Activin A receptor type 1C (ACVR1C) iRNA composition and method of use thereof

Abstract

The present invention relates to RNAi agents, such as double-stranded RNA (dsRNA) agents, that target the activin A receptor type 1C (ACVR1C) gene. The disclosure also relates to methods of using such RNAi agents to inhibit the expression of the ACVR1C gene, as well as methods for preventing and treating ACVR1C-related disorders, such as metabolic disorders, such as metabolic syndromes.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Application No. 63 / 539,682, filed September 21, 2024, and U.S. Provisional Application No. 63 / 694,377, filed September 13, 2024. The entire contents of each of the aforementioned applications are incorporated herein by reference.

[0002] Sequence List This application includes a sequence listing submitted electronically in .XML format, which is incorporated herein by reference in its entirety. The .XML copy was created on 16 September 2024, named "121301_22520_SL.xml", and has a size of 84,670,000 bytes. The sequence listing contained in this .XML file is part of the specification, which is incorporated herein by reference in its entirety. [Background technology]

[0003] With successful development of treatments for many infectious diseases in much of the world, non-communicable diseases, particularly metabolic disorders, are becoming a major health risk in the modern world. Increased consumption of high-calorie, low-fiber fast food and decreased physical activity due to mechanized transportation and sedentary lifestyles have led to the prevalence of obesity and metabolic disorders, such as metabolic syndromes, type 2 diabetes, hypertension, cardiovascular disease, stroke, and other functional impairments. Indeed, the incidence of individuals with metabolic disorders, such as metabolic syndromes, including numerous health conditions that put individuals at higher risk of heart disease, diabetes, stroke, and other diseases, has been increasing in recent years.

[0004] The activin A receptor type 1C (ACVR1C, which encodes the activin receptor-like kinase 7 protein, ALK7) is a type I receptor of the TGF-β family of signaling molecules. ACVR1C possesses intrinsic serine / threonine kinase activity in its cytoplasmic domain, inducing phosphorylation and activation of the SMAD2 / 3 / 4 complex, which then translocates to the nucleus where it binds to the SMAD-binding element and regulates gene transcription. ACVR1C expression levels vary considerably across tissues, with white and brown adipose tissue exhibiting the highest levels of expression.

[0005] ACVR1C functions to facilitate the storage and accumulation of energy reserves by antagonizing catabolic pathways of lipolysis and amino acid breakdown. Polymorphisms of ACVR1C have been found to be associated with an increased risk of metabolic syndromes in Chinese women and may be involved in cardiovascular remodeling in patients with metabolic syndromes [Zhang, W et al. Arq Bras Cardiol. 2013: 101(2):134-140]. In addition, mutants predicted to cause loss of ACVR1C gene function are thought to affect body fat distribution and provide protection from type 2 diabetes [Emdin CA et al. Diabetes. 2019: 68(1):226-234; Justice AE et al. Nat Genet. 2019: 51(3):452-69; Koprulu M et al. The Journal of Clincal Endocrinology & Metabolism. 2022: 107(4):1065-1077; Deaton AM et al. Nature Communications. 2022: 13:4319; Akabari P et al. Nature Communications. 2022 13:4844]. Studies performed on adipocytes of obese mouse strains demonstrated that ACVR1C dysfunction (resulting from nonsense mutations) led to increased lipolysis in adipocytes and decreased fat accumulation, while conversely, activation of ACVR1C inhibited lipolysis by suppressing the expression of lipolipase [Guo T et al. eLife 2014. 1-18 (DOI: 10.7554 / eLife.03245; Adam RC et al. Proc. Natl. Acad. Sci. USA 2023. 120(32)]. Furthermore, lower-weight ACVR1C-deficient mice showed enhanced glucose tolerance and insulin sensitivity, and metabolic rate measurements in these mice revealed increased O2 consumption, decreased respiratory quotient, and increased energy expenditure [Yogosawa, S et al. Diabetes 2013: 62(1):115-123].These findings suggest that specific inhibitors of ACVR1C may have therapeutic applications in metabolic disorders such as obesity and metabolic syndromes.

[0006] Current treatments for metabolic disorders include lifestyle changes, dietary therapy, exercise, and the use of lipid-lowering agents such as statins, incretin agonists, and other medications. However, these therapies and treatments are often limited by compliance and are not always effective (sometimes due to patient adherence), resulting in side effects and drug-drug interactions. [Overview of the project] [Problems that the invention aims to solve]

[0007] Therefore, there is a need in the industry for alternative treatments for individuals with metabolic disorders such as obesity and metabolic syndromes, as well as related diseases such as diabetes, hypertension, and cardiovascular disease. These alternatives include drugs that possess both high biological activity and in vivo stability, effectively inhibit the expression of the activin A receptor type 1C (ACVR1C) gene, and selectively and efficiently silence the activin ACVR1C gene using the cell's own RNAi mechanism. [Means for solving the problem]

[0008] The present invention provides iRNA compositions that perform RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of a target gene encoding activin A receptor type 1C (ACVR1C). The ACVR1C gene may be present in cells, for example, in cells of subjects such as human subjects. The present invention also provides methods of using the iRNA compositions of the present invention for inhibiting the expression of the ACVR1C gene and / or for treating subjects that are expected to benefit from inhibiting or reducing the expression of the ACVR1C gene, for example, for treating subjects suffering from or susceptible to ACVR1C-related disorders, such as metabolic disorders, such as metabolic syndromes, diabetes, hypertension, and / or cardiovascular diseases.

[0009] Accordingly, in one embodiment, the disclosure of the present invention relates to a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of activin A receptor type 1C (ACVR1C) in cells, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, which differ from the nucleotide sequence of SEQ ID NO: 2 by three or fewer, e.g., 3, 2, 1, or 0 nucleotides, and one or more C 22 The present invention provides a dsRNA agent in which a hydrocarbon chain is conjugated to a sense chain.

[0010] In one embodiment, the antisense chain comprises at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, which differ from any one of the antisense chain nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12 by three or fewer nucleotides, e.g., 3, 2, 1, or 0 nucleotides.

[0011] In another embodiment, the present disclosure relates to a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of activin A receptor type 1C (ACVR1C) in cells such as adipocytes and / or hepatocytes, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand having one nucleotide sequence of any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27 and a small number of nucleotides different from 0, 1, 2, or 3 or fewer. The present invention provides a dsRNA agent comprising at least 15, for example, 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides, wherein the antisense strand comprises at least 15, for example, 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides, where 0, 1, 2, or 3 or fewer nucleotides differ from the corresponding portion of any one nucleotide sequence of SEQ ID NOs. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28.

[0012] In another embodiment, the disclosure of the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of activin A receptor type 1C (ACVR1C) in cells such as adipocytes and / or liver cells, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising a region complementary to the mRNA encoding ACVR1C, the complementary region comprising at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, which differ from any one of the antisense nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12 by 0, 1, 2, or 3 or fewer nucleotides.

[0013] In yet another embodiment, the disclosure of the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of activin A receptor type 1C (ACVR1C) in cells such as adipocytes and / or liver cells, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, or 21, which differ from any one of the sense nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12 by 0, 1, 2, or 3 or fewer nucleotides, and the antisense strand comprising at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, which differ from any one of the antisense nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12 by 0, 1, 2, or 3 or fewer nucleotides. In some embodiments, these dsRNA agents have one or more C molecules conjugated at one or more positions on at least one strand of the dsRNA agent, for example, at an internal position. 22 It further contains hydrocarbon chains.

[0014] In another embodiment, the disclosure of the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of activin A receptor type 1C (ACVR1C) in cells such as adipocytes, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, or 21, which differ from any one of the sense nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12 by 0, 1, 2, or 3 or fewer nucleotides, and the antisense strand comprising at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, which differ from any one of the antisense nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12 by 0, 1, 2, or 3 or fewer nucleotides.

[0015] In one embodiment, the dsRNA agent includes a sense strand comprising a continuous nucleotide sequence having at least 85%, for example 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity over its entire length with any one of the sense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12; and an antisense strand comprising a continuous nucleotide sequence having at least 85%, for example 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity over its entire length with any one of the antisense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12.

[0016] In one embodiment, the dsRNA agent includes a sense strand containing at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, or 21, which differ from any one of the nucleotide sequences of the sense strand from any one of Tables 2-5, 7, 8, 11, and 12 by three or fewer nucleotides, and an antisense strand containing at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, which differ from any one of the nucleotide sequences of the antisense strand from any one of Tables 2-5, 7, 8, 11, and 12 by three or fewer nucleotides.

[0017] In one embodiment, the dsRNA agent includes a sense strand containing at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, or 21, which differ from any one of the nucleotide sequences of the sense strand from any one of Tables 2-5, 7, 8, 11, and 12 by two or fewer nucleotides, and an antisense strand containing at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, or 23, which differ from any one of the nucleotide sequences of the antisense strand from any one of Tables 2-5, 7, 8, 11, and 12 by two or fewer nucleotides.

[0018] In one embodiment, the dsRNA agent includes a sense strand containing at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, or 21, which differ from any one of the sense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12 by 11 or fewer nucleotides, and an antisense strand containing at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, which differ from any one of the antisense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12 by 11 or fewer nucleotides.

[0019] In one embodiment, the dsRNA agent comprises a sense strand containing or comprising a nucleotide sequence selected from the group consisting of one of the sense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12, and an antisense strand containing or comprising a nucleotide sequence selected from the group consisting of one of the antisense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12.

[0020] In one embodiment, the disclosure of the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of activin A receptor type 1C (ACVR1C) in cells, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising a region complementary to the mRNA encoding ACVR1C, the complementary region comprising at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides that differ from any one of the antisense nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12, by three or fewer nucleotides, e.g., 3, 2, 1, or 0 nucleotides.

[0021] In one embodiment, the sense strand comprises at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, or 21, which differ from one of the sense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12 by three or fewer nucleotides, e.g., 3, 2, 1, or 0 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, which differ from one of the antisense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12 by three or fewer nucleotides, e.g., 3, 2, 1, or 0 nucleotides.

[0022] In some embodiments, the antisense strand comprises one of the nucleotide sequences of a double-stranded antisense strand selected from the group consisting of AD-2640052, AD-2640053, AD-2640060, AD-2509191, and AD-2508176, and at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, which differ by three or fewer nucleotides, e.g., 3, 2, 1, or 0.

[0023] In some embodiments, the sense strand and antisense strand include one of the nucleotide sequences of a double-stranded antisense strand selected from the group consisting of AD-2640052, AD-2640053, AD-2640060, AD-2509191, or AD-2508176, and at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, which differ by three or fewer nucleotides, e.g., 3, 2, 1, or 0.

[0024] In another embodiment, the present invention relates to a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of activin A receptor type 1C (ACVR1C) in cells, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, the antisense strand being (a) 5'-UUAATUAUCAUCAUUAGGCUUUG-3' of sequence number 14921; (b) 5'-UUUGACACAAAGUUGAGAUAUAG-3' of sequence number 14922; (c) 5'-UAACAUAAUUAUCAUCAUUAGGC-3' of sequence number 14925; (d) 5'-UAGGACUCAAAGAUAUUCACAUU-3' of sequence number 9291 and (e) 5'-UCACACAAAAGACAUACACACUU-3' of sequence number 8956 The present invention provides a dsRNA agent comprising one of the nucleotide sequences of an antisense strand selected from the group consisting of and at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, with three or fewer nucleotides different, e.g., 3, 2, 1, or 0 nucleotides.

[0025] In some embodiments, the sense strand comprises at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, which differ from one of the sense and antisense strand nucleotide sequences selected from the group consisting of (a) to (e) below, by three or fewer nucleotides, e.g., 3, 2, 1, or 0 nucleotides; and the antisense strand comprises at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, which differ from one of the sense and antisense strand nucleotide sequences selected from the group consisting of (a) to (e) below, by three or fewer nucleotides, e.g., 3, 2, 1, or 0 nucleotides: (a) 5'-AAGCCUAAUGAUGAUAAUUAA-3' of SEQ ID NO: 14909 and 5'-UUAATUAUCAUCAUUAGGCUUUG-3' of SEQ ID NO: 14921; (b) 5'-AUAUCUCAACUUUGUGUCAAA-3' of SEQ ID NO: 14910 and 5'-UUUGACACAAAGUUGAGAUAUAG-3' of SEQ ID NO: 14922; (c) 5'-CUAAUGAUGAUAAUUAUGUUA-3' of SEQ ID NO: 14913 and 5'-UAACAUAAUUAUCAUCAUUAGGC-3' of SEQ ID NO: 14925; (d) 5'-UGUGAAUAUCUUUGAGUCCUA-3' of SEQ ID NO: 7805 and 5'-UAGGACUCAAAGAUAUUCACAUU-3' of SEQ ID NO: 9291; and (e) 5'-GUGUGUAUGUCUUUUGUGUGA-3' of SEQ ID NO: 7470 and 5'-UCACACAAAAGACAUACACACUU-3' of SEQ ID NO: 8956.

[0026] In one embodiment, the dsRNA agent comprises 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. In one embodiment, all of the nucleotides in the sense strand are modified nucleotides; all of the nucleotides in the antisense strand are modified nucleotides; or all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand are modified nucleotides.

[0028] In one embodiment, at least one of the modified nucleotides is a deoxynucleotide, a 3'-terminated deoxythimidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally fixed nucleotide, a restricted ethyl nucleotide, a debasalized nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, or a 2'-O-A Selected from the group consisting of lucyl-modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimics, thermally destabilized nucleotides, glycol-modified nucleotides (GNAs), nucleotides containing 2'-phosphates, and 2-O-(N-methylacetamide)-modified nucleotides; and combinations thereof.

[0029] 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.

[0030] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, glycol modified nucleotides (GNAs), e.g., Ggn, Cgn, Tgn, or Agn, nucleotides having a 2' phosphate, e.g., G2p, C2p, A2p, or U2p, nucleotides containing a phosphorothioate group, and vinyl phosphonate nucleotides; and combinations thereof.

[0031] In another embodiment, at least one of the modified nucleotides is a nucleotide having a thermally destabilizing nucleotide modification. In one embodiment, the thermally destabilizing nucleotide modification is selected from the group consisting of debase modification; mismatch with opposing nucleotides in the double helix; destabilizing sugar modification, 2'-deoxy modification, acyclic nucleotide, unlocked nucleic acid (UNA), and glycerol nucleic acid (GNA).

[0032] In some embodiments, the modified nucleotide includes a short sequence of a 3'-terminal deoxythymidine nucleotide (dT).

[0033] In some embodiments, the dsRNA agent further comprises a phosphate or phosphate mimetic at the 5' end of the antisense strand. In some embodiments, the phosphate mimetic is 5'-vinylphosphonate (VP). In some embodiments, the 5' end of the antisense strand of the dsRNA agent does not contain 5'-vinylphosphonate (VP).

[0034] In some embodiments, the dsRNA agent further comprises at least one terminal chiral phosphorus atom. Site-specific chiral modifications to internucleotide linkages may be present at the 5' end, 3' end, or both the 5' and 3' ends of the strand. This is referred to herein as a “terminal” chiral modification. The terminal modification may be present at the 3' or 5' terminal position in the terminal region, for example, at a position on the terminal nucleotide, or within the last 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the strand. The chiral modification may be present on the sense strand, the antisense strand, or both the sense and antisense strands. Each chiral pure phosphorus atom may be in either an Rp configuration or an Sp configuration, or a combination thereof. Further details regarding chiral modification and chiralally modified dsRNA agents can be found in PCT / US18 / 67103, filed December 21, 2018, entitled “Chirally-Modified Double-Stranded RNA Agents,” which is incorporated herein by reference in its entirety.

[0035] In some embodiments, the dsRNA agent further comprises: a terminal chiral modification present in the first internucleotide linkage at the 3' end of an antisense strand having a phosphorus atom linked in the Sp configuration; a terminal chiral modification present in the first internucleotide linkage at the 5' end of an antisense strand having a phosphorus atom linked in the Rp configuration; and a terminal chiral modification present in the first internucleotide linkage at the 5' end of a sense strand having a phosphorus atom linked in either the Rp or Sp configuration.

[0036] In one embodiment, the dsRNA agent further comprises: terminal chiral modifications present in the first and second nucleotide linkages at the 3' end of an antisense strand having a phosphorus atom linked in the Sp configuration; terminal chiral modifications present in the first nucleotide linkage at the 5' end of an antisense strand having a phosphorus atom linked in the Rp configuration; and terminal chiral modifications present in the first nucleotide linkage at the 5' end of a sense strand having a phosphorus atom linked in either the Rp or Sp configuration.

[0037] In one embodiment, the dsRNA agent further comprises: terminal chiral modifications present in the first, second, and third nucleotide linkages at the 3' end of an antisense strand having a phosphorus atom linked in the Sp configuration; terminal chiral modifications present in the first nucleotide linkage at the 5' end of an antisense strand having a phosphorus atom linked in the Rp configuration; and terminal chiral modifications present in the first nucleotide linkage at the 5' end of a sense strand having a phosphorus atom linked in either the Rp or Sp configuration.

[0038] In one embodiment, the dsRNA agent further comprises: terminal chiral modifications present in the first and second nucleotide linkages at the 3' end of an antisense strand having a phosphorus atom linked in the Sp configuration; terminal chiral modifications present in the third nucleotide linkage at the 3' end of an antisense strand having a phosphorus atom linked in the Rp configuration; terminal chiral modifications present in the first nucleotide linkage at the 5' end of an antisense strand having a phosphorus atom linked in the Rp configuration; and terminal chiral modifications present in the first nucleotide linkage at the 5' end of a sense strand having a phosphorus atom linked in either the Rp or Sp configuration.

[0039] In one embodiment, the dsRNA agent further comprises: terminal chiral modifications present in the first and second nucleotide linkages at the 3' end of an antisense strand having a phosphorus atom linked in the Sp configuration; terminal chiral modifications present in the first and second nucleotide linkages at the 5' end of an antisense strand having a phosphorus atom linked in the Rp configuration; and terminal chiral modifications present in the first nucleotide linkage at the 5' end of a sense strand having a phosphorus atom linked in either the Rp or Sp configuration.

[0040] In some embodiments, the 3' end of the sense chain is protected by a terminal 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]dioxolanil, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuranyl, and decalinyl.

[0041] In one embodiment, the dsRNA agent further comprises at least one internucleotide linkage of phosphorothioate or methylphosphonate. In one embodiment, the internucleotide linkage of phosphorothioate or methylphosphonate is located at the 3' end of one strand, for example, an antisense strand or a sense strand. In another embodiment, the internucleotide linkage of phosphorothioate or methylphosphonate is located at the 5' end of one strand, for example, an antisense strand or a sense strand. In one embodiment, the internucleotide linkage of phosphorothioate or methylphosphonate is located at both the 5' and 3' ends of one strand. In one embodiment, the strand is an antisense strand.

[0042] In one embodiment, the base pair at position 1 of the 5' end of the antisense strand of the double helix is ​​an AU base pair.

[0043] The double-stranded region may consist of nucleotide pairs of 19 to 30 nucleotides in length; nucleotide pairs of 19 to 25 nucleotides in length; nucleotide pairs of 19 to 23 nucleotides in length; nucleotide pairs of 23 to 27 nucleotides in length; or nucleotide pairs of 21 to 23 nucleotides in length.

[0044] In one embodiment, each chain independently consists of 30 or fewer nucleotides in length.

[0045] In one embodiment, the sense strand has a length of 21 nucleotides, and the antisense strand has a length of 23 nucleotides.

[0046] The complementary region may have a length of at least 17 nucleotides; a length of 19 to 23 nucleotides; or a length of 19 nucleotides.

[0047] In one embodiment, at least one strand includes a 3' overhang of at least one nucleotide. In another embodiment, at least one strand includes a 3' overhang of at least two nucleotides.

[0048] In some embodiments, one or more C 22 The hydrocarbon chain is conjugated at one or more internal positions on at least one of the dsRNA agent strands.

[0049] In some embodiments, one or more C 22 The lipophilicity of a hydrocarbon chain is expressed by the octanol-water partition coefficient, logK. ow Measured by logK and greater than 0. The lipophilic portion is greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10. ow It may have.

[0050] In some embodiments, the hydrophobicity of the dsRNA agent is measured by the percentage of unbound dsRNA in a plasma protein binding assay of the dsRNA agent and is greater than 0.2. In one embodiment, the plasma protein binding assay used for determination is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. The hydrophobicity of the dsRNA agent is measured by the percentage of unbound dsRNA in the binding assay and 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 with respect to enhanced in vivo delivery of dsRNA.

[0051] C 22 The hydrocarbon chain may be saturated or unsaturated.

[0052] C 22 The hydrocarbon chain may be linear or branched.

[0053] In some embodiments, the internal positions include all positions except for the three terminal positions from each end of at least one strand. In some embodiments, the internal positions exclude the region of the cleavage site of the sense strand. In some embodiments, the internal positions exclude positions 9 to 12 or 11 to 13 counted from the 5' end of the sense strand. In some embodiments, the internal positions exclude the region of the cleavage site of the antisense strand. In some embodiments, the internal positions exclude positions 12 to 14 counted from the 5' end of the antisense strand.

[0054] In some embodiments, one or more C 22 The hydrocarbon chain is conjugated to one or more of the following internal positions: counted from the 5' end of each strand, positions 4 to 8 and 13 to 18 in the sense strand, and positions 6 to 10 and 15 to 18 in the antisense strand. In some embodiments, one or more C 22 The hydrocarbon chain is conjugated to one or more of the following internal positions: counted from the 5' end of each strand, positions 5, 6, 7, 15, 16 and 17 in the sense strand, and positions 15 and 17 in the antisense strand. In some embodiments, one or more C 22 The hydrocarbon chain is conjugated to position 6 in the sense strand counted from the 5' end of the sense strand. In some embodiments, one or more C 22 The hydrocarbon chain is conjugated to position 16 in the sense strand counted from the 5' end of the sense strand.

[0055] In some embodiments, one or more C 22 The hydrocarbon chain is an aliphatic, alicyclic, or polyalicyclic compound, for example, one or more C 22The hydrocarbon chain contains a functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0056] In some embodiments, one or more C 22 The hydrocarbon chain is a C22 acid, for example, selected from the group consisting of docosanic 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.

[0057] [ka]

[0058] In some embodiments, one or more C 22 The hydrocarbon chain is a C22 alcohol, for example, selected from the group consisting of 1-docosanol, 6-octyltetradecane-1-ol, 10-hexylhexadecane-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.

[0059] [ka]

[0060] In some embodiments, one or more C 22The 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-docosenaamide, (Z)-docosa-13-enamide, (Z)-N-hydroxy-13-docosenaamide, (E)-docosa-14-enamide, 6-cis-docosenaamide, 14-docosenaamide, docosa-11-enamide, (4E,13E)-docosa-4,13-dienamide, and (5E,13E)-docosa-5,13-dienamide.

[0061] One or more C 22 The hydrocarbon chain may be conjugated to the dsRNA agent via direct linkage to the ribosaccharide 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 phosphate linkages between nucleotides.

[0062] [ka]

[0063] In a particular embodiment, one or more C 22 The hydrocarbon chain is conjugated to the dsRNA agent via one or more linkers (tethers), for example, via a carrier that replaces one or more nucleotides at internal positions.

[0064] In some embodiments, one or more C 22The hydrocarbon chain is conjugated to the dsRNA agent via a linker containing ethers, thioethers, ureas, carbonates, amines, amides, maleimide-thioethers, disulfides, phosphodiesters, sulfonamide linkages, click reaction products (e.g., triazoles from azido-alkyne cycloaddition), or carbamates.

[0065] In some embodiments, at least one of the linkers (tethers) is a redox-cleavable linker (e.g., a reductively cleavable linker; e.g., 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).

[0066] In other embodiments, at least one of the linkers (tethers) is a biologically clevable linker selected from the group consisting of DNA, RNA, disulfides, amides, galactosamine, glucosamine, glucose, galactose, mannose-functionalized monosaccharides or oligosaccharides, and combinations thereof.

[0067] In a particular embodiment, one or more C 22A hydrocarbon chain is conjugated to a dsRNA agent via a carrier that replaces one or more nucleotides. The carrier may be a cyclic or 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, pyridadinyl, tetrahydrofuryl, and decalin. In one embodiment, the acyclic group is a serinol backbone or a diethanolamine backbone-based portion. In some embodiments, the carrier replaces one or more nucleotides at an internal position within the dsRNA agent.

[0068] In some embodiments, the dsRNA agent further comprises a targeted ligand that targets a receptor that mediates delivery to adipose tissue. In one embodiment, the targeted ligand is selected from the group consisting of angiopep-2, lipoprotein receptor-associated protein (LRP) ligand, bEnd.3 cell-binding ligand, transferrin receptor (TfR) ligand, manose receptor ligand, glucose transporter protein, LDL receptor ligand, trans-retinol, RGD peptide, LDL receptor ligand, CD63 ligand, and carbohydrate-based ligands.

