selected pH, release cationic lipids from ligands within the cell or into desired compartments within the cell.

[0489] Linkers may contain cleavable linking groups that can be cleaved by specific enzymes. The type of cleavable linking group incorporated into the linker may depend on the target cell. For example, liver targeting ligands can link to cationic lipids via linkers containing ester groups. Because hepatocytes are rich in esterases, linkers are cleaved more efficiently in hepatocytes than in cell types that are not rich in esterases. Other cell types rich in esterases include lung, renal cortex, and testicular cells.

[0490] When targeting peptidase-rich cell types such as hepatocytes and synovial cells, linkers containing peptide bonds can be used.

[0491] In general, the suitability of a candidate cleavable linker can be evaluated by testing the ability (or conditions) under which a degrading agent cleaves the candidate linker. It would also be desirable to test the candidate cleavable linker for its ability to resist cleavage in blood or in contact with other non-target tissues. Thus, the relative sensitivity to cleavage between the first and second conditions can be determined, where the first condition is selected to show cleavage in target cells, and the second condition is selected to show cleavage in other tissues or biological fluids, for example, in blood or serum. Evaluations can be carried out in cell-free systems, in cells, in cell culture media, in organ or tissue culture media, or in whole animals. It may be useful to perform an initial evaluation in cell-free or culture conditions and confirm it by further evaluation in whole animals. In certain embodiments, useful candidate compounds are cleaved at least 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster 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).

[0492] i. Redox-cleavable linking groups In certain embodiments, the cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of a reductively cleavable linking group is a disulfide linking group (-SS-). To determine whether a candidate cleavable linking group is a suitable “reductively cleavable linking group” or suitable for use with, for example, a specific iRNA moiety and a specific targeting agent, one can turn to the methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT) or other reducing agent using reagents known in the art, in a cell and mimicking the rate of cleavage that would be observed, for example, in a target cell. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. Under certain conditions, a candidate compound is cleaved by up to approximately 10% in blood. In other embodiments, useful candidate compounds are degraded at a rate at least 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in cells (or under in vitro conditions selected to mimic extracellular conditions) compared to in blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using a standard enzyme kinetics assay under conditions selected to mimic an intracellular medium and compared to conditions selected to mimic an extracellular medium.

[0493] ii. Cleavable linking groups of phosphate systems In other embodiments, the cleavable linker includes a phosphate-based cleavable linking group. The phosphate-based cleavable linking group is cleaved by a reagent that decomposes or hydrolyzes the phosphate group. Examples of reagents that cleave phosphate groups in cells include enzymes such as phosphatases in cells. Examples of phosphate linking groups include -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-, and -OP(S)(Rk)-S-, where Rk can be independently a C1-C20 alkyl, a C1-C20 haloalkyl, a C6-C10 aryl, or a C7-C12 aralkyl in each of its appearances. Exemplary embodiments include -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-, and -OP(S)(H)-S-. In certain embodiments, the phosphate-based linking group is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0494] iii. Acid-cleavable linking groups In other embodiments, the cleavable linker includes an acid-cleavable linking group. An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In certain embodiments, the acid-cleavable linking group is cleaved in an acidic environment where the pH is about 6.5 or lower (e.g., about 6.0, 5.5, 5.0 or lower), or by a reagent such as an enzyme that can act as a general acid. In cells, certain low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for acid-cleavable linking groups. Examples of acid-cleavable linking groups, but not limited to, include hydrazones, esters, and amino acid esters. Acid-cleavable groups may have the general formula -C=NN-, C(O)O, or -OC(O). Exemplary embodiments include cases where the carbon is bonded to the oxygen of the ester (alkoxy group), aryl groups, substituted alkyl groups, or tertiary alkyl groups, such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0495] iv. Ester linking groups In other embodiments, the cleavable linker comprises an ester-based cleavable linking group. The ester-based cleavable linking group is cleaved in cells by enzymes such as esterases and amidases. Examples of ester-based cleavable linking groups, but not limited to them, include esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0496] v. Peptide-based cleavage groups In further embodiments, the cleavable linker includes a cleavable linking group of the peptide system. The cleavable linking group of the peptide system is cleaved in cells by enzymes such as peptidases and proteases. The cleavable linking group of the peptide system is a peptide bond formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The cleavable group of the peptide system does not contain an amide group (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkylene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The cleavable group of the peptide system is generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to yield peptides and proteins, and does not include all amide functional groups. The cleavable linking group of the peptide system has the general formula -NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0497] In some embodiments, the iRNA of the present invention is conjugated to a carbohydrate via a linker. Examples of iRNA carbohydrate conjugates having linkers of the compositions and methods of the present invention, but not limited to these, include: [ka] (formula XXXVII), [ka] (formula XXXVIII), [ka] (formula XXXIX), [ka] (formula XL), [ka] (formula XLI), [ka] (formula XLII), [ka] (Formula XLIII), and [ka] (Formula XLIV) is given, where if one of X or Y is an oligonucleotide, the other is hydrogen.

[0498] In certain embodiments of the compositions and methods of the present invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives linked via a divalent or trivalent branched linker.

