Metabolic disorder-related target gene IRNA compositions and methods of use thereof

Abstract

The present invention relates to RNAi agents, e.g., double-stranded RNA (dsRNA) agents, that target metabolic disorder-related target genes, e.g., inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), or inhibin subunit beta C (INHBC) genes. The present invention also relates to methods of using such RNAi agents to inhibit the expression of metabolic disorder-related target gene genes, as well as methods of preventing and treating metabolic disorders, e.g., metabolic syndrome.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 223,995, filed July 21, 2021, U.S. Provisional Patent Application No. 63 / 278,126, filed November 11, 2021, U.S. Provisional Patent Application No. 63 / 285,143, filed December 2, 2021, U.S. Provisional Patent Application No. 63 / 287,578, filed December 9, 2021, U.S. Provisional Patent Application No. 63 / 321,799, filed March 21, 2022, and U.S. Provisional Patent Application No. 63 / 323,543, filed March 25, 2022. The entire contents of each of the foregoing applications are incorporated herein by reference. [Background technology]

[0002] With the steady conquest of numerous infectious diseases across much of the globe, non-communicable diseases, particularly metabolic disorders, have become a major health risk in the modern world. The increased consumption of high-calorie, low-fiber fast food and decreased physical activity resulting from mechanized transportation and sedentary lifestyles have led to the spread of metabolic disorders such as metabolic syndrome, type 2 diabetes, hypertension, cardiovascular disease, stroke, and other disorders. Indeed, the incidence of subjects with metabolic disorders, such as metabolic syndrome, who have numerous health conditions that place them at higher risk for heart disease, diabetes, stroke, and other diseases, has increased in recent years.

[0003] Current treatments for metabolic disorders include lifestyle changes, diet, exercise, and treatment with medications such as lipid-lowering agents (e.g., statins) and other drugs. However, these therapies and treatments are often limited by compliance, are not always effective, cause side effects, and result in drug-drug interactions. As a result, there is a need in the art for alternative treatments for subjects with metabolic disorders, such as metabolic syndrome and related diseases (e.g., diabetes, hypertension, and cardiovascular disease), that can selectively and efficiently silence metabolic disorder-related target genes, i.e., drugs that use the cell's own RNAi machinery with both high biological activity and in vivo stability and can effectively inhibit the expression of metabolic disorder-related target INHBE genes, such as inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), or inhibin subunit beta C (INHBC). Summary of the Invention

[0004] The present invention provides iRNA compositions that affect RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of genes encoding metabolic disorder-related target genes selected from the group consisting of inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), and inhibin subunit beta C (INHBC). The target genes can be located within a cell, e.g., within a cell of a subject, such as a human subject. The present invention also provides methods of using an iRNA composition of the present invention selected from the group consisting of inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), and inhibin subunit beta C (INHBC) to inhibit expression of a metabolic disorder-associated target gene, and / or methods of using an iRNA composition of the present invention selected from the group consisting of inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), and inhibin subunit beta C (INHBC) to treat a subject that would benefit from inhibiting or reducing expression of a metabolic disorder-associated target gene, e.g., the subject is suffering from or susceptible to a metabolic disorder, e.g., metabolic syndrome and / or cardiovascular disease.

[0005] Accordingly, in certain aspects, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of a metabolic disorder-associated target gene selected from the group consisting of inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), and inhibin subunit beta C (INHBC) in a cell, such as an adipocyte and / or hepatocyte, wherein the dsRNA agent comprises a sense strand and an antisense strand that form a duplex region, and the sense strand is selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, or comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides, contiguous nucleotides that differ by no more than 0, 1, 2, or 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by no more than 0, 1, 2, or 3 nucleotides from the corresponding portion of the nucleotide sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56.

[0006] In another aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of a metabolic disorder-associated target gene selected from the group consisting of inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), and inhibin subunit beta C (INHBC) in a cell, such as an adipocyte and / or hepatocyte, wherein the dsRNA agent comprises a sense strand and an antisense strand that form a duplex region, wherein the antisense strand comprises a region of complementarity to an mRNA encoding the target gene, and wherein the region of complementarity comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 0, 1, 2, or 3 or fewer nucleotides from any one of the antisense nucleotide sequences in any one of Tables 2-17, 19, and 20.

[0007] In yet another aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of a metabolic disorder-associated target gene selected from the group consisting of inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), and inhibin subunit beta C (INHBC) in a cell, such as an adipocyte and / or a hepatocyte, wherein the dsRNA agent comprises a sense strand and an antisense strand that form a duplex region, and the sense strand is selected from the group consisting of an agonist, an antisense strand ... and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by 0, 1, 2, or 3 or less nucleotides from any one of the sense nucleotide sequences in any one of Tables 2-17, 19, and 20, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 0, 1, 2, or 3 or less nucleotides from any one of the antisense nucleotide sequences in any one of Tables 2-17, 19, and 20. In some embodiments, these dsRNA agents further comprise one or more C22 hydrocarbon chains conjugated to one or more positions, e.g., internal positions, on at least one strand of the dsRNA agent.

[0008] In another aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of a metabolic disorder-associated target gene selected from the group consisting of inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), and inhibin subunit beta C (INHBC) in a cell, such as an adipocyte and / or hepatocyte, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a duplex region, and the sense strand is selected from the group consisting of a nucleotide sequence ... and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by no more than 0, 1, 2, or 3 nucleotides from any one of the sense nucleotide sequences in any one of Tables 2-17, 19, and 20, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by no more than 0, 1, 2, or 3 nucleotides from any one of the antisense nucleotide sequences in any one of Tables 2-17, 19, and 20. In some embodiments, these dsRNA agents further comprise one or more GalNAc ligands conjugated to at least one strand of the dsRNA agent, e.g., through a bivalent or trivalent branched linker.

[0009] In one embodiment, the dsRNA agent comprises a sense strand comprising a contiguous nucleotide sequence having at least 85%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, nucleotide sequence identity over its entire length to any one of the sense strand nucleotide sequences of any one of Tables 2-17, 19, and 20, and an antisense strand comprising a contiguous nucleotide sequence having at least 85%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, nucleotide sequence identity over its entire length to any one of the antisense strand nucleotide sequences of any one of Tables 2-17, 19, and 20.

[0010] In one embodiment, a dsRNA agent comprises a sense strand that includes at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the sense strand nucleotide sequences of any one of Tables 2-17, 19, and 20, and an antisense strand that includes at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense strand nucleotide sequences of any one of Tables 2-17, 19, and 20.

[0011] In one embodiment, a dsRNA agent comprises a sense strand that includes at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by no more than two nucleotides from any one of the sense strand nucleotide sequences of any one of Tables 2-17, 19, and 20, and an antisense strand that includes at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, or 23 contiguous nucleotides that differ by no more than two nucleotides from any one of the antisense strand nucleotide sequences of any one of Tables 2-17, 19, and 20.

[0012] In one embodiment, a dsRNA agent comprises a sense strand that includes at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by no more than one nucleotide from any one of the sense strand nucleotide sequences of any one of Tables 2-17, 19, and 20, and an antisense strand that includes at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by no more than one nucleotide from any one of the antisense strand nucleotide sequences of any one of Tables 2-17, 19, and 20.

[0013] In one embodiment, the dsRNA agent comprises a sense strand that comprises, or consists of, a nucleotide sequence selected from the group consisting of any one of the sense strand nucleotide sequences in any one of Tables 2-17, 19, and 20, and an antisense strand that comprises, or consists of, a nucleotide sequence selected from the group consisting of any one of the antisense strand nucleotide sequences in any one of Tables 2-17, 19, and 20.

[0014] In one embodiment, the target gene is INHBE.

[0015] In one embodiment, the target gene is ACVR1C.

[0016] In one embodiment, the target gene is PLIN1.

[0017] In one embodiment, the target gene is PDE3B.

[0018] In one embodiment, the target gene is INHBC.

[0019] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of inhibin subunit beta E (INHBE) in a cell, such as an adipocyte and / or a hepatocyte, the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand is located between nucleotides 400-422, 410-432, 518-540, 519-541, 640-662, 1430-1452, 1863-1885, or 1886 of SEQ ID NO: 1. The antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by 0, 1, 2, or 3 or fewer nucleotides from any one of the nucleotide sequences of SEQ ID NO:64 to 1886, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 0, 1, 2, or 3 or fewer nucleotides from the corresponding nucleotide sequence of SEQ ID NO:2.

[0020] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of inhibin subunit beta E (INHBE) in a cell, such as an adipocyte and / or a hepatocyte, the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand is nucleotides 400-422, 410-432, 518-540, 519-541, 640-662, 1430-1452, 1863-1885, or 1864-1888 of SEQ ID NO: 1. 6 nucleotide sequences that differ by no more than 0, 1, 2, or 3 nucleotides, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by no more than 0, 1, 2, or 3 nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 2, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by no more than 0, 1, 2, or 3 nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 2. In some embodiments, these dsRNA agents further comprise one or more C22 hydrocarbon chains conjugated to one or more positions, e.g., internal positions, on at least one strand of the dsRNA agent.

[0021] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of inhibin subunit beta E (INHBE) in a cell, such as an adipocyte and / or a liver cell, the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand is located between nucleotides 400-422, 410-432, 518-540, 519-541, 640-662, 1430-1452, 1863-1885, or 1864-1886 of SEQ ID NO:1. 86 nucleotide sequences, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by 0, 1, 2, or 3 or fewer nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 2, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 0, 1, 2, or 3 or fewer nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 2. In some embodiments, these dsRNA agents further comprise one or more GalNAc ligands conjugated to at least one strand of the dsRNA agent, e.g., through a bivalent or trivalent branched linker.

[0022] In some embodiments, the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 0, 1, 2, or 3 or fewer nucleotides from any one of the antisense strand nucleotide sequences of the duplex selected from the group consisting of: AD-1706583, AD-1711744, AD-1706593, AD-1708473, AD-1706662, AD-1706761, AD-1707306, AD-1707639, AD-1707640.

[0023] In some embodiments, the sense and antisense strands comprise at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 0, 1, 2, or 3 or fewer nucleotides from any one of the sense and antisense strand nucleotide sequences of a duplex selected from the group consisting of: AD-1706583, AD-1711744, AD-1706593, AD-1708473, AD-1706662, AD-1706761, AD-1707306, AD-1707639, AD-1707640.

[0024] In some embodiments, the antisense strand comprises: (a)5'-AGUUAUTCUGGGACGACUGGUCA-3', (b)5'-AGUUAUTCUGGGACGACUGGUCU-3', (c)5'-ATGGAGGAUGAGUUAUUCUGGGA-3', (d)5'-AUGAAGTGGAGUCUGUGACAGUA-3', (e)5'-ACUGAAGUGGAGUCUGUGACAGU-3', (f)5'-ACGGAAGAUCCTCAAGCAAAGAG-3', (g)5'-ACAGACAAGAAAGUGCCCAUUUG-3', (h) 5'-AAGAAAGUAUAAAUGCUUGUCUC-3', and (i) comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 0, 1, 2, or 3 or fewer nucleotides from any one of the antisense strand nucleotide sequences of the duplex selected from the group consisting of 5'-AAAGAAAGUAUAAAUGCUUGUCU-3'.

[0025] In some embodiments, (a) 5'-ACCAGUCGUCCCAGAAUAACU-3' and 5'-AGUUAUTCUGGGACGACUGGUCA-3', (b) 5'-ACCAGUCGUCCCAGAAUAACU-3' and 5'-AGUUAUTCUGGGACGACUGGUCU-3', (c) 5'-CCAGAAUAACUCAUCCUCCAU-3' and 5'-ATGGAGGAUGAGUUAUUCUGGGA-3', (d) 5'-CUGUCACAGACUCCACUUCAU-3' and 5'-AUGAAGTGGAGUCUGUGACAGUA-3', (e) 5'-UGUCACAGACUCCACUUCAGU-3' and 5'-ACUGAAGUGGAGUCUGUGACAGU-3', (f) 5'-CUUUGCUUGAGGAUCUUCCGU-3' and 5'-ACGGAAGAUCCTCAAGCAAAGAG-3', (g) 5'-AAUGGGCACUUUCUUGUCUGU-3' and 5'-ACAGACAAGAAAGUGCCCAUUUG-3', (h) 5'-GACAAGCAUUUAUACUUUCUU-3' and 5'-AAGAAAGUAUAAAUGCUUGUCUC-3', and (i) 5'-ACAAGCAUUUAUACUUUCUUU-3' and 5'-AAAGAAAGUAUAAAUGCUUGUCU-3', and the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by 0, 1, 2, or 3 or less nucleotides, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, or 23 contiguous nucleotides that differ by 0, 1, 2, or 3 or less nucleotides.

[0026] In one embodiment, the dsRNA agent includes at least one modified nucleotide.

[0027] In one embodiment, substantially all of the nucleotides in the sense strand are modified nucleotides, substantially all of the nucleotides in the antisense strand are modified nucleotides, or substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides.

[0028] In one embodiment, all nucleotides in the sense strand are modified nucleotides, all nucleotides in the antisense strand are modified nucleotides, or all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides.

[0029] 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, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-C-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, The nucleotide is selected from the group consisting of morpholino nucleotides, phosphoramidates, non-natural base 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-methylacetamido) modified nucleotides, and combinations thereof.

[0030] In one embodiment, at least one of the modified nucleotides 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.

[0031] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a glycol-modified nucleotide (GNA), such as Ggn, Cgn, Tgn, or Agn, a nucleotide containing a 2' phosphate, such as G2p, C2p, A2p, or U2p, a nucleotide containing a phosphorothioate group, and a vinyl-phosphonate nucleotide, and combinations thereof.

[0032] In another embodiment, at least one of the modified nucleotides is a nucleotide having a thermally destabilizing nucleotide modification.

[0033] In one embodiment, the thermally destabilizing nucleotide modification is selected from the group consisting of an abasic modification; a mismatch with the opposing nucleotide in the duplex; a destabilizing sugar modification, a 2'-deoxy modification, an acyclic nucleotide, an unlocked nucleic acid (UNA), and a glycerol nucleic acid (GNA).

[0034] In some embodiments, the modified nucleotides include a short sequence of 3'-terminal deoxythymidine nucleotides (dT).

[0035] In some embodiments, the dsRNA agent further comprises a phosphate or a phosphate mimic at the 5' end of the antisense strand.

[0036] In some embodiments, the phosphate mimic is a 5'-vinylphosphonate (VP).

[0037] In some embodiments, the 5'-end of the antisense strand of a dsRNA agent does not contain a 5'-vinylphosphonate (VP).

[0038] In some embodiments, the dsRNA agent further comprises at least one terminal, chiral phosphorus atom.

[0039] Site-specific chiral modifications to internucleotide bonds can occur at the 5'-end, 3'-end, or both the 5'-end and 3'-end of the strand. This is referred to herein as a "terminal" chiral modification. Terminal modifications can occur at the 3'-end or 5'-end positions within the terminal region, for example, at the terminal nucleotide of the strand, or within the last 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the strand. Chiral modifications can occur on the sense strand, the antisense strand, or both the sense and antisense strands. Each chirally pure phosphorus atom can be in either the Rp or Sp configuration, and combinations thereof. Further details related to chiral modifications and chiral modified dsRNA agents can be found in International Patent Application No. PCT / US18 / 67103, entitled "Chirally-Modified Double-Stranded RNA Agents," filed December 21, 2018, the entire contents of which are incorporated herein by reference.

[0040] In some embodiments, the dsRNA agent further includes a terminal chiral modification occurring at a first internucleotide linkage at the 3'-end of the antisense strand, the first internucleotide linkage having a linking phosphorus atom in the Sp configuration; a terminal chiral modification occurring at a first internucleotide linkage at the 5'-end of the antisense strand, the first internucleotide linkage having a linking phosphorus atom in the Rp configuration; and a terminal chiral modification occurring at a first internucleotide linkage at the 5'-end of the sense strand, the first internucleotide linkage having a linking phosphorus atom in either the Rp or Sp configuration.

[0041] In one embodiment, the dsRNA agent further includes a terminal chiral modification occurring at the first and second internucleotide linkages at the 3'-end of the antisense strand, with the linking phosphorus atom in the Sp configuration; a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration; and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration.

[0042] In one embodiment, the dsRNA agent further includes a terminal chiral modification that occurs at the first, second, and third internucleotide linkages at the 3'-end of the antisense strand, with the linking phosphorus atom in the Sp configuration; a terminal chiral modification that occurs at the first internucleotide linkage at the 5'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration; and a terminal chiral modification that occurs at the first internucleotide linkage at the 5'-end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration.

[0043] In one embodiment, the dsRNA agent further includes a terminal chiral modification occurring at the first and second internucleotide linkages at the 3'-end of the antisense strand, with the linking phosphorus atom in the Sp configuration; a terminal chiral modification occurring at the third internucleotide linkage at the 3'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration; a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration; and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration.

[0044] In one embodiment, the dsRNA agent further includes a terminal chiral modification that occurs at the first and second internucleotide linkages at the 3'-end of the antisense strand, with the linking phosphorus atom in the Sp configuration; a terminal chiral modification that occurs at the first and second internucleotide linkages at the 5'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration; and a terminal chiral modification that occurs at the first internucleotide linkage at the 5'-end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration.

[0045] In some embodiments, the 3' end of the sense strand is protected via an end cap that is an amine-bearing cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

[0046] In one embodiment, the dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

[0047] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3' end of one strand, eg, the antisense strand or the sense strand.

[0048] In another embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5' end of one strand, for example, the antisense strand or the sense strand.

[0049] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkages are at both the 5' and 3' ends of one strand, hi one embodiment, the strand is the antisense strand.

[0050] In one embodiment, the base pair at position 1 of the 5'-end of the antisense strand of the duplex is an AU base pair.

[0051] The double-stranded region can be 19 to 30 nucleotide pairs in length, 19 to 25 nucleotide pairs in length, 19 to 23 nucleotide pairs in length, 23 to 27 nucleotide pairs in length, or 21 to 23 nucleotide pairs in length.

[0052] In one embodiment, each strand is independently 30 nucleotides or less in length.

[0053] In one embodiment, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

[0054] The region of complementarity can be at least 17 nucleotides in length, 19-23 nucleotides in length, or 19 nucleotides in length.

[0055] In one embodiment, at least one strand comprises a 3' overhang of at least 1 nucleotide, hi another embodiment, at least one strand comprises a 3' overhang of at least 2 nucleotides.

[0056] In some embodiments, one or more C22 hydrocarbon chains are conjugated to one or more internal positions on at least one strand of the dsRNA agent.

[0057] In some embodiments, the octanol-water partition coefficient, log K ow One or more C measured by 22 The lipophilicity of the hydrocarbon chain is greater than 0. The lipophilic moiety has a log K ow may have

[0058] In some embodiments, the hydrophobicity of the dsRNA agent measured by the unbound fraction in a plasma protein binding assay of the dsRNA agent is greater than 0.2. In one embodiment, the plasma protein binding assay measured is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. The hydrophobicity of the dsRNA agent measured by the unbound dsRNA fraction in a binding assay is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45, or greater than 0.5, for enhanced in vivo delivery of dsRNA.

[0059] C 22 The hydrocarbon chain may be saturated or unsaturated.

[0060] C 22 The hydrocarbon chain may be linear or branched.

[0061] In some embodiments, internal positions include all but the three terminal positions from each end of at least one strand.

[0062] In some embodiments, the internal position excludes the cleavage site region of the sense strand.

[0063] In some embodiments, internal positions exclude positions 9-12 or 11-13 counting from the 5' end of the sense strand.

[0064] In some embodiments, the internal position excludes the cleavage site region of the antisense strand.

[0065] In some embodiments, internal positions exclude positions 12-14 from the 5' end of the antisense strand.

[0066] In some embodiments, one or more C 22The hydrocarbon chains are conjugated to one or more of the following internal positions: positions 4-8 and 13-18 on the sense strand and positions 6-10 and 15-18 on the antisense strand, counting from the 5' end of each strand.

[0067] In some embodiments, one or more C 22 The hydrocarbon chains are conjugated to one or more of the following internal positions: positions 5, 6, 7, 15, and 17 on the sense strand and positions 15 and 17 on the antisense strand, counting from the 5' end of each strand.

[0068] In some embodiments, one or more C 22 The hydrocarbon chain is conjugated to position 6 on the sense strand, counting from the 5' end of the sense strand.

[0069] In some embodiments, one or more C 22 The hydrocarbon chain may be aliphatic, alicyclic, or polyalicyclic, e.g., one or more C 22 The hydrocarbon chain contains a functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0070] In some embodiments, one or more C 22 The hydrocarbon chain is a C22 acid, for example, the C22 acid is selected from the group consisting of docosanoic acid, 6-octyltetradecanoic acid, 10-hexylhexadecanoic acid, all-cis-7,10,13,16,19-docosapentaenoic acid, all-cis-4,7,10,13,16,19-docosahexaenoic acid, all-cis-13,16-docosadienoic acid, all-cis-7,10,13,16-docosatetraenoic acid, all-cis-4,7,10,13,16-docosapentaenoic acid, and cis-13-docosenoic acid. [ka]

[0071] In some embodiments, one or more C 22The hydrocarbon chain is a C22 alcohol, for example, the C22 alcohol is selected from the group consisting of 1-docosanol, 6-octyltetradecan-1-ol, 10-hexylhexadecan-1-ol, cis-13-docosene-1-ol, docosan-9-ol, docosan-2-ol, docosan-10-ol, docosan-11-ol, and cis-4,7,10,13,16,19-docosahexanol. [ka]

[0072] In some embodiments, one or more C 22 The hydrocarbon chain is a C22 amide, for example, the C22 amide is selected from the group consisting of (E)-docosa-4-enamide, (E)-docosa-5-enamide, (Z)-docosa-9-enamide, (E)-docosa-11-enamide, 12-docosenamid, (Z)-docosa-13-enamide, (Z)-N-hydroxy-13-docosenamid, (E)-docosa-14-enamide, 6-cis-docosenamid, 14-docosenamidocos-11-enamide, (4E,13E)-docosa-4,13-dienamide, and (5E,13E)-docosa-5,13-dienamide.

[0073] One or more C's 22 The hydrocarbon chain may be conjugated to the dsRNA agent via a direct bond to the ribosugar of the dsRNA agent. Alternatively, one or more C 22 The hydrocarbon chain may be conjugated to the dsRNA agent via a linker or carrier. In some embodiments, one or more C 22 The hydrocarbon chain may be conjugated to the dsRNA agent via an internucleotide phosphate linkage. [ka]

[0074] In certain embodiments, one or more C 22The hydrocarbon chain is conjugated to the dsRNA agent via one or more linkers (tethers), eg, carriers, that substitute one or more nucleotides at an internal position.

[0075] In some embodiments, one or more C 22 The hydrocarbon chain is conjugated to the dsRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide bond, the product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate.

[0076] In some embodiments, at least one of the linkers (tethers) is a redox-cleavable linker (e.g., a reductively cleavable linker, such as a disulfide group), an acid-cleavable linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group), an esterase-cleavable linker (e.g., an ester group), a phosphatase-cleavable linker (e.g., a phosphate group), or a peptidase-cleavable linker (e.g., a peptide bond).

[0077] In other embodiments, at least one of the linkers (tethers) is a biocleavable linker selected from the group consisting of DNA, RNA, disulfides, amides, and functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0078] In certain embodiments, one or more C 22The hydrocarbon chain is conjugated to dsRNA agent through the carrier that replaces one or more nucleotides.The carrier can be cyclic or acyclic.In one embodiment, the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalin.In one embodiment, the acyclic group is based on serinol skeleton or diethanolamine skeleton.

[0079] In some embodiments, the carrier replaces one or more nucleotides at an internal position of the dsRNA agent.

[0080] In some embodiments, the dsRNA agent further comprises a targeting ligand that targets a receptor that mediates delivery to adipose tissue. In one embodiment, the targeting ligand is selected from the group consisting of angiopep-2, lipoprotein receptor-related protein (LRP) ligand, bEnd.3 cell-binding ligand, transferrin receptor (TfR) ligand, mannose receptor ligand, glucose transporter protein, LDL receptor ligand, trans-retinol, RGD peptide, LDL receptor ligand, CD63 ligand, and carbohydrate-based ligand.

[0081] In some embodiments, the dsRNA agent further comprises a targeting ligand that targets liver tissue.

[0082] In one embodiment, the targeting ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

[0083] In some embodiments, the targeting ligand is a carbohydrate-based ligand.

[0084] In one embodiment, the targeting ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0085] In one embodiment, the targeting ligand is one or more GalNAc derivatives attached through a monovalent, divalent, or trivalent branched linker.

[0086] In one embodiment, the targeting ligand is: [ka]

[0087] In one embodiment, the dsRNA agent is conjugated to a targeting ligand as shown in the following schematic diagram. [ka] wherein X is O or S.

[0088] In one embodiment, X is O.

[0089] In some embodiments, the one or more C22 hydrocarbon chains or targeting ligands are conjugated via a biocleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, and functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0090] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of inhibin subunit beta E (INHBE), the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by 0, 1, 2, 3, or 4 or fewer nucleotides from the nucleotide sequence ascscagucgUfCfCfcagaauaacu (SEQ ID NO:), and the antisense strand differs by 0, 1, 2, 3, or 4 or fewer nucleotides from the nucleotide sequence asdGsuudAudTcuggdGaCfgacugguscsa (SEQ ID NO:). , 1, 2, 3, or 4 or less different contiguous nucleotides, wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U; dA, dG, dC, and dT are 2'-deoxy A, G, C, and T; s is a phosphorothioate linkage; and the dsRNA comprises one or more C22 hydrocarbon chains conjugated to one or more internal positions on at least one strand of the dsRNA agent.

[0091] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of inhibin subunit beta E (INHBE), the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by 0, 1, 2, 3, or 4 or fewer nucleotides from the nucleotide sequence ascscagucgUfCfCfcagaauaacu (SEQ ID NO:), and the antisense strand differs by 0, 1, 2, 3, or 4 or fewer nucleotides from the nucleotide sequence asdGsuudAudTcuggdGaCfgacugguscsu (SEQ ID NO:). , 1, 2, 3, or 4 or less different contiguous nucleotides, wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U; dA, dG, dC, and dT are 2'-deoxy A, G, C, and T; s is a phosphorothioate linkage; and the dsRNA comprises one or more C22 hydrocarbon chains conjugated to one or more internal positions on at least one strand of the dsRNA agent.

[0092] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of inhibin subunit beta E (INHBE), the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by 0, 1, 2, 3, or 4 or fewer nucleotides from the nucleotide sequence cscsagaauaAfCfUfcauccuccau (SEQ ID NO:), and the antisense strand differs by 0, 1, 2, 3, or 4 or fewer nucleotides from the nucleotide sequence asdTsggdAgdGaugadGuUfauucuggsgsa (SEQ ID NO:). , 1, 2, 3, or 4 or less different contiguous nucleotides, wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U; dA, dG, dC, and dT are 2'-deoxy A, G, C, and T; s is a phosphorothioate linkage; and the dsRNA comprises one or more C22 hydrocarbon chains conjugated to one or more internal positions on at least one strand of the dsRNA agent.

[0093] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of inhibin subunit beta E (INHBE), the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by 0, 1, 2, 3, or 4 nucleotides or less from the nucleotide sequence csusgucaCfaGfAfCfuccacuucau (SEQ ID NO:), and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by 0, 1, 2, 3, or 4 nucleotides or less from the nucleotide sequence asUfsgadAg(Tgn)ggagucUfgUfgacagsusa (SEQ ID NO:). the dsRNA agent comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 different contiguous nucleotides that differ by no more than 1 nucleotide, where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U; dA, dG, dC, and dT are 2'-deoxy A, G, C, and T; Tgn is a thymidine-glycol nucleic acid (GNA) S-isomer; and s is a phosphorothioate linkage; and the dsRNA agent comprises one or more C22 hydrocarbon chains conjugated to one or more internal positions on at least one strand of the dsRNA agent.

[0094] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of inhibin subunit beta E (INHBE), the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by 0, 1, 2, 3, or 4 or fewer nucleotides from the nucleotide sequence usgsucacagAfCfUfccacuucagu (SEQ ID NO:), and the antisense strand differs by 0, 1, 2, 3, or 4 or fewer nucleotides from the nucleotide sequence asdCsugdAadGuggadGuCfugugacasgsu (SEQ ID NO:). , 1, 2, 3, or 4 or less different contiguous nucleotides, wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U; dA, dG, dC, and dT are 2'-deoxy A, G, C, and T; s is a phosphorothioate linkage; and the dsRNA comprises one or more C22 hydrocarbon chains conjugated to one or more internal positions on at least one strand of the dsRNA agent.