[0069] In some embodiments, the dsRNA agent further comprises a targeting ligand that targets liver tissue. In one embodiment, the targeting ligand is conjugated to the 3' end of the sense strand of the dsRNA agent. In some embodiments, the targeting ligand is a carbohydrate-based ligand. In one embodiment, the targeting ligand is an N-acetylgalactosamine (GalNAc) derivative. In one embodiment, the targeting ligand is one or more GalNAc derivatives linked via a monovalent, divalent, or trivalent branched linker. In one embodiment, the targeting ligand is

[0070] [ka] In one embodiment, the dsRNA agent is conjugated to a target ligand as shown in the following diagram.

[0071] [ka] In the formula, X is either O or S. In one embodiment, X is O.

[0072] In some embodiments, one or more C 22 The hydrocarbon chain or targeted ligand is conjugated via a biologically cleavable linker selected from the group consisting of DNA, RNA, disulfides, amides, galactosamine, glucosamine, glucose, galactose, mannose-functionalized monosaccharides or oligosaccharides, and combinations thereof.

[0073] In one embodiment, the disclosure of the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of activin A receptor type 1C (ACVR1C) in cells, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, or 21, which differ from any one of the sense and antisense strand nucleotide sequences selected from the group consisting of (a) to (e) below, and four or fewer, e.g., 4, 3, 2, 1, or 0 bases, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides, which differ from any one of the sense and antisense strand nucleotide sequences selected from the group consisting of (a) to (e) below: (a) 5'-asasgcc(Uda)AfaUfGfAfugauaauusasa-3' from sequence number 14933 and 5'-VPusUfsaadTu(Agn)ucaucaUfuAfggcuususg-3' from sequence number 14945; (b) 5'-asusauc(Uda)CfaAfCfUfuugugucasasa-3' of SEQ ID NO: 14934 and 5'-VPusUfsugdAc(Agn)caaaguUfgAfgauausasg-3' of SEQ ID NO: 14946; (c) 5'-csusaaugAfuGfAfUfaauu(Ada)ugususa-3' of SEQ ID NO: 14937 and 5'-VPusAfsacaUfaauuaucAfuCfauuagsgsc-3' of SEQ ID NO: 14949; (d) 5'-usgsuga(Ada)UfaUfCfUfuugaguccsusa-3' of SEQ ID NO: 10777 and 5'-VPusAfsggaCfucaaagaUfaUfucacasusu-3' of SEQ ID NO: 12263; and (e) 5'-gsusgug(Uda)AfuGfUfCfuuuugugusgsa-3' of sequence number 10442 and 5'-VPusCfsacaCfaaaagacAfuAfcacacsusu-3' of sequence number 11928.

[0074] In one embodiment, the dsRNA agent of the present invention is a pharmaceutically acceptable salt thereof. Examples of "pharmaceutically acceptable salts" of each RNAi agent as used herein include, but are not limited to, sodium salts, calcium salts, lithium salts, potassium salts, ammonium salts, magnesium salts, and mixtures thereof. Those skilled in the art will understand that RNAi agents may be provided as polycationic salts having one cation per free acid group of an optionally modified phosphodiester backbone and / or any other acidic modification (e.g., a phosphonate group at the 5' terminus). For example, an oligonucleotide of length "n" contains n-1 optionally modified phosphodiesters, such that an oligonucleotide of length 21nt can be provided as a salt having up to 20 cations (e.g., 20 sodium cations). Similarly, an RNAi agent having a sense strand of length 21nt and an antisense strand of length 23nt may be provided as a salt having up to 42 cations (e.g., 42 sodium cations). In the above example, if the RNAi agent also contains a phosphate or vinylphosphonate group at the 5' terminus, the RNAi agent may be provided as a salt having up to 44 cations (e.g., 44 sodium cations).

[0075] The disclosures of the present invention also provide cells containing any of the dsRNA agents of the present disclosure, and pharmaceutical compositions containing any of the dsRNA agents of the present disclosure.

[0076] The pharmaceutical compositions of this disclosure may include an unbuffered solution, for example, a dsRNA agent in physiological saline or water, or the pharmaceutical compositions of this disclosure may include a buffered solution, for example, a buffered solution containing an acetate, citrate, prolamin, carbonate, or phosphate or any combination thereof; or a dsRNA agent in phosphate-buffered saline (PBS).

[0077] In one aspect, the present disclosure provides a method of inhibiting the expression of the activin A receptor type 1C (ACVR1C) gene in cells, such as adipocytes and / or liver cells. The method comprises contacting the cell with any of the dsRNAs of the present disclosure or any of the pharmaceutical compositions of the present disclosure, thereby inhibiting the expression of the ACVR1C gene in the cell.

[0078] In one embodiment, the cell is within a subject, for example a human subject, such as a subject having a metabolic disorder, for example diabetes, metabolic syndrome, cardiovascular disease, or hypertension.

[0079] In one embodiment, the cell is an adipocyte. In one embodiment, the cell is a hepatocyte.

[0080] In certain embodiments, the expression of the ACVR1C gene is inhibited by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In one embodiment, inhibiting the expression of the ACVR1C gene reduces the level of ALK7 protein in the serum of the subject by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0081] In one aspect, the present disclosure provides a method of treating a subject having a disorder that is expected to benefit from reduced ALK7 expression. The method comprises administering to the subject a therapeutically effective amount of any of the dsRNAs of the present disclosure or any of the pharmaceutical compositions of the present disclosure, thereby treating the subject having a disorder that is expected to benefit from reduced ACVR1C expression.

[0082] In another embodiment, the disclosure of the present invention provides a method for preventing at least one symptom in a subject having a disorder in which a reduction in ACVR1C expression is expected to be beneficial. The method comprises administering a prophylactic effective amount of any of the dsRNAs of the present disclosure or any of the pharmaceutical compositions of the present disclosure to a subject, thereby preventing at least one symptom in a subject having a disorder in which a reduction in ACVR1C expression is expected to be beneficial.

[0083] In certain embodiments, the disorder is an ACVR1C-related disorder. In some embodiments, the ACVR1C-related disorder is a metabolic disorder. Metabolic disorders may include, for example, metabolic syndromes, carbohydrate disorders such as type II diabetes, prediabetes, lipid metabolism disorders such as hyperlipidemia, hypertension, lipodystrophy, renal disease, cardiovascular disease, and weight disorders. In some embodiments, the ACVR1C-related disorder is a metabolic syndrome. In some embodiments, the ACVR1C-related disorder is a cardiovascular disease. In some embodiments, the ACVR1C-related disorder is hypertension. In some embodiments, the ACVR1C-related disorder is a renal disease.

[0084] In one embodiment, the subject is a human being.

[0085] In one embodiment, administration of dsRNA to a subject reduces the body mass index-adjusted waist-to-hip ratio in the subject.

[0086] In certain embodiments, administration of dsRNA to a subject results in a reduction in the accumulation of ALK7 protein in the subject.

[0087] In one embodiment, the dsRNA agent is administered subcutaneously to the subject.

[0088] In one embodiment, the method of the present disclosure further includes determining the level of ACVR1C in a sample from a subject. In one embodiment, the level of ACVR1C in the subject sample is the ALK7 protein level in a blood, serum, or adipose tissue sample.

[0089] In certain embodiments, the method of the present disclosure further includes administering an additional therapeutic agent. In certain embodiments, the additional therapeutic agent is selected from the group consisting of insulin, glucagon-like peptide 1 agonists, sulfonylurea, seglitinide, biguanide, thiazolidinedione, alpha-glucosidase inhibitors, SGLT2 inhibitors, DPP-4 inhibitors, HMG-CoA reductase inhibitors, statins, and any combination thereof.

[0090] The disclosure of the present invention also provides a kit comprising any of the dsRNAs of the present disclosure or any of the pharmaceutical compositions of the present disclosure, and optionally instructions for use. In one embodiment, the present disclosure provides a kit for performing a method of inhibiting the expression of the ACVR1C gene in cells by contacting cells with a double-stranded RNA agent of the present disclosure effective in inhibiting the expression of the ACVR1C gene in cells. The kit comprises the RNAi agent and instructions for use, and may include means for administering the RNAi agent to a subject.

[0091] The disclosure of the present invention further provides RNA-induced silencing complexes (RISCs) comprising the antisense strand of any of the dsRNA agents of this disclosure.

[0092] The disclosures of the present invention are further illustrated by the following detailed description. [Brief explanation of the drawing]

[0093] [Figure 1] Figure 1 is a graph showing mouse ACVR1C mRNA levels, analyzed by RT-qPCR, from gonads, subcutaneous white and brown adipose tissue (n=3 / group) 21 days after subcutaneous administration of dsRNA double-stranded AD-1735913 at a single dose of either 0.5 mg / kg, 2 mg / kg, or 5 mg / kg. Mouse ACVR1C mRNA levels were calculated relative to a PBS-treated control group and normalized to the PPIA (peptidyl prolyl cis-trans isomerase) gene. [Figure 2] Figure 2 is a graph showing the effect on ACVR1C mRNA levels in mouse brown and white adipose tissue (gonads and subcutaneous tissue) measured by RT-qPCR 21 days after a single subcutaneous administration of 5 mg / kg of either AD-2410459.1, AD-2410460.1, AD-1735894, AD-2410461.1, or AD-2410462.1. Data are expressed as residual ACVR1C mRNA% compared to the PBS control group and are normalized to the UBC (ubiquitin C) gene. [Figure 3] Figure 3 is a graph showing the percentage of residual ACVR1C mRNA in NHP after administration of AD-2640052, AD-2640053, AD-2640060, or PBS, compared to the mean pre-administration levels on day -15 and day -2. [Figure 4] Figure 4 is a graph showing the percentage of residual ACVR1C mRNA in NHP adipose tissue at day 85 relative to the mean PBS group, after administration of AD-2640052, AD-2640053, AD-2640060, or PBS. [Figure 5] Figure 5 is a graph showing the percentage of the ALK7 peptide region in NHP at day -15, day -2, day 43, and day 57 for AD-2640052, AD-2640053, AD-2640060, AD-2509191, AD-2508176, or the PBS group. [Figure 6] Figure 6 is a graph showing the percentage ratio of the ALK7 peptide region in NHP to the mean pre-administration time point on day -15 and day -2 for the AD-2640052, AD-2640053, AD-2640060, and PBS groups. [Figure 7] Figure 7 is a graph showing the percentage ratio of the ALK7 peptide region in NHP to the average PBS of AD-2640052, AD-2640053, AD-2640060, and the PBS group in peripheral adipose tissue on day 85. [Modes for carrying out the invention]

[0094] The present disclosure provides iRNA compositions that affect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the activin A receptor type 1C (ACVR1C) gene. This gene may be present in a cell, e.g., an adipocyte, e.g., a cell in a subject such as a human. The use of these iRNAs enables targeted degradation of the corresponding gene (ACVR1C gene) mRNA in mammals.

[0095] The iRNAs of the present disclosure are designed to target the human ACVR1C gene, including portions of the gene that are conserved in orthologs of other mammalian species. Without intending to be limited by theory, it is believed that the aforementioned properties and specific target sites or combinations or subcombinations of specific modifications in these iRNAs confer improved efficacy, stability, potency, durability, and safety to the iRNAs of the present disclosure.

[0096] Accordingly, the present disclosure uses dsRNA compositions that effect RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the ACVR1C gene to treat and prevent ACVR1C-associated disorders, e.g., metabolic disorders, e.g., metabolic syndrome; carbohydrate disorders, e.g., type II diabetes, pre-diabetes; lipid metabolism disorders, e.g., hyperlipidemia, hypertension, lipodystrophy; renal diseases; cardiovascular diseases, and weight disorders.

[0097] The iRNAs of this disclosure include an RNA strand (antisense strand) having a region of up to approximately 30 nucleotides or less in length, for example, a region of 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which is substantially complementary to at least a portion of the mRNA transcript of the ACVR1C gene.

[0098] In certain embodiments, one or both strands of the double-stranded RNA agent of the Disclosure have a length of up to 66 nucleotides, for example, 36–66, 26–36, 25–36, 31–60, 22–43, or 27–53 nucleotides, and have a region of at least 19 consecutive nucleotides substantially complementary to at least a portion of the mRNA transcript of a metabolic disorder-related target gene. In some embodiments, a dsRNA agent having such a longer antisense strand may also include a second RNA strand (sense strand) which is, for example, 20–60 nucleotides in length, in which case the sense and antisense strands form a double helix of 18–30 consecutive nucleotides.

[0099] The use of the iRNAs of this disclosure enables targeted degradation of ACVR1C mRNA in mammals. Using in vitro and in vivo assays, the inventors have demonstrated that gene-targeting iRNAs can strongly mediate RNAi, resulting in significant inhibition of ACVR1C gene expression. Therefore, methods and compositions comprising these iRNAs are useful for treating subjects with ACVR1C-related disorders, such as metabolic disorders (e.g., metabolic syndromes), carbohydrate disorders (e.g., type II diabetes, prediabetes), lipid metabolism disorders (e.g., hyperlipidemia, hypertension, lipodystrophy), renal disease, cardiovascular disease, or weight disorders, or for treating subjects at risk of developing ACVR1C-related disorders.

[0100] Accordingly, the disclosure of the present invention provides methods and combination therapies for treating subjects having disorders, such as ACVR1C-related disorders, such as metabolic disorders, such as metabolic syndromes; carbohydrate disorders, such as type II diabetes, prediabetes; lipid metabolism disorders, such as hyperlipidemia, hypertension, lipodystrophy; renal diseases; cardiovascular diseases; and weight disorders, using iRNA compositions that perform RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the ACVR1C gene.

[0101] The disclosure of the present invention also provides a method for preventing at least one symptom in subjects having disorders that are expected to benefit from inhibiting or reducing the expression of the ACVR1C gene, such as ACVR1C-related disorders, such as metabolic disorders, such as metabolic syndromes; carbohydrate disorders, such as type II diabetes, prediabetes; lipid metabolism disorders, such as hyperlipidemia, hypertension, lipodystrophy; renal disease; cardiovascular disease; and weight disorders.

[0102] The following detailed description discloses how to prepare and use compositions containing iRNA for inhibiting ACVR1C expression, as well as compositions, uses, and methods for treating subjects who are expected to benefit from the inhibition and / or reduction of ACVR1C expression, such as subjects who are susceptible to or diagnosed with ACVR1C-related disorders, such as metabolic disorders.

[0103] I. Definition To make the disclosure of this invention easier to understand, certain terms are first defined. In addition, it should be noted that whenever a parameter value or range of values ​​is described, values ​​and ranges between the described values ​​are also intended to be part of this disclosure.

[0104] The articles “a” and “an” are used herein to refer to one or more (i.e., at least one) grammatical object of the article. For example, “an element” means one or more elements, e.g., multiple elements.

[0105] In this specification, the term "including" is used to mean "including, but not limited to, the following."

[0106] The term "or" is used herein to mean and is synonymous with the term "and / or" unless the context clearly indicates otherwise. For example, "sense strand or antisense strand" is understood as "sense strand or antisense strand or sense strand and antisense strand."

[0107] The term “approximately” is used herein to mean a typical range of tolerances in the industry. For example, “approximately” may be understood as approximately 2 standard deviations from the mean. In certain embodiments, “approximately” means ±10%. In certain embodiments, “approximately” means ±5%. When “approximately” precedes a set of numbers or ranges, it is understood that “approximately” can modify each of those numbers or ranges.

[0108] The terms “at least,” “greater than,” or “more than” accompanying a number or a range of numbers are understood to include the number adjacent to the term “at least” and all subsequent numbers or integers that may be clearly and logically included from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 19 nucleotides of a nucleic acid molecule with 21 nucleotides” means that 19, 20, or 21 nucleotides are present in the given characteristic. If “at least” precedes a range of numbers or numbers, it is understood that “at least” may modify each of the numbers or ranges.

[0109] When used herein, “less than or equal to” or “or less than” is understood to mean a value or integer that is logically lower than the value adjacent to that phrase and logically lower than zero, from the context. For example, a double helix with an overhang of “two or fewer nucleotides” has an overhang of two, one, or zero nucleotides. When “less than or equal to” is present in conjunction with a set of numbers or ranges, it is understood that “less than or equal to” can modify each of those numbers or ranges. When used herein, a range includes both an upper and lower limit.

[0110] When used herein, the detection method may include determining that the amount of analyte present is below the detection level of the method.

[0111] If there is a conflict between the indicated target site and the nucleotide sequence of the sense or antisense strand, the indicated sequence takes precedence.

[0112] In the event of any conflict between a given sequence and its indicated site on a transcript or other sequence, the nucleotide sequence described herein shall prevail.

[0113] "Activin A receptor type 1C" is used herein synonymously with the term "ACVR1C" and refers to the type I receptor of the TGF-β family of signaling molecules. ACVR1C possesses intrinsic serine / threonine kinase activity in its cytoplasmic domain, inducing phosphorylation and activation of the SMAD2 / 3 / 4 complex, which then translocates to the nucleus where it binds to the SMAD-binding element (SBE) and regulates gene transcription (e.g., activation and / or repression). ACVR1C expression levels vary considerably across tissues, with white and brown adipose tissue exhibiting the highest levels of expression. ACVR1C is also known as "activin receptor-like kinase 7" (ALK-7).

[0114] The sequences of human ACVR1C mRNA transcripts can be found, for example, under GenBank accession numbers GI:1519315475 (NM_145259.3, SEQ ID NO: 1; reverse complement, SEQ ID NO: 2), GI:1890343165 (NM_001111031.2, SEQ ID NO: 3; reverse complement, SEQ ID NO: 4), GI:1676439980 (NM_001111032.2, SEQ ID NO: 5, reverse complement, SEQ ID NO: 6), and GI:1676318472 (NM_001111033.2, SEQ ID NO: 7, reverse complement, SEQ ID NO: 8). The sequence of mouse ACVR1C mRNA can be found, for example, under GenBank accession number GI:161333830 (NM_001111030.1, SEQ ID NO: 9; reverse complement, SEQ ID NO: 10) or GI:161333829 (NM_001033369.3, SEQ ID NO: 11; reverse complement, SEQ ID NO: 12). The sequence of rat ACVR1C mRNA can be found, for example, under GenBank accession number GI:1937875934 (NM_139090.2; SEQ ID NO: 13; reverse complement, SEQ ID NO: 14). The sequence of rhesus monkey (Macaca mulatta) ACVR1C mRNA can be found, for example, under GenBank accession number GI:388454445 (NM_001266690.1; SEQ ID NO: 15; reverse complement, SEQ ID NO: 16). The sequence of the cynomolgus monkey (Macaca fascicularis) ACVR1C mRNA can be found, for example, under GenBank accession number GI:2161825093 (XM_005573226.3; SEQ ID NO: 17; reverse complement, SEQ ID NO: 18). The sequence of the cynomolgus monkey ACVR1C mRNA can also be found, for example, under XM_015432222.1; (SEQ ID NO: 19; reverse complement, SEQ ID NO: 21); or XM_005573228.2 (SEQ ID NO: 21; reverse complement, SEQ ID NO: 22); or XM_005573225.2 (SEQ ID NO: 23; reverse complement, SEQ ID NO: 24). The sequence of cynomolgus monkey ACVR1C mRNA can be found, for example, under GenBank accession number GI:2161825091 (XM_005573224.3; SEQ ID NO: 25; reverse complement, SEQ ID NO: 26).The sequence of cynomolgus monkey ACVR1C mRNA can be found, for example, under GenBank accession number GI:2161825094 (XM_005573230.3; SEQ ID NO: 27; reverse complement, SEQ ID NO: 28).

[0115] Further examples of ACVR1C mRNA sequences are readily available through publicly accessible databases, such as GenBank, UniProt, OMIM, the UCSC Genome Browser, and the Macaca Genome Project website. Further information on ACVR1C can be found at www.ncbi.nlm.nih.gov / gene / ?term=ACVR1C.

[0116] The term ACVR1C also, as used herein, refers to variations of the ACVR1C gene, including variants provided to SNP databases. A great many 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). The entire contents of the aforementioned GenBank accession numbers and gene database numbers are incorporated herein by reference as of the filing date of this application.

[0117] "Target sequence" or "target nucleic acid," as used herein, refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the ACVR1C gene, which contains mRNA that is the product of RNA processing of the primary transcript. In one embodiment, the target portion of the sequence is expected to be long enough to serve as a substrate for RNAi-directed cleavage at or near that portion of the nucleotide sequence of the mRNA molecule formed during the transcription of the ACVR1C gene. In one embodiment, the target sequence is within the protein-coding region of the ACVR1C gene. In another embodiment, the target sequence is within the 3'UTR of the ACVR1C gene. The target nucleic acid may be a cellular gene (or mRNA transcribed from that gene) whose expression is associated with a particular disorder or pathological condition.

[0118] The target sequence may be approximately 19 to 36 nucleotides in length, for example, approximately 19 to 30 nucleotides in length. For example, the target sequence may be approximately 19 to 30 nucleotides in length, and may be 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 certain embodiments, the target sequence may be 19 to 23 nucleotides in length, or 21 to 23 nucleotides in length. Ranges and lengths intermediate to those described above are also expected to be part of this disclosure.

[0119] The term “sequence-containing chain” as used herein refers to an oligonucleotide containing a chain of nucleotides described by a sequence referenced using standard nucleotide nomenclature.

[0120] "G," "C," "A," "T," and "U" generally refer to nucleotides containing guanine, cytosine, adenine, thymidine, and uracil as bases, respectively. However, it will be understood that the terms "ribonucleotide" or "nucleotide" may also refer to modified nucleotides or substitute portions (see, for example, Table 1), as detailed below. Those skilled in the art will be well aware that guanine, cytosine, adenine, and uracil can be replaced with other portions that do not substantially alter the base-pairing properties of oligonucleotides containing such substitute portions. For example, but not limited to, nucleotides containing inosine as a base can base-pair with nucleotides containing adenine, cytosine, or uracil. Thus, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA described herein with, for example, nucleotides containing inosine. In another example, any adenine and cytosine in the oligonucleotide can be replaced with guanine and uracil, respectively, to form GU fluctuation base pairs with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods described herein.

[0121] The terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interference agent,” as used synonymously herein, refer to agents containing RNA, as defined herein, that mediate targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. iRNA directs sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA modulates, for example, inhibits the expression of the ACVR1C gene in cells within a subject such as a mammalian subject, for example, in adipocytes.

[0122] In one embodiment, the RNAi agent of the present disclosure comprises a single-stranded RNA that interacts with a target RNA sequence, such as an ACVR1C mRNA sequence, to direct the cleavage of the target RNA. While we do not wish to be constrained by theory, it is thought that long double-stranded RNA introduced into cells is degraded into siRNA by type III endonucleases known as Dicers [Sharp et al. (2001) Genes Dev. 15:485]. Dicers, i.e., ribonuclease-III-like enzymes, process dsRNA into short interfering RNAs of 19-23 base pairs with a characteristic two-base 3' overhang [Bernstein, et al., (2001) Nature 409:363]. The siRNA is then incorporated into an RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA double helix, allowing the complementary antisense strand to guide target recognition [Nykanen, et al., (2001) Cell 107:309]. Once bound to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target, inducing silencing [Elbashir, et al., (2001) Genes Dev. 15:188]. Thus, in one embodiment, this disclosure relates to single-stranded RNA (siRNA) that is generated in cells and facilitates the formation of a RISC complex for performing silencing of the ACVR1C gene. Accordingly, the term “siRNA” is also used herein to refer to iRNA as described above.

[0123] In certain embodiments, the RNAi agent may be a single-stranded siRNA (ssRNAi) introduced into a cell or organism to inhibit a target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaut-2 and then cleaves the target mRNA. Single-stranded siRNAs typically consist of 15 to 30 nucleotides and are chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein may be used as single-stranded siRNA as described herein or chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

[0124] In certain embodiments, “iRNA” for use in the compositions, uses, and methods of this disclosure is double-stranded RNA and is referred herein to as “double-stranded RNA agent,” “double-stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA.” The term “dsRNA” refers to a complex of ribonucleic acid molecules having a double-stranded structure containing two antiparallel, substantially complementary nucleic acid strands, which are referred to as having “sense” and “antisense” directions with respect to the target RNA, i.e., the ACVR1C mRNA sequence. In certain embodiments of this disclosure, double-stranded RNA (dsRNA) initiates the degradation of the target RNA, e.g., mRNA, via a post-transcriptional gene silencing mechanism referred herein to as RNA interference or RNAi.

[0125] Generally, the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. In addition, as used herein, “iRNA” may contain ribonucleotides with chemical modifications, and iRNA may contain substantial modifications in multiple nucleotides. The term “modified nucleotide” as used herein independently refers to a nucleotide having a modified sugar moiety, a modified internucleotide linkage, or a modified nucleic acid base, or any combination thereof. Thus, the term modified nucleotide includes, for example, the substitution, addition, or removal of functional groups or atoms to internucleoside linkages, sugar moieties, or nucleic acid bases. Suitable modifications for use in the agents of this disclosure include all types of modifications disclosed herein or known in the art. Any such modifications, when used in an siRNA-type molecule, are encompassed in “iRNA” or “RNAi agent” for the purposes of this specification and the claims.