[0499] In one embodiment, the dsRNA of the present invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of the following formulas (XLV) to (XLVI): Formula XXXXV Formula XLVI [ka] Formula XLVII Formula XLVIII During the ceremony, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C each represent a number from 0 to 20 independently, and the repeating unit may be the same or different. P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T4B , T 4A , T 5B , T 5C Each instance is independently either nonexistent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O. Q 2A Q 2B Q 3A Q 3B Q 4A Q 4B Q 5A Q 5B Q 5C Each instance is independently a non-existent alkylene or substituted alkylene, and one or more methylenes in the formula are O, S, S(O), SO2, N(R) N ), C(R')=C(R''), C≡C or C(O) may interrupt or terminate by one or more of these. R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C Each instance is independent of the non-existent NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R) a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocycline, L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B , and L 5C represents a ligand, that is, each instance is independently a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharid...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent or a pharmaceutically acceptable salt thereof for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) in cells, The dsRNA agent or a pharmaceutically acceptable salt thereof comprises a sense strand and an antisense strand that form a double-stranded region. The sense strand comprises the nucleotide sequence 5'-AAGCUCCUUCUUUUUAUUGUU-3' of SEQ ID NO: 46, and the antisense strand comprises the nucleotide sequence 5'-AACAAUAAAAAAGAAGGAGCUUGG-3' of SEQ ID NO:

661. All nucleotides in the sense strand and all nucleotides in the antisense strand include nucleotide modifications selected from the group consisting of 2'-O-methyl nucleotide modifications, 2'-fluoronucleotide modifications, and deoxynucleotide modifications, and At least one of the modifications is a 2'-deoxynucleotide modification. The antisense strand includes two phosphorothioate nucleotide linkages at its 5' end and two phosphorothioate nucleotide linkages at its 3' end, and the sense strand includes two phosphorothioate nucleotide linkages at its 5' end, and At least one chain is conjugated to a ligand containing an N-acetylgalactosamine (GalNAc) derivative. dsRNA preparations or pharmaceutically acceptable salts thereof.

2. The dsRNA agent or a pharmaceutically acceptable salt thereof according to Claim 1, wherein the sense strand is 21 nucleotides long and the antisense strand is 23 nucleotides long.

3. The dsRNA agent or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent or a pharmaceutically acceptable salt thereof.

4. The dsRNA agent or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the ligand is one or more GalNAc derivatives bound via a monovalent, divalent, or trivalent branched linker.

5. The dsRNA agent or a pharmaceutically acceptable salt thereof according to claim 4, wherein the ligand is one of the following. 【Chemistry 1】

6. The dsRNA agent or a pharmaceutically acceptable salt thereof is conjugated to the ligand as shown in the schematic diagram below, 【Chemistry 2】 The dsRNA agent or a pharmaceutically acceptable salt thereof according to claim 5, wherein X is O or S.

7. The dsRNA agent or a pharmaceutically acceptable salt thereof according to claim 6, wherein X is O.

8. The dsRNA agent or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, wherein the sense strand consists of the nucleotide sequence 5'-AAGCUCCUUCUUUUUAUUGUU-3' of SEQ ID NO: 46, and the antisense strand consists of the nucleotide sequence 5'-AACAAUAAAAAAGAAGGAGCUUGG-3' of SEQ ID NO:

661.

9. A cell containing the dsRNA agent according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof.

10. A pharmaceutical composition for inhibiting the expression of a gene encoding angiopoietin-like 3 (ANGPTL3), comprising a dsRNA agent according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof.

11. The pharmaceutical composition according to claim 10, wherein the dsRNA agent or a pharmaceutically acceptable salt thereof is in a non-buffered solution.

12. The pharmaceutical composition according to claim 10, wherein the dsRNA agent or a pharmaceutically acceptable salt thereof is present in a buffer solution.

13. An in vitro method for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) gene in a cell, the method comprising contacting the cell with a dsRNA agent according to any one of claims 1-8 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 10-12, thereby inhibiting the expression of the ANGPTL3 gene in the cell.

14. The method according to claim 13, wherein the expression of ANGPTL3 is inhibited by at least 50%, 60%, 70%, 80%, 90%, or 95% by contacting the cells with the dsRNA agent or a pharmaceutically acceptable salt thereof.

15. Use of a dsRNA agent according to any one of claims 1-8 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to any one of claims 10-12 in the manufacture of a pharmacopoecilist for the treatment of a subject with a disorder who would benefit from reduced angiopoietin-like 3 (ANGPTL3) expression.

16. Use of a dsRNA agent according to any one of claims 1-8 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to any one of claims 10-12 in the manufacture of a pharmacopoecilist for the prevention of at least one symptom in a disabled subject who would benefit from reduced angiopoietin-like 3 (ANGPTL3) expression.

17. The use according to claim 15 or 16, wherein the failure is an ANGPTL3-related failure.

18. The use according to claim 17, wherein the ANGPTL3-related disorder is a disorder of lipid metabolism.

19. The use according to claim 18, wherein the lipid metabolism disorder is hyperlipidemia or hypertriglyceridemia.

20. The use according to any one of claims 15-19, wherein the subject is a human.

21. The use according to any one of claims 15-20, wherein administration of the dsRNA agent or a pharmaceutically acceptable salt thereof causes a decrease in one or more serum lipids and / or a decrease in ANGPTL3 protein accumulation.

22. The use according to any one of claims 15-21, wherein the dsRNA agent or a pharmaceutically acceptable salt thereof is administered subcutaneously.

23. The use according to any one of claims 15-22, further comprising administering an additional therapeutic agent to the subject in order to treat an ANGPTL3-related disorder.

24. A kit, vial, or syringe comprising a dsRNA agent according to any one of claims 1-8 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 10-12.

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