[0095] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of inhibin subunit beta E (INHBE), the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by 0, 1, 2, 3, or 4 or fewer nucleotides from the nucleotide sequence csusuugcuuGfAfGfgaucuuccgu (SEQ ID NO:), and the antisense strand differs by 0, 1, 2, 3, or 4 or fewer nucleotides from the nucleotide sequence asdCsggdAadGauccdTcAfagcaaagsasg (SEQ ID NO:). , 1, 2, 3, or 4 or less different contiguous nucleotides, wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U; dA, dG, dC, and dT are 2'-deoxy A, G, C, and T; s is a phosphorothioate linkage; and the dsRNA comprises one or more C22 hydrocarbon chains conjugated to one or more internal positions on at least one strand of the dsRNA agent.

[0096] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of inhibin subunit beta E (INHBE), the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by 0, 1, 2, 3, or 4 or fewer nucleotides from the nucleotide sequence asasugggcaCfUfUfucuugucugu (SEQ ID NO:), and the antisense strand differs from the nucleotide sequence asdCsagdAcdAagaadAgUfgcccauususg (SEQ ID NO:). , 1, 2, 3, or 4 or less different contiguous nucleotides, wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U; dA, dG, dC, and dT are 2'-deoxy A, G, C, and T; s is a phosphorothioate linkage; and the dsRNA comprises one or more C22 hydrocarbon chains conjugated to one or more internal positions on at least one strand of the dsRNA agent.

[0097] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of inhibin subunit beta E (INHBE), the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by 0, 1, 2, 3, or 4 or fewer nucleotides from the nucleotide sequence gsascaagcaUfUfUfauacuuucuu (SEQ ID NO:), and the antisense strand comprises the nucleotide sequence asdAsgadAadGuauadAaUfgcuugucsusc (SEQ ID NO:). and at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 different contiguous nucleotides that differ by 0, 1, 2, 3, or 4 or fewer nucleotides from: a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U; dA, dG, dC, and dT are 2'-deoxy A, G, C, and T; s is a phosphorothioate linkage; and the dsRNA comprises one or more C22 hydrocarbon chains conjugated to one or more positions on at least one strand of the dsRNA agent.

[0098] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of inhibin subunit beta E (INHBE), the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by 0, 1, 2, 3, or 4 or fewer nucleotides from the nucleotide sequence ascsaagcauUfUfAfuacuuucuuu (SEQ ID NO:), and the antisense strand differs by 0, 1, 2, 3, or 4 or fewer nucleotides from the nucleotide sequence asdAsagdAadAguaudAaAfugcuuguscsu (SEQ ID NO:). , 1, 2, 3, or 4 or less different contiguous nucleotides, wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U; dA, dG, dC, and dT are 2'-deoxy A, G, C, and T; s is a phosphorothioate linkage; and the dsRNA comprises one or more C22 hydrocarbon chains conjugated to one or more internal positions on at least one strand of the dsRNA agent.

[0099] In some embodiments, the octanol-water partition coefficient, log K ow One or more C measured by 22 The lipophilicity of the hydrocarbon chain is greater than 0. The lipophilic moiety has a log K ow may have

[0100] In some embodiments, the hydrophobicity of the dsRNA agent measured by the unbound fraction in a plasma protein binding assay of the dsRNA agent is greater than 0.2. In one embodiment, the plasma protein binding assay measured is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. The hydrophobicity of the dsRNA agent measured by the unbound dsRNA fraction in a binding assay is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45, or greater than 0.5, for enhanced in vivo delivery of dsRNA.

[0101] C 22 The hydrocarbon chain may be saturated or unsaturated.

[0102] C 22 The hydrocarbon chain may be linear or branched.

[0103] In some embodiments, internal positions include all but the three terminal positions from each end of at least one strand.

[0104] In some embodiments, the internal position excludes the cleavage site region of the sense strand.

[0105] In some embodiments, internal positions exclude positions 9-12 or 11-13 counting from the 5' end of the sense strand.

[0106] In some embodiments, the internal location excludes the cleavage site region of the antisense strand.

[0107] In some embodiments, internal positions exclude positions 12-14 from the 5' end of the antisense strand.

[0108] In some embodiments, one or more C 22The hydrocarbon chains are conjugated to one or more of the following internal positions: positions 4-8 and 13-18 on the sense strand and positions 6-10 and 15-18 on the antisense strand, counting from the 5' end of each strand.

[0109] In some embodiments, one or more C 22 The hydrocarbon chains are conjugated to one or more of the following internal positions: positions 5, 6, 7, 15, and 17 on the sense strand and positions 15 and 17 on the antisense strand, counting from the 5' end of each strand.

[0110] In some embodiments, one or more C 22 The hydrocarbon chain is conjugated to position 6 on the sense strand, counting from the 5' end of the sense strand.

[0111] In some embodiments, one or more C 22 The hydrocarbon chain may be aliphatic, alicyclic, or polyalicyclic, e.g., one or more C 22 The hydrocarbon chain contains a functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0112] In some embodiments, one or more C 22 The hydrocarbon chain is a C22 acid, for example, the C22 acid is selected from the group consisting of docosanoic acid, 6-octyltetradecanoic acid, 10-hexylhexadecanoic acid, all-cis-7,10,13,16,19-docosapentaenoic acid, all-cis-4,7,10,13,16,19-docosahexaenoic acid, all-cis-13,16-docosadienoic acid, all-cis-7,10,13,16-docosatetraenoic acid, all-cis-4,7,10,13,16-docosapentaenoic acid, and cis-13-docosenoic acid. [ka]

[0113] In some embodiments, one or more C 22The hydrocarbon chain is a C22 alcohol, for example, the C22 alcohol is selected from the group consisting of 1-docosanol, 6-octyltetradecan-1-ol, 10-hexylhexadecan-1-ol, cis-13-docosene-1-ol, docosan-9-ol, docosan-2-ol, docosan-10-ol, docosan-11-ol, and cis-4,7,10,13,16,19-docosahexanol. [ka]

[0114] In some embodiments, one or more C 22 The hydrocarbon chain is a C22 amide, for example, the C22 amide is selected from the group consisting of (E)-docosa-4-enamide, (E)-docosa-5-enamide, (Z)-docosa-9-enamide, (E)-docosa-11-enamide, 12-docosenamid, (Z)-docosa-13-enamide, (Z)-N-hydroxy-13-docosenamid, (E)-docosa-14-enamide, 6-cis-docosenamid, 14-docosenamidocos-11-enamide, (4E,13E)-docosa-4,13-dienamide, and (5E,13E)-docosa-5,13-dienamide.

[0115] One or more C's 22 The hydrocarbon chain may be conjugated to the dsRNA agent via a direct bond to the ribosugar of the dsRNA agent. Alternatively, one or more C 22 The hydrocarbon chain may be conjugated to the dsRNA agent via a linker or carrier. In some embodiments, one or more C 22 The hydrocarbon chain may be conjugated to the dsRNA agent via an internucleotide phosphate linkage. [ka]

[0116] In certain embodiments, one or more C 22The hydrocarbon chain is conjugated to the dsRNA agent via one or more linkers (tethers), eg, carriers, that substitute one or more nucleotides at an internal position.

[0117] In some embodiments, one or more C 22 The hydrocarbon chain is conjugated to the dsRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide bond, the product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate.

[0118] In some embodiments, at least one of the linkers (tethers) is a redox-cleavable linker (e.g., a reductively cleavable linker, such as a disulfide group), an acid-cleavable linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group), an esterase-cleavable linker (e.g., an ester group), a phosphatase-cleavable linker (e.g., a phosphate group), or a peptidase-cleavable linker (e.g., a peptide bond).

[0119] In other embodiments, at least one of the linkers (tethers) is a biocleavable linker selected from the group consisting of DNA, RNA, disulfides, amides, and functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0120] In certain embodiments, one or more C 22The hydrocarbon chain is conjugated to dsRNA agent through the carrier that replaces one or more nucleotides.The carrier can be cyclic or acyclic.In one embodiment, the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalin.In one embodiment, the acyclic group is based on serinol skeleton or diethanolamine skeleton.

[0121] In some embodiments, the carrier replaces one or more nucleotides at an internal position of the dsRNA agent.

[0122] In some embodiments, the dsRNA agent further comprises a targeting ligand that targets a receptor that mediates delivery to adipose tissue. In one embodiment, the targeting ligand is selected from the group consisting of angiopep-2, lipoprotein receptor-related protein (LRP) ligand, bEnd.3 cell-binding ligand, transferrin receptor (TfR) ligand, mannose receptor ligand, glucose transporter protein, LDL receptor ligand, trans-retinol, RGD peptide, LDL receptor ligand, CD63 ligand, and carbohydrate-based ligand.

[0123] In some embodiments, the dsRNA agent further comprises a targeting ligand that targets liver tissue.

[0124] In one embodiment, the targeting ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

[0125] In some embodiments, the targeting ligand is a carbohydrate-based ligand.

[0126] In one embodiment, the targeting ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0127] In one embodiment, the targeting ligand is one or more GalNAc derivatives attached through a monovalent, divalent, or trivalent branched linker.

[0128] In one embodiment, the targeting ligand is: [ka]

[0129] In one embodiment, the dsRNA agent is conjugated to a targeting ligand as shown in the following schematic diagram. [ka] wherein X is O or S.

[0130] In one embodiment, X is O.

[0131] In some embodiments, the one or more C22 hydrocarbon chains or targeting ligands are conjugated via a biocleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, and functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0132] The invention also provides cells containing any of the dsRNA agents of the invention, and pharmaceutical compositions comprising any of the dsRNA agents of the invention.

[0133] Pharmaceutical compositions of the invention can include a dsRNA agent in an unbuffered solution, such as, for example, saline or water, or they can include a dsRNA agent in a buffered solution, such as, for example, a buffered solution containing acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof, or phosphate buffered saline (PBS).

[0134] In one aspect, the present invention provides a method for inhibiting the expression of a metabolic disorder-related target gene selected from the group consisting of inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), and inhibin subunit beta C (INHBC) in a cell, such as an adipocyte and / or hepatocyte. The method comprises contacting the cell 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 target gene in the cell.

[0135] In one embodiment, the target gene is INHBE.

[0136] In one embodiment, the target gene is ACVR1C.

[0137] In one embodiment, the target gene is PLIN1.

[0138] In one embodiment, the target gene is PDE3B.

[0139] In one embodiment, the target gene is INHBC.

[0140] In one embodiment, the cell is in a subject, eg, a human subject, eg, the subject has a metabolic disorder, such as diabetes, metabolic syndrome, cardiovascular disease, or hypertension.

[0141] In one embodiment, the cells are adipocytes.

[0142] In one embodiment, the cells are hepatocytes.

[0143] In certain embodiments, target gene expression is inhibited by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In one embodiment, inhibiting target gene expression reduces target gene protein levels in the subject's serum by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0144] In one aspect, the present invention provides a method for treating a metabolic disorder, the method comprising administering a therapeutically 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 treating the subject with a metabolic disorder.

[0145] In another aspect, the present invention provides the method for preventing at least one symptom in the subject with metabolic disorder.This method includes administering to subject any of the dsRNA of the present invention or any of the pharmaceutical compositions of the present invention with a preventively effective amount, thereby preventing at least one symptom in the subject with metabolic disorder.

[0146] In one embodiment, the target gene is INHBE.

[0147] In one embodiment, the target gene is ACVR1C.

[0148] In one embodiment, the target gene is PLIN1.

[0149] In one embodiment, the target gene is PDE3B.

[0150] In one embodiment, the target gene is INHBC.

[0151] In one embodiment, administration of a therapeutically or prophylactically effective amount reduces waist-to-hip ratio adjusted for body mass index in a subject.

[0152] The metabolic disorder may be, for example, metabolic syndrome, carbohydrate disorders such as type II diabetes, prediabetes, lipid metabolism disorders such as hyperlipidemia, hypertension, lipodystrophy, renal disease, cardiovascular disease, weight disorders.

[0153] In some embodiments, the metabolic disorder is metabolic syndrome.

[0154] In some embodiments, the metabolic disorder is type 2 diabetes.

[0155] In some embodiments, the metabolic disorder is obesity.

[0156] In some embodiments, the metabolic disorder is elevated triglyceride levels.

[0157] In some embodiments, the metabolic disorder is lipodystrophy.

[0158] In some embodiments, the metabolic disorder liver inflammation.

[0159] In some embodiments, the metabolic disorder is fatty liver disease.

[0160] In some embodiments, the metabolic disorder is hypercholesterolemia.

[0161] In some embodiments, the metabolic disorder is elevated liver enzymes.

[0162] In some embodiments, the metabolic disorder is non-alcoholic steatohepatitis (NASH).

[0163] In some embodiments, the metabolic disorder is a cardiovascular disease.

[0164] In some embodiments, the metabolic disorder is hypertension.

[0165] In some embodiments, the metabolic disorder is cardiomyopathy.

[0166] In some embodiments, the metabolic disorder is heart failure.

[0167] In some embodiments, the metabolic disorder is a kidney disease.

[0168] In certain embodiments, administration of the dsRNA to a subject causes a decrease in target gene protein accumulation in the subject.

[0169] In a further aspect, the present invention also provides a method for inhibiting the expression of a metabolic disorder-related target gene selected from the group consisting of inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), and inhibin subunit beta C (INHBC) in a subject. The method comprises administering a therapeutically effective amount of any of the dsRNAs provided herein to the subject, thereby inhibiting the expression of the target gene in the subject.

[0170] In one embodiment, the subject is a human.

[0171] In one embodiment, the dsRNA agent is administered to the subject at a dose of about 0.01 mg / kg to about 50 mg / kg.

[0172] In one embodiment, the dsRNA agent is administered to the subject subcutaneously.

[0173] In one embodiment, the method of the present invention further comprises determining the level of the target gene in a sample from the subject.

[0174] In one embodiment, the level of the target gene in a sample from a subject is the target gene protein level in a blood or serum or liver tissue sample.

[0175] In certain embodiments, the methods of the present invention further comprise administering to the subject an additional therapeutic agent.

[0176] In certain embodiments, the additional therapeutic agent is selected from the group consisting of insulin, glucagon-like peptide 1 agonists, sulfonylureas, seglitinides, biguanides, thiazolidinediones, alpha-glucosidase inhibitors, SGLT2 inhibitors, DPP-4 inhibitors, HMG-CoA reductase inhibitors, statins, and combinations of any of the foregoing.

[0177] 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 metabolic disorder-related target genes selected from the group consisting of inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), and inhibin subunit beta C (INHBC) in cells by contacting cells with the double-stranded RNAi agent of the present invention in an amount effective to inhibit the expression of the target gene in cells.The kit comprises an RNAi agent and instructions for use, and optionally includes a means for administering the RNAi agent to a subject.

[0178] The invention further provides an RNA-induced silencing complex (RISC) comprising the antisense strand of any of the dsRNA agents of the invention. [Brief explanation of the drawings]

[0179] [Figure 1] FIG. 1 is a schematic representation of the study design to determine the pharmacodynamic activity of subject duplexes targeting INHBE in non-human primates (NHPs). [Figure 2A] FIG. 2A is a graph illustrating the levels of INHBE mRNA in the liver of non-human primates administered a single 3 mg / kg dose of the indicated duplex subcutaneously at 28 days post-administration. [Figure 2B]FIG. 2B is a graph illustrating the levels of INHBC mRNA in the liver of non-human primates administered a single 3 mg / kg dose of the indicated duplex subcutaneously at 28 days post-administration. DETAILED DESCRIPTION OF THE INVENTION

[0180] The present invention provides iRNA compositions that affect RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcripts of metabolic disorder-related target genes selected from the group consisting of inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), and inhibin subunit beta C (INHBC). The genes may be located in cells, such as adipocytes and / or hepatocytes, within a subject, such as a human. The use of these iRNAs allows for targeted degradation of the mRNA of the corresponding gene (INHBE, ACVR1C, PLIN1, PDE3B, or INHBC) in a mammal.

[0181] The iRNAs of the present invention are designed to target metabolic disorder-related target genes selected from the group consisting of inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), and inhibin subunit beta C (INHBC), including portions of genes conserved in orthologs of other mammalian species. Without intending to be limited by theory, it is believed that combinations or subcombinations of the aforementioned features and specific target sites or specific modifications in these iRNAs improve the efficacy, stability, potency, durability, and safety of the iRNAs of the present invention.

[0182] As a result, the present invention provides methods for treating and preventing metabolic disorders, e.g., metabolic syndrome, carbohydrate disorders, e.g., type II diabetes, prediabetes, lipid metabolism disorders, e.g., hyperlipidemia, hypertension, lipodystrophy, renal disease, cardiovascular disease, weight disorders, using iRNA compositions that affect RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of metabolic disorder-associated target genes selected from the group consisting of inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), and inhibin subunit beta C (INHBC).

[0183] The iRNAs of the present invention can be up to about 30 nucleotides in length, e.g., 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, The RNA strand (antisense strand) comprises an RNA strand having a region that is 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least a portion of an mRNA transcript of a metabolic disorder-associated target gene.

[0184] In certain embodiments, one or both strands of a double-stranded RNAi agent of the invention are up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, or 27-53 nucleotides in length, and have a region of at least 19 contiguous nucleotides that are substantially complementary to at least a portion of an mRNA transcript of a metabolic disorder-associated target gene. In some embodiments, such iRNA agents with longer antisense strands can include a second RNA strand (sense strand) that is, for example, 20-60 nucleotides in length, where the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

[0185] The use of iRNAs of the present invention allows for targeted degradation of the mRNA of the corresponding gene (INHBE, ACVR1C, PLIN1, PDE3B, or INHBC gene) in mammals. Using in vitro assays, the inventors have demonstrated that iRNAs targeting genes can potently mediate RNAi, resulting in significant inhibition of target gene expression. Therefore, methods and compositions comprising these iRNAs are useful for treating subjects with metabolic disorders, such as metabolic syndrome, carbohydrate disorders, such as type II diabetes, prediabetes, lipid metabolism disorders, such as hyperlipidemia, hypertension, lipodystrophy, renal disease, cardiovascular disease, and weight disorders.

[0186] As a result, the present invention provides methods and combination therapies using iRNA compositions that affect RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), and inhibin subunit beta C (INHBC) to treat subjects with metabolic disorders, e.g., metabolic syndrome, carbohydrate disorders, e.g., type II diabetes, prediabetes, lipid metabolism disorders, e.g., hyperlipidemia, hypertension, lipodystrophy, renal disease, cardiovascular disease, weight disorders, who would benefit from inhibiting or reducing the expression of metabolic disorder-associated target genes selected from the group consisting of INHBE, ACVR1C, PLIN1, PDE3B, or INHBC.

[0187] The present invention also provides a method for preventing at least one symptom in a subject having a disorder, e.g., metabolic syndrome, a carbohydrate disorder, e.g., type II diabetes, prediabetes, a lipid metabolism disorder, e.g., hyperlipidemia, hypertension, lipodystrophy, renal disease, cardiovascular disease, or a weight disorder, that would benefit from inhibiting or reducing the expression of a metabolic disorder-related target gene selected from the group consisting of inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), and inhibin subunit beta C (INHBC).

[0188] The following detailed description discloses compositions, uses, and methods for treating a subject who would benefit from inhibiting or reducing the expression of a metabolic disorder-associated target gene selected from the group consisting of inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), and inhibin subunit beta C (INHBC), as well as how to make and use compositions containing iRNA to inhibit the expression of a metabolic disorder-associated target gene selected from the group consisting of inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), and inhibin subunit beta C (INHBC), for example, the subject is susceptible to or has been diagnosed with a metabolic disorder.

[0189] I. Definition In order that the present invention may be more readily understood, certain terms are first defined. Additionally, it should be noted that whenever a value or range of values ​​for a parameter is listed, it is intended that values ​​and ranges intermediate to the listed values ​​are also intended to be part of the invention.

[0190] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or to more than one element, e.g., a plurality of elements.

[0191] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0192] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly indicates otherwise. For example, "the sense strand or the antisense strand" is understood as "the sense strand or the antisense strand, or the sense strand and the antisense strand."

[0193] The term "about" is used herein to mean within a typical tolerance in the art. For example, "about" can be understood as about 2 standard deviations from the mean. In certain embodiments, about means ±10%. In certain embodiments, about means ±5%. When about precedes a series of numbers or ranges, it is understood that "about" can modify each of the consecutive numbers or ranges.

[0194] The terms "at least," "greater than," or "or more" preceding a number or series of numbers, when clear from the context, are understood to include the number adjacent to the term "at least," and all subsequent numbers or integers that may logically be included. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 19 nucleotides of a 21-nucleotide nucleic acid molecule" means that 19, 20, or 21 nucleotides have the specified property. When at least precedes a series of numbers or ranges, it is understood that "at least" can modify each of the series of numbers or ranges.

[0195] As used herein, "less than" or "or less than" refers to the value adjacent to the phrase and, if logical from the context, to zero, any logically smaller value or integer than the value.For example, a duplex with an overhang of "2 nucleotides or less" has an overhang of 2, 1, or 0 nucleotides.When "less than" is used before a series of numbers or ranges, it is understood that "less than" can modify each of the series of numbers or ranges.As used herein, a range includes both upper and lower limits.

[0196] As used herein, a method of detection can include determining that the amount of analyte present is below the detection level of the method.

[0197] In the event of a conflict between a given target site and the nucleotide sequence for either the sense or antisense strand, the given sequence controls.

[0198] In the event of a discrepancy between a sequence on a transcript or other sequence and its indicated site, the nucleotide sequence listed herein takes precedence.

[0199] As used herein, the term "metabolic disorder-associated target gene" or "target gene" refers to a gene encoding "inhibin subunit beta E" ("INHBE"), "activin A receptor type 1C" ("ACVR1C"), "perilipin-1" ("PLIN1"), "phosphodiesterase 3B" ("PDE3B"), or "inhibin subunit beta C" ("INHBC").

[0200] In one embodiment, the metabolic disorder-associated target gene is inhibin subunit beta E (INHBE).

[0201] As used herein, "inhibin subunit beta E" is used interchangeably with the term "INHBE"; INHBE refers to a growth factor belonging to the transforming growth factor-β (TGF-β) family. INHBE mRNA is primarily expressed in the liver (Fang J. et al. Biochemical & Biophysical Res. Comm. 1997;231(3):655-61), and INHBE is involved in regulating hepatocyte growth and differentiation (Chabicovsky M. et al. Endocrinology. 2003;144(8):3497-504). INHBE is also known as inhibin beta E chain, activin E, inhibin beta E subunit, inhibin beta E, and MGC4638. More specifically, INHBE is a hepatokine that has been shown to positively correlate with insulin resistance and body mass index in humans. Quantitative real-time PCR analysis also demonstrated increased INHBE gene expression in liver samples from insulin-resistant human subjects. Additionally, Inhbe gene expression has been shown to be increased in the liver of an art-recognized animal model of metabolic disorder, namely the db / db mouse model of type 2 diabetes. Inhibition of Inhbe expression in db / db mice has been demonstrated to suppress weight gain due to loss of fat but not lean mass.

[0202] The sequence of human INHBE mRNA transcript can be found, for example, in GenBank Accession No. GI:1877089956 (NM_031479.5, SEQ ID NO:1, reverse complement, SEQ ID NO:2). The sequence of mouse INHBE mRNA can be found, for example, in GenBank Accession No. GI:1061899809 (NM_008382.3, SEQ ID NO:3, reverse complement, SEQ ID NO:4). The sequence of rat INHBE mRNA can be found, for example, in GenBank Accession No. GI:148747589 (NM_031815.2, SEQ ID NO:5, reverse complement, SEQ ID NO:6). The predicted sequence of rhesus INHBE mRNA can be found, for example, in GenBank Accession No. GI:1622845604 (XM_001115958.3, SEQ ID NO:7, reverse complement, SEQ ID NO:8).

[0203] Additional examples of INHBE mRNA sequences are readily available through public databases such as GenBank, UniProt, OMIM, and the Macaca Genome Project website.

[0204] Further information regarding INHBE can be found, for example, at www.ncbi.nlm.nih.gov / gene / ?term=INHBE.

[0205] The entire contents of each of the foregoing GenBank Accession Numbers and Gene Database Numbers are incorporated herein by reference as of the filing date of this application.

[0206] As used herein, the term INHBE also refers to variations of the INHBE gene, including variants provided in SNP databases. Numerous sequence variations within the INHBE gene have been identified and may be found, for example, in NCBI dbSNP and UniProt (see, e.g., www.ncbi.nlm.nih.gov / snp / ?term=INHBE, the entire contents of which are incorporated herein by reference as of the filing date of this application).

[0207] In one embodiment, the metabolic disorder-associated target gene is activin A receptor type 1C (ACVR1C).

[0208] As used herein, "activin A receptor type 1C," which is used interchangeably with the term "ACVR1C," refers to a type I receptor for the TGF-β family of signaling molecules. ACVR1C possesses intrinsic serine / threonine kinase activity within its cytoplasmic domain, inducing phosphorylation and activation of the SMAD2 / 3 / 4 complex, which translocates to the nucleus where it binds to SMAD-binding elements (SBEs) and activates gene transcription. ACVR1C expression levels vary significantly among tissues, with white and brown adipose tissue possessing the highest expression levels. In addition to the full-length protein, ACVR1C variants are also expressed in adipose tissue, brain, and ovary (Murakami M et al. Biochem Genet. 2013;51(3-4):202-210). ACVR1C is also known as "activin receptor-like kinase 7" (ALK-7). Polymorphisms in ACVR1C are associated with an increased risk of metabolic syndrome in Chinese women and may be involved in cardiovascular remodeling in patients with metabolic syndrome (Zhang, W et al. Arq Bras Cardiol. 2013:101(2):134-140). Additionally, variants predicted to lead to loss of ACVR1C gene function are thought to affect body fat distribution and protect against type 2 diabetes (Emdin CA et al. Diabetes. 2019:68(1):226-234). Studies performed in adipocytes of obese mouse strains demonstrated that ACVR1C dysfunction (caused by a nonsense mutation) increased lipolysis in adipocytes and reduced fat accumulation, and conversely, ACVR1C activation inhibited lipolysis by suppressing the expression of adipose lipase. Furthermore, lower body weight ACVR1C-deficient mice exhibited increased glucose tolerance and insulin sensitivity, and measurements of metabolic rate in these mice revealed increased O2 consumption, decreased respiratory quotient, and increased energy expenditure (Yogosawa, S et al. Diabetes 2013:62(1):115-123).

[0209] The sequence of the human ACVR1C mRNA transcript is, for example, GenBank accession number GI:1519315475 (NM_145259.3, SEQ ID NO:9, reverse complement, SEQ ID NO:10), GI:1890343165 (NM_001111031.2, SEQ ID NO:11, reverse complement, SEQ ID NO:12), GI: 1676439,980 (NM_001111032.2, SEQ ID NO: 13, reverse complement, SEQ ID NO: 14), and The sequence of mouse ACVR1C mRNA can be found in, for example, GenBank accession number GI: 1676318472 (NM_001111033.2, SEQ ID NO: 15, reverse complement, SEQ ID NO: 16). The sequence of mouse ACVR1C mRNA can be found in, for example, GenBank accession number GI: 161333830 (NM_001111030.1, SEQ ID NO: 17, reverse complement, SEQ ID NO: 18) or GI: 161333829 (NM_001033369.3, SEQ ID NO: 19, reverse complement, SEQ ID NO: 20). The sequence of rat ACVR1C mRNA can be found in, for example, GenBank accession number GI: 1937875934 (NM_139090.2, SEQ ID NO: 21, reverse complement, SEQ ID NO: 22). The sequence of rhesus monkey ACVR1C mRNA can be found, for example, in GenBank Accession No. GI:388454445 (NM_001266690.1, SEQ ID NO: 23, reverse complement, SEQ ID NO: 24).

[0210] Additional examples of ACVR1C mRNA sequences are readily available through public databases such as GenBank, UniProt, OMIM, and the Macaca Genome Project website.

[0211] Further information regarding ACVR1C can be found, for example, at www.ncbi.nlm.nih.gov / gene / ?term=ACVR1C.