[0126] In certain embodiments of the disclosure of the present invention, the inclusion of a deoxyribonucleotide can be considered to constitute a modified nucleotide when present in an RNAi agent.

[0127] The double-stranded region may be of any length as long as it allows for the specific degradation of the desired target RNA via the RISC pathway, for example, a length of approximately 19–36 base pairs, for example, a length of approximately 19–30 base pairs, for example, a length of approximately 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs, for example, a length of The range may be approximately 19–30, 19–29, 19–28, 19–27, 19–26, 19–25, 19–24, 19–23, 19–22, 19–21, 19–20, 20–30, 20–29, 20–28, 20–27, 20–26, 20–25, 20–24, 20–23, 20–22, 20–21, 21–30, 21–29, 21–28, 21–27, 21–26, 21–25, 21–24, 21–23, or 21–22 base pairs. In certain embodiments, the double-stranded region is 19–21 base pairs in length, for example, 21 base pairs in length. Ranges and lengths intermediate to the ranges and lengths described above are also expected to be part of this disclosure.

[0128] The two strands forming a double helix structure may be different parts of one larger RNA molecule, or they may be separate RNA molecules. When the two strands are part of one larger molecule and are therefore linked by an unbroken chain of nucleotides between the 3' end of one strand and the 5' end of the other strand to form a double helix structure, the linked RNA strands are called a “hairpin loop.” A hairpin loop may contain at least one unpaired nucleotide. In some embodiments, a hairpin loop may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 23 or more unpaired nucleotides. In some embodiments, a hairpin loop may contain 10 or fewer nucleotides. In some embodiments, a hairpin loop may contain 8 or fewer unpaired nucleotides. In some embodiments, a hairpin loop may contain 4 to 10 unpaired nucleotides. In some embodiments, a hairpin loop may contain 4 to 8 nucleotides.

[0129] In certain embodiments, the two chains of a double-stranded oligomer compound may be linked together. The two chains may be linked at both ends or at only one end. Linking at one end means that the 5' end of the first chain is linked to the 3' end of the second chain, or the 3' end of the first chain is linked to the 5' end of the second chain. If the two chains are linked at both ends, the 5' end of the first chain is linked to the 3' end of the second chain, and the 3' end of the first chain is linked to the 5' end of the second chain. The two chains may be linked together by an oligonucleotide linker such as (N)n, but are not limited to this; where N is independently a modified or unmodified nucleotide, and n is 3 to 23. In some embodiments, n is 3 to 10, for example, 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 may be involved in base-pair interactions with other nucleotides in the linker. The two strands may also be linked together by a non-nucleoside linker, such as the linkers described herein. Those skilled in the art will understand that any chemical modification or variation of any of the oligonucleotides described herein can be used in the oligonucleotide linker.

[0130] Hairpin and dumbbell-shaped oligomeric compounds are expected to have a double-stranded region equal to or at least 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The double-stranded region may have a length equal to or less than 200, 100, or 50. In some embodiments, the double-stranded region ranges from 15 to 30, 17 to 23, 19 to 23, and 19 to 21 nucleotide pairs in length.

[0131] The hairpin oligomer compound may have a single-stranded overhang or unpaired terminal region at 3' in some embodiments, and on the antisense side of the hairpin in some embodiments. In some embodiments, the overhang is 1 to 4, more commonly 2 to 3, nucleotides in length. The hairpin oligomer compound capable of inducing RNA interference is also referred to herein as “shRNA”.

[0132] If the two substantially complementary strands of dsRNA are composed of separate RNA molecules, these molecules may, though not necessarily, be covalently linked. If the two strands are covalently linked by means other than an unbroken chain of nucleotides between the 3' end of one strand and the 5' end of the other strand to form a double-stranded structure, the linked structure is called a “linker.” RNA strands may have the same or different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA, minus all overhangs present in the double-stranded structure. In addition to the double-stranded structure, RNAi may contain one or more nucleotide overhangs. In one embodiment of an RNAi agent, at least one strand contains a 3' overhang of at least one nucleotide. In another embodiment, at least one strand contains a 3' overhang of at least two nucleotides, 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 includes a 5' overhang of at least one nucleotide. In certain embodiments, at least one strand includes a 5' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet another embodiment, both the 3' and 5' ends of one strand of the RNAi agent include an overhang of at least one nucleotide.

[0133] In certain embodiments, the iRNA agent of the present disclosure is a dsRNA in which each strand comprises 19 to 23 nucleotides and interacts with a target RNA sequence, such as an ACVR1C mRNA sequence, to direct the cleavage of the target RNA.

[0134] In some embodiments, the iRNA of this disclosure is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, such as an ACVR1C mRNA sequence, to direct the cleavage of the target RNA.

[0135] The term “nucleotide overhang,” as used herein, refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of a double-stranded iRNA. For example, a nucleotide overhang occurs when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. A dsRNA may contain at least one nucleotide overhang; alternatively, the overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang may contain, or consist of, a nucleotide / nucleoside analog such as a deoxynucleotide / nucleoside. The overhang may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotides of the overhang may be located at the 5' end, the 3' end, or both ends of either the antisense or sense strand of the dsRNA.

[0136] In one embodiment, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at its 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more of the nucleotides in the overhang are replaced with a nucleoside thiophosphate.

[0137] In certain embodiments, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at its 3' or 5' end, e.g., 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides at its 3' or 5' end, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more of the nucleotides in the overhang are replaced with a nucleoside thiophosphate.

[0138] In certain embodiments, the antisense strand of dsRNA has an overhang of 1 to 10 nucleotides at its 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In certain embodiments, the overhang in the sense strand or the antisense strand, or both, may include an extended length of more than 10 nucleotides, for example, 1 to 30 nucleotides, 2 to 30 nucleotides, 10 to 30 nucleotides, 10 to 25 nucleotides, 10 to 20 nucleotides, or 10 to 15 nucleotides. In certain embodiments, the extended overhang is located on the double-stranded sense strand. In certain embodiments, the extended overhang is located at the 3' end of the double-stranded sense strand. In certain embodiments, the extended overhang is located at the 5' end of the double-stranded sense strand. In certain embodiments, the extended overhang is located on the double-stranded antisense strand. In certain embodiments, the extended overhang is located at the 3' end of the antisense strand of the double helix. In certain embodiments, the extended overhang is located at the 5' end of the antisense strand of the double helix. In certain embodiments, one or more nucleotides in the extended overhang are replaced with a nucleoside thiophosphate. In certain embodiments, the overhang includes a self-complementary portion so that the overhang can form a stable hairpin structure under physiological conditions.

[0139] "Blunt" or "blunt-ended" means that a double-stranded RNA agent has no unpaired nucleotides at its ends, i.e., no nucleotide overhangs. A "blunt-ended" double-stranded RNA agent is double-stranded along its entire length, i.e., has no nucleotide overhangs at either end of the molecule. The RNAi agents of this disclosure include RNAi agents with no nucleotide overhang at one end (i.e., agents with one overhang and one blunt end), or RNAi agents with no nucleotide overhangs at either end. In most cases, such molecules are expected to be double-stranded along their entire length.

[0140] The terms "antisense strand" or "guide strand" refer to a strand of iRNA, such as dsRNA, that contains a region substantially complementary to the target sequence, for example, ACVR1C mRNA.

[0141] The term “complementary region,” as used herein, refers to a region in the antisense strand that is substantially complementary to a sequence as defined herein, such as a target sequence, such as the ACVR1C nucleotide sequence. If the complementary region is not perfectly complementary to the target sequence, mismatches may be located in the interior or terminal regions of the molecule. Generally, most acceptable mismatches are in the terminal regions, for example, within 5, 4, or 3 nucleotides from the 5' or 3' end of the iRNA. In some embodiments, the double-stranded RNA agent of this disclosure contains nucleotide mismatches in the antisense strand. In some embodiments, the antisense strand of the double-stranded RNA agent of this disclosure contains four or fewer mismatches with the target mRNA, for example, the antisense strand contains four, three, two, one, or zero mismatches with the target mRNA. In some embodiments, the antisense strand of the double-stranded RNA agent of this disclosure contains four or fewer mismatches with the sense strand, for example, 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 Disclosure contains nucleotide mismatches in the sense strand. In some embodiments, the sense strand of the double-stranded RNA agent of the Disclosure 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 mismatches are, for example, within 5, 4, or 3 nucleotides from the 3' end of the iRNA. In another embodiment, the nucleotide mismatches are, for example, in the 3' terminal nucleotide of the iRNA agent. In some embodiments, the mismatches are not in the seed region.

[0142] Therefore, 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., 3, 2, 1, or 0 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 a particular embodiment, if the antisense strand of the RNAi agent contains a mismatch to the target sequence, the mismatch may be limited to the last five nucleotides from either the 5' or 3' end of the complementary region. For example, in such an embodiment, for a 23-nucleotide RNAi agent, the strand complementary to the ACVR1C region 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 the target gene. Considering the efficacy of RNAi agents with a mismatch in inhibiting ACVR1C gene expression is particularly important when it is known that specific complementary regions in the target gene exhibit polymorphic sequence variations within the population.

[0143] The terms “sense strand” or “passenger strand,” as used herein, refer to a strand of iRNA containing a region substantially complementary to the antisense strand region, and the term “antisense strand” is as defined herein.

[0144] When used herein, "substantially all nucleotides are modified" may imply that a large portion, but not all, of the nucleotides are modified, and that the product contains five or fewer, four or fewer, three or fewer, two or fewer, or one or fewer unmodified nucleotides.

[0145] The term “cleavage region,” as used herein, refers to a region located directly adjacent to a cleavage site. A cleavage site is a site on the target where cleavage occurs. In some embodiments, the cleavage region includes three bases at either end of the cleavage site, directly adjacent to the cleavage site. In some embodiments, the cleavage region includes two bases at either end of the cleavage site, directly adjacent to the cleavage site. In some embodiments, the cleavage site occurs specifically at the site where nucleotides 10 and 11 of the antisense strand are bound, and the cleavage region includes nucleotides 11, 12, and 13.

[0146] As used herein, and unless otherwise specified, the term “complementary” means, as will be understood by those skilled in the art, the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence under certain conditions to form a double-stranded structure, when used to describe a first nucleotide sequence in relation to a second nucleotide sequence. Those skilled in the art will be able to determine the set of conditions most appropriate for testing the complementarity of the two sequences according to the end use of the hybridized nucleotides.

[0147] The complementary sequences within iRNAs, for example, dsRNAs, described herein include base pairing of an oligonucleotide or polynucleotide containing a first nucleotide sequence to an oligonucleotide or polynucleotide containing a second nucleotide sequence, spanning the full length of one or both nucleotide sequences. Such sequences may be referred to herein as “fully complementary.” However, where the first sequence is referred to herein as “substantially complementary” to the second sequence, the two sequences may be fully complementary, or they may form one or more mismatched base pairs, but generally five or fewer, four or fewer, three or fewer, or two or fewer, in the case of double-strand hybridization of up to 30 base pairs, but retain the ability to hybridize under conditions best suited to their end use, e.g., inhibition of gene expression in vitro or in vivo. However, if two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, such overhangs shall not be considered mismatches for the purpose of determining complementarity. For example, a dsRNA containing one oligonucleotide of 21 nucleotides in length and another oligonucleotide of 23 nucleotides in length can still be described as “fully complementary” for the purposes described herein, if the longer oligonucleotide contains a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide.

[0148] "Complementary" sequences, as used herein, may also include, or be formed entirely from, base pairs formed from non-Watson-Crick base pairs or non-naturally modified nucleotides, provided that the above requirements regarding their ability to hybridize are met. Examples of such non-Watson-Crick base pairs include, but are not limited to, G:U fluctuations or Hoogsteen base pairings.

[0149] The terms “complementary,” “fully complementary,” and “substantially complementary” can be used herein, as will be understood from the context of their use, in relation to base matching between the sense strand and antisense strand of a dsRNA, or between two oligonucleotides or polynucleotides, for example, between the antisense strand and target sequence of a double-stranded RNA agent.

[0150] A polynucleotide "substantially complementary to at least a portion of" messenger RNA (mRNA), as used herein, refers to a polynucleotide substantially complementary to a contiguous portion of the mRNA of interest (e.g., the mRNA encoding the ALK7 protein). For example, a polynucleotide is complementary to at least a portion of ACVR1C mRNA if its sequence is substantially complementary to an uninterrupted portion of the mRNA encoding ALK7.

[0151] Therefore, in some embodiments, the antisense strand polynucleotides disclosed herein are perfectly complementary to the target gene sequence.

[0152] In some embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to the target gene sequence and include a contiguous nucleotide sequence that is at least about 80% complementary over its entire length to the nucleotide sequence of SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27 relating to ACVR1C, or to an equivalent region of the fragment of SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27, for example, a contiguous nucleotide sequence that is about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary.

[0153] 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 over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12, or any one fragment of any one of the sense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12, for example, a contiguous nucleotide sequence that is about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary.

[0154] In some embodiments, the sense and antisense strands are selected from one of the double-stranded AD-1735894, AD-1735913, AD-2410459, AD-2410460, AD-2410461, and AD-2410462.

[0155] In some embodiments, the sense and antisense strands are selected from the double-stranded AD-1735894.

[0156] In some embodiments, the sense and antisense strands are selected from the double-stranded AD-1735913.

[0157] In some embodiments, the sense and antisense strands are selected from the double-stranded AD-2410459.

[0158] In some embodiments, the sense and antisense strands are selected from the double-stranded AD-2410460.

[0159] In some embodiments, the sense and antisense strands are selected from the double-stranded AD-2410461.

[0160] In some embodiments, the sense and antisense strands are selected from the double-stranded AD-2410462.

[0161] In one embodiment, the RNAi agent of the Disclosure comprises a sense strand substantially complementary to an antisense polynucleotide, wherein the antisense polynucleotide is identical to the target ACVR1C sequence, and the sense strand polynucleotide comprises a nucleotide sequence of SEQ ID NOs. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28, or an equivalent region of any one of the fragments of SEQ ID NOs. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28, and a contiguous nucleotide sequence that is at least about 80% complementary over its entire length, for example, comprising a contiguous nucleotide sequence that is about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary.

[0162] In some embodiments, the iRNA of the Disclosure comprises a sense strand substantially complementary to an antisense polynucleotide, the antisense polynucleotide being complementary to a target ACVR1C sequence, the sense strand polynucleotide comprising one of the antisense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12, or a fragment of one of the antisense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12, and a contiguous nucleotide sequence that is at least about 80% complementary over its entire length, for example, comprising a contiguous nucleotide sequence that is about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary.

[0163] In some embodiments, the double-stranded region of the double-stranded iRNA agent is equal in length 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, or at least 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.

[0164] In some embodiments, the antisense strand of the double-stranded iRNA agent is equal in length to 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0165] In some embodiments, the sense strand of the double-stranded iRNA agent is equal in length 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, or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0166] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent each independently have a length of 15 to 30 nucleotides.

[0167] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent each independently have a length of 19 to 25 nucleotides.

[0168] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent each independently have a length of 21 to 23 nucleotides.

[0169] In one embodiment, the sense strand of the iRNA agent has a length of 21 nucleotides, and the antisense strand has a length of 23 nucleotides, in which case the strands form a double-stranded region of 21 consecutive base pairs having a single-stranded overhang of 2 nucleotides at the 3' end.

[0170] Generally, “iRNA” includes ribonucleotides with chemical modifications. Such modifications include all types of modifications disclosed herein or known in the art. Any such modifications, when used in a dsRNA molecule, are included in “iRNA” for the purposes of this specification and the claims.

[0171] In certain embodiments of the disclosure of the present invention, the inclusion of a deoxyribonucleotide can be considered to constitute a modified nucleotide when present in an RNAi agent.

[0172] In one aspect of this disclosure, the agent for use in the methods and compositions of this disclosure is a single-stranded antisense oligonucleotide molecule that inhibits target mRNA via an antisense inhibition mechanism. The single-stranded antisense oligonucleotide molecule is complementary to a sequence in the target mRNA. The single-stranded antisense oligonucleotide can inhibit translation stoichiometrically by base-pairing with the mRNA and physically blocking the translation mechanism. See Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense oligonucleotide molecule may be about 14 to about 30 nucleotides in length and may have a sequence complementary to the target sequence. For example, the single-stranded antisense oligonucleotide molecule may contain a sequence that is at least about 14, 15, 16, 17, 18, 19, 20, or more consecutive nucleotides from any one of the antisense sequences described herein.

[0173] The phrase "bringing cells into contact with iRNA," for example, "bringing cells into contact with dsRNA," as used herein, includes bringing cells into contact by any possible means. Bringing cells into contact with iRNA includes bringing cells into contact with iRNA in vitro or in vivo. The contact may be made directly or indirectly. For example, iRNA may be brought into physical contact with cells by the individual performing the method, or alternatively, iRNA may be placed in a situation where it can later come into contact with cells or cause contact.

[0174] In vitro cell contact may be performed, for example, by incubating cells with iRNA. In vivo cell contact may be performed, for example, by injecting iRNA into or near the tissue in which the cells are located, or by injecting iRNA into another region, for example, the bloodstream or subcutaneous space, so that the drug reaches the tissue in which the cells to be later contacted are located. For example, the iRNA may contain, or be coupled to, a targeted ligand, such as GalNAc, that directs the iRNA to a site of interest, for example, the liver. In other embodiments, the RNAi agent may contain, or be coupled to, one or more C22 hydrocarbon chains and one or more GalNAc derivatives. In other embodiments, the RNAi agent may contain, or be coupled to, one or more C22 hydrocarbon chains and not contain, or be uncoupled to, one or more GalNAc derivatives. Combinations of in vitro and in vivo contact methods are also possible. For example, cells may also be contacted with the RNAi agent in vitro and then transplanted into a subject.

[0175] In certain embodiments, contacting cells with iRNA includes “introducing” or “delivering” iRNA to cells by facilitating or performing uptake or absorption into the cells. Absorption or uptake of iRNA can be carried out by spontaneous diffusion or active cellular processes, or by adjuvants or devices. Introduction of iRNA into cells may be in vitro or in vivo. For example, in the case of in vivo introduction, iRNA may be injected into a tissue site or administered systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Further approaches are described below herein or are known in the art.

[0176] The term “lipid nanoparticle” or “LNP” refers to a vesicle containing a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule, such as iRNA, or a plasmid on which iRNA is transcribed. LNPs are described, for example, in U.S. Patents 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0177] "Subject," as used herein, is an animal that expresses the target gene either endogenously or heterologously, such as mammals, e.g., primates (e.g., humans, non-human primates, e.g., monkeys, and chimpanzees), non-primates (e.g., cattle, pigs, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, or mice), or birds. In one embodiment, the subject is a human, e.g., a human being treated or evaluated for a disease or disorder in which a reduction in ACVR1C expression is expected to benefit as described herein; a human being at risk of a disease or disorder in which a reduction in ACVR1C expression is expected to benefit; a human being having a disease or disorder in which a reduction in ACVR1C expression is expected to benefit; or a human being being treated for a disease or disorder in which a reduction in ACVR1C expression is expected to benefit. 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 child subject.

[0178] The term “to treat” or “treatment,” as used herein, means reducing a beneficial or desired outcome in a subject, such as reducing at least one sign or symptom associated with ACVR1C expression or ALK7 protein production, such as ACVR1C-related disease, such as metabolic disorder. Treatment also includes reducing one or more signs or symptoms associated with unwanted ACVR1C expression; reducing the degree of unwanted ACVR1C activation or stabilization; and mitigating or temporarily mitigating unwanted ACVR1C activation or stabilization. “Treatment” also means extending survival compared to the survival predicted in the absence of treatment.

[0179] In the context of ACVR1C levels or disease markers or symptom levels in a subject, the term “lowering” refers to a statistically significant reduction in such levels. The reduction may be, for example, at least 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater. In certain embodiments, the reduction is at least 20%. In certain embodiments, the reduction in disease markers, for example, protein or gene expression levels, is at least 50%. In the context of ACVR1C levels in a subject, “lowering” means reducing them to a level acceptable as within the normal range for an individual without such disorder. In certain embodiments, “lowering” means reducing the difference between the level of a marker or symptom in a subject suffering from the disease and an acceptable level within the normal range for the individual. The term “lowering” can also be used in relation to normalizing the symptoms of a disease or condition, i.e., reducing the difference between levels in a subject with ACVR1C-related disorder toward or to the level in a normal subject without ACVR1C-related disorder. As used herein, “normal” is considered the upper limit of normal if the disease is associated with an increase in the value of the symptoms. “Normal” is considered the lower limit of normal if the disease is associated with a decrease in the value of the symptoms.

[0180] When used herein, “prevention” or “prevention” refers to a reduction in the likelihood of developing symptoms associated with such disease, disorder, or condition, e.g., symptoms of ACVR1C-related disorders, e.g., metabolic disorders, if used in relation to a disease, disorder, or condition that can be treated or mitigated by reducing the expression of the ACVR1C gene or the production of the ALK7 protein. Effective prevention is considered to be the prevention of the development of symptoms associated with such disease, disorder, or condition (e.g., a reduction of at least about 10% on a clinically acceptable scale for that disease or disorder), or a delay in the onset of delayed symptoms (e.g., a delay of several days, weeks, months, or years).

[0181] The term “ACVR1C-related disorder” or “ACVR1C-related disease” is, as used herein, a disease or disorder in which a reduction in mRNA expression or ACVR1C activity is expected to be beneficial. The term “ACVR1C-related disease” is a disease or disorder caused by or associated with ACVR1C mRNA expression or ALK7 protein production. The term “ACVR1C-related disease” includes diseases, disorders, or conditions in which a reduction in ACVR1C mRNA expression or ALK7 protein activity is expected to be beneficial.

[0182] In some embodiments, ACVR1C-related disorders are metabolic disorders.

[0183] A "metabolic disorder" is a disorder that disrupts normal metabolism, that is, the process of converting food into energy at the cellular level. Metabolic disorders affect the ability of cells to perform important biochemical reactions, including the processing or transport of proteins (amino acids), carbohydrates (sugars and starches), or lipids (fatty acids). Non-limiting examples of metabolic disorders include carbohydrate disorders, e.g., diabetes mellitus, type 1 diabetes mellitus, type 2 diabetes mellitus, galactosemia, hereditary fructose intolerance, fructose-1,6-diphosphatase deficiency, impaired glycogen storage, congenital glycosylation disorders, insulin resistance, insulin deficiency, insulin hyperinsulinemia, impaired glucose tolerance (IGT), abnormal glycogen metabolism; amino acid metabolism disorders, e.g., maple syrup urine disease (MSUD), or homocystinuria; organic acid metabolism disorders, e.g., methylmalonic aciduria, 3-methylglutaconic aciduria - Baas syndrome, glutaric aciduria, or 2-hydroxyglutaric aciduria - D and L types; fatty acid beta-oxidation disorders, e.g., medium-chain acyl- CoA dehydrogenase deficiency (MCAD), long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHAD), very long-chain acyl-CoA dehydrogenase deficiency (VLCAD); lipid metabolism disorders, e.g., GM1 gangliosidosis, Tay-Sachs disease, Sandhoff disease, Fabry disease, Gaucher disease, Niemann-Pick disease, Krabbe disease, mucolipidosis, or mucopolysaccharidosis; lipid distribution and / or storage disorders, e.g., lipodystrophy; mitochondrial disorders, e.g., mitochondrial cardiomyopathy; Leigh disease; mitochondrial encephalopathy, lactic acidosis, and stroke-like seizures (MELAS); myoclonus epilepsy with red ragged fibers (myoclonic Epilepsy with ragged-red fibers (MERRF); neuropathy, ataxia, and retinitis pigmentosa (NARP); Barth syndrome; peroxisome diseases, such as Zellweger syndrome (cerebral-hepatorenal syndrome), X-linked adrenoleukodystrophy, or Refsum disease.

[0184] In one embodiment, metabolic disorders are metabolic syndromes. The term “metabolic syndrome,” as used herein, is a disorder comprising a combination of components reflecting overnutrition, a sedentary lifestyle, genetic factors, aging, and the resulting excessive obesity. Metabolic syndromes include a combination of abdominal obesity, insulin resistance, dyslipidemia, and elevated blood pressure, and are associated with other comorbidities such as pro-thrombogenic conditions, pro-inflammatory conditions, non-alcoholic fatty liver disease, and reproductive disorders. The prevalence of metabolic syndromes is increasing to epidemic levels not only in the United States and other urbanized countries of the world, but also in developing countries. Metabolic syndromes are associated with nearly doubling the risk of cardiovascular disease and increasing the risk of concomitant type 2 diabetes fivefold.