[0212] As used herein, the term ACVR1C also refers to variations of the ACVR1C gene, including variants provided in SNP databases. Numerous sequence variations within the ACVR1C 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=ACVR1C, the entire contents of which are incorporated herein by reference as of the filing date of this application).

[0213] The entire contents of each of the foregoing GenBank Accession Numbers and Gene Database Numbers are incorporated herein by reference as of the filing date of this application.

[0214] In one embodiment, the metabolic disorder-associated target gene is perilipin-1 (PLIN1).

[0215] As used herein, "perilipin-1," used interchangeably with the term "PLIN1," refers to a protein that coats lipid storage droplets in adipocytes, thereby protecting them until they can be degraded by hormone-sensitive lipase. PLIN1 is primarily expressed in adipose tissue. PLIN1 is also known as perilipin, lipid droplet-associated protein, PERI, PLIN, and FPLD4.

[0216] Constitutive overexpression of PLIN1 in cultured adipocytes has been shown to block the ability of TNF-α to increase lipolysis. In animals, separate laboratories independently generated PLIN1-null mouse strains and observed that the mice were lean and developed systemic insulin resistance with age. A study comparing lipolysis in PLIN1-null and wild-type mice revealed that PLIN1-null adipocytes had increased rates of constitutive (unstimulated) lipolysis and decreased catecholamine-stimulated lipolysis. Several studies have found that polymorphisms in the PLIN1 gene affect body weight and metabolic disease risk. Interestingly, one PLIN1 polymorphism was found to be associated with decreased PLIN1 expression and increased rates of basal and stimulated adipocyte lipolysis, and individuals with this polymorphism tended to have reduced body weight and fat mass (Greenberg, AS et al. J Clin Invest. 2011:121(6):2102-2110). Heterozygous frameshift variants in PLIN1 also implicate familial partial lipodystrophy, a rare disorder characterized by a limited capacity of peripheral fat to store triglycerides, which leads to metabolic abnormalities including insulin resistance, hypertriglyceridemia, and hepatic steatosis (Gandotra S, Le Dour C, Bottomley W, et al. N Engl J Med 2011;364:740-748).

[0217] The sequence of human PLIN1 mRNA transcript can be found, for example, in GenBank accession numbers GI:1519242647 (NM_002666.5, SEQ ID NO:25, reverse complement, SEQ ID NO:26) and GI:1675042447 (NM_001145311.2, SEQ ID NO:27, reverse complement, SEQ ID NO:28). The sequence of mouse PLIN1 mRNA can be found, for example, in GenBank accession numbers GI:164698407 (NM_175640.2, SEQ ID NO:29, reverse complement, SEQ ID NO:30) and GI:164698412 (NM_001113471.1, SEQ ID NO:31, reverse complement, SEQ ID NO:32). The sequence of rat PLIN1 mRNA can be found, for example, in GenBank Accession No. GI:815890869 (NM_001308145.1, SEQ ID NO:33, reverse complement, SEQ ID NO:34). The predicted sequence of rhesus monkey PLIN1 mRNA can be found, for example, in GenBank Accession No. GI:1622954660 (XM_028851317.1, SEQ ID NO:35, reverse complement, SEQ ID NO:36).

[0218] Additional examples of PLIN1 mRNA sequences are readily available through public databases such as GenBank, UniProt, OMIM, and the Macaca Genome Project website.

[0219] Further information regarding PLIN1 can be found, for example, at www.ncbi.nlm.nih.gov / gene / ?term=PLIN1.

[0220] The entire contents of each of the foregoing GenBank Accession Numbers and Gene Database Numbers are incorporated herein by reference as of the filing date of this application.

[0221] As used herein, the term PLIN1 also refers to variations of the PLIN1 gene, including variants provided in SNP databases. Numerous sequence variations within the PLIN1 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=PLIN1, the entire contents of which are incorporated herein by reference as of the filing date of this application).

[0222] In one embodiment, the metabolic disorder-associated target gene is phosphodiesterase 3B (PDE3B).

[0223] As used herein, "phosphodiesterase 3B" is used interchangeably with the term "PDE3B"; PDE3B is a phosphodiesterase that hydrolyzes cAMP and cGMP and is expressed in cells important for regulating energy homeostasis, including adipocytes, hepatocytes, hypothalamic cells, and beta cells. PDE3B is also known as cGMP-inhibited 3',5'-cyclic phosphodiesterase B, cyclic GMP-inhibited phosphodiesterase B, CGIPDE1, CGIP1, and cyclic nucleotide phosphodiesterase.

[0224] PDE3B protein is phosphorylated and activated in hepatocytes and adipocytes in response to stimulation by insulin and / or cAMP-increasing drugs.The activation of PDE3B leads to increased hydrolysis of cAMP, and thereby leads to the inhibition of catecholamine-induced lipolysis.Mice that specifically overexpress PDE3B in β cells show reduced glucose-induced insulin secretion.PDE3B knockout (KO) mice, when maintained on a high-fat diet, demonstrate numerous alterations in the regulation of energy homeostasis, including reduced fat mass, reduced adipocytes, and reduced weight gain compared to control mice (Degerman, E. et al.CurrOpin Pharmaco.2011:11(6):676-682).

[0225] The sequence of human PDE3B mRNA transcript can be found, for example, in GenBank accession number GI:1889438535 (NM_001363570.2, SEQ ID NO:37, reverse complement, SEQ ID NO:38), GI:1519241942 (NM_000922.4, SEQ ID NO:39, reverse complement, SEQ ID NO:40), and GI:1889636835 (NM_001363569.2, SEQ ID NO:41, reverse complement, SEQ ID NO:42).The sequence of mouse PDE3B mRNA can be found, for example, in GenBank accession number GI:112983647 (NM_011055.2, SEQ ID NO:43, reverse complement, SEQ ID NO:44). The sequence of rat PDE3B mRNA can be found, for example, in GenBank Accession No. GI:1939401976 (NM_017229.2, SEQ ID NO:45, reverse complement, SEQ ID NO:46). The predicted sequence of rhesus monkey PDE3B mRNA can be found, for example, in GenBank Accession No. GI:1622864110 (XM_015114810.2, SEQ ID NO:47, reverse complement, SEQ ID NO:48).

[0226] Additional examples of PDE3B mRNA sequences are readily available through public databases such as GenBank, UniProt, OMIM, and the Macaca Genome Project website.

[0227] Further information regarding PDE3B can be found, for example, at www.ncbi.nlm.nih.gov / gene / ?term=PDE3B.

[0228] The entire contents of each of the foregoing GenBank Accession Numbers and Gene Database Numbers are incorporated herein by reference as of the filing date of this application.

[0229] As used herein, the term PDE3B also refers to variations of the PDE3B gene, including variants provided in SNP databases.A large number of sequence variations within the PDE3B 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=PDE3B, the entire contents of which are incorporated herein by reference as of the filing date of this application).

[0230] In one embodiment, the metabolic disorder-associated target gene is inhibin subunit beta C (INHBC).

[0231] As used herein, "inhibin subunit beta C" is used interchangeably with the term "INHBC"; INHBC refers to the beta C chain of inhibin, a member of the TGF-β superfamily. INHBC mRNA is primarily expressed in the liver, and INHBC is involved in regulating hepatocyte growth and differentiation (Chabicovsky M. et al. Endocrinology. 2003;144(8):3497-504). INHBC is also known as inhibin beta C chain, inhibin beta C subunit, inhibin beta C, activin C, activin beta-C chain, and IHBC. In mice, overexpression of INHBC increased total liver weight as a percentage of body weight and increased both hepatocyte proliferation and apoptosis. INHBC has been demonstrated to be significantly upregulated in obese, insulin-resistant subjects (Choi, et al. Front Physiol. 2019;10:379). A SNP in the INHBC locus has been identified as having genome-wide significance with serum urate levels and is associated with increased risk of gout (Yang Q, et al. 2010, Circ. Cardiovasc. Genet., 3:523-530). In addition, the INHBC locus also co-localizes with a GWAS signal for estimated glomerular filtration rate (eGFR), a marker of renal function (Gudjonsson A. et al., 2022, Nature Communication, 13:480).

[0232] The sequence of human INHBC mRNA transcript can be found, for example, in GenBank Accession No. GI:1519246544 (NM_005538.4, SEQ ID NO:49, reverse complement, SEQ ID NO:50). The sequence of mouse INHBC mRNA can be found, for example, in GenBank Accession No. GI:1049480142 (NM_010565.4, SEQ ID NO:51, reverse complement, SEQ ID NO:52). The sequence of rat INHBC mRNA can be found, for example, in GenBank Accession No. GI:59709462 (NM_022614.2, SEQ ID NO:53, reverse complement, SEQ ID NO:54). The predicted sequence of rhesus INHBC mRNA can be found, for example, in GenBank Accession No. GI:1622845603 (XM_001115940.4, SEQ ID NO:55, reverse complement, SEQ ID NO:56).

[0233] Additional examples of INHBC mRNA sequences are readily available through public databases such as GenBank, UniProt, OMIM, and the Macaca Genome Project website.

[0234] Further information regarding INHBC can be found, for example, at www.ncbi.nlm.nih.gov / gene / ?term=INHBC.

[0235] The entire contents of each of the foregoing GenBank Accession Numbers and Gene Database Numbers are incorporated herein by reference as of the filing date of this application.

[0236] As used herein, the term INHBC also refers to variations of the INHBC gene, including variants provided in SNP databases. Numerous sequence variations within the INHBC 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=INHBC, the entire contents of which are incorporated herein by reference as of the filing date of this application).

[0237] As used herein, "target sequence" or "target nucleic acid" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of a target gene, including mRNA that is a product of RNA processing of a primary transcript. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for RNAi-dependent cleavage at or near that portion of the nucleotide sequence of the mRNA molecule formed during transcription of the target gene. In one embodiment, the target sequence is within the protein-coding region of the target gene. In another embodiment, the target sequence is within the 3'UTR of the target gene. The target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or pathology.

[0238] The target sequence can be about 19 to 36 nucleotides in length, for example, about 19 to 30 nucleotides in length. For example, the target sequence can be about 19-30 nucleotides in length, 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 in length. In certain embodiments, the target sequence is 19 to 23 nucleotides in length, optionally 21 to 23 nucleotides in length. Ranges and lengths between the ranges and lengths listed above are also intended to be part of this disclosure.

[0239] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by a sequence referenced using standard nucleotide nomenclature.

[0240] Generally, "G", "C", "A", "T" and "U" respectively represent the nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as base.However, it is understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides, as described in more detail below, or alternative replacement moieties (see, for example, Table 1).Those skilled in the art are well aware that guanine, cytosine, adenine and uracil can be substituted with other moieties without substantially changing the base pairing properties of the oligonucleotide that contains the nucleotide with such replacement moieties.For example, but not limited to, the nucleotide that contains inosine as its base can base pair with the nucleotide that contains adenine, cytosine or uracil.Therefore, the nucleotide that contains uracil, guanine or adenine can be substituted with, for example, the nucleotide that contains inosine in the nucleotide sequence of the dsRNA of the present invention. In another example, adenine and cytosine anywhere within an oligonucleotide can be substituted with guanine and uracil, respectively, to form a GU wobble base pair with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods featured herein.

[0241] The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interfering agent," as used interchangeably herein, refer to an agent that contains RNA, as that term is defined herein, and mediates cleavage of a target of RNA transcription via the RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA regulates, e.g., inhibits, expression of an INHBE, ACVR1C, PLIN1, PDE3B, or INHBC gene in cells, e.g., hepatocytes and / or adipocytes, within a subject, such as a mammalian subject.

[0242] In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence, for example, a metabolic disorder-related target mRNA sequence, and controls the cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNA introduced into cells is degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, an RNase III-like enzyme, processes this dsRNA into 19-23 base pair small interfering RNAs with a characteristic two-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases can unwind the siRNA duplex, thereby inducing target recognition for the complementary antisense strand (Nykanen, et al., (2001) Cell 107:309). When bound to the appropriate target mRNA, one or more endonucleases in the RISC cleave the target and induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Therefore, in one aspect, the present invention relates to a single-stranded RNA (siRNA) that is produced in cells and promotes the formation of a RISC complex to achieve the silencing of a target gene, namely, a metabolic disorder-related target gene selected from the group consisting of inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), and inhibin subunit beta C (INHBC). Thus, the term "siRNA" is also used herein to refer to the iRNA described above.

[0243] In certain embodiments, the RNAi agent can be a single-stranded siRNA (ssRNAi) that is introduced into a cell or organism to inhibit target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNA is generally 15-30 nucleotides and chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein can be used as the single-stranded siRNA described herein or as the single-stranded siRNA that is chemically modified by the method described in Lima et al., (2012) Cell 150:883-894.

[0244] In certain embodiments, the "iRNA" used in the compositions, uses, and methods of the present invention is double-stranded RNA, and is referred to herein as a "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 duplex structure comprising two antiparallel and substantially complementary nucleic acid strands, and is referred to as having a "sense" or "antisense" conformation with respect to a target RNA, i.e., a metabolic disorder-related target gene selected from the group consisting of inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), and inhibin subunit beta C (INHBC). In some embodiments of the present invention, the double-stranded RNA (dsRNA) induces the degradation of the target RNA, for example, mRNA, via a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0245] Generally, the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides; however, as described in detail herein, each strand 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 ribonucleotides with chemical modifications, and iRNA may contain substantial modifications in multiple nucleotides.As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide bond, or a modified nucleobase, or any combination thereof.Therefore, the term modified nucleotide includes the substitution, addition, or removal of, for example, functional groups or atoms, to the internucleoside bond, sugar moiety, or nucleobase.The modifications suitable for use in the agent of the present invention include all types of modifications disclosed herein or known in the art.When used in siRNA-type molecules, all such modifications are encompassed by "iRNA" or "RNAi agent" for the purposes of this specification and claims.

[0246] In certain embodiments of the present disclosure, the inclusion of deoxy-nucleotides, if present within an RNAi agent, can be considered to constitute modified nucleotides.

[0247] The duplex region can be of any length that allows for specific degradation of the desired target RNA via the RISC pathway, and can range from about 19 to 36 base pairs in length, such as about 19 to 30 base pairs in length, e.g., about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, The duplex region may be 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 about 21-22 base pairs in length, etc. In certain embodiments, the duplex region is 19-21 base pairs in length, e.g., 21 base pairs in length. Ranges and lengths between the ranges and lengths listed above are also contemplated as part of this disclosure.

[0248] The two strands forming the double-stranded structure can be different portions of a larger RNA molecule, or they can be separate RNA molecules. When the two strands are part of a larger molecule and are therefore connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the corresponding 5' end of the other strand, the connected RNA strands are called "hairpin loops." A hairpin loop can contain at least one unpaired nucleotide. In some embodiments, a hairpin loop can contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 23, or more unpaired nucleotides. In some embodiments, a hairpin loop can be 10 or fewer nucleotides. In some embodiments, a hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, a hairpin loop can be 4 to 10 unpaired nucleotides. In some embodiments, a hairpin loop can be 4 to 8 nucleotides.

[0249] In certain embodiments, the two strands of a double-stranded oligomeric compound may be linked to each other. The two strands may be linked to each other at both ends or only one end. Linked at one end means that the 5' end of the first strand is linked to the 3' end of the second strand, or the 3' end of the first strand is linked to the 5' end of the second strand. When the two strands are linked to each other at both ends, the 5' end of the first strand is linked to the 3' end of the second strand, and the 3' end of the first strand is linked to the 5' end of the second strand. The two strands may be linked together by an oligonucleotide linker, including, but not limited to, (N)n, where N is independently a modified or unmodified nucleotide, and n is 3 to 23. In some embodiments, n is 3 to 10, e.g., 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the oligonucleotide linker is selected from the group consisting of GNRA, (G)4, (U)4, and (dT)4, where N is a modified or unmodified nucleotide, and R is a modified or unmodified purine nucleotide. Some of the nucleotides in the linker can participate in base pairing interactions with other nucleotides in the linker. The two strands can also be linked together by a non-nucleoside linker, for example, by a linker described herein. Those skilled in the art will understand that any chemical modification or variation of the oligonucleotides described herein can be used in the oligonucleotide linker.

[0250] Hairpin and dumbbell oligomeric compounds will have a double-stranded region of at least or equal to 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The double-stranded region can be up to 200, 100, or 50 nucleotide pairs in length. In some embodiments, the ranges for the double-stranded region are 15-30, 17-23, 19-23, and 19-21 nucleotide pairs in length.

[0251] Hairpin oligomeric compounds may have a single-stranded overhang or terminal unpaired region, in some embodiments at the 3' end, and in some embodiments on the antisense side of the hairpin. In some embodiments, the overhang is 1 to 4 nucleotides in length, more generally 2 to 3 nucleotides in length. Hairpin oligomeric compounds capable of inducing RNA interference are also referred to herein as "shRNAs."

[0252] When the two substantially complementary strands of dsRNA are composed of separate RNA molecules, they can, but do not necessarily, be covalently linked. When the two strands are covalently linked by a means other than an uninterrupted chain of nucleotides between the 3'-end of one strand and the 5'-end of the corresponding strand that forms a duplex structure, the connecting structure is called a "linker." The RNA strands can have the same or different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, RNAi can also contain one or more nucleotide overhangs. In one embodiment of an RNAi agent, at least one strand comprises a 3' overhang of at least one nucleotide. In another embodiment, at least one strand comprises a 3' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5' overhang of at least one nucleotide. In certain embodiments, at least one strand comprises a 5' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3' end and the 5' end of one strand of the RNAi agent comprise an overhang of at least one nucleotide.

[0253] In certain embodiments, an iRNA agent of the invention is a dsRNA, each strand of which comprises 19-23 nucleotides, that interacts with a target RNA sequence, e.g., a metabolic disorder-associated target gene, and directs cleavage of the target RNA.

[0254] In some embodiments, the iRNA of the present invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, for example, a metabolic disorder-associated target gene mRNA sequence, and directs cleavage of the target RNA.

[0255] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex 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 can contain at least one nucleotide overhang, or the overhang can contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang can comprise or consist of a nucleotide / nucleoside analog, such as a deoxynucleotide / nucleoside. An overhang can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, a certain overhanging nucleotide can be present on the 5'-end, the 3'-end, or both ends of either the antisense strand or the sense strand of a dsRNA.

[0256] In one embodiment, the antisense strand of dsRNA has an overhang of 1-10 nucleotides at 3'-end or 5'-end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides.In one embodiment, the sense strand of dsRNA has an overhang of 1-10 nucleotides at 3'-end or 5'-end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides.In another embodiment, one or more of the nucleotides in the overhang are replaced with nucleoside thiophosphate.

[0257] In certain embodiments, the antisense strand of dsRNA has 1-10 nucleotides at 3'-end or 5'-end, for example, 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides overhang.In one embodiment, the sense strand of dsRNA has 1-10 nucleotides at 3'-end or 5'-end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides overhang.In another embodiment, one or more nucleotides in the overhang are replaced with nucleoside thiophosphate.

[0258] In certain embodiments, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In certain embodiments, the overhang on the sense strand, the antisense strand, or both strands can comprise an extended length greater than 10 nucleotides, e.g., 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 length. In certain embodiments, the extended overhang is on the sense strand of the duplex. In certain embodiments, the extended overhang is on the 3' end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the 5' end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the antisense strand of the duplex. In certain embodiments, the extended overhang is present on the 3'-end of the antisense strand of the double strand.In certain embodiments, the extended overhang is present on the 5'-end of the antisense strand of the double strand.In certain embodiments, one or more of the nucleotides in the extended overhang are replaced with nucleoside thiophosphate.In certain embodiments, the overhang comprises a self-complementary portion, so that the overhang can form a stable hairpin structure under physiological conditions.

[0259] " Blunt " or " blunt end " means that there is no unpaired nucleotide at the end of a double-stranded RNA agent, i.e., there is no nucleotide overhang.A " blunt-ended " double-stranded RNA agent is double-stranded throughout its entire length, i.e., there is no nucleotide overhang at either end of the molecule.The RNAi agent of the present invention includes an RNAi agent that does not have a nucleotide overhang at one end (i.e., an agent that has one overhang and one blunt end), or an RNAi agent that does not have a nucleotide overhang at either end.In most cases, these molecules will be double-stranded throughout their entire length.

[0260] The term "antisense strand" or "guide strand" refers to the strand of an iRNA, e.g., a dsRNA, that includes a region that is substantially complementary to a target sequence, e.g., a metabolic disorder-associated target gene mRNA.

[0261] As used herein, the term "region of complementarity," as defined herein, refers to a region on the antisense strand that is substantially complementary to a sequence, e.g., a target sequence, e.g., an INHBE nucleotide sequence. If the region of complementarity is not perfectly complementary to the target sequence, the mismatch can be in an internal or terminal region of the molecule. Generally, the most tolerable mismatches are in the terminal regions, e.g., within 5, 4, or 3 nucleotides of the 5' or 3' end of the iRNA. In some embodiments, a double-stranded RNA agent of the invention contains nucleotide mismatches in the antisense strand. In some embodiments, the antisense strand of a double-stranded RNA agent of the invention contains four or fewer mismatches with the target mRNA, e.g., the antisense strand contains four, three, two, one, or zero mismatches with the target mRNA. In some embodiments, the antisense strand of a double-stranded RNA agent of the invention contains four or fewer mismatches with the sense strand, e.g., the antisense strand contains four, three, two, one, or zero mismatches with the sense strand. In some embodiments, the double-stranded RNA agent of the present invention contains a nucleotide mismatch in the sense strand. In some embodiments, the sense strand of the double-stranded RNA agent of the present invention contains four 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 mismatch is, for example, within 5, 4, or 3 nucleotides from the 3' end of the iRNA. In another embodiment, the nucleotide mismatch is, for example, in the 3' terminal nucleotide of the iRNA agent. In some embodiments, the mismatch is not in the seed region.

[0262] Thus, the RNAi agents described herein may contain one or more mismatches to the target sequence. In one embodiment, the RNAi agents described herein contain three or fewer mismatches (i.e., three, two, one, or zero mismatches). 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, when the antisense strand of an RNAi agent contains a mismatch to the target sequence, the mismatch can be optionally limited to be within the last five nucleotides from either the 5'-end or the 3'-end of the complementary region. For example, in such an embodiment, for a 23-nucleotide RNAi agent, the strand complementary to a region of a metabolic disorder-related target gene generally does not contain any mismatches within the central 13 nucleotides. Methods described herein or known in the art can be used to determine whether an RNAi agent containing a mismatch to the target sequence is effective in inhibiting the expression of a target gene. Consideration of the efficacy of mismatched RNAi agents in inhibiting expression of INHBE, ACVR1C, PLIN1, PDE3B, or INHBC target genes is important, especially when the particular complementary region in the target gene is known to have polymorphic sequence variation within the population.

[0263] The term "sense strand" or "passenger strand," as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand, as those terms are defined herein.

[0264] As used herein, "substantially all of the nucleotides are modified" means extensively but not entirely modified and may include no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0265] As used herein, the term "cleavage region" refers to the region located directly adjacent to the cleavage site.Cleavage site is the site on the target where cleavage occurs.In some embodiments, the cleavage region comprises three bases on either end of the cleavage site and directly adjacent to the cleavage site.In some embodiments, the cleavage region comprises two bases on either end of the cleavage site and directly adjacent to the cleavage site.In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and this cleavage region comprises nucleotides 11, 12, and 13.

[0266] As used herein, unless otherwise specified, the term "complementary," when used to describe a first nucleotide sequence in the context of a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize to form a duplex structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under specified conditions, as would be understood by one of skill in the art. Such conditions may be, for example, stringent conditions, such as 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12-16 hours followed by a wash (see, e.g., "Molecular Cloning: A Laboratory Manual," Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions that may be encountered in an organism, may be applied. Those skilled in the art can determine the most appropriate set of conditions for testing the complementarity of two sequences depending on the ultimate use of the hybridized nucleotides.

[0267] A complementary sequence in an iRNA, such as in a dsRNA described herein, includes base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence with an oligonucleotide or polynucleotide comprising a 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, when a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences may be fully complementary, or they may form one or more, but generally no more than 5, 4, 3, or 2, mismatched base pairs during hybridization for a duplex of up to 30 base pairs, while maintaining the ability to hybridize under conditions most relevant to its final application, for example, in vitro or in vivo, inhibiting gene expression. However, if two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, these overhangs shall not be considered mismatches when determining complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, where the longer oligonucleotide comprises a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, may still be referred to as "fully complementary" for purposes described herein.

[0268] "Complementary" sequences, as used herein, may also include or be formed entirely of non-Watson-Crick base pairs or base pairs formed from non-naturally occurring modified nucleotides, including, but not limited to, G:U wobble base pairs or Hoogsteen base pairs, so long as they meet the above requirements regarding their ability to hybridize.

[0269] The terms "complementary," "fully complementary," and "substantially complementary" herein may be used in reference to base matching between two oligonucleotides or polynucleotides, such as between the sense and antisense strands of a dsRNA, or the antisense strand of a double-stranded RNA agent and a target sequence, as will be understood from the context of their use.

[0270] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a continuous portion of an mRNA of interest (e.g., an mRNA encoding a metabolic disorder-associated target gene). For example, a polynucleotide is complementary to at least a portion of a metabolic disorder-associated target gene mRNA if the sequence is substantially complementary to an uninterrupted portion of the mRNA encoding the metabolic disorder-associated target gene.

[0271] As a result, in some embodiments, the antisense strand polynucleotides disclosed herein are perfectly complementary to the target gene sequence.

[0272] In some embodiments, the antisense strand polynucleotides disclosed herein comprise a contiguous nucleotide sequence that is substantially complementary to a target gene sequence and is at least about 80% complementary, such as about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary, over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO: 1, 3, 5, or 7 for INHBE, or a fragment of SEQ ID NO: 1, 3, 5, or 7.

[0273] In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of a target INHBE sequence and comprise a contiguous nucleotide sequence that is at least 80% complementary, such as about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary, over its entire length, to a fragment of SEQ ID NO:1 selected from the group consisting of nucleotides 400-442, 410-432, 518-540, 519-541, 640-662, 1430-1452, 1863-1885, or 1864-1886 of SEQ ID NO:1.

[0274] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target INHBE sequence and comprise a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 2-3 or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 2-3.

[0275] In one embodiment, an RNAi agent of the present disclosure comprises a sense strand that is substantially complementary to an antisense polynucleotide that is also identical to a target INHBE sequence, and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, such as 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary, over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO: 2, 4, 6, or 8, or a fragment of any one of SEQ ID NO: 2, 4, 6, or 8.

[0276] In some embodiments, the iRNA of the invention comprises a sense strand that is substantially complementary to an antisense polynucleotide that is also complementary to a target INHBE sequence, and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, such as about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary, over its entire length to any one of the antisense strand nucleotide sequences in any one of Tables 2-3, or a fragment of any one of the antisense strand nucleotide sequences in any one of Tables 2-3.

[0277] In some embodiments, the sense and antisense strands are selected from any one of the duplexes AD-1706583, AD-1711744, AD-1706593, AD-1708473, AD-1706662, AD-1706761, AD-1707306, AD-1707639, and AD-1707640.

[0278] In some embodiments, the sense and antisense strands are selected from the duplex AD-1706583.

[0279] In some embodiments, the sense and antisense strands are selected from the duplex AD-1711744.

[0280] In some embodiments, the sense and antisense strands are selected from the duplex AD-1706593.

[0281] In some embodiments, the sense and antisense strands are selected from the duplex AD-1708473.

[0282] In some embodiments, the sense and antisense strands are selected from the duplex AD-1706662.

[0283] In some embodiments, the sense and antisense strands are selected from the duplex AD-1706761.

[0284] In some embodiments, the sense and antisense strands are selected from the duplex AD-1707306.

[0285] In some embodiments, the sense and antisense strands are selected from the duplex AD-1707639.

[0286] In some embodiments, the sense and antisense strands are selected from the duplex AD-1707640.

[0287] In other embodiments, the antisense strand polynucleotides disclosed herein comprise a contiguous nucleotide sequence that is substantially complementary to the target gene sequence and is at least about 80% complementary, such as about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary, over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO: 9, 11, 13, 15, 17, 19, 21, or 23 for ACVR1C, or a fragment of SEQ ID NO: 9, 11, 13, 15, 17, 19, 21, or 23.