[0185] Abdominal fat accumulation [e.g., large waist circumference (high waist-to-hip ratio)], high blood pressure, insulin resistance, and lipid metabolism disorders are central to metabolic syndromes and their individual components (e.g., central obesity, fasting blood glucose (FBG) / prediabetes / diabetes, hypercholesterolemia, hypertriglyceridemia, and hypertension).

[0186] In one embodiment, the metabolic disorder is a carbohydrate disorder. In one embodiment, the carbohydrate disorder is diabetes.

[0187] The term “diabetes” as used herein refers to a group of metabolic disorders characterized by high blood glucose levels resulting from a defect in insulin secretion, action, or both. Exemplary symptoms of diabetes include frequent urination, increased thirst, blurred vision, slow wound healing, numbness or tingling in the hands and feet, dry skin, and frequent infections, such as those of the gums, skin, and vagina. There are two most common types of diabetes, namely type 1 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 an increase in blood glucose levels.

[0188] The term "Type 1 diabetes," as used herein, refers to a chronic disease that occurs when the pancreas produces too little insulin to adequately regulate blood glucose levels. Type 1 diabetes is also known as insulin-dependent diabetes, IDDM, and juvenile diabetes. People with Type 1 diabetes (insulin-dependent diabetes) produce little or no insulin. Although about 6 percent of the US population has some form of diabetes, only about 10 percent of all diabetes cases are Type 1 diabetes. Most people with Type 1 diabetes develop the condition before the age of 30. Type 1 diabetes is a typical example of the progressive autoimmune destruction of pancreatic beta cells, resulting in insulin deficiency. More than 90 percent of the insulin-producing cells (beta cells) in the pancreas are permanently destroyed. The resulting insulin deficiency is severe, and people with Type 1 diabetes must regularly inject insulin to survive. People with Type 1 diabetes may also experience nausea, vomiting, or stomach pain.

[0189] In type 2 diabetes (also known as non-insulin-dependent diabetes mellitus or NDDM), the pancreas continues to produce insulin, sometimes at levels significantly higher than normal. However, the body develops tolerance to its effects, resulting in relative insulin deficiency. While type 2 diabetes can occur in children and young adults, it usually develops after age 30 and becomes increasingly common with age: approximately 15 percent of people over 70 have type 2 diabetes. Obesity is a risk factor for type 2 diabetes, and 80-90 percent of people with this condition are obese.

[0190] In some embodiments, diabetes includes prediabetes. “Prediabetes” refers to one or more early diabetic conditions, such as abnormal glucose utilization, abnormal or abnormal fasting glucose levels, impaired glucose tolerance, and abnormal insulin sensitivity and insulin resistance. Prediabetes is a major risk factor for developing type 2 diabetes, cardiovascular disease, and death.

[0191] In some embodiments, diabetes mellitus includes secondary or other specific types of diabetes mellitus, e.g., monogenic deficiency of beta-cell function, gene deficiency of insulin action, exocrine pancreatic disease, endocrine disease, drug / chemical-induced, infectious, and rare immune-mediated and genetic syndromes associated with diabetes mellitus. Exemplary genetic deficiencies of beta-cell function include, but are not limited to, juvenile-onset adult-type diabetes mellitus 3 (MODY3) (chromosome 12, HNF-1α), MODY1 (chromosome 20, HNF-4α), MODY2 (chromosome 7, glucokinase), other very rare forms of MODY (e.g., MODY4: chromosome 13, insulin promoter factor-1; MODY6: chromosome 2, NeuroD1; MODY7: chromosome 9, carboxyl ester lipase); transient neonatal diabetes mellitus (most commonly ZAC / HYAMI imprinting deficiency at 6q24), or persistent neonatal diabetes mellitus (most commonly the KCNJ11 gene encoding the Kir6.2 subunit of the beta-cell KATP channel). Exemplary genetic defects in insulin action include, but are not limited to, type A insulin resistance, pyelonephrosis, Rabson-Mendenhall syndrome, lipoatrophic diabetes mellitus, or mutant insulin. Exemplary diseases of the pancreatic exocrine glands include, but are not limited to, pancreatitis, trauma / pancreatectomy, neoplasia, cystic fibrosis, hemochromatosis, or fibrocalculous pancreatopathy. Exemplary endocrine diseases include, but are not limited to, acromegaly, Cushing's syndrome, glucagonoma, chromaffin cell tumor, somatostatin-producing tumor, aldosterone-producing adenoma, or hyperthyroidism. Exemplary drug / chemical-induced diabetes mellitus include, but are not limited to, diabetes mellitus induced by vacor, pentamidine, nicotinic acid, glucocorticoids, thyroid hormones, diazoxide, β-adrenergic agonists, thiazides, dilantine, or γ-interferon.Exemplary diabetes-related infections include, but are not limited to, congenital rubella, cytomegalovirus, or mumps. Rare immune-mediated and genetic syndromes associated with exemplary diabetes include, but are not limited to, Stiffman syndrome, anti-insulin receptor antibody syndrome, Down syndrome, Klinefelter syndrome, Turner syndrome, Wolfram syndrome, Friedreich ataxia, Huntington's chorea, Laurence-Moon-Beadle syndrome, myotonic dystrophy, porphyria, or Prader-Willi syndrome. Diabetes can be diagnosed by applying a glucose tolerance test. Clinically, diabetes is often divided into several basic categories. Major examples of these categories include autoimmune diabetes, non-insulin-dependent diabetes mellitus (type 1 NDDM), insulin-dependent diabetes mellitus (type 2 IDDM), non-autoimmune diabetes mellitus, non-insulin-dependent diabetes mellitus (type 2 NIDDM), and juvenile-onset adult-onset diabetes mellitus (MODY). A further category, often referred to as secondary diabetes, is diabetes caused by several identifiable conditions that cause or enable the development of diabetic diabetes. Examples of secondary diabetes include diabetes caused by pancreatic disease, hormonal abnormalities, drug or chemical-induced diabetes, diabetes caused by insulin receptor abnormalities, diabetes associated with genetic syndromes, and diabetes of other causes. [See, for example, Harrison's (1996) 14th ed., New York, McGraw-Hill].

[0192] In one embodiment, the metabolic disorder is a lipid metabolism disorder. “Lipid metabolism disorder” or “lipid metabolism disorder” as used herein means any disorder related to or caused by a disorder in lipid metabolism. The term also includes any disorder, disease or condition that may cause hyperlipidemia, or any condition characterized by an abnormally elevated level of any or all lipids and / or lipoproteins in the blood. The term also includes hereditary disorders, e.g., familial hypertriglyceridemia, familial partial lipodystrophy types (FPLDs) (e.g., familial partial lipodystrophy type 1 (FPLD1) or Kobberling type, FPLD2 or Dunnigan type, FPLD3 or PPARG-related FPLD, FPLD4 or PLIN1-related FPLD, FPLD5 or CIDEC-related FPLD, FPLD6 or LIPE-related FPLD, CAV1-related FPLD, ADRA2A-related FPLD, AKT2-related FPLD), progeria syndrome, MAD This term refers to disorders induced or acquired as a result of disease, disability or condition (e.g., renal failure), diet, or ingestion of certain drugs (e.g., as a result of highly active antiretroviral therapy (HAART) used to treat AIDS or HIV). The term also refers to disorders of fat distribution / storage, e.g., lipodystrophy.

[0193] Further examples of lipid metabolism disorders include, but are not limited to, atherosclerosis, dyslipidemia, hypertriglyceridemia (including drug-induced hypertriglyceridemia, diuretic-induced hypertriglyceridemia, alcohol-induced hypertriglyceridemia, β-adrenergic blocker-induced hypertriglyceridemia, estrogen-induced hypertriglyceridemia, postmenopausal lipid metabolism disorders, glucocorticoid-induced hypertriglyceridemia, retinoid-induced hypertriglyceridemia, cimetidine-induced hypertriglyceridemia, and familial hypertriglyceridemia), and conditions related to hypertriglyceridemia. Examples include acute pancreatitis, chylomicron syndrome, familial chylomicronemia, apoE deficiency or resistance, LPL deficiency or decreased activity, hyperlipidemia (including familial combined hyperlipidemia), hypercholesterolemia, lipodystrophy, gout associated with hypercholesterolemia, xanthomatous cereals (subcutaneous cholesterol deposits), hyperlipidemia with heterogeneous LPL deficiency, hyperlipidemia with high LDL and heterogeneous LPL deficiency, fatty liver disease or non-alcoholic stetohepatitis (NASH), or skin conditions associated with lipid metabolism disruption (e.g., UV-induced lipid metabolism disruption and associated skin disorders, sensitive skin, or psoriasis).

[0194] Cardiovascular diseases are also considered “metabolic disorders” as defined herein. These diseases include coronary artery disease (also known as ischemic heart disease), hypertension, inflammation and restenosis associated with coronary artery disease, peripheral vascular disease, and stroke.

[0195] Kidney disease is also considered a “metabolic disorder” as defined herein. Such diseases include chronic kidney disease, diabetic nephropathy, diabetic nephropathy, or gout.

[0196] Weight-related disorders are also considered “metabolic disorders” as defined herein. Such disorders include obesity, hypometabolism, hypothyroidism, uremia, and other conditions associated with weight gain (including rapid weight gain), weight loss, maintaining weight loss, or the risk of weight regrowth after weight loss.

[0197] Blood glucose disorders are further considered “metabolic disorders” as defined herein. Examples of such disorders include diabetes mellitus, hypertension, and polycystic ovary syndrome with respect to insulin resistance. Other exemplary metabolic disorders also include kidney transplantation, nephrotic syndrome, Cushing’s syndrome, acromegaly, systemic lupus erythematosus, abnormal globulinemia, lipodystrophy, glycogen storage disease type 1, and Addison’s disease.

[0198] 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 medical cause).

[0199] In one embodiment, the metabolic disorder is secondary hypertension. "Secondary hypertension" has an identifiable underlying disorder that may have multiple etiologies, including causes of the kidneys, blood vessels, and endocrine system, such as renal parenchymal disease (e.g., polycystic kidney disease, glomerulovascular or interstitial disease), renovascular disease (e.g., renal artery stenosis, fibromuscular dysplasia), endocrine disorders (e.g., excess corticosteroids or mineralocorticoids, chromaffin cell tumor, hyperthyroidism or hypothyroidism, excess growth hormone, hyperparathyroidism), aortic coarctation, or use of oral contraceptives.

[0200] In one embodiment, the metabolic disorder is resistant hypertension. "Resistant hypertension" is a condition in which blood pressure remains above the target (e.g., systolic pressure above 130 mmHg or diastolic pressure above 90 mmHg) despite the parallel use of three different classes of antihypertensive agents, one of which is a thiazide diuretic. Patients whose blood pressure is controlled by four or more drug therapies are also considered to have resistant hypertension.

[0201] Additional diseases or conditions relating to metabolic disorders will be obvious to those skilled in the art and are within the scope of this disclosure.

[0202] Symptoms of ACVR1C-related disorders, such as metabolic disorders, include, for example, insulin resistance, inability to regulate blood glucose, loss of lean or fat-free muscle mass, excess fat mass, lower metabolic rate, weight gain, and / or increased body mass index. Further details regarding the signs and symptoms of various diseases or conditions are provided herein and are well known in the industry.

[0203] When used herein, “therapeutic effective dose” is intended to include an amount of RNAi agent sufficient to treat the disease (e.g., by reducing, alleviating, or maintaining the symptoms of a pre-existing disease or one or more diseases) when administered to a subject with ACVR1C-associated disorder. “Therapeutic effective dose” may vary depending on the RNAi agent, how the drug is administered, the disease and its severity, as well as the patient’s medical history, age, weight, family history, genetic makeup, previous or concurrent treatments, if any, of the type, and other individual characteristics of the subject to be treated.

[0204] When used herein, “Prophylactic effective dose” is intended to contain an amount of RNAi agent sufficient to prevent or alleviate the symptoms of the disease or one or more diseases when administered to a subject with ACVR1C-associated disorder. Alleviating the disease means slowing the course of the disease or reducing the severity of later-onset disease. The “Prophylactic effective dose” may be modified depending on the RNAi agent, how the drug is administered, the degree of the disease risk, as well as the patient’s medical history, age, weight, family history, genetic makeup, previous or concurrent treatments, if any, of the type, and other individual characteristics of the patient being treated.

[0205] The “therapeutic effective dose” or “preventive effective dose” also includes the amount of RNAi agent that produces several desired effects with a reasonable benefit-risk ratio applicable to either treatment. The iRNA employed in the method of the disclosure of the present invention may be administered in an amount sufficient to produce a reasonable benefit-risk ratio applicable to such treatment.

[0206] The term "pharmaceutically acceptable" is used herein to mean, within the bounds of sound medical judgment, a compound, material, composition, or dosage form suitable for use in contact with human and animal tissues, with a reasonable benefit-to-risk ratio that does not cause excessive toxicity, irritation, allergic response, or other problems or complications.

[0207] The term “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, manufacturing aid [e.g., lubricant, talc, magnesium stearate, calcium stearate or zinc stearate, or steric acid], or a material that encapsulates a solvent involved in transporting or delivering a compound of interest from one organ or part of the body to another. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the composition and is not harmful to the subject being treated. Such carriers are known in the art. Examples of pharmaceutically acceptable carriers include carriers for administration by injection.

[0208] The term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, in addition to fluids, cells, or tissues present within the subject. Examples of biological fluids include blood, serum and serous fluids, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, and saliva. Tissue samples may include samples from tissues, organs, or local areas. For example, a sample may be derived from a specific organ, a part of an organ, or fluids or cells within those organs. In certain embodiments, a sample may be derived from fat (e.g., a specific segment of adipose tissue, or a specific type of cell in fat, e.g., adipocytes). In some embodiments, a sample may be derived from subcutaneous white adipose tissue (scWAT) (e.g., abdomen or groin), visceral white adipose tissue (VWAT) (e.g., mesentery, gonads, perirenal), or brown adipose tissue (BAT) (e.g., interscapular, periaortic). In some embodiments, “subject-derived sample” refers to urine obtained from the subject. "Sample derived from the subject" may also refer to blood, or blood-derived serum or plasma, from the subject.

[0209] II. iRNAs of the Disclosure The disclosure of the present invention provides iRNAs that inhibit the expression of the ACVR1C gene. In certain embodiments, the iRNAs include double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the ACVR1C gene in cells (e.g., adipocytes) in subjects prone to developing ACVR1C-related diseases, such as metabolic disorders, e.g., metabolic syndromes; carbohydrate disorders, e.g., type II diabetes, prediabetes; lipid metabolism disorders, e.g., hyperlipidemia, hypertension, lipodystrophy; renal disease; cardiovascular disease; and weight disorders, such as cells in mammals, e.g., humans.

[0210] The dsRNA agent contains an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed by the expression of the ACVR1C gene. The complementary region is approximately 19 to 30 nucleotides in length (for example, approximately 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides).

[0211] Upon contact with cells expressing the ACVR1C gene, the iRNA inhibits the expression of the ACVR1C gene (e.g., human, primate, non-primate, or rat gene) by at least about 50% when assayed by, for example, PCR or branched DNA (bDNA)-based methods, or by protein-based methods, such as Western blotting or flow cytometry techniques, such as immunofluorescence analysis or LCMS. In certain embodiments, inhibition of expression is determined by the qPCR method provided in the examples herein, using appropriate biological cell lines provided therein, with siRNA at a concentration of, for example, 10 nM. In certain embodiments, inhibition of expression in vivo is determined by knockdown of the human gene in rodents expressing the human gene, such as mice expressing a human target gene or mice infected with AAV, when administered as a single dose, for example, 3 mg / kg, at the lowest point of RNA expression.

[0212] A dsRNA comprises two RNA strands, which are complementary and hybridize to form a double-stranded structure under the conditions in which the dsRNA is expected to be used. One strand of the dsRNA (the antisense strand) contains a complementary region that is substantially, generally, fully complementary to the target sequence. The target sequence can be obtained from the sequence of mRNA formed during the expression of the ACVR1C gene. The other strand (the sense strand) contains a region complementary to the antisense strand such that, when combined under favorable conditions, the two strands hybridize to form a double-stranded structure. As described elsewhere herein and as is known in the art, the complementary sequence of the dsRNA may also be contained as a self-complementary region of a single nucleic acid molecule, as opposed to being located on a separate oligonucleotide.

[0213] Generally, a double-stranded structure consists of 15-30 base pairs, for example, lengths of 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, 1 These are 9-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. In certain embodiments, the double-stranded structure is a base pair of 18–25 base pairs of 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 24–25 base pairs of length, e.g., a base pair of 19–21 base pairs. Ranges and lengths intermediate between the above-described ranges and lengths are also expected to be part of this disclosure.

[0214] Similarly, regions complementary to the target sequence are nucleotides of length 15-30, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19- 27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides, for example, nucleotides with a length of 19-23 or nucleotides with a length of 21-23. Ranges and lengths intermediate between the above-described ranges and lengths are also expected to be part of this disclosure.

[0215] In some embodiments, the double-stranded structure has a length of 19 to 30 base pairs. Similarly, the region complementary to the target sequence has a length of 19 to 30 nucleotides.

[0216] In some embodiments, the dsRNA is approximately 19–23 nucleotides long, or approximately 25–30 nucleotides long. Generally, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNA longer than approximately 21–23 nucleotides can serve as a substrate for Dicer. Also, as those skilled in the art would expect to recognize, the RNA region targeted for cleavage is expected in most cases to be part of a larger RNA molecule, often part of an mRNA molecule. Where applicable, the “part” of the mRNA target is a contiguous sequence of the mRNA target that is long enough to allow it to serve as a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

[0217] Those skilled in the art will also recognize that the double-stranded region is the main functional part of dsRNA and is a double-stranded region of approximately 19 to 30 base pairs, for example, approximately 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Therefore, in one embodiment, an RNA molecule or complex of RNA molecules having a double-stranded region of more than 30 base pairs to the extent that it is processed into a functional double helix targeting the desired RNA for cleavage, for example, 15 to 30 base pairs, is a dsRNA. Therefore, in one embodiment, it will be recognized by those skilled in the art that a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful for targeting ACVR1C gene expression is not generated in the target cell by cleaving a larger dsRNA.

[0218] The dsRNAs described herein may further contain one or more single-stranded nucleotide overhangs, e.g., 1-4, 2-4, 1-3, 2-3, 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang may exhibit superior inhibitory properties compared to their blunt-terminated counterparts. The nucleotide overhang may contain, or consist of, a nucleotide / nucleoside analog, such as a deoxynucleotide / nucleoside. The overhang may be on the sense strand, on the antisense strand, or in any combination thereof. Furthermore, the nucleotides of the overhang may be present at the 5' end, 3' end, or both ends of the antisense or sense strand of the dsRNA.

[0219] dsRNA may be synthesized by standard methods known in the art. The double-stranded RNAi compounds of this disclosure may be prepared using a two-step procedure. First, the individual strands of the double-stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compound can be prepared using liquid-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage of readily preparing oligonucleotide chains containing nucleotides that are not naturally occurring or are modified nucleotides. Similarly, the single-stranded oligonucleotides of this disclosure can be prepared using liquid-phase or solid-phase organic synthesis or both.

[0220] In one embodiment, the dsRNA of this disclosure comprises a sense sequence and an antisense sequence, which are at least two nucleotide sequences. The sense strand is selected from the group of sequences provided in any one of Tables 2-5, 7, 8, 11, and 12, and the corresponding antisense strand of the sense strand is selected from the group of sequences provided in any one of Tables 2-5, 7, 8, 11, and 12. In this embodiment, one of the two sequences is complementary to the other of the two sequences, and one of the sequences is substantially complementary to the sequence of mRNA produced by the expression of the relevant target gene. Thus, in this embodiment, the dsRNA is expected to comprise two oligonucleotides, in which case one oligonucleotide is described as the sense strand in any one of Tables 2-5, 7, 8, 11, and 12, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in any one of Tables 2-5, 7, 8, 11, and 12.

[0221] In certain embodiments, a substantially complementary sequence of the dsRNA is contained in a separate oligonucleotide. In other embodiments, a substantially complementary sequence of the dsRNA is contained in a single oligonucleotide.

[0222] In some embodiments, the sense or antisense strand is selected from one of the following double-stranded sense or antisense strands: AD-1735894, AD-1735913, AD-2410459, AD-2410460, AD-2410461, AD-2410462, AD-2509512, AD-2640052, AD-2640053, AD-2640060, AD-2509191, AD-2509494, AD-2509477, and AD-2508176.

[0223] For example, although the sequences in Table 2 are not described as modified or conjugated sequences, it will be understood that the RNA of the iRNA in this disclosure, for example, the dsRNA in this disclosure, may include any one of the sequences listed in any one of Tables 2-5, 7, 8, 11, and 12, which are unmodified, unconjugated, or modified or conjugated in a manner different from those described herein. In other words, this disclosure includes the dsRNAs of Tables 2-5, 7, 8, 11, and 12, which are unmodified, unconjugated, modified as described herein, or conjugated.

[0224] Those skilled in the art are well aware that dsRNAs having a double-strand structure of approximately 20–23 base pairs, for example, 21 base pairs, have been valued as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA double-strand structures may also be effective [Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226]. Based on the properties of the oligonucleotide sequences provided in any one of Tables 2–5, 7, 8, 11, and 12 in the embodiments described above, the dsRNAs described herein may include at least one strand having a minimum length of 21 nucleotides. It is reasonably predictable that shorter double helices having any one of the sequences in Tables 2-5, 7, 8, 11, and 12, with only a few nucleotides removed from one or both ends, may be equally effective compared to the dsRNAs described above. Therefore, dsRNAs having at least 19, 20, or more consecutive nucleotide sequences derived from any one of the sequences in Tables 2-5, 7, 8, 11, and 12, and whose ability to inhibit ACVR1C gene expression differs by about 5% or less, 10% or less, 15% or less, 20% or less, 25% or less, or 30% or less compared to dsRNAs containing the complete sequence, are expected to be within the scope of the disclosure of this invention.

[0225] In addition, the RNAs provided in Tables 2-5, 7, 8, 11, and 12 identify sites in the ACVR1C transcript that are susceptible to RISC-mediated cleavage. Thus, the disclosure of the present invention further features iRNAs that target one or less of these sites. As used herein, an iRNA is said to target a specific site within the RNA transcript if it promotes cleavage of the transcript somewhere within that particular site. Such iRNAs are generally expected to contain at least about 19 consecutive nucleotides from any one of the sequences provided in any one of Tables 2-5, 7, 8, 11, and 12, coupled to an additional nucleotide sequence obtained from a region adjacent to a selected sequence in the ACVR1C gene.

[0226] III. Modification of the RNAi agents disclosed herein In certain embodiments, the RNA of the iRNA of the Disclosure, e.g., dsRNA, is unmodified and does not include, for example, chemical modifications or conjugations known in the art and described herein. In other embodiments, the RNA of the iRNA of the Disclosure, e.g., dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the Disclosure, substantially all of the nucleotides of the iRNA of the Disclosure are modified. In other embodiments of the Disclosure, all or substantially all of the nucleotides of the iRNA are modified, i.e., five or fewer, four or fewer, three or fewer, two or fewer, or one or fewer unmodified nucleotides are present in the iRNA chain.

[0227] In some embodiments, the dsRNA agents of this disclosure include at least one nucleic acid modification as described herein. For example, the at least one modification is selected from the group consisting of modified nucleoside linkages, modified nucleic acid bases, modified sugars, and any combination thereof. Such modifications may be present anywhere in the dsRNA agents of this disclosure. For example, the modification may be present on one side of the RNA molecule.

[0228] In one embodiment, the dsRNA agent of the present disclosure comprises one or more C22 hydrocarbon chains conjugated at one or more internal positions in at least one of the strands, and the remaining positions of the sense and antisense strands are not art-known and are not subject to any additional chemical modifications described herein.

[0229] In some embodiments, the dsRNA agents of this disclosure comprise one or more C22 hydrocarbon chains conjugated at one or more internal positions on at least one strand, and comprise at least one additional nucleic acid modification as described herein. For example, the at least one modification is selected from the group consisting of modified internucleoside linkages, modified nucleic acid bases, modified sugars, and any combination thereof. Such modifications may be located anywhere in the dsRNA agents of this disclosure. For example, the modifications may be located on one side of the RNA molecule.

[0230] Modifications include, for example, terminal modifications, such as 5' terminal modifications (phosphorylation, conjugation, inverted ligation) or 3' terminal modifications (conjugation, DNA nucleotide, inverted ligation, etc.); base modifications, such as stabilizing bases, destabilizing bases, or replacement with bases that base-pair with the partner's extended repertoire, base removal (debasing nucleotide), or conjugated bases; sugar modifications (e.g., at the 2' or 4' position) or sugar replacement; or backbone modifications, such as modifications or replacements of phosphodiester linkages. Specific examples of RNAi agents useful in the embodiments described herein, but not limited to these, include RNAs containing a modified backbone or lacking natural internucleoside linkages. RNAs having a modified backbone include those lacking phosphorus atoms in the backbone. For the purposes of this specification, as sometimes referenced in the art, modified RNAs lacking phosphorus atoms in their internucleoside backbone can also be considered oligonucleosides. In some embodiments, the modified RNAi agent is expected to have a phosphorus atom in the main chain between its nucleosides.