[0288] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target ACVR1C sequence and comprise a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary, over its entire length, to any one of the sense strand nucleotide sequences in any one of Tables 4-7 or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 4-7.

[0289] In one embodiment, the RNAi agent of the present disclosure comprises a sense strand that is substantially complementary to an antisense polynucleotide that is also identical to the target ACVR1C sequence, and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, such as 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary, over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, or 24, or a fragment of any one of SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, or 24.

[0290] In some embodiments, the iRNA of the invention comprises a sense strand that is substantially complementary to an antisense polynucleotide that is also complementary to a target ACVR1C sequence, and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, such as about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary, over its entire length to any one of the antisense strand nucleotide sequences in any one of Tables 4-7, or a fragment of any one of the antisense strand nucleotide sequences in any one of Tables 4-7.

[0291] In other embodiments, the antisense strand polynucleotides disclosed herein comprise a contiguous nucleotide sequence that is substantially complementary to a target gene sequence and is at least about 80% complementary, such as about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary, over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO: 25, 27, 29, 31, 33, or 35 for PLIN1, or a fragment of SEQ ID NO: 25, 27, 29, 31, 33, or 35.

[0292] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target PLIN1 sequence and comprise a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 8-11 or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 8-11.

[0293] In one embodiment, an RNAi agent of the present disclosure comprises a sense strand that is substantially complementary to an antisense polynucleotide that is also identical to the target PLIN1 sequence, and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, such as 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary, over its entire length to an equivalent region of the nucleotide sequence of SEQ ID NO: 26, 28, 30, 32, 34, or 36, or a fragment of any one of SEQ ID NO: 26, 28, 30, 32, 34, or 36.

[0294] In some embodiments, the iRNA of the invention comprises a sense strand that is substantially complementary to an antisense polynucleotide that is also complementary to a target PLIN1 sequence, and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, such as about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary, over its entire length to any one of the antisense strand nucleotide sequences in any one of Tables 8-11, or a fragment of any one of the antisense strand nucleotide sequences in any one of Tables 8-11.

[0295] In other embodiments, the antisense strand polynucleotides disclosed herein comprise a contiguous nucleotide sequence that is substantially complementary to the target gene sequence and is at least about 80% complementary, such as about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary, over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO: 37, 39, 41, 43, 45, or 47 for PDE3B, or a fragment of SEQ ID NO: 37, 39, 41, 43, 45, or 47.

[0296] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target PDE3B sequence and comprise a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 12-15 or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 12-15.

[0297] In one embodiment, the RNAi agent of the present disclosure comprises a sense strand that is substantially complementary to an antisense polynucleotide that is also identical to the target PDE3B sequence, and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, such as 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary, over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO: 38, 40, 42, 44, 46, or 48, or a fragment of any one of SEQ ID NO: 38, 40, 42, 44, 46, or 48.

[0298] In some embodiments, the iRNA of the invention comprises a sense strand that is substantially complementary to an antisense polynucleotide that is also complementary to a target PDE3B sequence, and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, such as about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary, over its entire length to any one of the antisense strand nucleotide sequences in any one of Tables 12-15, or a fragment of any one of the antisense strand nucleotide sequences in any one of Tables 12-15.

[0299] In other embodiments, the antisense strand polynucleotides disclosed herein comprise a contiguous nucleotide sequence that is substantially complementary to a target gene sequence and is at least about 80% complementary, such as about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary, over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO: 49, 51, 53, or 55 for INHBC, or a fragment of SEQ ID NO: 49, 51, 53, or 55.

[0300] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target INHBC sequence and comprise a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 16-17 or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 16-17.

[0301] In one embodiment, an RNAi agent of the present disclosure comprises a sense strand that is substantially complementary to an antisense polynucleotide that is also identical to the target INHBC sequence, and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, such as 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary, over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO: 50, 52, 54, or 56, or a fragment of any one of SEQ ID NO: 50, 52, 54, or 56.

[0302] In some embodiments, the iRNA of the invention comprises a sense strand that is substantially complementary to an antisense polynucleotide that is also complementary to a target INHBC sequence, and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, such as about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary, over its entire length to any one of the antisense strand nucleotide sequences in any one of Tables 16-17, or a fragment of any one of the antisense strand nucleotide sequences in any one of Tables 16-17.

[0303] In some embodiments, the double-stranded region of a double-stranded iRNA agent is equal to or at least equal to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotide pairs in length.

[0304] In some embodiments, the antisense strand of the double-stranded iRNA agent is equal to or at least equal to 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0305] In some embodiments, the sense strand of a double-stranded iRNA agent is equal to or at least equal to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0306] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each independently 15 to 30 nucleotides in length.

[0307] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each independently 19 to 25 nucleotides in length.

[0308] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each independently 21 to 23 nucleotides in length.

[0309] In one embodiment, the sense strand of the iRNA agent is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, the strands forming a double-stranded region of 21 contiguous base pairs with a 2-nucleotide long single-stranded overhang at the 3' end.

[0310] Generally, "iRNA" includes ribonucleotides with chemical modifications. Such modifications can include all types of modifications disclosed herein or known in the art. All of these modifications, when used in dsRNA molecules, are encompassed by "iRNA" for the purposes of this specification and claims.

[0311] In certain embodiments of the present disclosure, the inclusion of deoxy-nucleotides, if present within an RNAi agent, can be considered to constitute modified nucleotides.

[0312] In one embodiment of the present invention, an agent for use in the methods and compositions of the present invention is a single-stranded antisense oligonucleotide molecule that inhibits a target mRNA via an antisense inhibition mechanism. The single-stranded antisense oligonucleotide molecule is complementary to a sequence within the target mRNA. The single-stranded antisense oligonucleotide can stoichiometrically inhibit translation by base pairing with the mRNA and physically interfering with the translation machinery. See Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense oligonucleotide molecule may be about 14 to about 30 nucleotides in length and may have a sequence complementary to the target sequence. For example, the single-stranded antisense oligonucleotide molecule may comprise a sequence that is at least about 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense sequences described herein.

[0313] The phrase "contacting a cell with an iRNA" such as dsRNA as used herein includes contacting a cell by any possible means. Contacting a cell with an iRNA includes contacting a cell with an iRNA in vitro or contacting a cell with an iRNA in vivo. Contacting can be performed directly or indirectly. Thus, for example, an iRNA can be physically contacted with a cell by performing a method separately, or the iRNA can be placed in a situation that allows or causes it to contact a cell later.

[0314] Contacting cells in vitro can be achieved, for example, by incubating the cells with iRNA. Contacting cells in vivo can be achieved, for example, by injecting iRNA into or near the tissue in which the cells reside, 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 reside. For example, the iRNA may contain or be bound to a targeting ligand, such as GalNAc, that directs the iRNA to a target site, such as the liver. In other embodiments, the RNAi agent may contain or be bound to one or more C22 hydrocarbon chains or one or more GalNAc derivatives. In other embodiments, the RNAi agent contains or is bound to one or more C22 hydrocarbon chains, and does not contain or is not bound to one or more GalNAc derivatives. A combination of in vitro and in vivo contacting methods is also possible. For example, cells may be contacted with an RNAi agent in vitro and then transferred to a subject.

[0315] In certain embodiments, contacting a cell with an iRNA includes "introducing" or "delivering the iRNA into a cell" by promoting or causing uptake or absorption into the cell. Absorption or uptake of the iRNA can occur through spontaneous diffusion or active intracellular processes, or by auxiliary agents or devices. Introduction of the iRNA into a cell can be in vitro or in vivo. For example, for in vivo introduction, the iRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell can include methods known in the art, such as electroporation and lipofection. Additional approaches are described herein below or known in the art.

[0316] The term "lipid nanoparticle" or "LNP" refers to a vesicle comprising a lipid layer that encapsulates a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an iRNA or a plasmid from which the iRNA is transcribed. LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0317] As used herein, a "subject" refers to an animal, such as a mammal, including a primate (such as a human, a monkey, or a non-human primate, e.g., a chimpanzee), a non-primate (such as a cow, pig, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, or mouse), or a bird that expresses a target gene either endogenously or heterologously. In one embodiment, the subject is a human, such as a human being treated or evaluated for a disease or disorder that would benefit from reduced metabolic disorder-associated target gene expression, a human being at risk for a disease or disorder that would benefit from reduced metabolic disorder-associated target gene expression, a human being with a disease or disorder that would benefit from reduced metabolic disorder-associated target gene expression, or a human being treated for a disease or disorder that would benefit from reduced metabolic disorder-associated target gene expression, as described herein. In some embodiments, the subject is a female human. In other embodiments, the subject is a male human. In one embodiment, the subject is an adult subject. In another embodiment, the subject is a pediatric subject.

[0318] As used herein, the term "treat" or "treatment" refers to a beneficial or desired result, such as reducing at least one sign or symptom of a metabolic disorder in a subject. Treatment also includes reducing one or more signs or symptoms associated with undesired metabolic disorder-related target gene expression, reducing the degree of activation or stabilization of undesired metabolic disorder-related target genes, or improving or alleviating the activation or stabilization of undesired metabolic disorder-related target genes. "Treatment" can also mean extending survival time compared to the expected survival time if no treatment is performed.

[0319] The term "lower" in the context of the level of a metabolic disorder-related target gene, or a disease marker or symptom in a subject, refers to a statistically significant decrease in such level. The decrease can 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 decrease is at least 20%. In certain embodiments, the decrease is at least 50% in a disease marker, for example, in the protein level or gene expression level. In the context of the level of a metabolic disorder-related target gene in a subject, "lower" preferably refers to a decrease to a level that is accepted as being within the normal range in an individual without such a disorder. In certain embodiments, "lower" refers to a decrease in the difference between the level of the marker or symptom in a subject suffering from the disease and the level that is accepted as being within the normal range for the individual. The term "reduce" can also be used in reference to normalizing the symptoms or pathology of a disease, i.e., reducing the difference between the level of a subject suffering from a metabolic disorder and the level of a normal subject not suffering from a metabolic disorder toward or to the level of a normal subject not suffering from a metabolic disorder. As used herein, if the disease involves an elevated value for a symptom, "normal" refers to the upper limit of normal. If the disease involves a decreased value for a symptom, "normal" refers to the lower limit of normal.

[0320] As used herein, "prevention" or "preventing," when used in reference to a disease, disorder, or condition thereof that may be treated or ameliorated by reducing the expression of a metabolic disorder-associated target gene, refers to a reduction in the likelihood that a subject will develop such a disease, disorder, or condition, for example, a symptom of a metabolic disorder, for example, a symptom associated with diabetes. A failure to develop a disease, disorder, or condition, or a reduction in the onset of symptoms associated with such a disease, disorder, or condition (e.g., a reduction of at least about 10% on a scale clinically acceptable for the disease or disorder), or a delay in the onset of symptoms (e.g., a delay of several days, weeks, months, or years) is considered effective prevention.

[0321] The therapeutic and prophylactic methods of the present invention are useful for treating any disease or disorder, including diseases, disorders, or conditions caused by or associated with INHBE, ACVR1C, PLIN1, PDE3B, and / or INHBC gene expression or INHBE, ACVR1C, PLIN1, PDE3B, and / or INHBC protein production, and that would benefit from a decrease in INHBE, ACVR1C, PLIN1, PDE3B, and / or INHBC gene expression, replication, or protein activity, such as a metabolic disorder. In some embodiments, the metabolic disorder is metabolic syndrome.

[0322] A "metabolic disorder" is a disorder that interferes with normal metabolism, the process that converts food into energy at the cellular level. Metabolic diseases affect the ability of cells to carry out important biochemical reactions that involve the processing or transport of proteins (amino acids), carbohydrates (sugars and starches), or lipids (fatty acids).

[0323] For example, metabolic disorders may be associated with a body fat distribution characterized by a higher accumulation of fat around the waist (e.g., greater abdominal fat or greater waist circumference) and / or a lower accumulation of fat around the hips (e.g., lower gluteal-thigh fat or smaller hip circumference), resulting in a larger waist-to-hip ratio (WHR) and higher cardiometabolic risk independent of body mass index (BMI).

[0324] Non-limiting examples of metabolic diseases include carbohydrate disorders, such as diabetes, type I diabetes, type II diabetes, galactosemia, hereditary fructose intolerance, fructose 1,6-diphosphatase deficiency, glycogen storage disorders, congenital glycosylation disorders, insulin resistance, insulin deficiency, hyperinsulinemia, impaired glucose tolerance (IGT), abnormal glycogen metabolism; disorders of amino acid metabolism, such as maple syrup urine disease (MSUD), or homocystinuria; disorders of organic acid metabolism, such as methylmalonic aciduria, 3-methylglutaconic aciduria-Barth syndrome, glutaric aciduria, or 2-hydroxyglutaric aciduria-D and L forms; disorders of fatty acid beta-oxidation, such as medium-chain acyl-CoA dehydrogenase deficiency (MCAD), long-chain 3-l-hydroxyacyl-CoA dehydrogenase deficiency (LCHAD). , very long-chain acyl-CoA dehydrogenase deficiency (VLCAD), disorders of lipid metabolism, such as GM1 gangliosidosis, Tay-Sachs disease, Sandhoff disease, Fabry disease, Gaucher disease, Niemann-Pick disease, Krabbe disease, mucolipidosis, or mucopolysaccharidoses; disorders of lipid distribution and / or storage, such as lipodystrophy, mitochondrial disorders; mitochondrial disorders, such as mitochondrial cardiomyopathy; Leigh's disease; mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS); myoclonic epilepsy with ragged-red fibers (MERRF); neuropathy, ataxia, and retinitis pigmentosa (NARP); Barth syndrome; or peroxisomal disorders, such as Zellweger syndrome (cerebrohepatorenal syndrome), X-linked adrenoleukodystrophy, or Refsum disease.

[0325] In certain embodiments, metabolic disorders are associated with body fat distribution and include metabolic syndrome, type 2 diabetes, hyperlipidemia or hyperlipidemia (high or altered circulating levels of low-density lipoprotein cholesterol (LDL-C), triglycerides, very-low-density lipoprotein cholesterol (VLDL-C), apolipoprotein B or other lipid fractions), obesity (particularly abdominal obesity), lipodystrophy (such as the inability of fat to be deposited in fat depots, either localized (segmental lipodystrophy) or generalized (lipoatrophy)), insulin resistance during fasting or metabolic challenge, or higher or altered levels of insulin. These include, but are not limited to, elevated insulin levels, hepatic fat deposits or fatty liver disease and its complications (e.g., cirrhosis, fibrosis, or inflammation of the liver), non-alcoholic steatohepatitis, other types of liver inflammation, higher liver enzyme levels or other markers of liver damage or elevations or changes in liver enzyme levels or other markers of liver damage, inflammation or fat deposits in the liver, higher blood pressure and / or hypertension, higher blood sugar or glucose or hyperglycemia, metabolic syndrome, coronary artery disease, and other atherosclerotic conditions, and complications of each of the foregoing conditions.

[0326] In one embodiment, the metabolic disorder is metabolic syndrome. As used herein, the term "metabolic syndrome" refers to a disorder that includes a cluster of components reflecting overnutrition, a sedentary lifestyle, genetic factors, aging, and resulting excess adiposity. Metabolic syndrome includes a cluster of abdominal obesity, insulin resistance, dyslipidemia, and elevated blood pressure, and is associated with other comorbidities, including a prothrombotic state, a proinflammatory state, nonalcoholic fatty liver disease, and reproductive disorders. The prevalence of metabolic syndrome is rising to epidemic proportions in the United States and the rest of the urbanized world, as well as in developing countries. Metabolic syndrome is associated with an approximately two-fold increase in the risk of cardiovascular disease and a five-fold increase in the risk of developing type 2 diabetes.

[0327] Abdominal adiposity (e.g., large waist circumference (high waist-to-hip ratio)), hypertension, insulin resistance, and hyperlipidemia are central to metabolic syndrome and its individual components (e.g., central obesity, fasting blood glucose (FBG) / prediabetes / diabetes, hypercholesterolemia, hypertriglyceridemia, and hypertension).

[0328] In one embodiment, the metabolic disorder is a carbohydrate disorder, hi one embodiment, the carbohydrate disorder is diabetes.

[0329] As used herein, the term "diabetes" refers to a group of metabolic disorders characterized by high blood sugar (glucose) levels resulting from defects in insulin secretion or action, or both. There are two most common types of diabetes: type 1 diabetes and type 2 diabetes, both of which result from the body's inability to regulate insulin. Insulin is a hormone released by the pancreas in response to increased blood sugar (glucose) levels.

[0330] As used herein, the term "type 1 diabetes" refers to a chronic disease that occurs when the pancreas produces too little insulin to properly regulate blood glucose levels. Type 1 diabetes is also known as insulin-dependent diabetes mellitus, IDDM, and juvenile-onset diabetes. People with type 1 diabetes (insulin-dependent diabetes) produce little or no insulin. Approximately 6 percent of the U.S. population has some form of diabetes, but only about 10 percent of all diabetic patients have type 1 disease. Most people with type 1 diabetes develop the disease before the age of 30. Type 1 diabetes represents the result of progressive autoimmune destruction of pancreatic beta cells with subsequent insulin deficiency. More than 90% of the pancreas' insulin-producing cells (beta cells) are permanently destroyed. The resulting insulin deficiency is severe, and type 1 diabetic patients must receive regular insulin injections to survive.

[0331] In type II diabetes (also called non-insulin-dependent diabetes mellitus, or NDDM), the pancreas continues to produce insulin, sometimes at higher-than-normal levels. However, the body develops resistance to its effects, resulting in relative insulin deficiency. Type II diabetes can develop in children and adolescents but usually begins after age 30 and becomes progressively more common with age; approximately 15% of people over 70 have type II diabetes. Obesity is a risk factor for type II diabetes; 80–90% of people with this disorder are obese.

[0332] In some embodiments, diabetes comprises prediabetes.Prediabetes refers to one or more early diabetic conditions, including impaired glucose utilization, abnormal or impaired fasting glucose level, impaired glucose tolerance, impaired insulin sensitivity and insulin resistance.Prediabetes is the main risk factor for the development of type 2 diabetes mellitus, cardiovascular disease and death.Many focus on the development of therapeutic interventions that can effectively treat prediabetes and prevent the development of type 2 diabetes.

[0333] Diabetes can be diagnosed by performing a glucose tolerance test. Clinically, diabetes is often divided into several basic categories. Primary examples of these categories include autoimmune diabetes mellitus, non-insulin-dependent diabetes mellitus (NDDM type 1), insulin-dependent diabetes mellitus (IDDM type 2), non-autoimmune diabetes mellitus, non-insulin-dependent diabetes mellitus (NIDDM type 2), and maturity-onset diabetes of the young (MODY). A further category, often referred to as secondary, refers to diabetes resulting from some identifiable condition that causes or allows the development of a diabetic syndrome. Examples of secondary categories include diabetes caused by pancreatic disease, diabetes induced by hormonal abnormalities, drugs, or chemicals, diabetes caused by insulin receptor abnormalities, diabetes associated with genetic syndromes, and diabetes of other causes (see, e.g., Harrison's (1996) 14th ed., New York, McGraw-Hill).

[0334] In one embodiment, the metabolic disorder is a lipid metabolism disorder. As used herein, "lipid metabolism disorder" or "lipid metabolism disorder" refers to any disorder related to or caused by lipid metabolism disorder. This term also includes any disorder, disease or condition that can lead to hyperlipidemia, or a condition characterized by abnormally elevated levels of any or all of lipids and / or lipoproteins in the blood. This term refers to induced or acquired disorders, such as genetic disorders such as familial hypertriglyceridemia, familial partial lipodystrophy type 1 (FPLD1), or disorders induced or acquired as a result of disease, disorder or condition (e.g., renal failure), diet, or certain drugs (e.g., as a result of highly active antiretroviral therapy (HAART) used to treat AIDS or HIV). This term also refers to disorders of fat distribution / storage, such as lipodystrophy.

[0335] Additional examples of disorders of lipid metabolism include atherosclerosis, hyperlipidemia, hypertriglyceridemia (including drug-induced hypertriglyceridemia, diuretic-induced hypertriglyceridemia, alcoholic hypertriglyceridemia, beta-adrenergic blocker-induced hypertriglyceridemia, estrogen-induced hypertriglyceridemia, glucocorticoid-induced hypertriglyceridemia, retinoid-induced hypertriglyceridemia, cimetidine-induced hypertriglyceridemia, and familial hypertriglyceridemia), disorders associated with hypertriglyceridemia, and disorders associated with urinary tract infections. These conditions include, but are not limited to, associated acute pancreatitis, chylomicron syndrome, familial chylomicronemia, Apo-E deficiency or resistance, LPL deficiency or hypoactivity, hyperlipidemia (including familial combined hyperlipidemia), hypercholesterolemia, lipodystrophy, gout associated with hypercholesterolemia, xanthomatosis (subcutaneous cholesterol deposits), hyperlipidemia with heterogeneous LPL deficiency, and hyperlipidemia with hyperlipidemia and heterogeneous LPL deficiency, fatty liver disease, or nonalcoholic steatohepatitis (NASH).

[0336] Cardiovascular diseases, as defined herein, are also considered "metabolic disorders." These diseases can include coronary artery disease (also called ischemic heart disease), hypertension, inflammation associated with coronary artery disease, restenosis, peripheral vascular disease, and stroke.

[0337] Kidney disease is also considered a "metabolic disorder" as defined herein. Such diseases may include chronic kidney disease, diabetic nephropathy, diabetic kidney disease, or gout.

[0338] Weight-related disorders are also considered "metabolic disorders" as defined herein and may include obesity, hypometabolic states, hypothyroidism, uremia, and other conditions associated with weight gain (including rapid weight gain), weight loss, maintaining weight loss, or the risk of regaining weight after weight loss.

[0339] Blood glucose disorders, as defined herein, are also considered "metabolic disorders." Such disorders may include diabetes, hypertension, and polycystic ovarian syndrome associated with insulin resistance. Other exemplary metabolic disorders may include kidney transplantation, nephrotic syndrome, Cushing's syndrome, acromegaly, systemic lupus erythematosus, dysglobulinemia, lipodystrophy, glycogenosis type I, and Addison's disease.

[0340] In one embodiment, the metabolic disorder is primary hypertension. "Primary hypertension" is the result of environmental or genetic causes (e.g., the result of no apparent underlying disease cause).

[0341] In one embodiment, the metabolic disorder is secondary hypertension. "Secondary hypertension" has an identifiable underlying disease that can have multiple etiologies, including renal, vascular, and endocrine causes, such as renal parenchymal disease (e.g., polycystic kidney disease, glomerular, or interstitial disease), renal vascular disease (e.g., renal artery stenosis, fibromuscular dysplasia), endocrine disorders (e.g., corticosteroid or mineralocorticoid excess, pheochromocytoma, hyper- or hypothyroidism, growth hormone excess, hyperparathyroidism), aortic stenosis, and oral contraceptive use.

[0342] In one embodiment, the metabolic disorder is resistant hypertension. "Resistant hypertension" is blood pressure that remains above target (for example, systolic above 130 mmHg or diastolic above 90 mmHg) despite the simultaneous use of three antihypertensive drugs of different classes, one of which is a thiazide diuretic. Subjects whose blood pressure is controlled with four or more drugs are also considered to have resistant hypertension.

[0343] Additional diseases or conditions associated with metabolic disorders will be apparent to one of skill in the art and are within the scope of the present disclosure.

[0344] A "therapeutically effective amount," as used herein, is intended to include an amount of an RNAi agent that, when administered to a subject with a metabolic disorder, is sufficient to treat the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of the disease). A "therapeutically effective amount" can vary depending on the RNAi agent, the method of administration of the agent, the disease and its severity and medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, if any, and other personal characteristics of the subject to be treated.

[0345] As used herein, a "prophylactically effective amount" is intended to include an amount of an RNAi agent that, when administered to a subject with a metabolic disorder, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Amelioration of the disease includes slowing the progression of the disease or reducing the severity of any future disease. A "prophylactically effective amount" may vary depending on the RNAi agent, the method of administration of the agent, the degree of risk of the disease, and the patient's medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, if any, and other personal characteristics of the patient to be treated.

[0346] A "therapeutically effective amount" or "prophylactically effective amount" also includes that amount of an RNAi agent that produces some desired effect at a reasonable benefit / risk ratio applicable to any treatment. The iRNAs used in the methods of the invention can be administered in amounts sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0347] The phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions or dosage forms that are suitable for use in contact with the tissues of human and animal subjects, within the scope of sound medical judgment, without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0348] The phrase "pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition, or vehicle involved in the transport or transfer of the 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 or zinc stearate, or stearic acid), or solvent encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject being treated. Such carriers are known in the art. Pharmaceutically acceptable carriers include carriers for administration by injection.

[0349] The term "sample," as used herein, encompasses similar bodily fluids, cells, or tissues isolated from a subject, as well as collections of bodily fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum, and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva, and the like. Tissue samples can include samples from tissues, organs, or localized regions. For example, samples can be derived from specific organs, parts of organs, or bodily fluids or cells within those organs. In certain embodiments, samples can be derived from the liver (e.g., the entire liver or a specific segment of the liver, or a specific type of cell within the liver, such as hepatocytes). In some embodiments, a "sample derived from a subject" refers to urine obtained from a subject. A "sample derived from a subject" can also refer to blood from a subject or serum or plasma derived from blood.

[0350] II. iRNAs of the Invention The present invention provides iRNAs that inhibit the expression of metabolic disorder-related target genes, such as INHBE, ACVR1C, PLIN1, PDE3B, or INHBC. In certain embodiments, the iRNAs comprise double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of metabolic disorder-related target genes in cells (e.g., adipocytes and / or hepatocytes), such as cells in a subject (e.g., a mammal (e.g., a human susceptible to developing metabolic disorders (e.g., metabolic syndrome, carbohydrate disorders, e.g., type II diabetes, prediabetes, lipid metabolism disorders, e.g., hyperlipidemia, hypertensive lipodystrophy); kidney disease; cardiovascular disease, and weight disorders)). The dsRNAi agent comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during the expression of the metabolic disorder-related target gene. The region of complementarity is about 19-30 nucleotides in length (eg, about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides in length).

[0351] When contacted with cells expressing the target gene, the iRNA inhibits expression of the target gene (e.g., human, primate, non-primate, or rat INHBE, ACVR1C, PLIN1, PDE3B, or INHBC gene) by at least about 50%, as analyzed, for example, by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, such as immunofluorescence analysis, for example, using Western blotting or flow cytometry techniques. 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, for example, mice expressing the human target gene or AAV-infected mice, when administered, for example, as a single dose, at a minimum of 3 mg / kg of RNA expression.

[0352] dsRNA comprises two RNA strands, which are complementary and hybridize to form a duplex structure under the conditions in which dsRNA is used. One strand of dsRNA (antisense strand) comprises a complementary region that is substantially complementary to the target sequence, and is generally completely complementary. The target sequence can be derived from the sequence of mRNA formed during the expression of INHBE, ACVR1C, PLIN1, PDE3B, or INHBC gene. The other strand (sense strand) comprises a region that is complementary to the antisense strand, so that the two strands hybridize to form a duplex structure when combined under suitable conditions. As described elsewhere herein and known in the art, the complementary sequences of dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, so that they are opposite each other on separate oligonucleotides.

[0353] Generally, the duplex structure is 15 to 30 base pairs in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, They are 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. In certain embodiments, the duplex structure is 8 to 25 base pairs in length, e.g., 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 to 25 base pairs in length, e.g., 19 to 21 base pairs in length. Ranges and lengths between the above-listed ranges and lengths are also intended to be part of this disclosure.

[0354] Similarly, the region complementary to the target sequence may be 15 to 30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, e.g., 19-23 nucleotides in length or 21-23 nucleotides in length. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of this disclosure.

[0355] In some embodiments, the duplex structure is 19 to 30 base pairs in length. Similarly, the region of complementarity to the target sequence is 19 to 30 nucleotides in length.

[0356] In some embodiments, the dsRNA is about 19 to about 23 nucleotides in length, or about 25 to about 30 nucleotides in length. Generally, the dsRNA is long enough to function as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21 to 23 nucleotides in length can function as substrates for Dicer. As those skilled in the art will recognize, the region of an RNA targeted for cleavage is most often a portion of a longer RNA molecule, often an mRNA molecule. In relevant cases, a "portion" of an mRNA target is a contiguous sequence of the mRNA target that is long enough to allow it to serve as a substrate for RNAi-dependent cleavage (i.e., cleavage via the RISC pathway).