[0231] A. Modification of nucleic acid bases The naturally occurring base moieties of nucleosides are typically heterocyclic bases. Two of the most common classes of such heterocyclic bases are purines and pyrimidines. In the case of nucleosides containing pentofuranosyl sugars, the phosphate group may be linked to the 2', 3', or 5' hydroxyl portion of the sugar. In forming oligonucleotides, such phosphate groups covalently link adjacent nucleosides to each other, forming linear polymeric compounds. Within oligonucleotides, the phosphate group is generally referred to as forming the internucleoside backbone of the oligonucleotide. The naturally occurring linkage or backbone of RNA and DNA is a 3'-to-5' phosphodiester linkage.

[0232] In addition to “unmodified” or “natural” nucleic acid bases such as the purine nucleic acid bases adenine (A) and guanine (G), and the pyrimidine nucleic acid bases thymine (T), cytosine (C), and uracil (U), many modified nucleic acid bases or nucleic acid base mimetics known to those skilled in the art can be applied with the compounds described herein. Unmodified or natural nucleic acid bases may be modified or replaced to provide iRNAs with improved properties. For example, nuclease-resistant oligonucleotides may be prepared using these bases, or using any one of synthetic and natural nucleic acid bases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine) and any of the oligomer modifications described herein. Alternatively, substituted or modified analogs of any of the above bases and “universal bases” may be employed. When a natural base is replaced with a non-natural and / or universal base, the nucleotide is said to contain a modified nucleic acid base and / or a modification of a nucleic acid base as described herein. Modified nucleic acid bases and / or modifications of nucleic acid bases also include natural, unnatural, and universal bases, including conjugated moieties, such as ligands described herein. Conjugated moieties for conjugation with nucleic acid bases include cationic amino groups that can be conjugated to nucleic acid bases via linkers having appropriate alkyl, alkenyl, or amide links.

[0233] The oligomeric compounds described herein may also include modifications or substitutions of nucleic acid bases (often simply referred to in the industry as “bases”). “Unmodified” or “natural” nucleic acid bases as used herein include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Exemplary modified nucleic acid bases, but not limited to these, include other synthetic and natural nucleic acid bases such as inosine, xanthine, hypoxanthine, nebularine, isoguanisine, tubercidine, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl(aminoalkyl))adenine, 2-(aminopropyl)adenine, and 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 Nin, 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-(guanidinium alkyl)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-(methoxycarbonyl-methyl)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)psoidouracil, 4-(thio)psoidouracil, 2,4-(dithio)psoidouracil (psuedouracil), 5-(alkyl)psoidouracil, 5-(methyl)-2-(thio)psoidouracil, 5-(methyl)-2-(thio)psoidouracil, 5-(alkyl)-4-(thio)psoidouracil, 5-(methyl)-4-(thio)psoidouracil, 5-(alkyl)-2,4- (Dithio)psoidouracil, 5-(methyl)-2,4-(dithio)psoidouracil, 1-substituted pseudouracil, 1-substituted 2(thio)psoidouracil, 1-substituted 4(thio)psoidouracil, 1-substituted 2,4-(dithio)psoidouracil, 1-(aminocarbonylethylenyl)psoidouracil, 1-(aminocarbonylethylenyl)-2(thio)psoidouracil, 1-(aminocarbonylethylenyl)-4-(thio)psoidouracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)psoidouracil Douracil, 1-(aminoalkylaminocarbonylethylenyl)-psoidouracil, 1-(aminoalkylaminocarbonylethylenyl)-2(thio)-psoidouracil, 1-(aminoalkylaminocarbonylethylenyl)-4-(thio)psoidouracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)psoidouracil, 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 1,3-(dia (Aza)-2-(oxo)-phenothiazine(phenthiazin)-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenothiazine-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 7-substituted 1,3-(diaza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenothiazine-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenothiazine-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxadin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxadin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenothiazine-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazine-1-yl, 7-(guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxadin-1-yl, 7-(guanidinium alkylhydro Xy)-1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 7-(guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenothiazine-1-yl, 7-(guanidinium alkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazine-1-yl, 1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nebularin, tubercidine, isoguanisine, inosinyl, 2-azainosinyl, 7-deazainosinyl, nitroimidazo Ryl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrillyl, 5-(methyl)isocarbostyrillyl, 3-(methyl)-7-(propynyl)isocarbostyrillyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidazopyridinyl, 9-(methyl)-imidazopyridinyl, pyrrolopyriminyl, isocarbostyrillyl, 7-(propynyl)isocarbostyrillyl, propynyl-7-(aza) Indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenylon, tetracerenyl, pentaceryl, 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 pyrimidine, N,2 - Substitution purine, N 6 - Substitution purine, O 6 -substituted purines, substituted 1,2,4-triazoles, pyrrolopyrimidine-2-on-3-yl, 6-phenylpyrrolopyrimidine-2-on-3-yl, para-substituted 6-phenylpyrrolopyrimidine-2-on-3-yl, ortho-substituted 6-phenylpyrrolopyrimidine-2-on-3-yl, bis-ortho-substituted 6-phenylpyrrolopyrimidine-2-on-3-yl, para-(aminoalkylhydroxy)-6-phenylpyrrolopyrimidine-2-on- Examples include 3-yl, ortho-(aminoalkylhydroxy)-6-phenylpyrrolopyrimidine-2-on-3-yl, bis-ortho--(aminoalkylhydroxy)-6-phenylpyrrolopyrimidine-2-on-3-yl, pyridopyrimidine-3-yl, 2-oxo-7-aminopyridopyrimidine-3-yl, 2-oxo-pyridopyrimidine-3-yl, or any O-alkylated or N-alkylated derivative thereof. Alternatively, substituted or modified analogs of any of the above bases and “universal bases” may be employed.

[0234] A universal nucleobase, as used herein, is any nucleobase that can base pair with all four naturally occurring nucleobases without substantially affecting fusion behavior, recognition by intracellular enzymes, or iRNA double-strand activity. Some exemplary universal nucleobases, but not limited to these, include 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methylisocarbostyrilyl, 5-methylisocarbostyril, 3-methyl-7-propynylisocarbostyril, 7-azaindolyl, and 6-methyl-7-a Examples include zaindolyl, imidazopyridinyl, 9-methylimidazopyridinyl, pyrrolopyridinyl, isocarbostyryl, 7-propynylisocarbostyryl, propynyl-7-azaindyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracerenyl, pentacerenyl, and their structural derivatives (see, for example, Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

[0235] Further nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808; those disclosed in International Application No. PCT / US09 / 038425 filed on 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 by Sanghvi, YS, Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Eds., CRC Press, 1993. All of the above is incorporated herein by reference.

[0236] In certain embodiments, the modified nucleic acid base is a nucleic acid base that is structurally quite similar to the parent nucleic acid base, such as 7-deazapurine, 5-methylcytosine, or G-clamp. In certain embodiments, the nucleic acid base mimetic includes more complex structures, such as tricyclic phenoxazine nucleic acid base mimetic. Methods for preparing the modified nucleic acid bases described above are well known to those skilled in the art.

[0237] B. Modification of sugars The dsRNA agents of this disclosure provided herein may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) monomers comprising a nucleoside or nucleotide having a modified sugar moiety. For example, the furanosyl sugar ring of a nucleoside can be modified in numerous ways, including, but not limited to, the addition of substituents, or the cross-linking of two non-geminal ring atoms to form locked nucleic acids or bicyclic nucleic acids. In certain embodiments, the oligomeric compound comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) monomers that are LNAs.

[0238] In some embodiments of locked nucleic acids, the 2' position of furnaosyl 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)-; It is linked to the 4th position by a linker that is selected independently, During the ceremony, x is 0, 1, or 2; n is 1, 2, 3, or 4; Each R1 and R2 is independently H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1); Each of 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.

[0239] In some embodiments, each linker 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 linker 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 a C1-C12 alkyl group.

[0240] Specific LNAs have been prepared and disclosed in patent documents and 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; WO94 / 14226; WO2005 / 021570; Singh et al., J. Org. Chem., 1998, 63, Examples of issued U.S. patents and published applications disclosing 10035-10039;LNA 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; as well as pre-grant U.S. Publications Nos. 2004-0171570; 2004-0219565; 2004-0014959; 2003-0207841; 2004-0143114; and 20030082807.

[0241] LNAs in which the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring, forming a methyleneoxy(4'-CH2-O-2') linkage and thus forming a bicyclic sugar moiety, are also provided herein (reviewed in Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S. Patents 6,268,490 and 6,670,461). The linkage may also be a methylene(-CH2-) group that forms a bridge with the 2' oxygen atom and the 4' carbon atom, in which case the term methyleneoxy(4'-CH2-O-2')LNA is used for the bicyclic portion; in the case where this position is an ethylene group, the term ethyleneoxy(4'-CH2CH2-O-2')LNA is used (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 thermal stability (Tm=+3~+10°C) in double helix with complementary DNA and RNA, stability against degradation by 3' exonucleases, and excellent solubility. Promising and non-toxic antisense oligonucleotides containing BNA have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638).

[0242] Similarly discussed isomer of methyleneoxy(4'-CH2-O-2')LNA is alpha-L-methyleneoxy(4'-CH2-O-2')LNA, which has been shown to have excellent stability against 3'-exonucleases. Alpha-L-methyleneoxy(4'-CH2-O-2')LNA was incorporated into antisense gapmers and chimeras that showed strong antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0243] The synthesis and preparation of methyleneoxy(4'-CH2-O-2')LNA monomers, adenine, cytosine, guanine, 5-methylcytosine, thymine, and uracil, along with 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 WO98 / 39352 and WO99 / 14226.

[0244] Phosphothioate methyleneoxy(4'-CH2-O-2')LNA and 2'-thio-LNA, analogs of methyleneoxy(4'-CH2-O-2')LNA, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of locked nucleoside analogs containing oligodeoxyribonucleotide double helixes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO99 / 14226). Furthermore, the synthesis of 2'-amino-LNA, a novel conformationally restricted high-affinity oligonucleotide analog, has been described in the industry (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2'-amino- and 2'-methylamino-LNAs have been prepared, and their double-strand thermal stability with complementary RNA and DNA strands has been reported to date.

[0245] Modified sugar moieties are well known and can be used to alter, typically increase, the affinity of antisense compounds to their target, and / or increase nuclease resistance. A representative list of modified sugars, but not limited to these, includes bicyclic modified sugars, e.g., methyleneoxy(4'-CH2-O-2')LNA and ethyleneoxy(4'-(CH2)2-O-2' bridged)ENA; substituted sugars having 2'-F, 2'-OCH3, or 2'-O(CH2)2-OCH3 substituents, particularly 2'-substituted sugars; and 4'-thio-modified sugars. Sugars may also be substituted, among other things, with sugar mimetic groups. Methods for preparing modified sugars are well known to those skilled in the art.

[0246] 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(CH2CH2O) n CH2CH2OR, n=1~50; a "locked" nucleic acid (LNA) 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) n Examples include amines (n=1~10, amine=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, ethylenediamine or polyamino); and O-CH2CH2(NCH2CH2NMe2)2.

[0247] "Deoxy" modifications include hydrogen (i.e., deoxyribose sugar, which is particularly important for single-chain overhangs); halo (e.g., fluoro); amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH2CH2NH) nCH2CH2-amines (amine = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino);-NHC(O)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); cyano; mercapto; alkylthioalkyl; thioalkoxy; thioalkyl; alkyl; cycloalkyl; aryl; examples include alkenyls and alkynyls which may be substituted with amino functional groups.

[0248] Other suitable 2' modifications, such as a modified MOE, are described in U.S. Patent Application Publication No. 20130130378, the contents of which are incorporated herein by reference.

[0249] Modifications at the 2' position may be present in the arabinose configuration. The term "arabinose configuration" refers to the configuration of the substituent at C2' of ribose in the same configuration as the 2'-OH in arabinose.

[0250] The sugar may contain two different modifications, such as gem modifications, on the same carbon of the sugar. The sugar group may also contain one or more carbons having a stereochemical configuration opposite to that of the corresponding carbon of ribose. Thus, the oligomeric compound may contain one or more monomers, for example, arabinose as the sugar. The monomer may have an alpha linkage at the 1' position of the sugar, for example, an alpha-nucleoside. The monomer may also have an opposite configuration at the 4' position, for example, by exchanging C5' and H4' or substitutions between them. When C5' and H4' or substitutions between them are exchanged, the sugar is said to be modified at the 4' position.

[0251] Other examples of dsRNA agents disclosed herein include debasic sugars, i.e., sugars lacking a nucleic acid base at C-1' or having another chemical group in place of the nucleic acid base at C1'. See, for example, U.S. Patent No. 5,998,203, the contents of which are incorporated herein in whole. These debasic sugars may also further contain modifications to one or more of the sugar atoms that make up the composition. The dsRNA agents disclosed herein may also contain one or more sugars that are L-isomers, such as L-nucleosides. Modifications to the sugar group may also include sulfur, a substituted nitrogen, or the substitution of 4'-O at a CH2 group. In some embodiments, the linkage between C1' and the nucleic acid base is in an α configuration.

[0252] As a modification of sugars, acyclic nucleotides can also be cited, in which case the CC bonds between ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', C1'-O4') are absent, and / or at least one of the ribose carbons or oxygen atoms (e.g., C1', C2', C3', C4', or O4') is absent in the nucleotide, either independently or in combination. In some embodiments, acyclic nucleotides are,

[0253] [ka] In the formula, B is a modified or unmodified nucleic acid base, R1 and R2 are independently H, halogen, OR3, or alkyl; R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar.

[0254] 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'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), and gem 2'-OMe / 2'F having the stereochemistry of arabinose with 2'-O-Me.

[0255] When a particular nucleotide is linked to the next nucleotide via its 2' position, it will be understood that the sugar modifications described herein may be located at the 3' position of the sugar of the particular nucleotide, for example, the nucleotide linked via its 2' position. Modifications at the 3' position may be present in the xylose configuration. The term "xylose configuration" refers to the configuration of substituents at C3' of ribose in the same configuration as the 3'-OH in xylose sugar.

[0256] The hydrogens connected to C4' and / or C1' can be replaced by linear or branched optionally substituted alkyls, optionally substituted alkenyls, optionally substituted alkynyls, and the main chains of alkyls, alkenyls, and alkynyls may contain one or more of O, S, S(O), SO2, N(R'), C(O), N(R')C(O)O, O(O)N(R'), CH(Z'), phosphorus-containing linkages, optionally substituted aryls, optionally substituted heteroaryls, optionally substituted heterocyclics, or optionally substituted cycloalkyls, where R' is hydrogen, acyl, or optionally substituted aliphatic, and Z' is OR 11 COR 11 CO2R 11 ,

[0257]

Chemical Formula

[0258] In some embodiments, C4' and C5' together form an optionally substituted heterocyclic ring, which preferably comprises at least one -PX(Y)-, X is H, OH, OM, SH, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted alkylamino or optionally substituted dialkylamino, M is, independently at each occurrence, an alkali metal or transition metal with a total charge of +1; Y is O, S, or NR', and R' is hydrogen, optionally substituted aliphatic. Preferably, this modification is at the 5' end of the iRNA.

[0259] In certain embodiments, the LNA includes a bicyclic nucleoside having the formula:

[0260]

Chemical Formula

[0261] In some embodiments, each of the substituted groups is independently monosubstituted or polysubstituted with a protected substituent independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, and CN, where each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O, S, or NJ1.

[0262] In certain such embodiments, each of the substituted groups is independently monosubstituted or polysubstituted with a substituent 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 a substituted C1-C6 alkyl, and X is O or NJ1.

[0263] In certain embodiments, the Z group is a C1-C6 alkyl group substituted with one or more Xx, where each Xx is independently OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, or CN; each J1, J2, and J3 is independently H or a C1-C6 alkyl group, where X is O, S, or NJ1. In another embodiment, the Z group is a C1-C6 alkyl group substituted with one or more Xx, where each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O-), substituted alkoxy, or azide.

[0264] 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; 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., CH3O-), or azide.

[0265] In certain such embodiments, the Z group is in a (R)-configuration.

[0266] [ka]

[0267] In certain such embodiments, the Z group is in an (S)-configuration.

[0268] [ka]

[0269] 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, with a more preferred hydroxyl protecting group T1 being 4,4'-dimethoxytrityl.

[0270] 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.

[0271] In certain embodiments, the compounds of this disclosure are of the formula:

[0272] [ka] Or formula:

[0273] [ka] Or formula:

[0274] [ka] It includes at least one monomer among the following: During the ceremony, Bx is the heterocyclic base moiety; T3 is a H, a hydroxyl protecting group, a linked conjugate group, or an internucleoside linking group connected to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomeric subunit, or oligomeric compound; T4 is a H, a hydroxyl protecting group, a linked conjugate group, or an internucleoside linking group connected to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomeric subunit, or oligomeric compound; At least one of T3 and T4 is an internucleoside linking group connected to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomeric subunit, or oligomeric compound; Z is a 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 amide.

[0275] In some embodiments, each of the substituted groups is independently monosubstituted or polysubstituted with a protected substituent independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, and CN, where each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O, S, or NJ1.

[0276] In some embodiments, each of the substituted groups is independently monosubstituted or polysubstituted with a substituent 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 or C1-C6 alkyl, and X is O or NJ1.

[0277] In certain such embodiments, at least one Z is a C1-C6 alkyl or a substituted C1-C6 alkyl. In certain embodiments, each Z is independently a C1-C6 alkyl or a substituted C1-C6 alkyl. In certain embodiments, at least one Z is a C1-C6 alkyl. In certain embodiments, each Z is independently a C1-C6 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 a substituted C1-C6 alkyl. In certain embodiments, each Z is independently a substituted C1-C6 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.

[0278] In certain embodiments, at least one substituent is a C1-C6 alkoxy (for example, at least one Z is a C1-C6 alkyl substituted with one or more C1-C6 alkoxys). In other embodiments, each substituent is independently a C1-C6 alkoxy (for example, each Z is independently a C1-C6 alkyl substituted with one or more C1-C6 alkoxys).

[0279] In certain embodiments, at least one C1-C6 alkoxy substituent is CH3O- (for example, at least one Z is CH3OCH2-). In other embodiments, each C1-C6 alkoxy substituent is CH3O- (for example, each Z is CH3OCH2-).

[0280] In certain embodiments, at least one substituent is halogen (for example, at least one Z is a C1-C6 alkyl substituted with one or more halogens). In certain embodiments, each substituent is independently halogen (for example, each Z is independently a C1-C6 alkyl substituted with one or more halogens). In certain embodiments, at least one halogen substituent is fluoro (for example, at least one Z is CH2FCH2-, CHF2CH2-, or CF3CH2-). In certain embodiments, each halo substituent is fluoro (for example, each Z is independently CH2FCH2-, CHF2CH2-, or CF3CH2-).

[0281] In certain embodiments, at least one substituent is a hydroxyl group (for example, at least one Z is a C1-C6 alkyl group substituted with one or more hydroxyls). In certain embodiments, each substituent is independently a hydroxyl group (for example, each Z is independently a C1-C6 alkyl group substituted with one or more hydroxyls). In certain embodiments, at least one Z is HOCH2-. In other embodiments, each Z is HOCH2-.

[0282] In certain embodiments, at least one Z is CH3-, CH3CH2-, CH2OCH3-, CH2F-, or HOCH2-. In certain embodiments, each Z is independently CH3-, CH3CH2-, CH2OCH3-, CH2F-, or HOCH2-.

[0283] In a particular embodiment, at least one Z group is a C1-C6 alkyl group substituted with one or more Xx groups, where each Xx is independently OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, or CN; each J1, J2, and J3 is independently H or a C1-C6 alkyl group, where X is O, S, or NJ1. In another embodiment, at least one Z group is a C1-C6 alkyl group substituted with one or more Xx groups, where each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O-), or azide.

[0284] In a particular embodiment, each Z group is independently a C1-C6 alkyl group substituted with one or more Xx, where each Xx is independently OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, or CN; each J1, J2, and J3 is independently H or a C1-C6 alkyl group, where X is O, S, or NJ1. In another embodiment, each Z group is independently a C1-C6 alkyl group substituted with one or more Xx, where each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O-), or azide.

[0285] 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; 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., CH3O-), or azide.

[0286] In certain embodiments, each Z group is independently -CH2Xx, and each Xx is independently OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, or CN; 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, and each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O-), or azide.

[0287] In a particular embodiment, at least one Z is CH3-. In another embodiment, each Z is CH3-.

[0288] In certain embodiments, at least one Z group of the monomer is in a (R)-configuration, and the formula is:

[0289] [ka] Or formula:

[0290] [ka] Or formula:

[0291] [ka] It is represented by [this].

[0292] In certain embodiments, the Z group of each monomer in the formula is in the (R) configuration.

[0293] In certain embodiments, at least one Z group of the monomer is in a (S)-configuration, and the formula is:

[0294] [ka] Or formula:

[0295] [ka] Or formula:

[0296] [ka] It is represented by [this].

[0297] In certain embodiments, the Z group of each monomer in the formula is in an (S)- configuration.

[0298] In certain embodiments, T3 is an H or hydroxyl protecting group. In certain embodiments, T4 is an H or hydroxyl protecting group. In further embodiments, T3 is an internucleoside linking group attached to a nucleoside, nucleotide, or monomeric subunit. In certain embodiments, T4 is an internucleoside linking group attached to a nucleoside, nucleotide, or monomeric 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 phosphodiesters or phosphorothioates.

[0299] In a particular embodiment, the dsRNA agent of the present disclosure is of the formula:

[0300] [ka] Or formula:

[0301] [ka] Or formula:

[0302] [ka] It includes at least one region of at least two consecutive monomers having .

[0303] 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, as described below:

[0304] [ka]

[0305] In certain embodiments, the dsRNA agent of the Disclosure comprises at least two regions of at least two consecutive monomers having the above formula. In certain embodiments, the dsRNA agent of the Disclosure comprises a gapped motif. In certain embodiments, the dsRNA agent of the Disclosure comprises at least one region of about 8 to about 14 consecutive β-D-2'-deoxyribofuranosyl nucleosides. In certain embodiments, the dsRNA agent of the Disclosure comprises at least one region of about 9 to about 12 consecutive β-D-2'-deoxyribofuranosyl nucleosides.

[0306] In a particular embodiment, the dsRNA agent of the present disclosure is of the formula:

[0307] [ka] It comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) (S)-cEt monomer having, In the formula, Bx is the heterocyclic base portion.

[0308] In certain embodiments, the monomer comprises a sugar mimetic. In certain such embodiments, the mimetic is used in place of a sugar or a sugar-nucleoside linkage, and the nucleic acid base is maintained for hybridization to a selected target. Typical examples of sugar mimetics include, but are not limited to, cyclohexenyl or morpholino. Typical examples of sugar-nucleoside linkage mimetics include, but are not limited to, peptide nucleic acids (PNAs) and morpholino groups linked by uncharged achiral linkages. In some cases, the mimetic is used in place of a nucleic acid base. Typical nucleic acid base mimetics are well known in the art, and examples 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 nucleic acid base mimetics are well known to those skilled in the art.

[0309] C. Modification of intersugar linkages Linking groups that form oligomeric compounds, such as oligonucleotides, by linking monomers (including, but not limited to, modified and unmodified nucleosides and nucleotides) together are described herein. Such linking groups are also referred to as intersaccharide linkages. Two main 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, phosphodiesters (P=O), phosphate triesters, methylphosphonates, phosphoramidates, and phosphorothioates (P=S). Representative phosphorus-non-containing linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters (-OC(O)-S-), thionocarbamates (-OC(O)(NH)-S-); siloxanes (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Compared to natural phosphodiester linkages, modified linkages can be used to alter, typically increase, the nuclease resistance of oligonucleotides. In certain embodiments, linkages having chiral atoms may be prepared as racemic mixtures or as separate enantomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for preparing phosphorus-containing and phosphorus-free linkages are well known to those skilled in the art.

[0310] The phosphate group in the linking group may be modified by replacing one of the oxygen atoms with a different substituent. One result of this modification may be increased resistance of the oligonucleotide to nucleolytic degradation. Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and triesters. In some embodiments, one of the oxygen atoms of the unlinked phosphate during linking can be replaced with any of the following: S, Se, BR3 (where R is hydrogen, alkyl, or aryl), C (i.e., alkyl, aryl group, etc.), H, NR2 (where R is hydrogen, optionally substituted alkyl, or aryl), or OR (where R is optionally substituted alkyl or aryl). The phosphorus atom in the unmodified phosphate group is achiral. However, the substitution of one oxygen atom that does not form a bridge 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 thus modified is a chiral center. The phosphorus atom of the chiral center may have either an "R" configuration (Rp as herein) or an "S" configuration (Sp as herein).