[0357] Those skilled in the art will appreciate that the duplex region is the primary functional portion of the dsRNA, and may comprise, for example, about 19 to about 30 base pairs, e.g., about 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, Those skilled in the art will also recognize that a 21-24, 21-23, or 21-22 base pair duplex region is also a dsRNA. That is, in one embodiment, an RNA molecule or complex of RNA molecules having a duplex region of more than 30 base pairs is a dsRNA, as long as it is processed into, for example, a 15-30 base pair functional duplex that targets the desired RNA for cleavage. Thus, those skilled in the art will recognize that in one embodiment, an miRNA is a dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful for targeting INHBE, ACVR1C, PLIN1, PDE3B, or INHBC gene expression is not generated in the target cell by cleavage of a larger dsRNA.

[0358] The dsRNA described herein can further comprise one or more single-stranded nucleotide overhangs, such as 1-4, 2-4, 1-3, 2-3, 1, 2, 3, or 4 nucleotides. dsRNAs with at least one nucleotide overhang can have superior inhibitory properties compared to their blunt-ended counterparts. The nucleotide overhangs can comprise or consist of nucleotide / nucleoside analogs, such as deoxynucleotides / nucleosides. The overhangs can be on the sense strand, the antisense strand, or any combination thereof. Moreover, the overhanging nucleotides can be present at the 5'-end, 3'-end, or both ends of the antisense or sense strand of the dsRNA.

[0359] dsRNA can be synthesized by standard methods known in the art.Double-stranded RNAi compounds of the present invention can be prepared using a two-step method.First, each strand of double-stranded RNA molecules is prepared separately.Then, the strands of these components are annealed.The individual strands of siRNA compounds can be prepared using solution phase or solid phase organic synthesis or both.Organic synthesis has the advantage that it can easily prepare the oligonucleotide strands that contain unnatural nucleotides or modified nucleotides.Similarly, single-stranded oligonucleotides of the present invention can be prepared using solution phase or solid phase organic synthesis or both.

[0360] 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 group of sequences provided in any one of Tables 2-17, 19, and 20, and the corresponding antisense strand of the sense strand is selected from the group of sequences provided in any one of Tables 2-17, 19, and 20. In this embodiment, one of the two sequences is complementary to the other of the two sequences, where one of the sequences is substantially complementary to the sequence of mRNA produced upon expression of the associated target gene. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, one oligonucleotide described as the sense strand in any one of Tables 2-17, 19, and 20, and the second oligonucleotide described as the corresponding antisense strand of the sense strand in any one of Tables 2-17, 19, and 20.

[0361] In certain embodiments, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In other embodiments, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

[0362] In some embodiments, the sense or antisense strand is selected from the sense or antisense strand of any one of the duplexes AD-1706583, AD-1711744, AD-1706593, AD-1708473, AD-1706662, AD-1706761, AD-1707306, AD-1707639, and AD-1707640.

[0363] In some embodiments, the sense or antisense strand is selected from the sense or antisense strand of duplex AD-1706583.

[0364] In some embodiments, the sense or antisense strand is selected from the sense or antisense strand of duplex AD-1711744.

[0365] In some embodiments, the sense or antisense strand is selected from the sense or antisense strand of duplex AD-1706593.

[0366] In some embodiments, the sense or antisense strand is selected from the sense or antisense strand of duplex AD-1708473.

[0367] In some embodiments, the sense or antisense strand is selected from the sense or antisense strand of the duplex AD-1706662.

[0368] In some embodiments, the sense or antisense strand is selected from the sense or antisense strand of the duplex AD-1706761.

[0369] In some embodiments, the sense or antisense strand is selected from the sense or antisense strand of duplex AD-1707306.

[0370] In some embodiments, the sense or antisense strand is selected from the sense or antisense strand of the duplex AD-1707639.

[0371] In some embodiments, the sense or antisense strand is selected from the sense or antisense strand of the duplex AD-1707640.

[0372] It should be understood that, for example, the sequences in Table 2 are not described as modified or conjugated sequences, but the RNA of the iRNA of the present invention, e.g., the dsRNA of the present invention, may comprise any one of the sequences set forth in any one of Tables 2-17, 19, and 20, unmodified, unconjugated, or modified or conjugated differently from those set forth therein. In other words, the present invention encompasses the dsRNAs of Tables 2-17, 19, and 20 that are unmodified, unconjugated, modified, or conjugated as described herein.

[0373] Those skilled in the art are well aware that dsRNA with a duplex structure of about 20 to 23 base pairs, for example, 21 base pairs, has been hailed as being particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have discovered that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719, Kim et al. (2005) Nat Biotech 23:222-226). In the above-mentioned embodiment, due to the nature of the oligonucleotide sequences provided in any one of Tables 2-17, 19, and 20, the dsRNA described herein can comprise at least one strand with a minimum length of 21 nucleotides. It can be reasonably expected that shorter duplexes having any one of the sequences in any one of Tables 2-17, 19, and 20 minus only a few nucleotides at one or both ends can be similarly effective compared to the dsRNAs described above. Thus, dsRNAs having a sequence of at least 19, 20, or more contiguous nucleotides derived from any one of the sequences in any one of Tables 2-17, 19, and 20 that differ from dsRNAs containing the entire sequence in their ability to inhibit expression of the INHBE gene by no more than about 5, 10, 15, 20, 25, or 30% inhibition are contemplated as being within the scope of the present invention.

[0374] Additionally, the RNAs provided in Tables 2-17, 19, and 20 identify sites within metabolic disorder-associated target gene transcripts that are susceptible to RISC-mediated cleavage. Thus, the present invention further features iRNAs that target within one of these sites. As used herein, an iRNA is said to target within a specific site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within the specific site. Such iRNAs will generally comprise at least about 19 contiguous nucleotides from any one of the sequences provided in any one of Tables 2-17, 19, and 20, linked to an additional nucleotide sequence taken from a region adjacent to the selected sequence within a metabolic disorder-associated target gene.

[0375] III. Modifications to RNAi Agents of the Invention In certain embodiments, the RNA of the iRNA of the present invention, e.g., dsRNA, is unmodified and does not contain chemical modifications or conjugations, e.g., those 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 of the nucleotides of the iRNA, or substantially all of the nucleotides of the iRNA, are modified, i.e., no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 unmodified nucleotide is present in any strand of the iRNA.

[0376] In some embodiments, the dsRNA agent of the present invention comprises at least one nucleic acid modification as described herein.For example, at least one modification is selected from the group consisting of modified internucleoside bond, modified nucleobase, modified sugar, and any combination thereof.Not limited to, this modification can be present in any of the dsRNA agents of the present invention.For example, modification can be present in one of RNA molecules.

[0377] In one embodiment, a dsRNA agent of this disclosure contains one or more C22 hydrocarbon chains conjugated to one or more internal positions on at least one strand, and does not contain additional chemical modifications known in the art and described herein on the remainder of the sense and antisense strands.

[0378] In some embodiments, the dsRNA agent of the present invention comprises one or more C22 hydrocarbon chains conjugated to one or more internal positions on at least one chain, and also comprises at least one additional nucleic acid modification as described herein.For example, at least one modification is selected from the group consisting of modified internucleoside bond, modified nucleic acid base, modified sugar, and any combination thereof.Without being limited thereto, this modification can be present in any of the dsRNA agents of the present invention.For example, modification can be present in one of RNA molecules.

[0379] In one embodiment, a dsRNA agent of this disclosure comprises one or more targeting ligands, e.g., one or more GalNAc derivatives, and does not comprise additional chemical modifications known in the art and described herein in the remainder of the sense and antisense strands.

[0380] In some embodiments, the dsRNA agent of the present invention comprises one or more targeting ligands, for example, one or more GalNAc derivatives, and also comprises at least one additional nucleic acid modification as described herein.For example, at least one modification is selected from the group consisting of modified internucleoside linkage, modified nucleic acid base, modified sugar, and any combination thereof.Without being limited thereto, this modification can be present in any of the dsRNA agents of the present invention.For example, modification can be present in one of RNA molecules.

[0381] Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, reverse linkage) or 3'-end modifications (conjugation, DNA nucleotide, reverse linkage, etc.); base modifications, such as substitution with a stabilizing base, a destabilizing base, or a base that base-pairs with an extended repertoire partner, base removal (abasic nucleotide) or conjugated base; sugar modifications (e.g., at the 2'- or 4'-position) or sugar substitution; or backbone modifications, including modification or substitution of phosphodiester bond. Specific examples of RNAi agents useful in the embodiments described herein include, but are not limited to, RNAs that contain modified backbones or do not contain natural internucleoside linkages. RNAs with modified backbones include, among others, those that do not have a phosphorus atom in their backbone. For the purposes of this specification, and as sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered oligonucleosides. In some embodiments, the modified RNAi agent will have a phosphorus atom in its internucleoside backbone.

[0382] A. Nucleobase Modifications

[0383] The naturally occurring base portion of a nucleoside is typically a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. For those nucleosides containing a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3', or 5' hydroxyl moiety of the sugar. In the formation of oligonucleotides, these phosphate groups covalently link adjacent nucleosides to each other to form a linear polymeric compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The naturally occurring linkage or backbone of RNA and DNA is the 3' to 5' phosphodiester linkage.

[0384] In addition to "unmodified" or "natural" nucleobases, such as the purine nucleobases adenine (A) and guanine (G), and the pyrimidine nucleobases thymine (T), cytosine (C), and uracil (U), numerous modified nucleobases or nucleobase mimics known to those skilled in the art are suitable for use in the compounds described herein. Unmodified or natural nucleobases can be modified or substituted to provide iRNAs with improved properties. For example, nuclease-resistant oligonucleotides can be prepared using these bases, or synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidin), as well as any one of the oligomer modifications described herein. Alternatively, substituted or modified analogs of any of the above bases and "universal bases" can be employed. When a natural base is replaced with an unnatural and / or universal base, the nucleotide is said herein to contain a modified nucleobase and / or nucleobase modification. Modified nucleobases and / or nucleobase modifications also include natural bases, unnatural bases, and universal bases, including conjugate moieties such as, for example, the ligands described herein. Preferred conjugate moieties for conjugation to nucleobases include cationic amino groups that can be conjugated to the nucleobase via a linker having a suitable alkyl, alkenyl, or amide bond.

[0385] The oligomeric compounds described herein can also include modifications or substitutions of nucleobases (often simply referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Exemplary modified nucleobases include other synthetic and natural nucleobases such as inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N 6 -(Isopentenyl)adenine, 6-(Alkyl)adenine, 6-(Methyl)adenine, 7-(Deaza)adenine, 8-(Alkenyl)adenine, 8-(Alkyl)adenine, 8-(Alkynyl)adenine, 8-(Amino)adenine, 8-(Halo)adenine, 8-(Hydroxyl)adenine, 8-(Thioalkyl)adenine, 8-(Thiol)adenine, N 6 -(Isopentyl)adenine, N 6 -(methyl)adenine, N 6 ,N 6 -(Dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine cytosine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N 4-(Acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(aminoallyl)uracil , 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonylmethyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N 3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4- (Thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted 4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-(aminocarbonylethylenyl)-pseudouracil, 1-(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1-(aminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(aminoalkylaminocarbonyl ethylenyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo) )-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)- 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidin, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimino Dazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyridinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidines, N, 2 Substituted purines, N 6 Substituted purines, O 6 Substituted purines, substituted 1,2,4-triazoles, pyrrolo-pyrimidin-2-one-3-yl, 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one- The bases include, but are not limited to, ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl, 2-oxo-pyridopyrimidin-3-yl, or any O- or N-alkylated derivative thereof. Alternatively, substituted or modified analogs of any of the above bases and "universal bases" can be employed.

[0386] As used herein, a universal nucleobase is any nucleobase that can base pair with all four naturally occurring nucleobases without substantially affecting the melting behavior, intracellular enzyme recognition, or activity of an iRNA duplex. Some exemplary universal nucleobases include 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6-methylbenzimidazol, 4-methylbenzimidazol, 3-methylisocarbostyrilyl, 5-methylisocarbostyrilyl, 3-methyl-7-propynylisocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl, 9-methyl-imidizopyridinyl, and 10-methyl-11-isocarbostyrilyl. These include, but are not limited to, lysinyl, pyrrolopyridinyl, isocarbostyrylyl, 7-propynylisocarbostyrylyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylinolyl, 4,6-dimethylindolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, and structural derivatives thereof (see, e.g., Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

[0387] Additional nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in International Application No. PCT / US09 / 038425, filed March 26, 2009, those disclosed in Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990, those disclosed by English et al., Angewandte Chemie, International Edition, 1991, 30,613, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijin, P. Ed. Wiley-VCH, 2008, and those disclosed in Sanghvi, YS, Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Eds., CRC Press, 1993, the contents of all of the above are incorporated herein by reference.

[0388] In certain embodiments, modified nucleobase is the nucleobase that has a structure that is substantially similar to that of parent nucleobase, such as 7-deazapurine, 5-methylcytosine or G-clamp.In certain embodiments, nucleobase mimics include more complex structures, such as tricyclic phenoxazine nucleobase mimics.The preparation method of the above-mentioned modified nucleobase is well known to those skilled in the art.

[0389] B. Sugar modification The DsRNA agent of the present invention provided herein can comprise one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) monomers, which comprise the nucleoside or nucleotide with modified sugar moiety.For example, the furanosyl sugar ring of nucleoside can be modified in many ways, including but not limited to, by adding a substituent, or by bridging two non-geminal ring atoms, to form locked nucleic acid or bicyclic nucleic acid.In certain embodiments, oligomeric compound comprises one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) monomers that are LNA.

[0390] In some embodiments of the locked nucleic acid, the 2' position of the flunaosyl is -[C(R1)(R2)] n -, -[C(R1)(R2)] n -O-, -[C(R1)(R2)] n -N(R1)-, -[C(R1)(R2)] n -N(R1)-O-, -[C(R1R2)] n -ON(R1)-, -C(R1)=C(R2)-O-, -C(R1)=N-, -C(R1)=NO-, -C(=NR1)-, -C(=NR1)- O-, -C(=O)-, -C(=O)O-, -C(=S)-, -C(=S)O-, -C(=S)S-, -O-, -Si(R1)2-, -S(=O) x -, and -N(R1)-, connected to the 4' position by a linker independently selected from During the ceremony, x is 0, 1, or 2; n is 1, 2, 3, or 4; each R and R is independently H, a protecting group, hydroxyl, C-C alkyl, substituted C-C alkyl, C-C alkenyl, substituted C-C alkenyl, C-C alkynyl, substituted C-C alkynyl, C-C aryl, substituted C-C aryl, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, C-C alicyclic radical, substituted C-C alicyclic radical, halogen, OJ, NJJ, SJ, N, COOJ, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)-J), or sulfoxyl (S(=O)-J); Each J1 and J2 is independently H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or a protecting group.

[0391] In some embodiments, each of the linkers of the LNA compound is independently -[C(R1)(R2)]n-, -[C(R1)(R2)]nO-, -C(R1R2)-N(R1)-O-, or -C(R1R2)-ON(R1)-. In other embodiments, each of the linkers is independently 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2', 4'-(CH2)2-O-2', 4'-CH2-ON(R1)-2', and 4'-CH2-N(R1)-O-2'-, where each R1 is independently H, a protecting group, or C1-C12 alkyl.

[0392] Certain LNAs have been prepared and are widely documented in the patent as well as scientific literature (Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; WO 94 / 14226; WO 2005 / 021570; Singh ... al., J. Org. Chem., 1998, 63, 10035-10039. Examples of issued U.S. patents and published applications disclosing LNAs include, for example, U.S. Patent Nos. 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, and 6,525,191, and U.S. Pregrant Publication Nos. 2004-0171570, 2004-0219565, 2004-0014959, 2003-0207841, 2004-0143114, and 20030082807.

[0393] Also provided herein are LNAs in which the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring, thereby forming a methyleneoxy (4'-CH2-O-2') linkage to form a bicyclic sugar moiety (see also U.S. Patent Nos. 6,268,490 and 6,670,461, reviewed in Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 81-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243). The linkage may be a methylene (-CH2-) group bridging the 2' oxygen atom and the 4' carbon atom; the term methyleneoxy (4'-CH2-O-2') LNA is used for the bicyclic moiety, whereas the term ethyleneoxy (4'-CH2CH2-O-2') LNA is used when an ethylene group is present at this position (Singh et al., Chem. Commun., 1998, 4, 455-456; Morita et al., Bioorganic Medicinal Chemistry, 2003, 11, 2211-2226). Methyleneoxy (4'-CH2-O-2') LNA and other bicyclic sugar analogs exhibit very high duplex thermal stability for complementary DNA and RNA (Tm = +3 to +10°C), stability against 3'-exonuclease degradation, and good solubility properties. Potent and non-toxic antisense oligonucleotides, including BNAs, have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638).

[0394] An isomer of methyleneoxy(4'-CH2-O-2')LNA that is also being considered is alpha-L-methyleneoxy(4'-CH2-O-2')LNA, which has been shown to have superior stability against 3'-exonucleases. Alpha-L-methyleneoxy(4'-CH2-O-2')LNA has been incorporated into antisense gapmers and chimeras that have shown potent antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0395] The synthesis and preparation of methyleneoxy (4'-CH2-O-2') LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine, and uracil, as well as their oligomerization and nucleic acid recognition properties, have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNA and its preparation are also described in WO 98 / 39352 and WO 99 / 14226.

[0396] Analogs of methyleneoxy (4'-CH2-O-2') LNA, phosphorothioate-methyleneoxy (4'-CH2-O-2') LNA, and 2'-thio-LNA have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of locked nucleoside analogs containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99 / 14226). Moreover, the synthesis of a novel conformationally restricted, high-affinity oligonucleotide analog, 2'-amino-LNA, has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2'-amino- and 2'-methylamino-LNAs have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.

[0397] Modified sugar moieties are well known and can be used to alter, typically increase, the affinity of an antisense compound for its target and / or increase nuclease resistance. A representative list of preferred modified sugars includes, but is not limited to, bicyclic modified sugars, including methyleneoxy (4'-CH2-O-2') LNA and ethyleneoxy (4'-(CH2)2-O-2' bridged) ENA, substituted sugars, particularly 2'-substituted sugars with 2'-F, 2'-OCH3, or 2'-O(CH2)2-OCH3 substituents, and 4'-thio modified sugars. Sugars can also be substituted with, among other things, sugar mimetic groups. Methods for preparing modified sugars are well known to those skilled in the art. Some representative patents and publications that teach the preparation of such modified sugars include U.S. Patent 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; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; Nos. 5,700,920; 6,531,584, and 6,600,032, and WO 2005 / 121371.

[0398] Examples of "oxy"-2' hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), O(CH2CHO) n CH2CH2OR, n=1-50, "locked" nucleic acids (LNAs) in which the furanose portion of the nucleoside contains a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system; O-amine or O-(CH2) namines (n=1-10, amine=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, ethylenediamine, or polyamino); and O-CH2CH2(NCH2CH2NMe2)2.

[0399] Deoxy modifications include hydrogen (i.e., deoxyribose sugars, particularly associated with single-stranded overhangs), halo (e.g., fluoro), amino (e.g., NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CHCHNH) n Included are CH2CH2-amine (amine = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino), -NHC(O)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); cyano; mercapto; alkyl-thio-alkyl; thioalkoxy, thioalkyl; alkyl; cycloalkyl; aryl; alkenyl, and alkynyl, which can be optionally substituted, for example, with an amino functionality.

[0400] Other suitable 2'-modifications, such as modified MOEs, are described in US Patent Application Publication No. 20130130378, the contents of which are incorporated herein by reference.

[0401] Modifications at the 2' position can be in the arabinose configuration. The term "arabinose configuration" refers to the placement of the substituent on C2' of ribose in the same configuration as the 2'-OH is in arabinose.

[0402] A sugar can contain two different modifications, such as gem modifications, at the same carbon atom within the sugar. The sugar group can also contain one or more carbon atoms with an opposite stereochemical configuration to that of the corresponding carbon atom in ribose. Thus, an oligomeric compound can contain one or more monomers containing, for example, arabinose as the sugar. The monomer can have an alpha linkage at the 1' position on the sugar, for example, an alpha-nucleoside. The monomer can also have an opposite stereochemistry at the 4' position, for example, C5' and H4', or the substituents replacing them are interchanged. When C5' and H4' or the substituents replacing them are interchanged, the sugar is said to be modified at the 4' position.

[0403] The DsRNA agent of the present invention disclosed herein can also comprise abasic sugar, i.e., sugar that lacks a nucleobase at C-1', or sugar that has other chemical groups instead of a nucleobase at C1'.See, for example, U.S. Patent No. 5,998,203 (the contents of which are incorporated herein in their entirety).These abasic sugars can also further contain modifications to one or more of the component sugar atoms.The DsRNA agent of the present invention can also contain one or more sugars that are L-isomers, for example, L-nucleosides.Modifications to sugar groups can also include replacing 4'-O with sulfur, optionally substituted nitrogen, or CH2 group.In some embodiments, the bond between C1' and nucleobase is in α-configuration.

[0404] Sugar modifications can also include acyclic nucleotides in which the C-C bond between the ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') is absent, and / or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4') is independently present or in combination with the absence of a nucleotide. In some embodiments, the acyclic nucleotide is [ka] wherein B is a modified or unmodified nucleobase, R1 and R2 are independently H, halogen, OR3 or alkyl, and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar.

[0405] In some embodiments, the sugar modification is selected from the group consisting of 2'-H, 2'-O-Me (2'-O-methyl), 2'-O-MOE (2'-O-methoxyethyl), 2'-F, 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), 2'-S-methyl, 2'-O-CH2-(4'-C) (LNA), 2'-O-CH2CH2-(4'-C) (ENA), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-OD-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), and gem 2'-OMe / 2'F with 2'-O-Me in the arabinose configuration.

[0406] It is understood that when a particular nucleotide is linked to the next nucleotide through its 2' position, the sugar modifications described herein can be placed at the 3'-position of the sugar relative to that particular nucleotide, e.g., the nucleotide linked through its 2'-position. The modification at the 3' position can be in the xylose configuration. The term "xylose configuration" refers to the placement of the substituent on the C3' of the ribose in the same configuration as the C3'-OH is in a xylose sugar.

[0407] The hydrogen attached to C4' and / or C1' can be replaced by a straight or branched chain, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl, and the backbone of the alkyl, alkenyl, and alkynyl can contain one or more of O, S, S(O), SO2, N(R'), C(O), N(R')C(O)O, OC(O)N(R'), CH(Z'), a phosphorus-containing bond, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclic, or optionally substituted cycloalkyl, wherein R' is hydrogen, acyl, or optionally substituted aliphatic, and Z' is OR 11 , C.O.R. 11 , CO2R 11 , [ka] NR 21 R 31 ,CONR 21 R 31 , CON(H)NR 21 R 31 , ONR 21 R 31 , CON(H)N=CR 41 R 51 , N(R 21 )C(=NR 31 )NR 21 R 31 , N(R 21 )C(O)NR 21 R 31 , N(R 21 )C(S)NR 21 R 31 , OC(O)NR 21 R 31 , SC(O)NR 21 R 31 , N(R 21 )C(S)OR 11 , N(R 21 )C(O)OR 11 , N(R 21 )C(O)SR 11 , N(R 21 )N=CR 41 R 51 , ON=CR 41 R 51, SO2R 11 , SOR 11 , S.R. 11 and substituted or unsubstituted heterocyclic; R 21 and R 31 is independently for each occurrence hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR 11 , C.O.R. 11 , CO2R 11 , or NR 11 R 11 ' or R 21 and R 31 together with the atoms to which they are attached form a heterocyclic ring; R 41 and R 51 is, for each occurrence, independently selected from hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR 11 , C.O.R. 11 , or CO2R 11 , or NR 11 R 11 ' and R 11 and R 11 is independently hydrogen, aliphatic, substituted aliphatic, aryl, heteroaryl, or heterocyclic. In some embodiments, the hydrogen attached to C4' of the 5'-terminal nucleotide is replaced.

[0408] In some embodiments, C4' and C5' together form an optionally substituted heterocycle, preferably containing at least one -PX(Y)-, where X is H, OH, OM, SH, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted alkylamino, or optionally substituted dialkylamino, where M is, independently for each occurrence, an alkyl metal or transition metal having an overall charge of +1, and Y is O, S, or NR', where R' is hydrogen, optionally substituted aliphatic. Preferably, this modification is at the 5' end of the iRNA.

[0409] In certain embodiments, the LNA comprises a bicyclic nucleoside having the formula: [ka] During the ceremony, Bx is a heterocyclic base moiety; T1 is H or a hydroxyl protecting group; T2 is H, a hydroxyl protecting group, or a reactive phosphorus group; Z is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, or substituted amido.

[0410] In some embodiments, each of the substituents is independently mono- or poly-substituted with optionally protected substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, and CN, wherein each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O, S, or NJ1.

[0411] In certain such embodiments, each of the substituents is independently mono- or poly-substituted with optionally protected substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, and NJ3C(=X)NJ1J2, where each J1, J2, and J3 is independently H, C1-C6 alkyl, or substituted C1-C6 alkyl, and X is O or NJ1.

[0412] In certain embodiments, the Z group is C-C alkyl substituted with one or more X, where each X is independently OJ, NJJ, SJ, N, OC(=X)J, OC(=X)NJJ, NJC(=X)NJJ, or CN, where each J, J, and J is independently H or C-C alkyl, and X is O, S, or NJ. In another embodiment, the Z group is C-C alkyl substituted with one or more X, where each X is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO-), substituted alkoxy, or azido.

[0413] In certain embodiments, the Z group is -CH2Xx, where Xx is OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, or CN, where each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O, S, or NJ1. In another embodiment, the Z group is -CH2Xx, where Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO-), or azido.

[0414] In certain such embodiments, the Z group is in the (R)-configuration shown below. [ka]

[0415] In certain such embodiments, the Z group is in the (S)-configuration shown below. [ka]

[0416] In certain embodiments, each T1 and T2 is a hydroxyl protecting group. A preferred list of hydroxyl protecting groups includes benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, mesylate, tosylate, dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (MOX). In certain embodiments, T1 is a hydroxyl protecting group selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, and dimethoxytrityl, and a more preferred hydroxyl protecting group is 4,4'-dimethoxytrityl.

[0417] In certain embodiments, T2 is a reactive phosphorus group, and preferred reactive phosphorus groups include diisopropylcyanoethoxyphosphoramidite and H-phosphonate. In certain embodiments, T1 is 4,4'-dimethoxytrityl and T2 is diisopropylcyanoethoxyphosphoramidite.

[0418] In certain embodiments, the compounds of the present invention have the following formula: [ka] or the following expression: [ka] or the following expression: [ka] During the ceremony, Bx is a heterocyclic base moiety; T3 is H, a hydroxyl protecting group, an attached conjugate group, or an internucleoside linking group attached to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomeric subunit, or oligomeric compound; T4 is H, a hydroxyl protecting group, an attached conjugate group, or an internucleoside linking group attached to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomeric subunit, or oligomeric compound; wherein at least one of T3 and T4 is an internucleoside linking group attached to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomeric subunit, or oligomeric compound; Z is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, or substituted amido.

[0419] In some embodiments, each of the substituents is independently mono- or poly-substituted with optionally protected substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, and CN, wherein each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O, S, or NJ1.

[0420] In some embodiments, each of the substituents is independently mono- or poly-substituted with substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, and NJ3C(=X)NJ1J2, wherein each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O or NJ1.

[0421] In certain such embodiments, at least one Z is C-C alkyl or substituted C-C alkyl. In certain embodiments, each Z is independently C-C alkyl or substituted C-C alkyl. In certain embodiments, at least one Z is C-C alkyl. In certain embodiments, each Z is independently C-C alkyl. In certain embodiments, at least one Z is methyl. In certain embodiments, each Z is methyl. In certain embodiments, at least one Z is ethyl. In certain embodiments, each Z is ethyl. In certain embodiments, at least one Z is substituted C-C alkyl. In certain embodiments, each Z is independently substituted C-C alkyl. In certain embodiments, at least one Z is substituted methyl. In certain embodiments, each Z is substituted methyl. In certain embodiments, at least one Z is substituted ethyl. In certain embodiments, each Z is substituted ethyl.

[0422] In certain embodiments, at least one substituent is C-C alkoxy (e.g., at least one Z is C-C alkyl substituted with one or more C-C alkoxy). In other embodiments, each substituent is independently C-C alkoxy (e.g., each Z is independently C-C alkyl substituted with one or more C-C alkoxy).

[0423] In certain embodiments, at least one C1-C6 alkoxy substituent is CH3O- (e.g., at least one Z is CH3OCH2-). In other embodiments, each C1-C6 alkoxy substituent is CH3O- (e.g., each Z is CH3OCH2-).

[0424] In certain embodiments, at least one substituent is halogen (e.g., at least one Z is C-C alkyl substituted with one or more halogens). In certain embodiments, each substituent is independently halogen (e.g., each Z is independently C-C alkyl substituted with one or more halogens). In certain embodiments, at least one halogen substituent is fluoro (e.g., at least one Z is CHFCH-, CHFCH-, or CFCH-). In certain embodiments, each halo substituent is fluoro (e.g., each Z is independently CHFCH-, CHFCH-, or CFCH-).

[0425] In certain embodiments, at least one substituent is hydroxyl (e.g., at least one Z is C-C alkyl substituted with one or more hydroxyl). In certain embodiments, each substituent is independently hydroxyl (e.g., each Z is independently C-C alkyl substituted with one or more hydroxyl). In certain embodiments, at least one Z is HOCH2-. In other embodiments, each Z is HOCH2-.

[0426] In certain embodiments, at least one Z is CH3-, CH3CH2-, CHOCH3-, CH2F-, or HOCH2-. In certain embodiments, each Z is independently CH3-, CH3CH2-, CHOCH3-, CH2F-, or HOCH2-.

[0427] In certain embodiments, at least one Z group is C-C alkyl substituted with one or more X, where each X is independently OJ, NJJ, SJ, N, OC(=X)J, OC(=X)NJJ, NJC(=X)NJJ, or CN, where each J, J, and J is independently H or C-C alkyl, and X is O, S, or NJ. In another embodiment, at least one Z group is C-C alkyl substituted with one or more X, where each X is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO—), or azido.

[0428] In certain embodiments, each Z group is independently C-C alkyl substituted with one or more X, where each X is independently OJ, NJJ, SJ, N, OC(=X)J, OC(=X)NJJ, NJC(=X)NJJ, or CN, where each J, J, and J is independently H or C-C alkyl, and X is O, S, or NJ. In another embodiment, each Z group is independently C-C alkyl substituted with one or more X, where each X is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO—), or azido.

[0429] In certain embodiments, at least one Z group is -CH2Xx, where Xx is OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, or CN, where each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O, S, or NJ1. In certain embodiments, at least one Z group is -CH2Xx, where Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO-), or azido.

[0430] In certain embodiments, each Z group is independently -CH2Xx, where each Xx is independently OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, or CN, where each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O, S, or NJ1. In another embodiment, each Z group is independently -CH2Xx, where each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO-), or azido.

[0431] In certain embodiments, at least one Z is CH3-. In other embodiments, each Z is CH3-.

[0432] In certain embodiments, the Z group of at least one monomer has the following formula: [ka] or the following expression: [ka] or in the (R)-configuration represented by the following formula: [ka]

[0433] In certain embodiments, the Z group of each monomer of this formula is in the (R)-configuration.

[0434] In certain embodiments, the Z group of at least one monomer has the following formula: [ka] or the following expression: [ka] or in the (S)-configuration represented by the following formula: [ka]

[0435] In certain embodiments, the Z group of each monomer of this formula is in the (S)-configuration.

[0436] In certain embodiments, T3 is H or a hydroxyl protecting group. In certain embodiments, T4 is H or a hydroxyl protecting group. In further embodiments, T3 is an internucleoside linking group attached to a nucleoside, nucleotide, or monomer subunit. In certain embodiments, T4 is an internucleoside linking group attached to a nucleoside, nucleotide, or monomer subunit. In certain embodiments, T3 is an internucleoside linking group attached to an oligonucleoside or oligonucleotide. In certain embodiments, T4 is an internucleoside linking group attached to an oligonucleoside or oligonucleotide. In certain embodiments, T3 is an internucleoside linking group attached to an oligomeric compound. In certain embodiments, T4 is an internucleoside linking group attached to an oligomeric compound. In certain embodiments, at least one of T3 and T4 comprises an internucleoside linking group selected from phosphodiester or phosphorothioate.

[0437] In certain embodiments, the dsRNA agent of the invention has the following formula: [ka] or the following expression: [ka] or comprises at least one region of at least two consecutive monomers of the formula: [ka]

[0438] In certain such embodiments, LNAs include, but are not limited to, (A) α-L-methyleneoxy (4'-CH2-O-2') LNA, (B) β-D-methyleneoxy (4'-CH2-O-2') LNA, (C) ethyleneoxy (4'-(CH2)2-O-2') LNA, (D) aminooxy (4'-CH2-ON(R)-2') LNA, and (E) oxyamino (4'-CH2-N(R)-O-2') LNA. [ka]

[0439] In certain embodiments, a dsRNA agent of the invention comprises at least two regions of at least two consecutive monomers of the above formula. In certain embodiments, a dsRNA agent of the invention comprises a gap motif. In certain embodiments, a dsRNA agent of the invention comprises at least one region of about 8 to about 14 consecutive β-D-2'-deoxyribofuranosyl nucleosides. In certain embodiments, a dsRNA agent of the invention comprises at least one region of about 9 to about 12 consecutive β-D-2'-deoxyribofuranosyl nucleosides.

[0440] In certain embodiments, a dsRNA agent of the invention comprises an (S)-cEt monomer of at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) of the following formulas: [ka] Bx is a heterocyclic base moiety.

[0441] In certain embodiments, the monomer comprises a sugar mimetic. In certain such embodiments, the mimetic is used in place of the sugar or sugar-internucleoside linkage combination, while the nucleobase is maintained for hybridization to a selected target. Representative examples of sugar mimetics include, but are not limited to, cyclohexenyl or morpholino. Representative examples of mimetics for sugar-internucleoside linkage combinations include, but are not limited to, peptide nucleic acids (PNAs) and morpholino groups linked by uncharged achiral bonds. In some cases, a mimetic is used in place of the nucleobase. Representative nucleobase mimetics are well known in the art and include, but are not limited to, tricyclic phenoxazine analogs and universal bases (Berger et al., Nuc Acid Res. 2000, 28:2911-14, incorporated herein by reference). Methods for synthesizing sugar, nucleoside, and nucleobase mimetics are well known to those skilled in the art.

[0442] C. Sugar bond modification Described herein are linking groups that link monomers (including, but not limited to, modified and unmodified nucleosides and nucleotides) together to form oligomeric compounds, e.g., oligonucleotides. Such linking groups are also referred to as intersugar linkages. Two major classes of linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing linkages include, but are not limited to, phosphodiester (P=O), phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Representative non-phosphorus-containing linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thionocarbamate (-OC(O)(NH)-S-), siloxane (-O-Si(H)2-O-), and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Modified linkages can be used to modify, typically increase, the nuclease resistance of oligonucleotides compared to natural phosphodiester linkages.In certain embodiments, linkages with chiral atoms can be prepared as racemates, as separate enantiomers.Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates.Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known to those skilled in the art.

[0443] The phosphate group in the linking group can be modified by replacing one of the oxygen atoms with a different substituent. One result of this modification can be increased resistance to nucleolytic degradation of the oligonucleotide. Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in this bond can be replaced with any of the following: S, Se, BR3 (R is hydrogen, alkyl, aryl), C (i.e., alkyl group, aryl group, etc.), H, NR2 (R is hydrogen, optionally substituted alkyl, aryl), or OR (R is optionally substituted alkyl or aryl). The phosphorus atom in an unmodified phosphate group is achiral. However, replacing one of the non-bridging oxygen atoms with one of the above atoms or groups of atoms makes the phosphorus atom chiral; in other words, the phosphorus atom in the phosphate group modified in this way is a stereocenter. The chiral central phosphorus atom can have either the "R" configuration (herein Rp) or the "S" configuration (herein Sp).

[0444] Phosphorodithioates have both non-bridging oxygens replaced by sulfur. The phosphorus center in phosphorodithioates is achiral, preventing the formation of oligonucleotide diastereomers. Therefore, without wishing to be bound by theory, modifications to both non-bridging oxygens that eliminate chiral centers, such as forming phosphorodithioates, may be desirable in that they cannot produce diastereomeric mixtures. Therefore, the non-bridging oxygens can independently be any one of O, S, Se, B, C, H, N, or OR (R is alkyl or aryl).

[0445] The phosphate linker can also be modified by replacing the bridging oxygen (i.e., the oxygen that connects the phosphate to the sugar of the monomer) with nitrogen (bridging phosphoramidate), sulfur (bridging phosphorothioate), and carbon (bridging methylene phosphonate). The substitution can occur at either one of the linking oxygens or at both linking oxygens. When the bridging oxygen is the 3'-oxygen of the nucleoside, substitution with carbon is preferred. When the bridging oxygen is the 5'-oxygen of the nucleoside, substitution with nitrogen is preferred.

[0446] Modified phosphate linkages in which at least one of the oxygens linked to the phosphate is replaced, or the phosphate group is replaced with a non-phosphate group, are also referred to as "non-phosphodiester intersugar linkages" or "non-phosphodiester linkers."

[0447] In certain embodiments, the phosphate group can be replaced by a non-phosphorus-containing connector, such as a dephosphoryl linker. Dephosphoryl linkers are also referred to herein as non-phosphodiester linkers. Without wishing to be bound by theory, it is believed that the charged phosphodiester group is the reactive center in nucleotide degradation, and therefore replacement with a neutral structural mimic improves the stability of the nuclease. Again, without wishing to be bound by theory, in some embodiments, it may be desirable to introduce a modification in which the charged phosphate group is replaced with a neutral moiety.

[0448] Examples of moieties that can replace the phosphate group include amide (e.g., amide-3 (3'-CH2-C(=O)-N(H)-5'), and amide-4 (3'-CH2-N(H)-C(=O)-5')), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylate ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal (3'-S-CH2-O-5'), formacetal (3'-O-CH2-O-5'), oxime, methyleneimino, methylenecarbonylamino, methyl ... Examples of linkages include, but are not limited to, ethylenemethylimino (MMI, 3'-CH2-N(CH3)-O-5'), methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ether (C3'-O-C5'), thioether (C3'-S-C5'), thioacetamide (C3'-N(H)-C(=O)-CH2-S-C5', C3'-O-P(O)-O-SS-C5', C3'-CH2-NH-NH-C5', 3'-NHP(O)(OCH3)-O-5', and 3'-NHP(O)(OCH3)-O-5', as well as nonionic linkages containing mixed N, O, S, and CH2 constituent moieties. For example, Carbohydrate See Modifications in Antisense Research; YS Sanghvi and PDCook Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65). Preferred embodiments include methylenemethylimino (MMI), methylenecarbonylamino, amide, carbamate, and ethylene oxide linkers.

[0449] Those skilled in the art are well aware that in certain cases, substitution of a non-bridging oxygen can lead to enhanced intersugar bond cleavage by the adjacent 2'-OH, and therefore, in many cases, modification of a non-bridging oxygen can require modification of a 2'-OH, e.g., a modification that does not involve cleavage of the adjacent intersugar bond, e.g., arabinose sugars, 2'-O-alkyl, 2'-F, LNA, and ENA.

[0450] Preferred non-phosphodiester intersugar linkages include phosphorothioates, phosphorothioates with an enantiomeric excess of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the Sp isomer, phosphorothioates with an enantiomeric excess of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the Rp isomer, phosphorodithioates, phosphotriesters, aminoalkylphosphotrioesters, alkylphosphonates (e.g., methyl-phosphonates), selenophosphates, phosphoramidates (e.g., N-alkylphosphoramidates), and boranophosphonates.

[0451] In some embodiments, the dsRNA agent of the present invention comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, and up to and including all) modified or non-phosphodiester linkage. In some embodiments, the dsRNA agent of the present invention comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, and up to and including all) phosphorothioate linkage.

[0452] The dsRNA agent of the present invention can also be constructed by replacing phosphate linker and sugar with nuclease-resistant nucleoside or nucleotide substitute.Without wishing to be bound by theory, it is believed that the absence of repeatedly charged backbone reduces binding to proteins that recognize polyanions (e.g., nucleases).Again, without wishing to be bound by theory, in some embodiments, it may be desirable to introduce a modification in which bases are tethered by neutral backbone substitutes.Examples include morpholino, cyclobutyl, pyrrolidine, peptide nucleic acid (PNA), aminoethylglycyl PNA (aeg PNA), and back-non-extended pyrrolidine PNA (bep PNA) nucleoside substitutes.Preferred substitute is PNA substitute.

[0453] The dsRNA agents of the invention described herein can contain one or more asymmetric centers and can therefore give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined in terms of absolute stereochemistry, such as (R) or (S), such as sugar anomers, or (D) or (L), such as amino acids. The dsRNA agents of the invention provided herein include all such possible isomers, as well as their racemic and optically pure forms.

[0454] D. Terminal modification In some embodiments, the dsRNA agent further comprises a phosphate or a phosphate mimic at the 5'-end of the antisense strand. In one embodiment, the phosphate mimic is 5'-vinylphosphonate (VP).

[0455] In some embodiments, the 5'-end of the antisense strand of a dsRNA agent does not contain a 5'-vinylphosphonate (VP).

[0456] The termini of the iRNA agent of the present invention can be modified. Such modifications can be at one or both ends. For example, the 3' and / or 5' ends of the iRNA can be conjugated to a labeling moiety, such as a fluorophore (e.g., pyrene, TAMRA, fluorescein, Cy3, or Cy5 dye) or other functional molecular entity, such as a protecting group (e.g., sulfur-, silicon-, boron-, or ester-based). The functional molecular entity can be attached to the sugar through a phosphate group and / or a linker. The terminal atom of the linker can be connected to or replace the linking atom of the phosphate group or the C-3' or C-5' O, N, S, or C group of the sugar. Alternatively, the linker can be connected to or replace the terminal atom of a nucleotide surrogate (e.g., PNA).

[0457] When a linker / phosphate functional molecular entity-linker / phosphate array is interposed between the two strands of a double-stranded oligomeric compound, the array can replace a hairpin loop in a hairpin-type oligomeric compound.

[0458] Terminal modifications useful for regulating activity include modification of the 5'-end of iRNA with phosphate or phosphate analogs. In certain embodiments, the 5'-end of iRNA is phosphorylated or contains a phosphoryl analog. Exemplary 5'-phosphate modifications include modifications compatible with RISC-mediated gene silencing. Modifications at the 5'-end may also be useful for stimulating or inhibiting the immune system of a subject. In some embodiments, the 5'-end of an oligomeric compound is modified [ka] wherein W, X, and Y are selected from the group consisting of O, OR (wherein R is hydrogen, alkyl, or aryl), S, Se, BR3 (wherein R is hydrogen, alkyl, or aryl), BH3 -, C (i.e., alkyl group, aryl group, etc.), H, NR (R is hydrogen, alkyl, aryl), or OR (R is hydrogen, alkyl, or aryl); A and Z, for each occurrence, are each independently absent, O, S, CH, NR (R is hydrogen, alkyl, aryl), or optionally substituted alkylene, where the alkylene backbone can include one or more of O, S, SS, and NR (R is hydrogen, alkyl, aryl) internally and / or terminally; and n is 0-2. In some embodiments, n is 1 or 2. It is understood that A replaces the oxygen linked to the 5' carbon of the sugar. When n is 0, W and Y, together with the P to which they are attached, can form an optionally substituted 5-8 membered heterocycle, where W and Y are each independently O, S, NR', or alkylene. The heterocycle is preferably substituted with aryl or heteroaryl. In some embodiments, one or both hydrogens on the C5' of the 5'-terminal nucleotide are substituted with a halogen, eg, F.

[0459] Exemplary 5'-modifications include 5'-monophosphate ((HO)2(O)PO-5'), 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'), 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'), 5'-monothiophosphate (phosphorothioate, (HO)2(S)PO-5'), 5'-monodithiophosphate (phosphorodi ... phosphate, (HO)(HS)(S)PO-5'), 5'-phosphorothiolate ((HO)2(O)PS-5'), 5'-alpha-thiotriphosphate, 5'-beta-thiotriphosphate, 5'-gamma-thiotriphosphate, 5'-phosphoramidate ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'). Other 5'-modifications include 5'-alkylphosphonates (R(OH)(O)PO-5', R = alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc.), 5'-alkyl ether phosphonates (R(OH)(O)PO-5', R = alkyl ether, e.g., methoxymethyl (CHOMe), ethoxymethyl, etc.). Another exemplary 5'-modification is when Z is alkyl optionally substituted at least once, e.g., ((HO)2(X)PO[-(CH2) a -OP(X)(OH)-O] b - 5', ((HO)2(X)PO[-(CH2) a -P(X)(OH)-O] b -5', ((HO)2(X)P-[-(CH2) a -OP(X)(OH)-O] b -5', Dialkyl Terminal Phosphates and Phosphate Mimics: HO[-(CH2) a -OP(X)(OH)-O] b - 5', H2N[-(CH2) a -OP(X)(OH)-O] b - 5', H[-(CH2) a -OP(X)(OH)-O] b -5', Me2N[-(CH2) a -OP(X)(OH)-O] b -5', HO[-(CH2) a-P(X)(OH)-O] b -5', H2N[-(CH2) a -P(X)(OH)-O] b -5', H[-(CH2) a -P(X)(OH)-O] b -5', Me2N[-(CH2) a -P(X)(OH)-O] b -5', where a and b are each independently 1 to 10. Other embodiments include BH3, BH3 - and / or Se substitutions.

[0460] Terminal modifications can also be useful for monitoring distribution; in such cases, preferred groups added include fluorophores, such as fluorescein or Alexa dyes, such as Alexa 488. Terminal modifications can also be useful for enhancing uptake; modifications useful for this purpose include targeting ligands. Terminal modifications can also be useful for crosslinking the oligonucleotide to another moiety; modifications useful for this purpose include mitomycin C, psoralens, and their derivatives.

[0461] E. Thermal destabilization modification

[0462] Compounds of the invention, such as iRNA or dsRNA agents, can be optimized for RNA interference by increasing the propensity of the iRNA duplex to dissociate or melt (decreasing the free energy of duplex association) by introducing a thermally destabilizing modification into the sense strand at a site opposite the seed region of the antisense strand (i.e., positions 2-8 of the 5'-end of the antisense strand or positions 2-9 of the 5'-end of the antisense strand). This modification can increase the propensity of the duplex to dissociate or melt within the seed region of the antisense strand.

[0463] Thermally destabilizing modifications can include abasic modifications, mismatches with the opposing nucleotide in the opposing strand, and sugar modifications such as 2'-deoxy modifications or acyclic nucleotides, e.g., unlocked nucleic acids (UNAs) or glycerol nucleic acids (GNAs).

[0464] Exemplary abasic modifications are: [ka]

[0465] Exemplary sugar modifications are: [ka]

[0466] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, for example, where any of the bonds between the ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') are absent, and / or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4'), independently or in combination, is absent in the nucleotide. In some embodiments, an acyclic nucleotide is [ka] wherein B is a modified or unmodified nucleobase and R 1 and R 2are independently H, halogen, OR3, or alkyl, and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. The term "UNA" refers to an acyclic unlocked nucleic acid in which any of the sugar bonds has been removed to form an unlocked "sugar" residue. In one example, UNA also encompasses monomers in which the C1'-C4' bond has been removed (i.e., a carbon-oxygen-carbon covalent bond between the C1' and C4' carbons). In another example, the C2'-C3' bond of the sugar has been removed (i.e., a carbon-carbon covalent bond between the C2' and C3' carbons) (see Mikhailov et al., Tetrahedron Letters, 26 (17): 2059 (1985), and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), which are incorporated by reference in their entireties). Acyclic derivatives offer greater backbone flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be linked via 2'-5' or 3'-5' linkages.

[0467] The term "GNA" refers to glycol nucleic acid, which is a polymer similar to DNA or RNA, but differs in the composition of its "backbone" in that it is made up of repeating glycerol units linked by phosphodiester bonds. [ka]

[0468] The thermally destabilizing modification can be a mismatch (i.e., non-complementary base pair) between the thermally destabilizing nucleotide and the opposite nucleotide in the opposite strand of the dsRNA duplex.Exemplary mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or combinations thereof.Other mismatch base pairings known in the art are also contemplated by the present invention.Mismatches can occur between nucleotides that are either naturally occurring or modified nucleotides, i.e., mismatch base pairings can occur between the nucleobases derived from each nucleotide independently of the modification on the ribose sugar of the nucleotide.In certain embodiments, the compounds of the present invention, such as siRNA agents or iRNA agents, contain at least one nucleobase in the mismatch pairing that is a 2'-deoxynucleobase, for example, the 2'-deoxynucleobase is in the sense strand.

[0469] Many examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA) and mismatch modifications are described in detail in WO 2011 / 133876, which is incorporated herein by reference in its entirety.

[0470] Thermally destabilizing modifications can also include universal base and phosphate modifications that have reduced or eliminated ability to form hydrogen bonds with opposing bases.

[0471] Nucleobase modifications that impair or completely abolish the ability to form hydrogen bonds with bases in the opposing strand have been evaluated for destabilizing the central region of the dsRNA duplex, as described in International Publication No. 2010 / 0011895, the entire contents of which are incorporated herein by reference. Exemplary nucleobase modifications include: [ka]

[0472] Exemplary phosphate modifications that have been shown to reduce the thermal stability of dsRNA duplexes compared to natural phosphodiester linkages include the following: [ka]

[0473] In some embodiments, compounds of the invention can include 2'-5' linkages (having 2'-H, 2'-OH, and 2'-OMe, and also with P=O or P=S). For example, 2'-5' linkage modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5' end of the sense strand to prevent activation of the sense strand by RISC.

[0474] In another embodiment, the compounds of the present invention can include L-sugars (e.g., L-ribose, L-arabinose with 2'-H, 2'-OH, and 2'-OMe). For example, these L-sugar modifications can be used to promote nuclease resistance or to inhibit binding of the sense to antisense strand, or at the 5' end of the sense strand to prevent activation of the sense strand by RISC.

[0475] In one embodiment, an iRNA agent of the invention is conjugated to a ligand via a carrier, which can be a cyclic or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is selected from a serinol backbone or a diethanolamine backbone.

[0476] In some embodiments, at least one strand of an iRNA agent of the invention disclosed herein is 5' phosphorylated or contains a phosphoryl analog at the 5' prime end. 5'-phosphate modifications include modifications compatible with RISC-mediated gene silencing. Suitable modifications include 5'-monophosphate ((HO)2(O)PO-5'); 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)PO-(HO)(O)POP(HO )(O)-O-5'; 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)PO-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)PO-5'), 5 '-phosphorothiolates ((HO)2(O)PS-5'); any additional combination of oxygen / sulfur substituted monophosphates, diphosphates, and triphosphates (e.g., 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'), 5'-alkylphosphonates (R = alkyl = methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)-O-5'-, 5'-alkenylphosphonates (i.e., vinyl, substituted vinyl), (OH)2(O)P-5'-CH2-), 5'-alkyl ether phosphonates (R = alkyl ether = methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5'-).

[0477] IV. MODIFIED RNAI AGENTS OF THE INVENTION COMPRISING MOTIF In certain aspects of the present disclosure, the double-stranded RNAi agent of the present disclosure includes agents having chemical modifications disclosed in, for example, U.S. Patent Nos. 9,796,974 and 10,668,170, and U.S. Patent Publication Nos. 2014 / 288158, 2018 / 008724, 2019 / 038768, and 2020 / 353097, the entire contents of each of which are incorporated herein by reference.As shown therein and in PCT Publication WO 2013 / 074974 (the entire contents of which are incorporated herein by reference), one or more motifs of three identical modifications on three consecutive nucleotides can be introduced into the sense strand or antisense strand of the RNAi agent, particularly at or near the cleavage site.In some embodiments, the sense strand and antisense strand of the RNAi agent can be otherwise completely modified.Preferably, the 2nt overhang is at the 3' end of the antisense strand. An RNAi agent can optionally be modified with an (S)-glycol nucleic acid (GNA) modification, for example, at one or more residues of the antisense strand.

[0478] In one embodiment, an iRNA agent of the invention is a blunt-ended duplex 19 nt in length, the sense strand containing at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 7, 8, and 9 from the 5' end, and the antisense strand containing at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0479] In one embodiment, an iRNA agent of the invention is a 20 nt long, blunt-ended duplex in which the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0480] In one embodiment, an iRNA agent of the invention is a 21 nt long, blunt-ended duplex in which 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.

[0481] In one embodiment, an iRNA agent of the present invention comprises a 21-nucleotide (nt) sense strand and a 23-nucleotide (nt) 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, and one end of the iRNA is blunt and the other end contains a 2-nt overhang. Preferably, the 2-nt overhang is at the 3' end of the antisense strand. Optionally, the iRNA agent further comprises a ligand (e.g., GalNAc3).

[0482] In one embodiment, an iRNA agent of the invention comprises a sense strand and an antisense strand, the sense strand being 25 to 30 nucleotide residues in length and, starting from the 5'-terminal nucleotide (position 1), comprising at least 8 ribonucleotides at positions 1-23 of the first strand; the antisense strand being 36 to 66 nucleotide residues in length and, starting from the 3'-terminal nucleotide, comprising at least 8 ribonucleotides at positions 1-23 of the sense strand to form a duplex; at least the 3'-terminal nucleotide of the antisense strand is unpaired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are unpaired with the sense strand, thereby forming a 3' single-stranded overhang of 1 to 6 nucleotides; and the 5'-end of the antisense strand being at least 10 to 30 consecutive ribonucleotides that are not paired with the sense strand. The double-stranded nucleic acid comprises nucleotides corresponding to the 2'-F modification on three consecutive nucleotides, thereby forming a 10-30 nucleotide single-stranded 5' overhang, at least the 5'- and 3'-terminal nucleotides of the sense strand base-pair with nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplex region between the sense and antisense strands, and the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length, such that the double-stranded nucleic acid reduces target gene expression when introduced into a mammalian cell, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, at least one of the motifs occurring 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.

[0483] In one embodiment, an iRNA agent of the invention includes a sense strand and an antisense strand, the iRNA agent comprising 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 with 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 forming a blunt end, the second strand being 1-4 nucleotides longer at its 3' end than the first strand, the duplex region being at least 25 nucleotides in length, the second strand being sufficiently complementary to a target mRNA along at least 19 nt of the length of the second strand, such that the iRNA agent reduces target gene expression when introduced into a mammalian cell, and Dicer cleavage of the iRNA agent preferentially results in an siRNA that includes the 3' end of the second strand, thereby reducing target gene expression in the mammalian cell. Optionally, the iRNA agent further comprises a ligand (eg, GalNAc3).

[0484] In one embodiment, the sense strand of the iRNA agent contains at least one motif of three identical modifications on three consecutive nucleotides, one of which occurs at the cleavage site within the sense strand. For example, the sense strand can contain at least one motif of three 2'-F modifications on three consecutive nucleotides within positions 7-15 from the 5' end.

[0485] In one embodiment, the antisense strand of the iRNA agent can also contain a motif of at least one of three identical modifications on three consecutive nucleotides, one of which occurs at or near the cleavage site in the antisense strand. For example, the antisense strand can contain a motif of at least one of three 2'-O-methyl modifications on three consecutive nucleotides within positions 9-15 from the 5' end.

[0486] For iRNA agents having a duplex region 17 to 23 nt in length, the cleavage sites in the antisense strand are typically approximately 10, 11, and 12 nt from the 5' end. Thus, three identical modification motifs can occur at positions 9, 10, 11, 10, 11, 12, 11, 12, 13, 12, 13, 14, or 13, 14, 15 of the antisense strand, starting from the first nucleotide from the 5' end of the antisense strand, or starting from the first paired nucleotide within the duplex region from the 5' end of the antisense strand. The cleavage site in the antisense strand can also vary depending on the length of the duplex region of the iRNA from the 5' end.