[0311] Phosphorodithioates have oxygen atoms that do not form crosslinks, replaced by sulfur. The phosphorus center in phosphorodithioates is achiral, and therefore the formation of oligonucleotide diastereomers is impossible. Thus, although we do not wish to be bound by theory, modification to oxygen atoms that do not form crosslinks may be desirable in that it eliminates the formation of a chiral center, e.g., phosphorodithioate formation, and thus prevents the production of diastereomer mixtures. Therefore, the oxygen atoms that do not form crosslinks may independently be any one of O, S, Se, B, C, H, N, or OR (where R is alkyl or aryl).

[0312] Phosphate linkers may also be modified by substituting the crosslinking oxygen (i.e., the oxygen linking the phosphate to the monomeric sugar) with nitrogen (crosslinked phosphoramidate), sulfur (crosslinked phosphorothioate), and carbon (crosslinked methylenephosphonate). The substitution may be made with one or both of the linking oxygen atoms. Substitution with carbon is preferred when the crosslinking oxygen is the 3'-oxygen of the nucleoside. Substitution with nitrogen is preferred when the crosslinking oxygen is the 5'-oxygen of the nucleoside.

[0313] Modified phosphate linkages are also called "non-phosphodiester intersaccharide linkages" or "non-phosphodiester linkers" if at least one of the oxygen groups linked to the phosphate is replaced, or if a phosphate group is replaced by a non-phosphorus group.

[0314] In certain embodiments, the phosphate group can be replaced with a phosphorus-free connector, such as a dephospholinker. A dephospholinker is also referred to herein as a non-phosphodiester linker. While we do not wish to be bound by theory, since the charged phosphodiester group is the reaction center in nucleolytic degradation, its replacement with a neutral structural mimetic should confer enhanced nuclease stability. Again, while we do not wish 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.

[0315] Examples of moieties that can replace the phosphate group include, but are not limited to, amides (e.g., amide-3(3'-CH2-C(=O)-N(H)-5') and amide-4(3'-CH2-N(H)-C(=O)-5')), hydroxylaminos, siloxanes (dialkylsiloxanes), carboxamides, carbonates, carboxymethyls, carbamates, carboxylic acid esters, thioethers, ethylene oxide links, sulfides, sulfonates, sulfonamides, sulfonic acid esters, thioformacetal(3'-S-CH2-O-5'), formacetal(3'-O-CH2-O-5'), oximes, methyleneiminos, and methylenecals. Examples include methylcarbonylamino (methykenecarbonylamino), methylenemethylimino (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'-OP(O)-O-SS-C5', C3'-CH2-NH-NH-C5', 3'-NHP(O)(OCH3)-O-5' and 3'-NHP(O)(OCH3)-O-5'), and nonionic linkages containing mixed N, O, S and CH2 components. For example, Carbohydrate See Modifications in Antisense Research; YS Sanghvi and PD Cook Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65). Preferred embodiments include methylene methylimino (MMI), methylene carbonylamino, amide, carbamate, and ethylene oxide linker.

[0316] Those skilled in the art will recognize that, in certain cases, the substitution of non-crosslinking oxygen can result in enhanced cleavage of intersaccharide links by adjacent 2'-OH groups, and therefore, in many examples, modification of non-crosslinking oxygen may require modification of 2'-OH groups, such as modifications that do not participate in the cleavage of adjacent intersaccharide links, e.g., arabinose sugars, 2'-O-alkyl groups, 2'-F groups, LNA groups, and ENA groups.

[0317] Preferred nonphosphodiester sugar linkages include phosphorothioates, phosphorothioates having an enantiomer excess of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or higher of Sp isomers, phosphorothioates having an enantiomer excess of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or higher of Rp isomers, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, alkylphosphonates (e.g., methylphosphonate), selenophosphates, phosphoramidates (e.g., N-alkylphosphoamidate), and boranophosphonates.

[0318] In some embodiments, the dsRNA agents of the Disclosure comprise at least one modified or non-phosphodiester linkage (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, and up to all of them). In some embodiments, the dsRNA agents of the Disclosure comprise at least one phosphorothioate linkage (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, and up to all of them).

[0319] dsRNA agents of this disclosure can also be constructed in which the phosphate linker and sugar are replaced with nuclease-resistant nucleosides or nucleotide surrogates. While we do not wish to be constrained by theory, the absence of a backbone with repeating charges is thought to reduce binding to polyanion-recognizing proteins (e.g., nucleases). Again, while we do not wish to be constrained by theory, in some embodiments it may be desirable to introduce modifications in which the bases are tethered by a neutral surrogate backbone. Examples include morpholino, cyclobutyl, pyrrolidine, peptide nucleic acid (PNA), aminoethylglycyl PNA (aegPNA), and backnone-extended pyrrolidine PNA (bepPNA) nucleoside surrogates. The preferred surrogate is the PNA surrogate.

[0320] The dsRNA agents of this disclosure described herein may contain one or more chiral centers, thus resulting in enantiomers, diastereomers, and other stereoisomer configurations that can be defined as (R) or (S) from an absolute stereochemical standpoint, such as in the case of sugar anomers, or as (D) or (L) as in the case of amino acids. All such possible isomers, in addition to their racemic and optically pure forms, are included in the dsRNA agents of this disclosure provided herein.

[0321] D. End modification In some embodiments, the dsRNA agent further comprises a phosphate or phosphate mimetic at the 5' end of the antisense strand. In one embodiment, the phosphate mimetic is a 5'-vinyl phosphonate (VP).

[0322] In some embodiments, the 5' end of the antisense strand of the dsRNA agent does not contain 5'-vinylphosphonate (VP).

[0323] The ends of the iRNA agents of this disclosure may be modified. Such modifications may be made to one end or both ends. For example, the 3' and / or 5' ends of the iRNA may be conjugated to other functional molecular entities, such as labeling moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (e.g., sulfur, silicon, boron or ester-based). The functional molecular entities may be linked to a sugar via phosphate groups and / or linkers. The terminal atoms of the linker may be linked to or replace the atoms forming the phosphate group linkage, or the O, N, S or C groups of the C-3' or C-5' of the sugar. Alternatively, the linker may be linked to or replace the terminal atoms of a nucleotide surrogate (e.g., PNA).

[0324] Linker / phosphate functional molecular entities - When a linker / phosphate array is sandwiched between the two chains of a double-stranded oligomeric compound, this array can replace the hairpin loop of a hairpin-shaped oligomeric compound.

[0325] Useful terminal modifications for modulating activity include modifications of the 5' end of the iRNA with a phosphate or phosphate analog. In certain embodiments, the 5' end of the iRNA is phosphorylated or contains a phosphoryl analog. Exemplary 5'-phosphate modifications include those suitable for RISC-mediated gene silencing. Modifications at the 5' end may also be useful in stimulating or inhibiting the target immune system. In some embodiments, the 5' end of the oligomeric compound is modified

[0326] [ka] The formula includes, where W, X, and Y are independently O, OR (where R is hydrogen, alkyl, or aryl), S, Se, BR3 (where R is hydrogen, alkyl, or aryl), and BH3. -A is selected from the group consisting of C (i.e., alkyl group, aryl group, etc.), H, NR2 (where R is hydrogen, alkyl, or aryl), or OR (where R is hydrogen, alkyl, or aryl); A and Z are each independently, for each occurrence, absent, or optionally alkylene, which may contain one or more of O, S, CH2, NR (where R is hydrogen, alkyl, or aryl) internally and / or terminally; n is 0 to 2. In some embodiments, n is 1 or 2. A is understood to replace 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 connected, may form an optionally substituted 5 to 8-membered heterocyclic compound, where W and Y are each independently O, S, NR', or alkylene. Preferably, the heterocyclic compound is substituted with an aryl or heteroaryl. In some embodiments, one or both hydrogen atoms at the C5' end of the 5' terminal nucleotide are replaced with a halogen, such as fluorine (F).

[0327] Examples of 5' modifications include, but are not limited to, 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'- Examples of 5'-modifications include monodithiophosphates (phosphodithioates; (HO)(HS)(S)PO-5'), 5'-phosphorothiolates ((HO)2(O)PS-5'), 5'-alpha-thiotriphosphates, 5'-beta-thiotriphosphates, 5'-gamma-thiotriphosphates, and 5'-phosphoamidates ((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.) and 5'-alkyl ether phosphonates (R(OH)(O)PO-5', R=alkyl ether, e.g., methoxymethyl (CH2OMe), ethoxymethyl, etc.). Another exemplary 5' modification is when Z may be substituted at least once with an alkyl group, for example, ((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-terminated phosphates and phosphate mimetic: 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' is an example, where a and b are independently 1 to 10. Other embodiments include oxygen and / or sulfur, BH3, BH3 - This includes replacement with and / or Se.

[0328] Terminal modifications can also be useful for monitoring distribution, in which case preferred groups to be added include fluorophores, e.g., fluorescein or Alexa dyes, e.g., Alexa 488. Terminal modifications can also be useful for enhancing uptake, and useful modifications for this purpose include targeted ligands. Terminal modifications can also be useful for crosslinking oligonucleotides to other parts, and useful modifications for this purpose include mitomycin C, psoralens, and their derivatives.

[0329] E. Modifications that make things thermally unstable The compounds of this disclosure, such as iRNA or dsRNA agents, can be optimized for RNA interference by introducing a thermally destabilizing modification to the sense strand at a site opposite the seed region of the antisense strand (i.e., at positions 2–8 of the 5' end of the antisense strand, or at positions 2–9 of the 5' end of the antisense strand), thereby increasing the dissociation or melting properties of the iRNA double helix (reducing the free energy of double helix association). This modification can increase the dissociation or melting properties of the double helix in the seed region of the antisense strand.

[0330] Modifications that cause thermal instability include debase modification; mismatch with opposing nucleotides in opposing chains; and sugar modification, such as 2'-deoxy modification or acyclic nucleotides, such as unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA).

[0331] The excipient debase modification is,

[0332] [ka] That is the case.

[0333] The sugar modifications exemplified are,

[0334] [ka] That is the case.

[0335] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, for example, in which any of the bonds between 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 oxygen atoms (e.g., C1', C2', C3', C4', or O4') is absent in the nucleotide, either independently or in combination. In some embodiments, acyclic nucleotides are,

[0336] [ka] In the formula, B is a modified or unmodified nucleic acid base, and R 1 and R 2R3 is independently H, halogen, OR3, or alkyl; R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. The term "UNA" refers to an unlocked acyclic nucleic acid in which one of the sugar bonds has been removed to form an unlocked "sugar" residue. In one example, UNA also includes monomers in which the C1'-C4' bond (i.e., the carbon-oxygen-carbon bond of the covalent bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond of the sugar (i.e., the carbon-carbon bond of the covalent bond between the C2' and C3' carbons) has been removed [see Mikhailov et. al., Tetrahedron Letters, 26 (17): 2059 (1985); and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), which are incorporated herein by reference in their entirety]. Acyclic derivatives provide greater backchain flexibility without affecting Watson-Crick type pairing. Acyclic nucleotides may be linked via 2'-5' or 3'-5' ligatures.

[0337] The term "GNA" refers to glycol nucleic acids, which are polymers similar to DNA or RNA, but differ in the composition of their "backbone" in that it consists of repeating glycerol units linked by phosphodiester bonds.

[0338] [ka]

[0339] The thermally destabilizing modification may be a mismatch (i.e., a non-complementary base pair) between the thermally destabilizing nucleotide and the opposing nucleotide in the opposite strand within the dsRNA double helix. Examples of mismatched 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 mismatched base pairings known in the art may also be suitable for the disclosure of this invention. The mismatch may exist between nucleotides that are either naturally occurring or modified nucleotides; that is, the mismatched base pairing may exist between nucleic acid bases from each nucleotide, independent of any modification in the ribose sugar of the nucleotide. In certain embodiments, the compounds of this disclosure, e.g., siRNA or iRNA agents, contain at least one nucleic acid base in the mismatch pairing that is a 2'-deoxynucleotide, for example, the 2'-deoxynucleotide in the sense strand.

[0340] Further examples of debasalized nucleotides, acyclic nucleotide modifications (including UNAs and GNAs), and mismatch modifications are described in detail in WO2011 / 133876, which is incorporated herein by reference in its entirety.

[0341] Other examples of thermally destabilizing modifications include universal bases and phosphate modifications in which the ability to form hydrogen bonds with opposing bases is reduced or destroyed.

[0342] Nucleic acid base modifications that impair or completely destroy the ability to form base and hydrogen bonds in opposing strands have been evaluated for destabilization of the central region of the dsRNA double helix, as described in WO2010 / 0011895, which is incorporated herein in its entirety by reference. Exemplary nucleic acid base modifications are:

[0343] [ka] That is the case.

[0344] Exemplary phosphate modifications known to reduce the thermal stability of dsRNA double helix compared to natural phosphodiester linking are:

[0345] [ka] That is the case.

[0346] In some embodiments, the compounds of the present disclosure may include a 2'-5' linkage (having 2'-H, 2'-OH, and 2'-OMe, and having P=O or P=S). For example, the modification of the 2'-5' linkage may be used to promote nuclease resistance, or to inhibit the binding of the sense chain to the antisense chain, or to avoid RISC-mediated activation of the sense chain at its 5' end.

[0347] In another embodiment, the compounds of the present disclosure may contain L-sugars (e.g., L-ribose, L-arabinose, having 2'-H, 2'-OH, and 2'-OMe). For example, these L-sugar modifications may be used to promote nuclease resistance, inhibit the binding of the sense chain to the antisense chain, or, at the 5' end of the sense chain, to avoid RISC-mediated activation of the sense chain.

[0348] In one embodiment, the iRNA agent of the present disclosure is conjugated to a ligand via a carrier, the carrier may 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, pyridadinyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is selected from a selinol backbone or a diethanolamine backbone.

[0349] In some embodiments, at least one strand of the iRNA agent disclosed herein is 5' phosphorylated or contains a phosphoryl analog at the 5' prime terminus. Examples of 5'-phosphate modifications include those suitable for RISC-mediated gene silencing. Preferred 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'); and 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'-monoditithiophosphate (phosphorodithioate; (HO)(HS)(S)PO-5' Examples include 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-); and 5'-alkyl ether phosphonates (R=alkyl ether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5'-).

[0350] IV. Modified RNAi agents of this disclosure containing motifs In certain embodiments of this disclosure, the double-stranded RNAi agents of this disclosure include, for example, agents having chemical modifications as disclosed in U.S. Patent Nos. 9,796,974 and 10,668,170, whose entire contents are incorporated herein by reference, and in U.S. Patent Publications 2014 / 288158, 2018 / 008724, 2019 / 038768, and 2020 / 353097. Thus, as shown in and PCT Publication WO2013 / 074974 (whose entire contents are incorporated by reference), one or more motifs of three identical modifications in three consecutive nucleotides may be introduced into the sense or antisense strand of the RNAi agent, particularly at or near a cleavage site. In some embodiments, the sense and antisense strands of the RNAi agent may be otherwise completely modified. The introduction of these motifs disrupts the modification pattern of the sense or antisense strand, if such a pattern exists. The RNAi agent may be modified, for example, by (S)-glycol nucleic acid (GNA) modification in one or more residues of the antisense strand.

[0351] In one embodiment, the iRNA agent of the present disclosure is a bluntmer having 19nt lengths at both ends, wherein the sense strand contains three consecutive nucleotides at positions 7, 8, and 9 from the 5' end, with at least one motif of three 2'-F modification. The antisense strand contains three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, with at least one motif of three 2'-O-methyl modification.

[0352] In one embodiment, the iRNA agent of the present disclosure is a bluntomer having the same ends and a length of 20 nt, wherein the sense strand contains at least one motif of three 2'-F modification in three consecutive nucleotides at positions 8, 9, and 10 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modification in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0353] In one embodiment, the iRNA agent of the present disclosure is a bluntomer having the same ends and a length of 21nt, wherein the sense strand contains at least one motif of three 2'-F modification in three consecutive nucleotides at positions 9, 10, and 11 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modification in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0354] In one embodiment, the iRNA agent of the present disclosure comprises a sense strand of 21 nucleotides (nt) and an antisense strand of 23 nucleotides (nt), wherein the sense strand contains at least one motif of three 2'-F modification in 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 modification in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, wherein one end of the iRNA is blunt, while the other end contains a 2nt overhang. Preferably, the 2nt overhang is at the 3' end of the antisense. The iRNA agent may further contain a ligand (e.g., GalNAc3).

[0355] In one embodiment, the iRNA agent of the present disclosure comprises a sense and an antisense strand, wherein: the sense strand is 25 to 30 nucleotide residues in length, starting from the 5' terminal nucleotide (position 1), and positions 1 to 23 of the first strand contain at least 8 ribonucleotides; the antisense strand is 36 to 66 nucleotide residues in length, starting from the 3' terminal nucleotide, and contains at least 8 ribonucleotides at positions that pair with positions 1 to 23 of the sense strand, thereby forming a double helix; at least the 3' terminal nucleotide of the antisense strand does not pair with the sense strand, and up to 6 consecutive 3' terminal nucleotides do not pair with the sense strand, thereby forming a 3' single-stranded overhang of 1 to 6 nucleotides; the 5' end of the antisense strand does not pair with the sense strand. By containing 30 consecutive nucleotides, a single-stranded 5' overhang of 10 to 30 nucleotides is formed; when the sense and antisense strands are aligned with maximum complementarity, at least the 5' and 3' terminal nucleotides of the sense strand base-pair with the nucleotides of the antisense strand, thereby forming a substantially double-stranded region between the sense and antisense strands; in order to reduce target gene expression when the double-stranded nucleic acid is introduced into mammalian cells, the antisense strand is sufficiently complementary to the target RNA, with a length of at least 19 antisense strand ribonucleotides; the sense strand contains at least one motif of three 2'-F modification in three consecutive nucleotides, at least one of which is located at or near the cleavage site; the antisense strand contains at least one motif of three 2'-O-methyl modification in three consecutive nucleotides at or near the cleavage site.

[0356] In one embodiment, the iRNA agent of the present disclosure comprises sense and antisense strands, wherein the iRNA agent comprises a first strand having a length of at least 25 and a maximum of 29 nucleotides, and a second strand having a length of up to 30 nucleotides, having at least one motif of three 2'-O-methyl modification in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end; the 3' end of the first strand and the 5' end of the second strand form blunt ends, the second strand is 1 to 4 nucleotides longer than the first strand at its 3' end, and the double-stranded region is at least 25 nucleotides in length, and the second strand is sufficiently complementary to the target mRNA over a length of at least 19 nt of the second strand in order to reduce target gene expression when the iRNA agent is introduced into mammalian cells, and dicer cleavage of the iRNA preferentially yields an siRNA including the 3' end of the second strand, thereby reducing the expression of the target gene in mammals. The iRNA agent may further contain a ligand (e.g., GalNAc3).

[0357] In one embodiment, the sense strand of the iRNA agent contains three consecutive nucleotides with at least one motif of three identical modification, one of which is located at a cleavage site in the sense strand. For example, the sense strand may contain three consecutive nucleotides between positions 7 and 15 from the 5' end with at least one motif of three 2'-F modification.

[0358] In one embodiment, the antisense strand of the iRNA agent may also contain three consecutive nucleotides with at least one motif of three identical modification, one of which is located at or near a cleavage site in the antisense strand. For example, the antisense strand may contain three consecutive nucleotides between positions 9 and 15 from the 5' end with at least one motif of three 2'-O-methyl modification.

[0359] For iRNA agents having a double-stranded region of 17–23 nt in length, the cleavage sites on the antisense strand are typically around positions 10, 11, and 12 from the 5' end. Therefore, the three identical modification motifs may be located at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 on the antisense strand, counting from the first nucleotide from the 5' end of the antisense strand, or from the first paired nucleotide in the double-stranded region from the 5' end of the antisense strand. The cleavage sites in the antisense strand may also be modified according to the length of the iRNA double-stranded region from the 5' end.

[0360] In some embodiments, the iRNA agent comprises a sense strand and an antisense strand, each having 14 to 30 nucleotides, wherein the sense strand contains three consecutive nucleotides and at least two motifs of three identical modifications, at least one of which is located at or near a cleavage site in the strand, and at least one of which is located in another part of the strand separated from the motif at the cleavage site by at least one nucleotide. In one embodiment, the antisense strand also contains three consecutive nucleotides and at least one motif of three identical modifications, at least one of which is located at or near a cleavage site in the strand. Modifications in the motifs located at or near a cleavage site in the sense strand are different from modifications in the motifs located at or near a cleavage site in the antisense strand.

[0361] In some embodiments, the iRNA agent comprises 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 in three consecutive nucleotides, at least one of which is located at or near a cleavage site in the strand. In one embodiment, the antisense strand also contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at or near a cleavage site.

[0362] In some embodiments, the iRNA agent comprises 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 modification in 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 modification in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0363] In one embodiment, the iRNA agents of this disclosure contain mismatches within the target, within the double helix, or in combination thereof. The mismatches may be present in the overhang region or the double helix region. Base pairs can be ranked based on their property of promoting dissociation or dissolution (for example, based on the free energy of association or dissociation of a particular pair; the simplest approach is to examine pairs on a criterion of individual pairs, but the following adjacent or similar analyses can also be used). With respect to promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; I:C is preferred over G:C (I = inosine). Mismatches, e.g., non-regular or non-regular pairings (as described elsewhere herein) are preferred over regular (A:T, A:U, G:C) pairings; pairings containing universal bases are preferred over regular pairings.

[0364] In one embodiment, the iRNA agent of the present disclosure comprises at least one of the first 1, 2, 3, 4, or 5 base pairs in the double-stranded region from the 5' end of the antisense strand, such base pairs can be independently selected from the group of A:U, G:U, I:C, and mismatched pairs, e.g., non-normal or non-normal pairings or pairings containing universal bases, and can promote the dissociation of the antisense strand at the 5' end of the double-stranded region.

[0365] 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.

[0366] 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, a short sequence of deoxythymidine nucleotides, for example, two dT nucleotides, is present at the 3' end of the sense or antisense strand.

[0367] In certain embodiments, the compositions and methods of the Disclosure include vinyl phosphonate (VP) modifications of the RNAi agents described herein. In exemplary embodiments, the 5'-vinyl phosphonate modification of the Disclosure has the following structure:

[0368] [ka] It has, In the formula, X is either O or S; R is hydrogen, hydroxyl, fluoro, or C 1~20 It is an alkoxy (e.g., methoxy or n-hexadecyloxy); R 5’ This is =C(H)-P(O)(OH)2, with C5' carbon and R 5’ The double bond between them is E or Z oriented (e.g., E oriented); B is a nucleic acid base or a modified nucleic acid base, and B may be adenine, guanine, cytosine, thymine, or uracil.

[0369] The vinyl phosphonates of the present invention may be affixed to either the antisense or sense strand of the dsRNA of the present disclosure. In certain embodiments, the vinyl phosphonates of the present invention are affixed to the antisense strand of the dsRNA and may be affixed to the 5' end of the antisense strand of the dsRNA.

[0370] Modifications of vinyl phosphonates for the compositions and methods disclosed in the present invention are also anticipated. An exemplary vinyl phosphate structure is the structure described above, where R5' is =C(H)-OP(O)(OH)2, and the double bond between the C5' carbon and R5' is E or Z oriented (e.g., E oriented).

[0371] In one embodiment, this disclosure relates to double-stranded RNA (dsRNA) agents for inhibiting the expression of target genes having reduced off-target activity, as described in U.S. Patents 10,233448, 10,612,024, and 10,612,027, and U.S. Patent Publications 2017 / 275626, 2019 / 241891, 2019 / 241893, and 2021 / 017519, the entire contents of which are incorporated herein by reference. As exemplified herein, for example, a motif comprising a thermally destabilized nucleotide, e.g., i) a nucleotide that forms a mismatch pair with an opposing nucleotide in the antisense strand, ii) a nucleotide having a debasic modification, and / or iii) a sugar modification, with the nucleotide located at an opposing site (positions 2-8) in the seed region, may be introduced into the sense strand.

[0372] In one embodiment, the dsRNA agent of the Disclosure does not contain any 2'-F modification. In one embodiment, the dsRNA agent of the Disclosure contains only 2'-F and 2'-OMe modifications.

[0373] In one embodiment, the sense strand and / or antisense strand of the dsRNA agent comprises one or more blocks of internucleotide links of phosphorothioate or methylphosphonate. In one example, the sense strand comprises one block of internucleotide links of two phosphorothioate or methylphosphonate. In one example, the antisense strand comprises two blocks of internucleotide links of two phosphorothioate or methylphosphonate. For example, the two blocks of internucleotide links of phosphorothioate or methylphosphonate are separated by 16 to 18 phosphate nucleotide links.

[0374] In one embodiment, each of the sense and antisense strands of the dsRNA agent has 15 to 30 nucleotides. In one example, the sense strand has 19 to 22 nucleotides, and the antisense strand has 19 to 25 nucleotides. In another example, the sense strand has 21 nucleotides, and the antisense strand has 23 nucleotides.