[0487] In some embodiments, the iRNA agent includes a sense strand and an antisense strand, each having 14 to 30 nucleotides, wherein the sense strand contains at least two motifs of three identical modifications on three consecutive nucleotides, at least one of the motifs occurring at or near the cleavage site within the strand, and at least one of the motifs occurring in another portion of the strand separated from the motif at the cleavage site by at least one nucleotide. In one embodiment, the antisense strand also contains at least one motif of three identical modifications on three consecutive nucleotides, at least one of the motifs occurring at or near the cleavage site within the strand. The modification in the motif occurring at or near the cleavage site within the sense strand is different from the modification in the motif occurring at or near the cleavage site within the antisense strand.

[0488] In some embodiments, the iRNA agent includes a sense strand and an antisense strand, each having 14 to 30 nucleotides, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, at least one of the motifs occurring at or near the cleavage site within the strand. In one embodiment, the antisense strand also contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0489] In some embodiments, the iRNA agent includes a sense strand and an antisense strand, each having 14-30 nucleotides, 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.

[0490] In one embodiment, the iRNA agent of the present invention contains mismatches with the target, within the duplex, or a combination thereof. Mismatches can occur in overhang regions or within the duplex region. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., based on the free energy of association or dissociation of a particular pairing; the simplest approach is to examine each pair individually, but adjacent or similar analysis can also be used). In terms of promoting dissociation, A:U is preferred to G:C, G:U is preferred to G:C, and I:C is preferred to G:C (I = inosine). Mismatches, such as non-standard or non-standard pairings (described elsewhere herein), are preferred to standard (A:T, A:U, G:C) pairings, and pairings involving universal bases are preferred to standard pairings.

[0491] In one embodiment, an iRNA agent of the invention can have the first 1, 2, 3, 4, or 5 base pairs in the duplex region from the 5' end of the antisense strand independently selected from the group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical or non-canonical pairings, or pairings containing universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0492] In one embodiment, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U and dT.Alternatively, at least one of the first 1, 2 or 3 base pairs in the double-stranded region from the 5' end of the antisense strand is an AU base pair.For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0493] In another embodiment, the nucleotide at the 3' end of the sense strand is deoxythymidine (dT). In another embodiment, the nucleotide at the 3' end of the antisense strand is deoxythymidine (dT). In one embodiment, there is a short sequence of deoxythymidine nucleotides, for example, two dT nucleotides on the 3' end of the sense strand or antisense strand.

[0494] In certain embodiments, the compositions and methods of the present disclosure include vinyl phosphonate (VP) modification of an RNAi agent as described herein. In an exemplary embodiment, a 5'-vinyl phosphonate modified nucleotide of the present disclosure has the following structure: [ka] wherein X is O or S; R is hydrogen, hydroxy, fluoro, or C 1~20 alkoxy (e.g., methoxy or n-hexadecyloxy); R 5’ is =C(H)-P(O)(OH)2, and the C5' carbon and R 5’ and the double bond between B is a nucleobase or modified nucleobase, optionally B is adenine, guanine, cytosine, thymine, or uracil.

[0495] The vinyl phosphonate of the present disclosure can be attached to either the antisense or sense strand of the dsRNA of the present disclosure. In certain embodiments, the vinyl phosphonate of the present disclosure is attached to the antisense strand of the dsRNA, optionally at the 5' end of the antisense strand of the dsRNA.

[0496] Vinyl phosphate modifications are also contemplated for the compositions and methods of the present disclosure. Exemplary vinyl phosphate structures include those described above, where R5' is =C(H)-OP(O)(OH)2, and the double bond between the C5' carbon and R5' is in the E or Z configuration (e.g., E configuration).

[0497] In one aspect, the present invention relates to double-stranded RNA (dsRNA) agents for inhibiting the expression of target genes with reduced off-target effects, as described in U.S. Patent Nos. 10,233,448, 10,612,024, and 10,612,027, and U.S. Patent Publication Nos. 2017 / 275626, 2019 / 241891, 2019 / 241893, and 2021 / 017519, the entire contents of each of which are incorporated herein by reference. As exemplified therein, for example, a motif containing a thermally destabilized nucleotide, such as i) a nucleotide that forms a mismatch with the opposite nucleotide in the antisense strand, ii) a nucleotide with an abasic modification, and / or iii) a nucleotide with a sugar modification and positioned opposite the seed region (positions 2-8), may be introduced into the sense strand.

[0498] In one embodiment, a dsRNA agent of the invention does not include any 2'-F modifications.

[0499] In one embodiment, the sense strand and / or antisense strand of a dsRNA agent comprises one or more blocks of phosphorothioate or methylphosphonate internucleotide linkages. In one example, the sense strand comprises one block of two phosphorothioate or methylphosphonate internucleotide linkages. In one example, the antisense strand comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages. For example, the two blocks of phosphorothioate or methylphosphonate internucleotide linkages are separated by 16 to 18 phosphate internucleotide linkages.

[0500] In one embodiment, the sense and antisense strands of the dsRNA agent each have 15-30 nucleotides. In one example, the sense strand has 19-22 nucleotides and the antisense strand has 19-25 nucleotides. In another example, the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides.

[0501] In one embodiment, the nucleotide at position 1 of the 5' end of the antisense strand in the duplex is selected from the group consisting of A, dA, dU, U, and dT. In one embodiment, at least one of the first, second, and third base pairs from the 5' end of the antisense strand is an AU base pair.

[0502] In one embodiment, the antisense strand of the dsRNA agent of the present invention is 100% complementary to the target RNA, hybridizes to the target RNA, and inhibits its expression through RNA interference.In another embodiment, the antisense strand of the dsRNA agent of the present invention is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% complementary to the target RNA.

[0503] In one aspect, the present invention relates to a dsRNA agent, as defined herein, capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The sense strand contains at least one thermally destabilized nucleotide, at least one of which occurs at or near the site opposite the seed region of the antisense strand (i.e., positions 2 to 8 of the 5' end of the antisense strand, or positions 2 to 9 of the 5' end of the antisense strand). Each of the embodiments and aspects described herein regarding dsRNAs represented by Formula (I) can also be applied to dsRNAs containing thermally destabilized nucleotides.

[0504] For example, when the sense strand is 21 nucleotides long, the thermally destabilizing nucleotide can occur between positions 14 and 17 at the 5' end of the sense strand. The antisense strand contains at least two modified nucleic acids that are smaller than the sterically demanding 2'-OMe modification. Preferably, the two modified nucleic acids that are smaller than the sterically demanding 2'-OMe modification are separated by a length of 11 nucleotides. For example, the two modified nucleic acids are located at positions 2 and 14 at the 5' end of the antisense strand.

[0505] In one embodiment, the dsRNA agent further comprises at least one ASGPR ligand. For example, the ASGPR ligand is [ka] In one example, the ASGPR ligand is attached to the 3' end of the sense strand.

[0506] For example, a dsRNA agent as defined herein can include: i) a phosphorus-containing group at the 5'-end of the sense strand or antisense strand; ii) two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5'-end of the sense strand), two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5'-end of the antisense strand); and iii) a ligand, such as an ASGPR ligand (e.g., one or more GalNAc derivatives at the 5'-end or 3'-end of the sense strand or antisense strand). For example, the ligand can be at the 3'-end of the sense strand.

[0507] In certain embodiments, a dsRNA agent of the invention comprises: (a) a sense strand having: (I) 21 nucleotides in length; (II) an ASGPR ligand optionally attached to the 3' end, the ASGPR ligand comprising three GALNAC derivatives attached through a trivalent branched linker; and (III) a sense strand having 2'-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 17, 19, and 21, and 2'-OME modifications at positions 2, 4, 6, 8, 12, 14 to 16, 18, and 20 (counting from the 5' end); and (b) an antisense strand having: (I) 23 nucleotides in length; (II) 2'-OME modifications at positions 1, 3, 5, 9, 11-13, 15, 17, 19, 21, and 23, and 2'-F modifications at positions 2, 4, 6-8, 10, 14, 16, 18, 20, and 22 (counting from the 5' end), and (III) an antisense strand having phosphorothioate internucleotide linkages 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.

[0508] In another specific embodiment, the dsRNA agent of the invention comprises: (a) a sense strand having: (i) 21 nucleotides in length; (ii) an ASGPR ligand optionally attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached through a trivalent branched linker; (iii) 2'-F modifications at positions 1, 3, 5, 7, 9-11, 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), and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11-13, 15, 17, 19, and 21-23, and 2'F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5' end), and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, 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.

[0509] In another specific embodiment, the dsRNA agent of the invention comprises: (a) a sense strand, (i) 21 nucleotides in length; (ii) an ASGPR ligand optionally attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached through a trivalent branched linker; (iii) 2'-OMe modifications at positions 1-6, 8, 10, and 12-21, 2'-F modifications at positions 7 and 9, and a deoxy-nucleotide (e.g., dT) at position 11 (counting from the 5' end), and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 7, 9, 11, 13, 15, 17, and 19-23, and 2'-F modifications at positions 2, 4 to 6, 8, 10, 12, 14, 16, and 18 (counting from the 5' end), and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, 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.

[0510] In another specific embodiment, the dsRNA agent of the invention comprises: (a) a sense strand, (i) 21 nucleotides in length; (ii) an ASGPR ligand optionally attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached through a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, 10, 12, 14, and 16 to 21, and 2'-F modifications at positions 7, 9, 11, 13, and 15, and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 5, 7, 9, 11, 13, 15, 17, 19, and 21-23, and 2'-F modifications at positions 2 to 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counting from the 5' end), and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, 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.

[0511] In another specific embodiment, the dsRNA agent of the invention comprises: (a) a sense strand, (i) 21 nucleotides in length; (ii) an ASGPR ligand optionally attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached through a trivalent branched linker; and (iii) 2'-OMe modifications at positions 1 to 9, 12 to 21, and 2'-F modifications at positions 10 and 11; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11-13, 15, 17, 19, and 21-23, and 2'-F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5' end), and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, 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.

[0512] In another specific embodiment, the dsRNA agent of the invention comprises: (a) a sense strand, (i) 21 nucleotides in length; (ii) an ASGPR ligand optionally attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached through a trivalent branched linker; (iii) 2'-F modifications at positions 1, 3, 5, 7, 9-11, and 13, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, and 14-21; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5-7, 9, 11-13, 15, 17-19, and 21-23, and 2'-F modifications at positions 2, 4, 8, 10, 14, 16, and 20 (counting from the 5' end), and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, 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.

[0513] In another specific embodiment, the dsRNA agent of the invention comprises: (a) a sense strand, (i) 21 nucleotides in length; (ii) an ASGPR ligand optionally attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached through a trivalent branched linker; (iii) 2'-OMe modifications at positions 1, 2, 4, 6, 8, 12, 14, 15, 17, and 19-21, and 2'-F modifications at positions 3, 5, 7, 9-11, 13, 16, and 18, and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 25 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 4, 6, 7, 9, 11-13, 15, 17, and 19-23, 2'-F modifications at positions 2, 3, 5, 8, 10, 14, 16, and 18, and deoxy-nucleotides (e.g., dT) at positions 24 and 25 (counting from the 5' end), and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); The dsRNA 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.

[0514] In another specific embodiment, the dsRNA agent of the invention comprises: (a) a sense strand, (i) 21 nucleotides in length; (ii) an ASGPR ligand optionally attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached through a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2'-F modifications at positions 7 and 9 to 11, and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 8, 10-13, 15, and 17-23, and 2'-F modifications at positions 2, 6, 9, 14, and 16 (counting from the 5' end), and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, 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.

[0515] In another specific embodiment, the dsRNA agent of the invention comprises: (a) a sense strand, (i) 21 nucleotides in length; (ii) an ASGPR ligand optionally attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached through a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2'-F modifications at positions 7 and 9 to 11, and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 10-13, 15, and 17-23, and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end), and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, 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.

[0516] In another specific embodiment, the dsRNA agent of the invention comprises: (a) a sense strand, (i) 19 nucleotides in length; (ii) an ASGPR ligand optionally attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached through a trivalent branched linker; (iii) 2'-OMe modifications at positions 1-4, 6, and 10-19, and 2'-F modifications at positions 5 and 7-9, and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand, (i) 21 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 21, and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end), and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (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.

[0517] In one embodiment, a dsRNA agent of the invention comprises: (a) a sense strand, (i) a length of 18 to 23 nucleotides; (ii) a sense strand having three consecutive 2'-F modifications at positions 7 to 15; (b) an antisense strand, (i) a length of 18 to 23 nucleotides; (ii) at least a 2'-F modification anywhere on the strand, and (iii) an antisense strand having at least two phosphorothioate internucleotide linkages in the first five nucleotides (counting from the 5' end), A dsRNA agent has one or more lipophilic moieties conjugated to one or more positions on at least one strand, and has either a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand, or blunt ends at both ends of the duplex.

[0518] In one embodiment, a dsRNA agent of the invention comprises: (a) a sense strand, (i) a length of 18 to 23 nucleotides; (ii) a sense strand having fewer than four 2'-F modifications; and (b) an antisense strand, (i) a length of 18 to 23 nucleotides; (ii) fewer than 12 2'-F modifications, and (iii) an antisense strand having at least two phosphorothioate internucleotide linkages in the first five nucleotides (counting from the 5' end), A dsRNA agent has one or more lipophilic moieties conjugated to one or more positions on at least one strand, and has either a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand, or blunt ends at both ends of the duplex.

[0519] In one embodiment, a dsRNA agent of the invention comprises: (a) a sense strand, (i) a length of 19 to 35 nucleotides; (ii) a sense strand having fewer than four 2'-F modifications; and (b) an antisense strand, (i) a length of 19 to 35 nucleotides; (ii) fewer than 12 2'-F modifications, and (iii) an antisense strand having at least two phosphorothioate internucleotide linkages in the first five nucleotides (counting from the 5' end), The duplex region is 19 to 25 base pairs (preferably 19, 20, 21, or 22), and the dsRNA agent has one or more lipophilic moieties conjugated to one or more positions on at least one strand, and has either a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand, or blunt ends at both ends of the duplex.

[0520] In one embodiment, a dsRNA agent of the invention comprises a sense strand and an antisense strand that are 15-30 nucleotides in length, have at least two phosphorothioate internucleotide linkages in the first five nucleotides on the antisense strand (counting from the 5' end), the duplex region is 19-25 base pairs (preferably 19, 20, 21, or 22), the dsRNA agent has one or more lipophilic moieties conjugated to one or more positions on at least one strand, and the dsRNA agent contains less than 20%, less than 15%, and less than 10% non-natural nucleotides.

[0521] Examples of non-natural nucleotides include acyclic nucleotides, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-ON-methylacetamido (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F, and others.

[0522] In one embodiment, a dsRNA agent of the invention comprises a sense strand and an antisense strand that are 15-30 nucleotides in length, have at least two phosphorothioate internucleotide linkages in the first five nucleotides on the antisense strand (counting from the 5' end), the duplex region is 19-25 base pairs (preferably 19, 20, 21, or 22), the dsRNA agent has one or more lipophilic moieties conjugated to one or more positions on at least one strand, and the dsRNA agent has greater than 80%, greater than 85%, and greater than 90% natural nucleotides, such as 2'-OH, 2'-deoxy, and 2'-OMe.

[0523] In one embodiment, a dsRNA agent of the invention comprises a sense strand and an antisense strand that are 15-30 nucleotides in length, have at least two phosphorothioate internucleotide linkages in the first five nucleotides on the antisense strand (counting from the 5' end), the duplex region is 19-25 base pairs (preferably 19, 20, 21, or 22), the dsRNA agent has one or more lipophilic moieties conjugated to one or more positions on at least one strand, and the dsRNA agent has 100% naturally occurring nucleotides, such as 2'-OH, 2'-deoxy, and 2'-OMe.

[0524] Multimeric siRNAs are described in various publications, and all of them can be used with the iRNA of the present invention.Such publications include International Publication No. 2007 / 091269, US Patent No. 7858769, International Publication No. 2010 / 141511, International Publication No. 2007 / 117686, International Publication No. 2009 / 014887 and International Publication No. 2011 / 031520, which are incorporated herein by reference in their entirety.

[0525] In some embodiments, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of the iRNA agents of the invention are modified.

[0526] In some embodiments, each of the sense and antisense strands of an iRNA agent is independently modified with an acyclic nucleotide, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-ON-methylacetamido (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.

[0527] In some embodiments, the sense and antisense strands of an iRNA agent each contain at least two different modifications.

[0528] In some embodiments, a dsRNA agent of the invention does not contain any 2'-F modifications.

[0529] In some embodiments, a dsRNA agent of the invention contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 2'-F modifications. In one example, a dsRNA agent of the invention contains 9 or 10 2'-F modifications.

[0530] The iRNA agent of the present invention may further comprise at least one phosphorothioate or methylphosphonate internucleotide bond.The phosphorothioate or methylphosphonate internucleotide bond modification can occur on the nucleotide of either sense strand or antisense strand or both strands at any position of the strand.For example, internucleotide bond modification can occur on any nucleotide on sense strand or antisense strand, and each internucleotide bond modification can occur in an alternating pattern on sense strand or antisense strand, or sense strand or antisense strand can contain both internucleotide bond modifications in an alternating pattern.The alternating pattern of internucleotide bond modification on sense strand can be the same or different from that of antisense strand, and the alternating pattern of internucleotide bond modification on sense strand can have a relative shift with respect to the alternating pattern of internucleotide bond modification on antisense strand.

[0531] In one embodiment, the iRNA comprises a phosphorothioate or methylphosphonate internucleotide bond modification in the overhang region. For example, the overhang region can contain two nucleotides with a phosphorothioate or methylphosphonate internucleotide bond between the two nucleotides. The internucleotide bond modification can also be configured to link the overhang nucleotide to the terminal paired nucleotide in the duplex region. For example, at least 2, 3, 4, or all of the overhang nucleotides can be linked by a phosphorothioate or methylphosphonate internucleotide bond, and optionally, there can be an additional phosphorothioate or methylphosphonate internucleotide bond connecting the overhang nucleotide to the paired nucleotide adjacent to the overhang nucleotide. For example, there can be at least two phosphorothioate internucleotide bonds between the terminal three nucleotides, two of which are overhang nucleotides and the third is the paired nucleotide adjacent to the overhang nucleotide. Preferably, these terminal three nucleotides can be at the 3' end of the antisense strand.

[0532] In some embodiments, the sense strand and / or antisense strand of an iRNA agent include one or more blocks of phosphorothioate or methylphosphonate internucleotide linkages. In one example, the sense strand includes one block of two phosphorothioate or methylphosphonate internucleotide linkages. In one example, the antisense strand includes two blocks of two phosphorothioate or methylphosphonate internucleotide linkages. For example, the two blocks of phosphorothioate or methylphosphonate internucleotide linkages are separated by 16 to 18 phosphate internucleotide linkages.

[0533] In some embodiments, the antisense strand of an iRNA agent of the invention is 100% complementary to the target RNA, hybridizes to the target RNA, and inhibits its expression through RNA interference. In other embodiments, the antisense strand of an iRNA agent of the invention is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% complementary to the target RNA.

[0534] In one aspect, the present invention relates to an iRNA agent capable of inhibiting expression of a target gene. The iRNA agent includes a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The sense strand contains at least one thermally destabilized nucleotide, which occurs at or near the site opposite the seed region of the antisense strand (i.e., at positions 2 to 8 of the 5' end of the antisense strand or at positions 2 to 9 of the 5' end of the antisense strand). For example, the thermally destabilized nucleotide occurs between positions 14 and 17 of the 5' end of the sense strand when the sense strand is 21 nucleotides long. The antisense strand contains at least two modified nucleic acids that are less than sterically demanding 2'-OMe modifications. Preferably, the two modified nucleic acids that are less than sterically demanding 2'-OMe modifications are separated by a length of 11 nucleotides. For example, the two modified nucleic acids are at positions 2 and 14 of the 5' end of the antisense strand.

[0535] In some embodiments, the compounds of the present invention disclosed herein are miRNA mimics. In one design, miRNA mimics are double-stranded molecules (e.g., having a duplex region of about 16 to about 31 nucleotides in length) containing one or more sequences that share identity with the mature strand of a given miRNA. Double-stranded miRNA mimics have designs similar to those described above for double-stranded iRNAs. In some embodiments, miRNA mimics include a 16-31 nucleotide duplex region and one or more of the following chemical modification patterns: the sense strand contains 2'-O-methyl modifications of nucleotides 1 and 2 (counting from the 5' end of the sense oligonucleotide) and all Cs and Us; and the antisense strand modifications include 2'F modifications of all Cs and Us, phosphorylation of the 5' end of the oligonucleotide, and stabilized internucleotide linkages associated with a two-nucleotide 3' overhang.

[0536] VC 22 hydrocarbon chain As described in U.S. Provisional Patent Application No. 63 / 255,984, filed October 15, 2021, the entire contents of which are incorporated herein by reference, 22 The inclusion of a hydrocarbon chain, e.g., saturated or unsaturated, on one or more internal positions of a dsRNA agent increases the lipophilicity of the dsRNA agent and provides optimal hydrophobicity for enhanced in vivo delivery of the dsRNA, e.g., to muscle and / or adipose tissue.

[0537] One way to characterize lipophilicity is by the octanol-water partition coefficient, log K ow where K owis the ratio of the concentration of a chemical in the octanol phase to the concentration of the chemical in the aqueous phase of a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it can also be predicted by using coefficients attributable to the structural components of a chemical calculated using first principles or empirical methods (see, e.g., Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), incorporated herein by reference in its entirety). It provides a thermodynamic measure of a substance's tendency to prefer a non-aqueous or oily environment rather than water (i.e., its hydrophilic / lipophilic balance). In principle, a chemical can be expressed as its logK ow is greater than 0, the molecule is lipophilic in nature. Typically, a lipophilic moiety has a log K ow For example, the log K of 6-aminohexanol ow For example, the log K of cholesteryl N-(hexan-6-ol)carbamate is predicted to be approximately 0.7. ow is predicted to be 10.7.

[0538] The lipophilicity of a molecule can vary depending on the functional groups it contains. For example, C 22 C by adding a hydroxyl or amine group to the end of the hydrocarbon chain 22 Partition coefficients of hydrocarbon chains (e.g., log K ow ) can be increased or decreased.

[0539] Alternatively, one or more C 22 The hydrophobicity of the dsRNA agent that is conjugated with hydrocarbon chain can be measured by its protein binding property.For example, the unbound fraction of the plasma protein binding assay of dsRNA agent can be determined to be positively correlated with the relative hydrophobicity of dsRNA agent, and this can be positively correlated with the silencing activity of dsRNA agent.

[0540] In one embodiment, the plasma protein binding assay that is determined is electrophoretic mobility shift assay (EMSA) using human serum albumin protein.The hydrophobicity of dsRNA agent measured by the fraction of unbound dsRNA in binding assay is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45 or greater than 0.5 for enhancing siRNA in vivo delivery.

[0541] In certain embodiments, one or more C 22 The hydrocarbon chain may be aliphatic, cycloaliphatic, or polyaliphatic, and may be aliphatic, cyclic, such as cycloaliphatic, or polycyclic, such as polyaliphatic. The hydrocarbon chain may contain various substituents and / or one or more heteroatoms, such as oxygen or nitrogen atoms.

[0542] One or more C's 22 The hydrocarbon chain may be attached to the iRNA agent by any method known in the art, including through a functional group already present in the lipophilic moiety or through a functional group introduced into the iRNA agent, such as a hydroxy group (e.g., -CO-CH-OH). 22 Functional groups already present in the hydrocarbon chain or introduced into the dsRNA agent include, but are not limited to, hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0543] dsRNA agents and C 22Conjugation with a hydrocarbon chain can occur, for example, through the formation of an ether or carboxy or carbamoyl ester bond between a hydroxy and an alkyl group R-, an alkanoyl group RCO-, or a substituted carbamoyl group RNHCO-. The alkyl group R can be cyclic (e.g., cyclohexyl) or acyclic (e.g., straight-chain or branched-chain, and saturated or unsaturated). The alkyl group R can be butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, or octadecyl group, etc.

[0544] In some embodiments, C 22 The hydrocarbon chain is conjugated to the dsRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide bond, the product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate.

[0545] In one embodiment, one or more C 22 The hydrocarbon chain is C 22 Acids such as C 22 The acid is selected from the group consisting of docosanoic acid, 6-octyltetradecanoic acid, 10-hexylhexadecanoic acid, all-cis-7,10,13,16,19-docosapentaenoic acid, all-cis-4,7,10,13,16,19-docosahexaenoic acid, all-cis-13,16-docosadienoic acid, all-cis-7,10,13,16-docosatetraenoic acid, all-cis-4,7,10,13,16-docosapentaenoic acid, and cis-13-docosenoic acid. [ka]

[0546] In one embodiment, one or more C 22 The hydrocarbon chain is C 22 Alcohols, such as C 22The alcohol is selected from the group consisting of 1-docosanol, 6-octyltetradecan-1-ol, 10-hexylhexadecan-1-ol, cis-13-docosen-1-ol, docosan-9-ol, docosan-2-ol, docosan-10-ol, docosan-11-ol, and cis-4,7,10,13,16,19-docosahexanol. [ka]

[0547] In one embodiment, one or more C 22 The hydrocarbon chain is not cis-4,7,10,13,16,19-docosahexaenoic acid. 22 The hydrocarbon chain is not cis-4,7,10,13,16,19-docosahexanol. 22 The hydrocarbon chain is neither cis-4,7,10,13,16,19-docosahexaenoic acid nor cis-4,7,10,13,16,19-docosahexanol.

[0548] In one embodiment, one or more C 22 The hydrocarbon chain is C 22 Amides, e.g., C 22 The amide is selected from the group consisting of (E)-docosa-4-enamide, (E)-docosa-5-enamide, (Z)-docosa-9-enamide, (E)-docosa-11-enamide, 12-docosenamid, (Z)-docosa-13-enamide, (Z)-N-hydroxy-13-docosenamid, (E)-docosa-14-enamide, 6-cis-docosenamid, 14-docosenamidocos-11-enamide, (4E,13E)-docosa-4,13-dienamide, and (5E,13E)-docosa-5,13-dienamide.

[0549] In certain embodiments, in particular C 22 If the hydrocarbon chain has low lipophilicity or hydrophobicity, two or more C 22 A hydrocarbon chain can be incorporated into the double-stranded iRNA agent. In one embodiment, two or more C22 The hydrocarbon chains are incorporated into the same strand of the double-stranded iRNA agent. In one embodiment, each strand of the double-stranded iRNA agent has one or more incorporated C 22 In one embodiment, two or more C 22 The hydrocarbon chains are incorporated into the same position (i.e., the same nucleobase, the same sugar moiety, or the same internucleoside linkage) of the double-stranded iRNA agent. This can be achieved, for example, by incorporating two or more saturated or unsaturated C 22 Conjugating hydrocarbon chains and / or two or more C 22 Conjugating hydrocarbon chains and / or two or more C 22 The hydrocarbon chain is 22 This can be achieved by conjugating with one or more linkers that connect the hydrocarbon chains consecutively.

[0550] One or more C's 22 The hydrocarbon chain may be conjugated to the iRNA agent via a direct bond to the ribosugar of the iRNA agent. Alternatively, one or more C 22 The hydrocarbon chain may be conjugated to the double-stranded iRNA agent via a linker or carrier.

[0551] In certain embodiments, one or more C 22 The hydrocarbon chain may be conjugated to the iRNA agent via one or more linkers (tethers).

[0552] In one embodiment, one or more C 22 The hydrocarbon chain is conjugated to the dsRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide bond, the product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate.

[0553] A. Linker / Tether The linker / tether may have one or more C 22 The linker / tether is connected to a hydrocarbon chain. 100 Carbon-containing moieties (e.g., C1-C 75 , C1-C 50 , C1-C 20 , C1-C 10 , C1, C2, C3, C4, C5, C6, C7, C8, C9, or C 10 ), and may contain at least one nitrogen atom. In certain embodiments, the nitrogen atom forms part of a terminal amino or amido (NHC(O)-) group on the linker / tether, which may serve as an attachment point for a lipophilic moiety. Non-limiting examples of linkers / tethers (underlined) include TAP -(CH 2 ) n NH- ;TAP- C(O)(CH 2 ) n NH- ;TAP- NR''''(CH 2 ) n NH- , TAP- C(O)-(CH 2 ) n -C(O)- ;TAP- C(O)-(CH 2 ) n -C(O)O-; TAP- C(O)-O- ;TAP- C(O)-(CH 2 ) n -NH-C(O)- ;TAP- C(O)-(CH 2 ) n - ;TAP- C(O)-NH- ;TAP- C(O)- ;TAP- (CH 2 ) n -C(O)- ;TAP- (CH 2 ) n -C(O)O- ;TAP- (CH 2 ) n - ;, or TAP- (CH 2 ) n -NH-C(O)- where n is 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and R"" is C1-C6 alkyl. Preferably, N is 5, 6, or 11. In other embodiments, the nitrogen may form part of a terminal oxyamino group, e.g., -ONH2, or a hydrazino group, -NHNH2. The linker / tether may be optionally substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and / or may optionally be inserted with one or more additional heteroatoms, e.g., N, O, or S. Preferred tethering ligands include, for example, TAP -(CH 2 ) n NH (ligand) , TAP- C(O)(CH 2 ) n NH (ligand) , TAP- NR''''(CH 2 ) n NH (ligand) , TAP -(CH 2 ) n ONH (ligand) , TAP- C(O)(CH 2 ) n ONH (ligand) , TAP- NR''''(CH 2 ) n ONH (ligand) , TAP- (CH 2 ) n NHNH 2 (ligand) , TAP- C(O)(CH 2 ) n NHNH 2 (ligand) , TAP- NR''''(CH 2 ) n NHNH 2 (ligand) , TAP- C(O)-(CH 2 ) n -C(O)(ligand) , TAP- C(O)-(CH 2) n -C(O)O(ligand), TAP- C(O)-O(ligand) , TAP- C(O)-(CH 2 ) n -NH-C(O)(ligand) , TAP- C(O)-(CH 2 ) n (ligand) , TAP- C(O)-NH(ligand) , TAP- C(O)(ligand) , TAP- (CH 2 ) n -C(O)(ligand) , TAP- (CH 2 ) n -C(O)O(ligand) , TAP- (CH 2 ) n (ligand) , or TAP- (CH 2 ) n -NH-C(O)(ligand) In some embodiments, the amino-terminated linker / tether (e.g., NH, ONH, NHNH) can form an imino bond (i.e., C=N) with the ligand. In some embodiments, the amino-terminated linker / tether (e.g., NH, ONH, NHNH) can be acylated with, for example, C(O)CF.

[0554] In some embodiments, the linker / tether can terminate in a mercapto group (i.e., SH) or an olefin (e.g., CH=CH). For example, the tether can be -(CH 2 ) n -SH , TAP- C(O)(CH 2 ) n SH , TAP -(CH 2 ) n -(CH=CH 2 ) , or TAP- C(O)(CH 2 ) n (CH=CH 2 )where n can be as otherwise described. The tether can be optionally substituted, for example, with hydroxy, alkoxy, perhaloalkyl, and / or optionally inserted with one or more additional heteroatoms, for example, N, O, or S. The double bond can be cis or trans or E or Z.

[0555] In other embodiments, the linker / tether may include an electrophilic moiety, preferably at a terminal position of the linker / tether. Exemplary electrophilic moieties include, for example, an aldehyde, an alkyl halide, a mesylate, a tosylate, a nosylate, or a brosylate, or an activated carboxylic acid ester, for example, an NHS ester, or a pentafluorophenyl ester. Preferred linkers / tethers (underlined) include TAP -(CH 2 ) n CHO , TAP- C(O)(CH 2 ) n CHO , or TAP- NR''''(CH 2 ) n CHO where n is 1-6 and R"" is C1-C6 alkyl, or TAP -(CH 2 ) n C(O)ONHS That is, TAP- C(O)(CH 2 ) n C(O)ONHS , or TAP- NR''''(CH 2 ) n C(O)ONHS , where n is 1-6, and R"" is C1-C6 alkyl, TAP- (CH 2 ) n C(O)OC 6 F 5. TAP- C(O)(CH 2 ) n C(O)OC 6 F 5, or TAP- NR''''(CH 2) n C(O)OC 6 F 5, where n is 1-11 and R'''' is C1-C6 alkyl, or -(CH 2 ) n CH 2 LG , TAP- C(O)(CH 2 ) n CH 2 LG , or TAP- NR''''(CH 2 ) n CH 2 LG where n can be as described above and R''' is C-C alkyl (LG can be a leaving group, e.g., halide, mesylate, tosylate, nosylate, brosylate). Tethering can be achieved by coupling a nucleophilic group on the ligand, e.g., a thiol or amino group, with an electrophilic group on the tether.

[0556] In other embodiments, the monomer may have a phthalimide group (K) at the terminal position of the linker / tether. [ka] It may be desirable to include:

[0557] In other embodiments, other protected amino groups at the terminal positions of the linker / tether can be, for example, an alloc group, monomethoxytrityl (MMT), trifluoroacetyl, Fmoc, or arylsulfonyl (e.g., the aryl moiety can be ortho-nitrophenyl or ortho, para-dinitrophenyl).

[0558] Any of the linkers / tethers described herein may contain one or more additional linking groups, such as -O-(CH) n -, -(CH2) n -SS-, -(CH2) nIt may further contain - or -(CH=CH)-.

[0559] B. Cleavable Linker / Tether In some embodiments, at least one of the linkers / tethers can be a redox-cleavable linker, an acid-cleavable linker, an esterase-cleavable linker, a phosphatase-cleavable linker, or a peptidase-cleavable linker.

[0560] In one embodiment, at least one of the linkers / tethers can be a reductively cleavable linker (eg, a disulfide group).

[0561] In one embodiment, at least one of the linkers / tethers can be an acid-cleavable linker (eg, a hydrazone group, an ester group, an acetal group, or a ketal group).

[0562] In one embodiment, at least one of the linkers / tethers can be an esterase-cleavable linker (eg, an ester group).

[0563] In one embodiment, at least one of the linkers / tethers can be a phosphatase-cleavable linker (eg, a phosphate group).

[0564] In one embodiment, at least one of the linkers / tethers can be a peptidase-cleavable linker (eg, a peptide bond).

[0565] Cleavable linking groups are susceptible to cleaving agents, such as pH, redox potential, or the presence of degradative molecules. Cleavage agents are generally more widespread or found at higher levels or activity inside cells than in serum or blood. Examples of such degradative agents include redox agents that are selective for a specific substrate or have no substrate specificity, e.g., oxidases or reductases or reducing agents present in cells, such as mercaptans, that can degrade redox-cleavable linking groups by reduction; esterases; reagents that can create an endosome or acidic environment, e.g., reagents that result in 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.

[0566] Cleavable linking groups, such as disulfide bonds, can be pH sensitive. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some tethers will have a linking group that is cleaved at a preferred pH, thereby releasing the iRNA agent from an intracellular ligand (e.g., a targeting or cell-permeable ligand such as cholesterol) or into the interior of the cell or into a desired compartment of the cell.

[0567] The chemical bond (e.g., linking group) that connects the ligand to the iRNA agent can include a disulfide bond. When the iRNA agent / ligand complex is taken up into the cell by endocytosis, the acidic environment of the endosome cleaves the disulfide bond, thereby releasing the iRNA agent from the ligand (Quintana et al., Pharm Res. 19:1310-1316, 2002; Patri et al., Curr. Opin. Curr. Biol. 6:466-471, 2002). The ligand can be a targeting ligand or a second therapeutic agent that can complement the therapeutic effect of the iRNA agent.

[0568] The tether can include a cleavable linking group that can be cleaved by a specific enzyme. The type of linking group incorporated into the tether can depend on the cell targeted by the iRNA agent. For example, an iRNA agent targeting mRNA in liver cells can be conjugated to a tether containing an ester group. Liver cells are rich in esterases, and therefore the tether will be cleaved more efficiently in liver cells than in cell types that are not rich in esterases. Cleavage of the tether releases the iRNA agent from the ligand attached to the distal end of the tether, thereby potentially enhancing the silencing activity of the iRNA agent. Other cell types rich in esterases include lung, renal cortex, and testicular cells.

[0569] Tethers containing peptide bonds can be conjugated to iRNA agents that target cell types rich in peptidases, such as hepatocytes and synoviocytes. For example, iRNA agents that target synoviocytes, such as for the treatment of inflammatory diseases (e.g., rheumatoid arthritis), can be conjugated to tethers containing peptide bonds.

[0570] Generally, the suitability of a candidate cleavable linking group can be evaluated by testing the ability (or conditions) of a degradation agent to cleave the candidate linking group. It may also be desirable to test the candidate cleavable linking group for its ability to resist cleavage when in blood or other non-target tissues, such as the tissues to which the iRNA agent will be exposed when administered to a subject. Thus, the relative susceptibility to cleavage between a first condition and a second condition can be determined, where the first condition is selected to exhibit cleavage in target cells, and the second condition is selected to exhibit cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to perform initial evaluation in a cell-free or culture condition and confirm by further evaluation in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least 2, 4, 10, 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).

[0571] C. Redox-cleavable linking groups One class of cleavable linking groups is redox-cleavable linking groups, which cleave upon reduction or oxidation. An example of a reductively cleavable linking group is a disulfide bond (-SS-). To determine whether a candidate cleavable linking group is a suitable "reductively cleavable linking group," or suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, one can turn to the methods described herein. For example, candidates can be evaluated in cells by incubating with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate that would be observed in target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In preferred embodiments, candidate compounds are cleaved at most 10% in blood. In preferred embodiments, useful candidate compounds are degraded at least 2, 4, 10, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic the intracellular medium and compared to conditions selected to mimic the extracellular medium.

[0572] D. Phosphate-based cleavable linking groups Phosphate-based linking groups are cleaved by agents that decompose or hydrolyze phosphate groups. Examples of agents that cleave phosphate groups in cells include enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, and -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0573] E. Acid-cleavable linking groups

[0574] An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linking group is cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0 or less), or by an agent 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 the acid-cleavable linking group. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, ketals, acetals, esters, and esters of amino acids. Acid-cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). In a preferred embodiment, the carbon bonded to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group, such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0575] F. Ester-based bonding groups Ester-based linking groups are cleaved in cells by enzymes such as esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, 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.

[0576] G. Peptide-Based Cleavage Groups Peptide-based linking groups are cleaved intracellularly by enzymes such as peptidases and proteases. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to produce peptides and proteins. Peptide-based cleaving groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to produce peptides and proteins, and do not include all amide functional groups. Peptide cleavable linking groups have the general formula -NHCHR 1 C(O)NHCHR 2 C(O)—, wherein R 1 and R 2 are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0577] H. Biocleavable Linkers / Tethers

[0578] Linker can also include the biocleavable linker, which is a nucleotide linker and a non-nucleotide linker or a combination thereof, which connects two parts of a molecule, for example, one or both chains of two individual siRNA molecules to generate bis(siRNA).In some embodiments, the simple electrostatic interaction or stacking interaction between two individual siRNAs can represent linker.Non-nucleotide linkers include tethers or linkers derived from monosaccharides, disaccharides, oligosaccharides, and their derivatives, aliphatic, alicyclic, heterocyclic, and their combinations.

[0579] In some embodiments, at least one of the linkers (tethers) is a biocleavable linker selected from the group consisting of DNA, RNA, disulfides, amides, and functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, and mannose, and combinations thereof.

[0580] In one embodiment, the biocleavable carbohydrate linker may have 1-10 sugar units, with at least one anomeric linkage capable of connecting two siRNA units. When more than one sugar is present, the units can be linked via 1-3, 1-4, or 1-6 sugar linkages, or via an alkyl chain.

[0581] Exemplary biocleavable linkers include: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and [ka] Examples include:

[0582] Further discussion of biocleavable linkers may be found in PCT Application No. PCT / US18 / 14213, entitled "Endosomal Cleavable Linkers," filed January 18, 2018, the entire contents of which are incorporated herein by reference.

[0583] I. Carriers

[0584] In certain embodiments, one or more C 22 The hydrocarbon chain is conjugated to the iRNA agent via a carrier that replaces one or more nucleotides.

[0585] The carrier can be a cyclic group or an acyclic group. In one embodiment, the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin. In one embodiment, the acyclic group is a moiety based on a serinol skeleton or a diethanolamine skeleton.

[0586] In some embodiments, the carrier replaces one or more nucleotides at an internal position of the dsRNA agent.

[0587] In other embodiments, the carrier replaces the nucleotide at the end of sense strand or antisense strand.In one embodiment, the carrier replaces the terminal nucleotide on the 3'-end of sense strand, thereby functioning as the end cap that protects the 3'-end of sense strand.In one embodiment, the carrier is a cyclic group that has amine, for example, the carrier can be pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl or decalinyl.

[0588] A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modified subunit (PRMS). The carrier can be a cyclic or acyclic moiety and includes two "backbone attachment points" (e.g., hydroxyl groups) and a ligand (e.g., a lipophilic moiety). One or more C 22 The hydrocarbon chain can be directly attached to the carrier, as described above, or can be indirectly attached to the carrier by means of an intervening linker / tether. [ka]

[0589] The ligand-conjugated monomer subunit can be the 5'- or 3'-terminal subunit of an iRNA molecule, i.e., one of the two "W" groups can be a hydroxyl group and the other "W" group can be a chain of two or more unmodified or modified ribonucleotides. Alternatively, the ligand-conjugated monomer subunit can occupy an internal position and both "W" groups can be one or more unmodified or modified ribonucleotides. There can be more than one ligand-conjugated monomer subunit within an iRNA agent.

[0590] a. Sugar-substituted based monomers, e.g., ligand-conjugated monomers (cyclic)

[0591] Cyclic sugar-substituted based monomers, e.g., sugar-substituted based ligand-conjugated monomers, are also referred to herein as RRMS monomer compounds. The carriers may have the general formula (LCM-2) provided below (in which the...

Claims

Claim 1 A double-stranded ribonucleic acid (dsRNA) agent or a pharmaceutically acceptable salt thereof for inhibiting the expression of a metabolic disorder-related target gene selected from the group consisting of inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), and inhibin subunit beta C (INHBC), wherein the dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, a) when the metabolic disorder-related target gene is INHBE, the antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence 5'-ACAGACAAGAAAGUGCCCCAUUUG-3' by 3 or fewer nucleotides, wherein all nucleotides of the sense strand and all nucleotides of the antisense strand comprise nucleotide modifications; b) the sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence 5'-asasugggcaCfUfUfucuugucugu-3' by 4 or fewer modified or unmodified nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence 5'-asdCsagdAcdaagaadAgUfgccccaususg by 4 or fewer modified or unmodified nucleotides, wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively; Cf and Uf are 2'-fluoro C and U, respectively; dC is 2'-deoxycytidine-3'-phosphate nucleotide; dA is 2'-deoxyadenosine-3'-phosphate nucleotide; and s is a phosphorothioate bond, c) when the metabolic disorder-related target gene is INHBE, the antisense strand comprises at least 15 consecutive nucleotides that differ from any one of the antisense strand nucleotide sequences selected from the group consisting of 5'-AAAGACGG CAGAAUGGAAAGAGG-3', 5'-AAGAAA GUAUAAAUGCUUGUCUC-3', 5'-AGUU AUTCUGGGACGACUGGUCU-3', and 5'-AAAGCCAGAGUCUCAGACAAGA-3' by 3 or fewer nucleotides, All nucleotides of the sense strand and all nucleotides of the antisense strand contain nucleotide modifications; d) The sense strand and the antisense strand are (i) 5'-uscsuuuccauFfCfugccgucuuu-3' and 5'-asdAsagdAcddGgcaagdAaUfggaagagasg-3'; (ii) 5'-gsascagcaUfUfUfauacuuucuu-3' and 5'-asdAsgadAadGuauadAaUfgcuugucsusc; (iii) 5'-ascscagucgUfCfCfcagaauaac u-3' and 5'-asdGsuddAudTcuggdGaCf gacugguscs u; and (iv) 5'-csususguUfgAfGfAfcu cuggc uuu-3' and 5'-asAf sagdCc(Ag n)gaguc uCf aGf acaagsasa comprising at least 15 consecutive nucleotides that differ by no more than 4 nucleotides from any of the sense strand and antisense strand nucleotide sequences selected from the group consisting of wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively; Gf, Cf, and Uf are 2'-fluoro G, C, and U, respectively; dG is 2'-deoxyguanosine-3'-phosphate; dT is 2'-deoxythymidine-3'-phosphate; dC is 2'-deoxycytidine-3'-phosphate nucleotide; dA is 2'-deoxyadenosine-3'-phosphate nucleotide; (Agn) is adenosine-glycol nucleic acid (GNA); and s is a phosphorothioate bond, e) The target gene related to metabolic disorder is INHBE, and the antisense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the complement of any one of the nucleotide sequences of nucleotides 400-422, 1430-1452, 1863-1885, 410-432, 518-540, 519-541, 640-662, or 1864-1886 of SEQ ID NO: 1, All nucleotides of the sense strand and all nucleotides of the antisense strand contain nucleotide modifications; f) The sense strand contains at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, or 55, and the antisense strand contains at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the corresponding portion of any one of the nucleotide sequences of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56. All of the nucleotides of the sense strand and all of the nucleotides of the antisense strand contain nucleotide modifications; or g) The antisense strand contains a complementary region to the mRNA encoding the target gene, and the complementary region contains at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any one of the antisense nucleotide sequences in any one of the following tables 【Table 1-1】 【Table 1-2】 【Table 1-3】 【Table 1-4】 【Table 1-5】 【Table 2-1】 【Table 2-2】 【Table 2-3】 【Table 2-4】 【Table 2-5】 【Table 3-1】 【Table 3-2】 【Table 3-3】 【Table 3-4】 【Table 3-5】 【Table 3-6】 【Table 3-7】 【Table 3-8】 【Table 3-9】 【Table 3-10】 【Table 4-1】 【Table 4-2】 【Table 4-3】 【Table 4-4】 【Table 4-5】 【Table 4-6】 【Table 4-7】 【Table 4-8】 【Table 4-9】 【Table 4-10】 【Table 4-11】 【Table 4-12】 【Table 4-13】 【Table 4-14】 【Table 4-15】 【Table 4-16】 【Table 5-1】 【Table 5-2】 【Table 5-3】 【Table 5-4】 【Table 5-5】 【Table 5-6】 【Table 5-7】 【Table 5-8】 【Table 5-9】 【Table 5-10】 【Table 6-1】 【Table 6-2】 【Table 6-3】 【Table 6-4】 【Table 6-5】 【Table 6-6】 【Table 6-7】 【Table 6-8】 【Table 6-9】 【Table 6-10】 【Table 6-11】 【Table 6-12】 【Table 6-13】 【Table 6-14】 【Table 6-15】 【Table 6-16】 【Table 7-1】 【Table 7-2】 【Table 7-3】 【Table 7-4】 【Table 7-5】 【Table 8-1】 【Table 8-2】 【Table 8-3】 【Table 8-4】 【Table 8-5】 【Table 8-6】 【Table 8-7】 【Table 8-8】 【Table 9-1】 【Table 9-2】 【Table 9-3】 【Table 9-4】 【Table 9-5】 【Table 10-1】 【Table 10-2】 【Table 10-3】 【Table 10-4】 【Table 10-5】 【Table 10-6】 【Table 10-7】 【Table 10-8】 【Table 10-9】 【Table 11-1】 【Table 11-2】 【Table 11-3】 【Table 11-4】 【Table 11-5】 【Table 11-6】 【Table 11-7】 【Table 11-8】 【Table 11-9】 【Table 11-10】 【Table 12-1】 【Table 12-2】 【Table 12-3】 【Table 12-4】 【Table 12-5】 【Table 12-6】 【Table 12-7】 【Table 12-8】 【Table 12-9】 【Table 12-10】 【Table 12-11】 【Table 12-12】 【Table 12-13】 【Table 12-14】 【Table 12-15】 【Table 12-16】 【Table 13-1】 【Table 13-2】 【Table 13-3】 【Table 13-4】 【Table 13-5】 【Table 13-6】 【Table 13-7】 【Table 13-8】 【Table 13-9】 【Table 13-10】 【Table 14-1】 【Table 14-2】 【Table 14-3】 【Table 14-4】 【Table 14-5】 【Table 14-6】 【Table 14-7】 【Table 14-8】 【Table 14-9】 【Table 14-10】 【Table 14-11】 【Table 14-12】 【Table 14-13】 【Table 14-14】 【Table 14-15】 【Table 14-16】 【Table 15-1】 【Table 15-2】 【Table 15-3】 【Table 15-4】 【Table 15-5】 【Table 15-6】 【Table 15-7】 【Table 15-8】 【Table 16-1】 【Table 16-2】 【Table 16-3】 【Table 16-4】 【Table 16-5】 【Table 16-6】 【Table 16-7】 【Table 16-8】 【Table 17-1】 【Table 17-2】 【Table 17-3】 【Table 17-4】 【Table 17-5】 【Table 17-6】 【Table 17-7】 【Table 17-8】 【Table 17-9】 【Table 17-10】 【Table 17-11】 【Table 17-12】 【Table 17-13】 【Table 17-14】 【Table 17-15】 【Table 17-16】 【Table 17-17】 【Table 17-18】 【Table 17-19】 【Table 17-20】 【Table 17-21】 【Table 17-22】 【Table 17-23】 【Table 17-24】 【Table 17-25】 【Table 18-1】 【Table 18-2】 【Table 18-3】 【Table 18-4】 【Table 18-5】 【Table 18-6】 【Table 18-7】 【Table 18-8】 【Table 18-9】 【Table 18-10】 【Table 18-11】 【Table 18-12】 【Table 18-13】 【Table 18-14】 【Table 18-15】 【Table 18-16】 【Table 18-17】 【Table 18-18】 【Table 18-19】 【Table 18-20】 【Table 18-21】 【Table 18-22】 【Table 18-23】 【Table 18-24】 【Table 18-25】 and all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand contain nucleotide modifications. All of the nucleotides of the sense strand and all of the nucleotides of the antisense strand contain nucleotide modifications. A double-stranded ribonucleic acid (dsRNA) agent or a pharmaceutically acceptable salt thereof. Claim 2 At least one of the nucleotide modifications is selected from the group consisting of deoxy-nucleotide modification, 3'-terminal deoxythymidine (dT) nucleotide modification, 2'-O-methyl nucleotide modification, 2'-fluoro nucleotide modification, 2'-deoxy nucleotide modification, locked nucleotide modification, unlocked nucleotide modification, conformationally restricted nucleotide modification, constrained ethyl nucleotide modification, abasic nucleotide modification, 2'-amino nucleotide modification, 2'-O-allyl nucleotide modification, 2'-C-alkyl nucleotide modification, 2'-hydroxyl nucleotide modification, 2'-methoxyethyl nucleotide modification, 2'-O-alkyl nucleotide modification, morpholino nucleotide modification, phosphoramidate modification, nucleotide modification containing unnatural base, tetrahydropyran nucleotide modification, 1,5-anhydrohexitol nucleotide modification, cyclohexenyl nucleotide modification, nucleotide modification containing phosphorothioate group, nucleotide modification containing methylphosphonate group, nucleotide modification containing 5'-phosphate, nucleotide modification containing 5'-phosphate mimic, thermally destabilized nucleotide modification, glycol nucleotide (GNA) modification, nucleotide modification containing 2' phosphate, and 2-O-(N-methylacetamide) nucleotide modification, and combinations thereof, the dsRNA agent according to claim 1 or a pharmaceutically acceptable salt thereof.

3. The dsRNA agent according to claim 1 or a pharmaceutically acceptable salt thereof, further comprising a phosphate or a phosphate mimic at the 5'-end of the antisense strand.

4. The dsRNA agent according to claim 3 or a pharmaceutically acceptable salt thereof, wherein the phosphate mimic is 5'-vinylphosphonate (VP).

5. The 3'-end of the sense strand is protected via an end cap which is a cyclic group having an amine, and the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl, the dsRNA agent according to claim 1 or a pharmaceutically acceptable salt thereof.

6. The dsRNA agent according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the double-stranded region is 19 to 30 nucleotide pairs in length.

7. The dsRNA agent according to claim 1 or a pharmaceutically acceptable salt thereof, wherein each strand is independently 30 nucleotides or less in length.

8. The dsRNA agent according to claim 1 or a pharmaceutically acceptable salt thereof, wherein at least one strand contains a 3'-overhang of at least 1 nucleotide.

9. The dsRNA agent according to claim 1 or a pharmaceutically acceptable salt thereof, wherein at least one strand contains a 3'-overhang of at least 2 nucleotides.

10. Said one or more C ２２ The dsRNA agent or a pharmaceutically acceptable salt thereof according to claim 1, wherein the hydrocarbon chain is an aliphatic compound, an alicyclic compound, or a polyalicyclic compound.

11. The dsRNA agent according to claim 1 or a pharmaceutically acceptable salt thereof, further comprising a targeting ligand that targets liver tissue.

12. The dsRNA agent according to claim 11 or a pharmaceutically acceptable salt thereof, wherein the targeting ligand is conjugated to the 3'-end of the sense strand of the dsRNA agent.

13. The dsRNA agent according to claim 11 or a pharmaceutically acceptable salt thereof, wherein the targeting ligand is an N-acetylgalactosamine (GalNAc) derivative.

14. The dsRNA agent according to claim 11 or a pharmaceutically acceptable salt thereof, wherein the targeting ligand is one or more GalNAc derivatives linked through a monovalent, divalent, or trivalent branched linker.

15. The dsRNA agent according to claim 11 or a pharmaceutically acceptable salt thereof, wherein the targeting ligand is as follows. 【Chemical 1】

16. The dsRNA agent or a pharmaceutically acceptable salt thereof is conjugated to the ligand as shown in the following figure, [Chemical Formula 2] wherein X is O or S, the dsRNA agent according to claim 15 or a pharmaceutically acceptable salt thereof. The dsRNA agent or a pharmaceutically acceptable salt thereof according to claim 16, wherein X is O.

18. The dsRNA agent or a pharmaceutically acceptable salt thereof according to claim 1, further comprising at least one phosphorothioate nucleotide internucleotide linkage or methylphosphonate nucleotide internucleotide linkage.

19. A cell containing the dsRNA agent or a pharmaceutically acceptable salt thereof according to claim 1.

20. A pharmaceutical composition for inhibiting the expression of a metabolic disorder-related target gene selected from the group consisting of inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), and inhibin subunit beta C (INHBC), comprising the dsRNA agent or a pharmaceutically acceptable salt thereof according to claim 1 and a pharmaceutically acceptable carrier.

21. An in vitro method for inhibiting the expression of a metabolic disorder-related target gene selected from the group consisting of inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), and inhibin subunit beta C (INHBC) in a cell, the method comprising contacting the cell with the dsRNA agent or a pharmaceutically acceptable salt thereof according to claim 1, thereby inhibiting the expression of the metabolic disorder-related target gene in the cell.

22. The method according to claim 21, wherein the cell is a hepatocyte.

23. A pharmaceutical composition for treating a subject having a metabolic disorder, comprising a therapeutically effective amount of the dsRNA agent or a pharmaceutically acceptable salt thereof according to claim 1.

24. A pharmaceutical composition for preventing at least one symptom in a subject having a metabolic disorder, comprising a prophylactically effective amount of the dsRNA agent or a pharmaceutically acceptable salt thereof according to claim 1.

25. The pharmaceutical composition according to claim 23 or 24, wherein the metabolic disorder is selected from the group consisting of metabolic syndrome, type 2 diabetes, obesity, elevated triglyceride levels, lipodystrophy, liver inflammation, fatty liver disease, hypercholesterolemia, disorders associated with elevated liver enzymes, non-alcoholic steatohepatitis (NASH), cardiovascular disease, hypertension, cardiomyopathy, heart failure, and kidney disease.

26. The pharmaceutical composition according to claim 23 or 24, wherein the subject is a human.

27. The pharmaceutical composition according to claim 23 or 24, wherein the dsRNA agent or a pharmaceutically acceptable salt or pharmaceutical composition thereof is administered subcutaneously to the subject.

28. The pharmaceutical composition according to claim 23 or 24, further comprising the use of an additional therapeutic agent.

29. The pharmaceutical composition according to claim 28, wherein the additional therapeutic agent is selected from the group consisting of insulin, glucagon-like peptide 1 agonists, sulfonylureas, meglitinides, biguanides, thiazolidinediones, alpha-glucosidase inhibitors, SGLT2 inhibitors, DPP-4 inhibitors, HMG-CoA reductase inhibitors, statins, and any combination of the foregoing.

30. A kit, vial or syringe comprising the dsRNA agent according to claim 1 or a pharmaceutically acceptable salt thereof.

Images