[0375] In one embodiment, the nucleotide at position 1 of the 5' end of the antisense strand in the double helix 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.

[0376] In one embodiment, the antisense strand of the dsRNA agent of the Disclosure is 100% complementary to the target RNA that hybridizes to it and inhibits its expression via RNA interference. In another embodiment, the antisense strand of the dsRNA agent of the Disclosure 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.

[0377] In one embodiment, this disclosure 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, in which case at least one of the thermally destabilized nucleotides is located opposite or near 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). Each of the embodiments and aspects described herein relating to a dsRNA represented by formula (I) can also be applied to a dsRNA containing a thermally destabilized nucleotide.

[0378] The thermally destabilized nucleotides may be located between positions 14 and 17 of the 5' end of the sense strand, for example, if the sense strand is 21 nucleotides long. The antisense strand contains at least two modified nucleic acids smaller than the sterically bulky 2'-OMe modification. Preferably, the two modified nucleic acids smaller than the sterically bulky 2'-OMe are separated by a length of 11 nucleotides. For example, the two modified nucleic acids are located at positions 2 and 14 of the 5' end of the antisense strand.

[0379] In one embodiment, the dsRNA agent further comprises at least one ASGPR ligand. For example, the ASGPR ligand is one or more GalNAc derivatives linked via a divalent or trivalent branched linker, for example,

[0380] [ka] In one example, the ASGPR ligand is attached to the 3' end of the sense strand.

[0381] For example, a dsRNA agent as defined herein may include: i) a phosphorus-containing group at the 5' end of the sense or antisense strand; ii) modifications of two phosphorothioate nucleotide linkages within positions 1-5 (counting from the 5' end of the sense strand), and modifications of two phosphorothioate nucleotide linkages within positions 1 and 2 (counting from the 5' end of the antisense strand) and within positions 18-23; and iii) a ligand, such as an ASGPR ligand (e.g., one or more GalNAc derivatives), at the 5' or 3' end of the sense or antisense strand. For example, the ligand may be at the 3' end of the sense strand.

[0382] In certain embodiments, the dsRNA agent of the disclosure of the present invention is (a) (i) a nucleotide of length 21; (ii) optionally an ASGPR ligand attached to the 3' end, comprising three GalNAc derivatives linked via a trivalent branched linker; and (iii) a sense strand having 2'-F modifications at positions 1, 3, 5, 7, 9-11, 13, 17, 19, and 21, and 2'-OMe modifications (counting from the 5' end) at positions 2, 4, 6, 8, 12, 14-16, 18, and 20; and (b) (i) a nucleotide of length 23 (ii) comprising an antisense strand having 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) phosphorothioate internucleotide links between nucleotide positions 21-22 and between nucleotide positions 22-23 (counting from the 5' end), wherein 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.

[0383] In another specific embodiment, the dsRNA agent of the disclosure of the present invention is (a)(i) a 21-length nucleotide; (ii) optionally an ASGPR ligand attached to the 3' end, comprising three GalNAc derivatives linked via 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) a sense strand having phosphorothioate internucleotide links between nucleotide positions 1-2 and between nucleotide positions 2-3 (counting from the 5' end). Furthermore, the dsRNA agent comprises an antisense strand having (b)(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) phosphorothioate internucleotide links (counting from the 5' end) between nucleotide positions 1-2, between nucleotide positions 2-3, between nucleotide positions 21-22, and between nucleotide positions 22-23, wherein 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.

[0384] In another specific embodiment, the dsRNA agent of the disclosure of the present invention is (a)(i) a 21-length nucleotide; (ii) an ASGPR ligand optionally attached to the 3' end, comprising three GalNAc derivatives linked via a trivalent branched linker; (iii) a sense strand having 2'-OMe modifications at positions 1-6, 8, 10, and 12-21, 2'-F modifications at positions 7 and 9, and a deoxynucleotide (e.g., dT) at position 11 (counting from the 5' end); and (iv) a sense strand having phosphorothioate internucleotide links (counting from the 5' end) between nucleotide positions 1-2 and between nucleotide positions 2-3; The dsRNA agent comprises (b)(i) a 23-length nucleotide; (ii) 2'-OMe modifications at positions 1, 3, 7, 9, 11, 13, 15, 17, and 19-23, and 2'-F modifications at positions 2, 4-6, 8, 10, 12, 14, 16, and 18 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide links (counting from the 5' end) between nucleotide positions 1-2, between nucleotide positions 2-3, between nucleotide positions 21-22, and between nucleotide positions 22-23, wherein the dsRNA agent has a 2-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0385] In another specific embodiment, the dsRNA agent of the disclosure of the present invention is (a)(i) a nucleotide of length 21; (ii) an ASGPR ligand optionally attached to the 3' end, comprising three GalNAc derivatives linked via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1-6, 8, 10, 12, 14, and 16-21, and 2'-F modifications at positions 7, 9, 11, 13, and 15; and (iv) a sense strand having phosphorothioate internucleotide links (counting from the 5' end) between nucleotide positions 1-2 and between nucleotide positions 2-3; and (b)(i) a nucleotide of length 23 The dsRNA agent comprises an antisense strand having (ii) nucleotides; 2'-OMe modifications at positions 1, 5, 7, 9, 11, 13, 15, 17, 19, and 21-23, and 2'-F modifications at positions 2-4, 6, 8, 10, 12, 14, 16, 18, and 20 (counting from the 5' end); and (iii) phosphorothioate internucleotide links (counting from the 5' end) between nucleotide positions 1-2, between nucleotide positions 2-3, between nucleotide positions 21-22, and between nucleotide positions 22-23, wherein 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.

[0386] In another specific embodiment, the dsRNA agent of the disclosure of the present invention is (a)(i) a nucleotide of length 21; (ii) an ASGPR ligand optionally attached to the 3' end, comprising three GalNAc derivatives linked via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1-9 and 12-21, and 2'-F modifications at positions 10 and 11; and (iv) a sense strand having phosphorothioate internucleotide links (counting from the 5' end) between nucleotide positions 1-2 and between nucleotide positions 2-3; and (b)(i) a nucleotide of length 23; (ii) comprising an antisense strand having 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) an antisense strand having phosphorothioate internucleotide links (counting from the 5' end) between nucleotide positions 1-2, between nucleotide positions 2-3, between nucleotide positions 21-22, and between nucleotide positions 22-23, wherein 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.

[0387] In another specific embodiment, the dsRNA agent of the disclosure of the present invention is (a)(i) a nucleotide of length 21; (ii) an ASGPR ligand optionally attached to the 3' end, comprising three GalNAc derivatives linked via 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) a sense strand having phosphorothioate internucleotide links (counting from the 5' end) between nucleotide positions 1-2 and between nucleotide positions 2-3; and (b)(i) 23 The dsRNA agent comprises an antisense strand having nucleotides of 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) phosphorothioate internucleotide links (counting from the 5' end) between nucleotide positions 1-2, between nucleotide positions 2-3, between nucleotide positions 21-22, and between nucleotide positions 22-23, wherein 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.

[0388] In another specific embodiment, the dsRNA agent of the disclosure of the present invention is (a) (i) 21 nucleotides in length; (ii) optionally an ASGPR ligand attached to the 3' end, comprising three GalNAc derivatives linked via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1-6, 8, and 12-21, and 2'-F modifications at positions 7 and 9-11; and (iv) a sense strand having phosphorothioate internucleotide links (counting from the 5' end) between nucleotide positions 1-2 and between nucleotide positions 2-3; and (b) (i) 23 The dsRNA agent comprises an antisense strand having (ii) nucleotides of 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) phosphorothioate internucleotide links (counting from the 5' end) between nucleotide positions 1-2, between nucleotide positions 2-3, between nucleotide positions 21-22, and between nucleotide positions 22-23, wherein 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.

[0389] In another specific embodiment, the dsRNA agent of the disclosure of the present invention is (a) (i) 21 nucleotides in length; (ii) optionally an ASGPR ligand attached to the 3' end, comprising three GalNAc derivatives linked via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1-6, 8, and 12-21, and 2'-F modifications at positions 7 and 9-11; and (iv) a sense strand having phosphorothioate internucleotide links (counting from the 5' end) between nucleotide positions 1-2 and between nucleotide positions 2-3; and (b) (i) 23 The dsRNA agent comprises an antisense strand having (ii) nucleotides of 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) phosphorothioate internucleotide links (counting from the 5' end) between nucleotide positions 1-2, between nucleotide positions 2-3, between nucleotide positions 21-22, and between nucleotide positions 22-23, wherein 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.

[0390] In another specific embodiment, the dsRNA agent of the disclosure of the present invention is (a)(i) nucleotides of length 19; (ii) an ASGPR ligand optionally attached to the 3' end, comprising three GalNAc derivatives linked via 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) a sense strand having phosphorothioate internucleotide links (counting from the 5' end) between nucleotide positions 1-2 and between nucleotide positions 2-3; and (b)(i) nucleotides of length 21 The dsRNA agent comprises an antisense strand having (ii) 2'-OMe modifications at positions 1, 3-5, 7, 10-13, 15, and 17-21, and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end); and (iii) phosphorothioate internucleotide links (counting from the 5' end) between nucleotide positions 1-2, between nucleotide positions 2-3, between nucleotide positions 19-20, and between nucleotide positions 20-21, wherein 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.

[0391] In one embodiment, the dsRNA agent of the present invention comprises (a) a sense strand having (i) 18 to 23 nucleotides in length; (ii) three consecutive 2'-F modifications at positions 7 to 15; and (b) an antisense strand having (i) 18 to 23 nucleotides in length; (ii) at least one 2'-F modification anywhere on the strand; and (iii) at least two phosphorothioate nucleotide linkages (counting from the 5' end) in the first five nucleotides, wherein the dsRNA agent has one or more lipophilic moieties conjugated at one or more positions on at least one of the strands; either having 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 having blunt ends at both ends of the double helix.

[0392] In one embodiment, the dsRNA agent of the present invention comprises (a) a sense strand having (i) 18 to 23 nucleotides in length; (ii) fewer than 4 2'-F modifications; (b) an antisense strand having (i) 18 to 23 nucleotides in length; (ii) fewer than 12 2'-F modifications; and (iii) at least two phosphorothioate nucleotide linkages (counting from the 5' end) in the first 5 nucleotides, wherein the dsRNA agent has one or more lipophilic moieties conjugated at one or more positions on at least one of the strands; either having 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 having blunt ends at both ends of the double helix.

[0393] In one embodiment, the dsRNA agent of the present invention comprises (a) a sense strand having (i) 19 to 35 nucleotides in length; (ii) fewer than 4 2'-F modifications; (b) an antisense strand having (i) 19 to 35 nucleotides in length; (ii) fewer than 12 2'-F modifications; and (iii) at least two phosphorothioate nucleotide linkages (counting from the 5' end) in the first 5 nucleotides, wherein the double-stranded region has 19 to 25 base pairs (preferably 19, 20, 21 or 22); the dsRNA agent has one or more lipophilic moieties conjugated at one or more positions on at least one of the strands; either 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; or has blunt ends at both ends of the double helix.

[0394] In another embodiment, the dsRNA agent disclosed in the present invention comprises (a) (i) nucleotides of length 21; (ii) a liophilic moiety (e.g., C 22Hydrocarbon chain; (iii) 2'-OMe modifications at positions 1-6, 8, and 12-21, and 2'-F modifications at positions 7 and 9-11; and (iv) sense chain having phosphorothioate internucleotide links (counting from the 5' end) between nucleotide positions 1-2 and between nucleotide positions 2-3; and (b) (i) nucleotides of length 23; (ii) 2'-OMe modifications at positions 1, 3-4, 6, 8-13, 15, and 17-23, and 2'- The RNAi agent comprises an antisense strand having (iii) a modified F, a 2'-deoxy modified nucleotide at position 5, and a GNA at position 7 (counting from the 5' end); and (iii) phosphorothioate internucleotide links (counting from the 5' end) between nucleotide positions 1-2, between nucleotide positions 2-3, between nucleotide positions 21-22, and between nucleotide positions 22-23, wherein the RNAi 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.

[0395] In another embodiment, the dsRNA agent disclosed in the present invention comprises (a) (i) nucleotides of length 21; (ii) a lipophilic moiety (e.g., C 22 (iii) A sense strand having a hydrocarbon chain; (iii) 2'-OMe modifications at positions 1-6, 8, and 12-21, and 2'-F modifications at positions 7 and 9-11; and (iv) phosphorothioate internucleotide links (counted from the 5' end) between nucleotide positions 1-2 and between nucleotide positions 2-3; and (b) (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3-5, 7-13, 15, and 17-23, and 2'-F modifications (counted from the 5' end) at positions 2, 6, 14, and 16; and (iii) an antisense strand having phosphorothioate internucleotide links (counted from the 5' end) between nucleotide positions 1-2, between nucleotide positions 2-3, between nucleotide positions 21-22, and between nucleotide positions 22-23, wherein the RNAi 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.

[0396] In one embodiment, the dsRNA agent of the present invention comprises a sense strand and an antisense strand having 15 to 30 nucleotides in length; at least two phosphorothioate nucleotide linkages (counting from the 5' end) in the first five nucleotides of the antisense strand; the double-stranded region has 19 to 25 base pairs (preferably 19, 20, 21, or 22); the dsRNA agent has one or more lipophilic moieties conjugated at one or more positions on at least one of the strands; and the dsRNA agent has less than 20%, less than 15%, and less than 10% non-natural nucleotides. 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-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.

[0397] In one embodiment, the dsRNA agent of the present invention comprises a sense strand and an antisense strand having 15 to 30 nucleotides in length; at least two phosphorothioate nucleotide interlinks (counting from the 5' end) in the first five nucleotides of the antisense strand, the double-stranded region having 19 to 25 base pairs (preferably 19, 20, 21, or 22); the dsRNA agent has one or more lipophilic moieties conjugated at one or more positions on at least one of the strands; the dsRNA agent has more than 80%, more than 85%, and more than 90% native nucleotides, for example, 2'-OH, 2'-deoxy, and 2'-OMe are native nucleotides.

[0398] In one embodiment, the dsRNA agent of the present invention comprises a sense strand and an antisense strand having 15 to 30 nucleotides in length; at least two phosphorothioate nucleotide interlinks (counting from the 5' end) in the first five nucleotides of the antisense strand, the double-stranded region having 19 to 25 base pairs (preferably 19, 20, 21 or 22); the dsRNA agent has one or more lipophilic moieties conjugated at one or more positions on at least one of the strands; the dsRNA agent has 100% natural nucleotides, for example, 2'-OH, 2'-deoxy and 2'-OMe are natural nucleotides.

[0399] Various publications describe multimeric siRNAs, all of which can be used in conjunction with the iRNAs of this disclosure. Such publications include WO2007 / 091269, U.S. Patent No. 7,858,769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887, and WO2011 / 031520, which are incorporated herein by reference in their entirety.

[0400] In some embodiments, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of the iRNA agent of the Disclosure are modified.

[0401] In some embodiments, the sense and antisense strands of the iRNA agent are 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-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.

[0402] In some embodiments, each of the sense and antisense strands of the iRNA agent contains at least two different modifications.

[0403] In some embodiments, the dsRNA agents of the Disclosure contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 2'-F modifications. For example, the dsRNA agents of the Disclosure contain 9 or 10 2'-F modifications.

[0404] The iRNA agent of this disclosure may further comprise at least one internucleotide linkage of phosphorothioate or methylphosphonate. Modifications of the internucleotide linkage of phosphorothioate or methylphosphonate may be present on any nucleotide of either the sense strand or the antisense strand or both, at any position on the strand. For example, the internucleotide linkage modification may be present on each nucleotide of the sense strand or the antisense strand, each internucleotide linkage modification may be present on the sense strand or the antisense strand in an alternating pattern, or both the sense strand or the antisense strand may contain internucleotide linkage modifications in an alternating pattern. The alternating pattern of internucleotide linkage modifications on the sense strand may be the same as or different from that on the antisense strand, and the alternating pattern of internucleotide linkage modifications on the sense strand may have a shift relative to the alternating pattern of internucleotide linkage modifications on the antisense strand.

[0405] In one embodiment, the iRNA includes a modification of phosphorothioate or methylphosphonate internucleotide linkages in the overhang region. For example, the overhang region may contain two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. The modification of the internucleotide linkages may also be constructed to link the nucleotides in the overhang to the terminal paired nucleotides in the double-stranded region. For example, at least two, three, four, or all of the overhang nucleotides may be linked via phosphorothioate or methylphosphonate internucleotide linkages, and optionally, additional phosphorothioate or methylphosphonate internucleotide linkages may be present linking the overhang nucleotides to the paired nucleotides adjacent to the overhang nucleotides. For example, there may be at least two phosphorothioate internucleotide linkages between the three terminal nucleotides, where two of the three nucleotides are overhang nucleotides and the third nucleotide is the paired nucleotide adjacent to the overhang nucleotides. Preferably, these three terminal nucleotides may be at the 3' end of the antisense strand.

[0406] In some embodiments, the sense strand and / or antisense strand of the iRNA agent comprises one or more blocks of internucleotide links of phosphorothioate or methylphosphonate. In one example, the sense strand comprises one block of internucleotide links of two phosphorothioate or methylphosphonate. In one example, the antisense strand comprises two blocks of internucleotide links of two phosphorothioate or methylphosphonate. For example, the two blocks of internucleotide links of phosphorothioate or methylphosphonate are separated by 16 to 18 phosphate internucleotide links.

[0407] In one embodiment, the disclosure relates to an iRNA agent capable of inhibiting the expression of a target gene. The iRNA 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, the at least one of which is located opposite the seed region of the antisense strand, or near it (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). The thermally destabilized nucleotide is located, for example, between positions 14 to 17 of the 5' end of the sense strand, if the sense strand is 21 nucleotides long. The antisense strand contains at least two modified nucleic acids smaller than sterically bulky 2'-OMe modifications. Preferably, the two modified nucleic acids smaller than sterically bulky 2'-OMe are separated by a length of 11 nucleotides. For example, the two modified nucleic acids are located at positions 2 and 14 of the 5' end of the antisense strand. In some embodiments, the compounds of this disclosure disclosed herein are miRNA mimetic. In one design, the miRNA mimetic is a double-stranded molecule (e.g., having a double-stranded region of about 16 to about 31 nucleotides in length) and contains one or more sequences that are identical to a given strand of mature miRNA. A double-stranded miRNA mimetic has a design similar to that described above with respect to a double-stranded iRNA. In some embodiments, the miRNA mimetic comprises a double-stranded region of 16 to 31 nucleotides and one or more of the following chemical modification patterns: the sense strand contains nucleotides 1 and 2 (counting from the 5' end of the sense oligonucleotide), as well as all 2'-O-methyl modifications of C and U; the antisense strand modification may include all 2'F modifications of C and U, phosphorylation of the 5' end of the oligonucleotide, and stabilized internucleotide linkages related to the 3' overhangs of the two nucleotides.

[0408] V. Lipophilic part The lipophilic portion of this disclosure may contain at least one C10-C26 hydrocarbon chain. As described in U.S. Provisional Application No. 63 / 255,984 filed on 15 October 2021 (the entire content of which is incorporated herein by reference), one or more C10-C26 hydrocarbon chains may be located within the dsRNA agent. 22 The inclusion of hydrocarbon chains, such as saturated or unsaturated ones, increases the lipophilicity of the dsRNA agent, providing optimal hydrophobicity for enhanced in vivo delivery of dsRNA to, for example, muscle tissue and / or adipose tissue.

[0409] One way to characterize lipophilicity is by the octanol-water partition coefficient, logK ow This is due to the formula, and in the formula, K ow The logK coefficient is the ratio of the concentration of a chemical in the octanol phase to its concentration in the aqueous phase of the two-phase system at equilibrium. The octanol-water partition coefficient is a property of a substance measured in the laboratory. However, it can also be predicted by using a coefficient attributable to the constituent components of the chemical, calculated using first-principles methods or experimental methods (see, for example, the entirety of which is incorporated herein by reference [see Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001)]). This provides a thermodynamic measure of a substance's tendency to prefer non-aqueous or oily environments over water (i.e., its hydrophilic / lipophilic balance). In principle, a chemical substance has its logK coefficient. ow If logK is greater than 0, it has lipophilic properties. Typically, the lipophilic portion is greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10. ow It has, for example, the logK of 6-aminohexanol. ow For example, it is predicted to be approximately 0.7. Using the same method, the logK of cholesteryl N-(hexane-6-ol) carbamate ow It is predicted to be 10.7.

[0410] The lipophilicity of a molecule can change depending on the functional groups it possesses. For example, a hydroxyl group or an amine group can change C22 Adding to the end of a hydrocarbon chain is C 22 Partition coefficient of hydrocarbon chains (e.g., logK) ow The value can be increased or decreased.

[0411] Alternatively, one or more C 22 The hydrophobicity of dsRNA agents conjugated to hydrocarbon chains can be measured by their protein binding characteristics. For example, the proportion of unbound dsRNA agents in plasma protein binding assays may be determined to be positively correlated with the relative hydrophobicity of the dsRNA agent, which may be positively correlated with the silencing activity of the dsRNA agent.

[0412] In one embodiment, the plasma protein binding assay to be determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. The hydrophobicity of the dsRNA agent is measured by the percentage of unbound dsRNA in the binding assay, which, in the case of siRNA-enhanced in vivo delivery, 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.

[0413] In a particular embodiment, one or more C 22 The hydrocarbon chain is an aliphatic, alicyclic, or polyalicyclic compound, and is aliphatic, cyclic, e.g., alicyclic, or polycyclic, e.g., a polyalicyclic compound. The hydrocarbon chain may contain various substituents and / or one or more heteroatoms, e.g., oxygen or nitrogen atoms.

[0414] One or more C 22 The hydrocarbon chain may be attached to the iRNA agent by any method known in the art, such as via functional groups already present in the lipophilic portion or introduced into the iRNA agent, such as a hydroxyl group (e.g., -CO-CH2-OH). 22Functional groups already present in hydrocarbon chains or introduced into dsRNA agents include, but are not limited to, hydroxyls, amines, carboxylic acids, sulfonates, phosphates, thiols, azides, and alkynes.

[0415] dsRNA agents and C 22 Conjugation of hydrocarbon chains may occur, for example, through the formation of ether or carboxylic acid or carbamoyl ester links between a hydroxyl group and an alkyl group R-, an alkanoyl group RCO-, or a substituted carbamoyl group RNHCO-. The alkyl group R may be cyclic (e.g., cyclohexyl) or acyclic (e.g., linear or branched; and saturated or unsaturated). The alkyl group R may be butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, or octadecyl groups.

[0416] In some embodiments, C 22 The hydrocarbon chain is conjugated to the dsRNA agent via a linker containing ethers, thioethers, ureas, carbonates, amines, amides, maleimide-thioethers, disulfides, phosphodiesters, sulfonamide linkages, click reaction products (e.g., triazoles from azido-alkyne cycloaddition), or carbamates.

[0417] In one embodiment, one or more C 22 The hydrocarbon chain is C 22 It is an acid, for example, C 22The acid is selected from the group consisting of docosanic 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.

[0418] [ka]

[0419] In one embodiment, one or more C 22 The hydrocarbon chain is C 22 It is an alcohol, for example C 22 The alcohol is selected from the group consisting of 1-docosanol, 6-octyltetradecane-1-ol, 10-hexylhexadecane-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.

[0420] [ka]

[0421] In one embodiment, one or more C 22 The hydrocarbon chain is not cis-4,7,10,13,16,19-docosahexaenoic acid. In one embodiment, one or more C 22 The hydrocarbon chain is not cis-4,7,10,13,16,19-docosahexanol. In one embodiment, one or more C 22 The hydrocarbon chain is not cis-4,7,10,13,16,19-docosahexaenoic acid, nor is it cis-4,7,10,13,16,19-docosahexanol.

[0422] In one embodiment, one or more C 22 The hydrocarbon chain is C 22 It is an amide, for example, 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-docosenamamide, (Z)-docosa-13-enamide, (Z)-N-hydroxy-13-docosenamamide, (E)-docosa-14-enamide, 6-cis-docosenamamide, 14-docosenamamide, docosa-11-enamide, (4E,13E)-docosa-4,13-dienamide, and (5E,13E)-docosa-5,13-dienamide.

[0423] In one embodiment, the C22 hydrocarbon chain is not limited to these, but includes docosan-2-yl, docosan-3-yl, docosan-4-yl, docosan-5-yl, docosan-6-yl, docosan-7-yl, docosan-8-yl, docosan-9-yl, docosan-10-yl, docosan-11-yl, 2-(decyl)dodecane-1-yl, 2-(nonyl)tridecane-1-yl, 2-(octyl)tetradecane-1-yl, 2-(heptyl)pentadecane-1-yl, 2-(hexyl)hexadecane-1-yl, 2-(pentyl)heptadecane-1-yl 2-(butyl)octadecane-1-yl, 2-(propyl)nonadecane-1-yl, 2-(ethyl)eicosan-1-yl, 2-(methyl)henicosane-1-yl, 3-(nonyl)tridecane-1-yl, 3-(octyl)tetradecane-1-yl, 3-(heptyl)pentadecane-1-yl, 3-(hexyl)hexadecane-1-yl, 3-(pentyl)heptadecane-1-yl, 3-(butyl)octadecane-1-yl, 3-(propyl)nonadecane-1-yl, 3-(ethyl)eicosane-1-yl, 3-(methyl)henicosane-1-yl, 4-(octyl)tetradecane-1-yl, 4-(heptyl)pentadecane-1-yl, 4-(hexyl)hexadecane-1-yl, 4-(pentyl)heptadecane-1-yl, 4-(butyl)octadecane-1-yl, 4-(propyl)nonadecane-1-yl, 4-(ethyl)eicosane-1-yl, 4-(methyl)henicosane-1-yl, 5-(heptyl)pentadecane-1-yl, 5-(hexyl)hexadecane-1-yl, 5-(pentyl)heptadecane-1-yl, 5-(butyl)octadecane-1-yl, 5-(propyl)nonadecane-1- Il, 5-(ethyl)eicosan-1-yl, 5-(methyl)henicosane-1-yl, 6-(hexyl)hexadecane-1-yl, 6-(pentyl)heptadecane-1-yl, 6-(butyl)octadecane-1-yl, 6-(propyl)nonadecane-1-yl, 6-(ethyl)eicosane-1-yl, 6-(methyl)henicosane-1-yl, 7-(pentyl)heptadecane-1-yl, 7-(butyl)octadecane-1-yl, 7-(propyl)nonadecane-1-yl, 7-(ethyl)eicosane-1-yl, 7-(methyl)henicosane-1-yl,Examples include 8-(butyl)octadecane-1-yl, 8-(propyl)nonadecane-1-yl, 8-(ethyl)eicosane-1-yl, 8-(methyl)henicosane-1-yl, 9-(propyl)nonadecane-1-yl, 9-(ethyl)eicosane-1-yl, 9-(methyl)henicosane-1-yl, 10-(ethyl)eicosane-1-yl, 10-(methyl)henicosane-1-yl, and 11-(methyl)henicosane-1-yl, which are substituted with the 2'-oxygen of the oligonucleotide nucleoside as used herein.

[0424] In certain embodiments, particularly C 22 If the hydrocarbon chain has low lipophilicity or hydrophobicity, one more C 22 Hydrocarbon chains may be incorporated into the double-stranded iRNA agent. In one embodiment, two or more C 22 A hydrocarbon chain is incorporated into the same chain of the double-stranded iRNA agent. In one embodiment, each chain of the double-stranded iRNA agent contains one or more incorporated C 22 It has a hydrocarbon chain. In one embodiment, two or more C 22 The hydrocarbon chain is incorporated into the same position in the double-stranded iRNA agent (i.e., the same nucleic acid base, the same sugar moiety, or the same nucleoside linkage). This is, for example, two or more saturated or unsaturated C 22 Conjugate hydrocarbon chains via a support, and / or two or more C 22 Conjugate hydrocarbon chains via branched linkers, and / or two or more C 22 A hydrocarbon chain is conjugated via one or more linkers, and one or more linkers are C 22 This may also be achieved by continuously linking hydrocarbon chains.

[0425] One or more C 22 The hydrocarbon chain may be conjugated to the iRNA agent via direct linkage to the ribosaccharide of the iRNA agent. Alternatively, one or more C 22The hydrocarbon chain may be conjugated to the double-stranded iRNA agent via a linker or carrier. In certain embodiments, one or more C 22 The hydrocarbon chain may be conjugated to the iRNA agent via one or more linkers (tethers).

[0426] In one embodiment, one or more C 22 The hydrocarbon chain is conjugated to the dsRNA agent via a linker containing ethers, thioethers, ureas, carbonates, amines, amides, maleimide-thioethers, disulfides, phosphodiesters, sulfonamide linkages, click reaction products (e.g., triazoles from azido-alkyne cycloaddition), or carbamates.

[0427] In one embodiment, a lipophilic portion (for example, C 22 The hydrocarbon chain is linked at the 2' position in the internal nucleotides of the sense or antisense chain. In one embodiment, the lipophilic moiety (e.g., C) 22 The hydrocarbon chain is linked at the 2' position of a nucleotide at any of the 4-8 and 13-18 positions (e.g., positions 4, 5, 6, 7, 8, 13, 14, 15, 16, 17, 18) of the sense chain, counting from the 5' end. In one embodiment, the lipophilic moiety (e.g., C 22 The hydrocarbon chain is linked at the 2' position of the nucleotide at position 6 or 16 in the sense chain, counting from the 5' end.

[0428] A. Linker / Tether Linker / tether is a "Tethering Connection Point (TAP)" with one or more C 22 Linked to a hydrocarbon chain. The linker / tether is one of the C1-C12 100 carbon-containing portion (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) may contain and may have at least one nitrogen atom. In certain embodiments, the nitrogen atom forms part of the terminal amino or amide (NHC(O)-) group of the linker / tether, which may serve as a linking point for the lipophilic portion. Non-limiting examples of linkers / tethers (shown in underline) include TAP-(CH2) n NH-;TAP-C(O)(CH2) n NH-;TAP-NR''''(CH2) n NH-, TAP-C(O)-(CH2) n -C(O)-;TAP-C(O)-(CH2) n -C(O)O-;TAP-C(O)-O-;TAP-C(O)-(CH2) n -NH-C(O)-;TAP-C(O)-(CH2) n -;TAP-C(O)-NH-;TAP-C(O)-;TAP-(CH2) n -C(O)-;TAP-(CH2) n -C(O)O-;TAP-(CH2) n -; or TAP-(CH2) n Examples include -NH-C(O)-, where n is 1 to 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 a C1 to C6 alkyl group. Preferably, n is 5, 6, or 11. In other embodiments, nitrogen may form part of a terminal oxyamino group, e.g., -ONH2, or a hydrazino group, -NHNH2. The linker / tether may be substituted with, for example, hydroxy, alkoxy, or perhaloalkyl groups, and / or may have one or more additional heteroatoms, e.g., N, O, or S inserted. Ligands with preferred tethers include, for example, TAP-(CH2) n NH (ligand); TAP-C(O)(CH2) n NH (ligand); TAP-NR''''(CH2) n NH (ligand); TAP-(CH2) n ONH (ligand); TAP-C(O)(CH2) nONH (ligand); TAP-NR''''(CH2) n ONH (ligand); TAP-(CH2) n NHNH2 (ligand), TAP-C(O)(CH2) n NHNH2 (ligand); TAP-NR''''(CH2) n NHNH2 (ligand); TAP-C(O)-(CH2) n -C(O)(ligand);TAP-C(O)-(CH2) n -C(O)O(ligand);TAP-C(O)-O(ligand);TAP-C(O)-(CH2) n -NH-C(O)(ligand);TAP-C(O)-(CH2) n (Ligand); TAP-C(O)-NH(Ligand); TAP-C(O)(Ligand); TAP-(CH2) n -C(O)(ligand);TAP-(CH2) n -C(O)O(ligand);TAP-(CH2) n (Ligand); or TAP-(CH2) n Examples include -NH-C(O)(ligands). In some embodiments, amino-terminated linkers / tethers (e.g., NH2, ONH2, NH2NH2) can form an imino bond (i.e., C=N) with the ligand. In some embodiments, amino-terminated linkers / tethers (e.g., NH2, ONH2, NH2NH2) may be acylated with, for example, C(O)CF3.

[0429] In some embodiments, the linker / tether may be terminated with a mercapto group (i.e., SH) or an olefin (e.g., CH=CH2). For example, the tether may be TAP-(CH2) n -SH, TAP-C(O)(CH2) n SH, TAP-(CH2) n -(CH=CH2), or TAP-C(O)(CH2) nThe formula may also be (CH=CH2), where n may be as described elsewhere. The tether may be substituted with, for example, hydroxy, alkoxy, or perhaloalkyl, and / or may have one or more additional heteroatoms, such as N, O, or S, inserted. The double bond may be cis or trans or E or Z.

[0430] In other embodiments, the linker / tether may preferably contain an electrophilic component at the terminal position of the linker / tether. Examples of electrophilic components include, for example, aldehydes, alkyl halides, mesylates, tosylates, nosylates, or brosylates, or activated carboxylic acid esters, such as NHS esters or pentafluorophenyl esters. A preferred linker / tether (indicated by underlining) is TAP-(CH2) n CHO;TAP-C(O)(CH2) n CHO; or TAP-NR''''(CH2) n CHO is an example, where n is 1-6 and R'''' is a C1-C6 alkyl group; or TAP-(CH2) n C(O)ONHS;TAP-C(O)(CH2) n C(O)ONHS; or TAP-NR''''(CH2) n C(O)ONHS, where n is 1-6 and R'''' is C1-C6 alkyl; TAP-(CH2) n C(O)OC6F5;TAP-C(O)(CH2) n C(O)OC6F5; or TAP-NR''''(CH2) n The formula is C(O)OC6F5, where n is 1 to 11 and R'''' is a C1 to C6 alkyl group; or -(CH2) n CH2LG;TAP-C(O)(CH2) n CH2LG; or TAP-NR''''(CH2) nThe formula is CH2LG, where n may be as described elsewhere, and R'''' is a C1-C6 alkyl group (LG may be a leaving group, e.g., a halide, mesylate, tosylate, nosylate, or brosylate). Tethering may be performed by coupling the nucleophilic group of the ligand, e.g., a thiol or amino group, with the electrophilic group of the tether.

[0431] In other embodiments, it may be desirable for the monomer to contain a phthalimide group (K) at the terminal position of the linker / tether.

[0432] [ka]

[0433] In other embodiments, other protected amino groups may be located at the terminal positions of the linker / tether, such as alloc, monomethoxytrityl (MMT), trifluoroacetyl, Fmoc, or arylsulfonyl (for example, the aryl portion may be orthonitrophenyl or ortho,paradinitrophenyl).

[0434] Any of the linkers / tethers described herein may have one or more additional linking groups, for example, -O-(CH2) n -,-(CH2) n -SS-, -(CH2) n - or -(CH=CH)- may also be included.

[0435] B. Cuttable linker / tether In some embodiments, at least one of the linker / tether may be a redox-cleavable linker, an acid-cleavable linker, an esterase-cleavable linker, a phosphatase-cleavable linker, or a peptidase-cleavable linker.

[0436] In one embodiment, at least one of the linkers / tethers may be a reductively cleavable linker (e.g., a disulfide group). In one embodiment, at least one of the linkers / tethers may be an acid-cleavable linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group). In one embodiment, at least one of the linkers / tethers may be an esterase-cleavable linker (e.g., an ester group). In one embodiment, at least one of the linkers / tethers may be a phosphatase-cleavable linker (e.g., a phosphate group). In one embodiment, at least one of the linkers / tethers may be a peptidase-cleavable linker (e.g., a peptide bond).

[0437] Cleavable linking groups are susceptible to the presence of cleavage agents, such as pH, redox potential, or degradable molecules. Generally, cleavage agents are more prevalent or found at higher levels or with greater activity inside cells than in serum or blood. Examples of such degradable agents include redox agents that are selected for or do not exhibit substrate specificity for a particular substrate, such as oxidizers or reductases or reducing agents such as mercaptans that can degrade redoxably cleavable linking groups present in cells by reduction; esterases; agents that can create endosomes or acidic environments, such as those that result in a pH of 5 or lower; and enzymes, peptidases (which may be substrate-specific), and phosphatases that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids.

[0438] Cleavable linking groups, such as disulfide bonds, may be susceptible to pH fluctuations. While human serum has a pH of 7.4, the average intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes have a higher acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even higher acidic pH, as high as approximately 5.0. Some tethers are expected to have linking groups that cleave at a favorable pH, thereby releasing iRNA agents from intracellular ligands (e.g., targeted or cell-permeable ligands, such as cholesterol) or into desired compartments of the cell.

[0439] Disulfide bonds can be used as chemical junctions (e.g., linking groups) to link an iRNA agent to a ligand. When the iRNA agent / ligand complex is taken up by a cell via 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 may be a targeted ligand or a second therapeutic agent that can complement the therapeutic effect of the iRNA agent.

[0440] A tether can be a linking group that can be cleaved by a specific enzyme. The type of linking group incorporated into the tether may depend on the cell that will be targeted by the iRNA agent. For example, an iRNA agent that targets mRNA in liver cells may be conjugated to a tether containing an ester group. Liver cells are rich in esterases, and therefore the tether is expected to be cleaved more efficiently in liver cells than in cell types that are not rich in esterases. Cleavage of the tether may release the iRNA agent from the ligand attached to the distal end of the tether, thereby enhancing the silencing activity of the iRNA agent. Other cell types rich in esterases include lung, renal cortex, and testicular cells.

[0441] A tether containing a peptide bond may be conjugated to an iRNA agent targeted to peptidase-rich cell types such as liver cells or synovial cells. For example, an iRNA agent targeted to synovial cells for the treatment of inflammatory diseases (e.g., rheumatoid arthritis) may be conjugated to a tether containing a peptide bond.

[0442] Generally, the suitability of a candidate cleavable linker can be evaluated by testing the ability of a degradation agent (or condition) to cleave the candidate linker. It is also desirable to test the candidate cleavable linker for its ability to resist cleavage in the blood or in contact with other non-target tissues, such as tissues to which the iRNA agent is expected to be exposed when administered to a subject. Therefore, the relative sensitivity to cleavage between a first and second condition may be determined, in which case the first condition is selected to demonstrate cleavage in target cells, and the second condition is selected to demonstrate cleavage in other tissues or bodily fluids, such as blood or serum. Evaluations can be performed in cell-free systems, cells, cell cultures, organ or tissue cultures, or whole animals. It may be useful to perform the initial evaluation under cell-free or culture conditions and confirm it with further evaluation in whole animals. In a preferred embodiment, a useful candidate compound is 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).

[0443] C. Redox-cleavable linking groups One class of cleavable linking groups is redox-cleavable linking groups, which are cleaved by reduction or oxidation. An example of a redox-cleavable linking group is a disulfide linking group (-SS-). Methods described herein can be relied upon to determine whether a candidate cleavable linking group is a suitable “redox-cleavable linking group,” or whether it is suitable for use with, for example, a particular iRNA moiety and a particular targeting agent. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the rate of cleavage expected to be observed in cells, e.g., target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In a preferred embodiment, the candidate compound is cleaved up to 10% in blood. In a preferred embodiment, a useful candidate compound is degraded 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 under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of candidate compounds may be determined using a standard enzyme kinetic assay under conditions selected to mimic an intracellular medium and compared with conditions selected to mimic an extracellular medium.

[0444] D. Phosphate-based cleavable linking groups Phosphate-based linking groups are cleaved by agents that degrade 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(O)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0445] E. Acid-cleavable linking groups Acid-cleavable linking groups are linking groups that are cleaved under acidic conditions. In preferred embodiments, acid-cleavable linking groups are cleaved in an acidic environment having a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as common acids. In cells, organelles with particularly low pH, such as endosomes and lysosomes, can provide a cleavage environment for acid-cleavable linking groups. Examples of acid-cleavable linking groups, but not limited to these, include hydrazones, ketals, acetals, esters, and amino acid esters. Acid-cleavable groups may have the general formula -C=NN-, C(O)O, or -OC(O). In preferred embodiments, the carbon connected 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.

[0446] F. Ester-based linking group Ester-based linking groups are cleaved by enzymes such as esterases and amidases in cells. 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.

[0447] G. Peptide-based C-cleavage group Peptide-based linking groups are cleaved by enzymes such as peptidases and proteases in cells. 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 linking groups do not contain amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynelene. Peptide bonds are a special type of amide bond formed between amino acids to produce peptides and proteins. Peptide-based cleavable linking groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to produce peptides and proteins, and do not include the entire amide functional group. The general formula for peptide cleavable linking groups is -NHCHR 1 C(O)NHCHR 2 It contains C(O)-, in the formula, R 1 and R 2 These are the R groups of two adjacent amino acids. These candidates can be evaluated using a method similar to that described above.

[0448] H. Biocleavable linker / tether Linkers can also be bio-cleavable linkers, which are nucleotide and non-nucleotide linkers or combinations thereof that link two parts of a molecule, for example, one or both chains of two individual siRNA molecules, to produce a bis(siRNA). In some embodiments, a simple electrostatic or stacking interaction between two individual siRNAs can act as a linker. Non-nucleotide linkers include tethers or linkers derived from monosaccharides, disaccharides, oligosaccharides and their derivatives, aliphatic, alicyclic, heterocyclic, and combinations thereof.

[0449] In some embodiments, at least one of the linkers (tethers) is a biologically cleavable linker selected from the group consisting of functionalized monosaccharides or oligosaccharides of DNA, RNA, disulfides, amides, galactosamine, glucosamine, glucose, galactose, and mannose, as well as combinations thereof.

[0450] In one embodiment, a biologically cleavable carbohydrate linker may have 1 to 10 sugar units, each having at least one anomeric linkage capable of linking two siRNA units. If two or more sugars are present, these units may be linked via 1-3, 1-4, or 1-6 sugar linkages, or via alkyl chains.

[0451] Examples of biologically cleavable linkers include:

[0452] [ka] TIFF2026534213000038.tif169160 Includes TIFF2026534213000039.tif121160.

[0453] Further discussion of in vivo cleavable linkers can be found in PCT application PCT / US18 / 14213, filed on 18 January 2018, titled “Endosomal Cleavable Linkers,” the entire content of which is incorporated herein by reference.

[0454] I. Carrier In a particular embodiment, one or more C 22 The hydrocarbon chain is conjugated to the iRNA agent via a carrier that replaces one or more nucleotides.

[0455] The support may 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, pyridadinyl, tetrahydrofuryl, and decalin. In one embodiment, the acyclic group is a portion based on a selinol backbone or a diethanolamine backbone.

[0456] In some embodiments, the carrier replaces one or more nucleotides at a position within the dsRNA agent.

[0457] In other embodiments, the support replaces a nucleotide at the terminal end of the sense or antisense strand. In one embodiment, the support replaces a terminal nucleotide at the 3' end of the sense strand, thereby acting as a terminal cap to protect the 3' end of the sense strand. In one embodiment, the support is a cyclic group having an amine, and for example, the support may be pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanil, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuranil, or dekalinyl.

[0458] Ribonucleotide subunits in which the ribose sugar of the subunit is thus replaced are referred to herein as ribose-substituted modified subunits (RRMS). The support may be a cyclic or acyclic moiety and may include two “backbone connection points” (e.g., hydroxyl groups) and a ligand (e.g., a lipophilic moiety). One or more C 22 The hydrocarbon chain may be directly connected to the carrier, or it may be indirectly connected to the carrier by an intervening linker / tether as described above.

[0459] [ka]

[0460] The ligand-conjugated monomer subunit may be the 5' or 3' terminal subunit of the iRNA molecule, that is, one of the two "W" groups may be a hydroxyl group and the other "W" group may be a chain of two or more unmodified or modified ribonucleotides. Alternatively, the ligand-conjugated monomer subunit may occupy an internal position, and both "W" groups may be one or more unmodified or modified ribonucleotides. More than one ligand-conjugated monomer subunit may be present in the iRNA agent.

[0461] a. Monomers based on sugar substitution, such as ligand-conjugated monomers (cyclic) Monomers based on cyclic sugar substitution, such as monomers conjugated with a sugar substitution-based ligand, are also referred to herein as RRMS monomer compounds. The support may have the general formula (LCM-2) provided below [in which structure, preferred main chain connection points are R 1 or R 2 ;R 3 or R 4 You can choose from, or Y is CR 9 R 10 If R 9 and R 10 You may choose from (the two positions are chosen so that two main chain connection points are obtained, for example R 1 and R 4 , or R 4 and R 9 (This is the case). A preferred tethering connection point is when X is CH2, R 7 ;R 5 or R 6Examples include the carrier, which will be described later as an entity that can be incorporated into the chain. Therefore, this structure also has one (in the case of terminal positions) or two (in the case of internal positions) of the connection points, for example, R 1 Or R 2 ;R 3 Or R 4 ; or R 9 Or R 10 (Y is CR 9 R 10 It is understood that this also includes situations where the R group (if specified above) is linked to a phosphate or a modified phosphate, such as a sulfur-containing main chain. For example, one of the R groups specified above may be -CH2-, in which case one bond is linked to the support and the other bond is linked to a main chain atom, such as the linking oxygen or the central phosphorus atom.

[0462] [ka] During the ceremony, X is N(CO)R 7 , NR 7 or CH2; Y is NR 8 O, S, CR 9 R 10 and; Z is CR 11 R 12 or not; R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 Each of these is independently H, OR a , or (CH2) n Ure b And, however, R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 At least two of them are OR a and / or (CH2) n Ureb and; R 5 , R 6 , R 11 , and R 12 Each of these independently consists of a ligand, H, and 1-3 R 13 C1-C6 alkyl or C(O)NHR which may be substituted. 7 is; or R 5 and R 11 Together, R 14 A C3-C8 cycloalkyl group which may be substituted with; R 7 This could be a ligand, or for example, R 7 R d You may or R 7 This may be a ligand that is indirectly tethered to the carrier, for example, a ligand tethered via a tethering portion, for example, NR c R d C1~C replaced by 20 alkyl; or NHC(O)R d C1~C replaced by 20 Alkyl is also acceptable; R 8 is H or C1-C6 alkyl; R 13 These are hydroxyl, C1-C4 alkoxy, or halo; R 14 , NR c R 7 and; R 15 is a C1-C6 alkyl or C2-C6 alkenyl which may be substituted with cyano; R 16 C1~C 10 It is alkyl; R 17 It is a liquid-phase or solid-phase supported reagent; L is -C(O)(CH2) q C(O)-, or -C(O)(CH2) q It is S-; R aThis refers to a protecting group, for example, CAr3 (e.g., dimethoxytrityl group) or Si(X 5’ )(X 5” )(X 5”’ ) and (X 5’ ),(X 5” ), and (X 5”’ ) is as described elsewhere; R b is, P(O)(O - )H, P(OR 15 )N(R 16 )2 or LR 17 and; R c is H or C1-C6 alkyl; R d is H or a ligand; Each Ar may independently be substituted with a C1-C4 alkoxy, or a C6-C4 alkoxy. 10 It is an allele; n is between 1 and 4; q is between 0 and 4.

[0463] An example of a carrier is, for instance, X is N(CO)R 7 Or NR 7 And Y is CR 9 R 10 and Z does not exist; or X is N(CO)R 7 Or NR 7 And Y is CR 9 R 10 And Z is CR 11 R 12 That which is; or X is N(CO)R 7 Or NR 7 And Y is NR 8 And Z is CR 11 R 12 That which is; or X is N(CO)R 7 Or NR 7 And Y is O and Z is CR 11 R 12 Those which are; or those which are CH2; Y is CR 9 R 10 That which is; Z is CR 11 R 12 And R5 and R 11 However, together they form a C6 cycloalkyl (H, z=2) or indane ring system, for example, where X is CH2; Y is CR 9 R 10 That which is; Z is CR 11 R 12 And R 5 and R 11 However, when combined, they form a C5 cycloalkyl group (H, z=1).

[0464] In certain embodiments, the carrier may be based on a pyrroline ring system or a 4-hydroxyproline ring system, for example, X is N(CO)R 7 or NR 7 And Y is CR 9 R 10 Therefore, Z does not exist (D).

[0465] [ka] OFG 1 Preferably, it is attached to a primary carbon, for example, an extra-ring alkylene group, for example, a methylene group attached to one of the carbons of a five-membered ring (in D, -CH2OFG). 1 ) Connected to OFG 2 Preferably, it is directly connected to one of the carbon atoms of the 5-membered ring (D is -OFG). 2 ). In the case of pyrroline-based carriers, -CH2OFG 1 It may also be connected to C-2, OFG 2 It may also be connected to C-3; or -CH2OFG 1 It may also be connected to C-3, OFG 2 It may be connected to C-4. In certain embodiments, CH2OFG 1 and OFG 2 It may be geminally substituted on one of the carbons mentioned above. In the case of a 3-hydroxyproline-based support, -CH2OFG 1 It may also be connected to C-2, OFG 2It may be connected to C-4. Pyrroline and 4-hydroxyproline-based monomers may therefore contain linkages (e.g., carbon-carbon bonds), in which case the rotation of the bond is restricted with respect to that particular linkage, for example, the restriction arises from the presence of a ring. Thus, CH2OFG 1 and OFG 2 In any of the pairings detailed above, the atoms may be cis or trans relative to each other. Therefore, all cis / trans isomers are explicitly included. Monomers may also contain one or more chiral centers and therefore may exist as racemic compounds and racemic mixtures, single enantiomers, individual diastereomers and diastereomer mixtures. All such isomeric forms of monomers are explicitly included (e.g., CH2OFG 1 and OFG 2 Both centers having the R configuration may have the R configuration; or both may have the S configuration; or one center may have the R configuration and the other center may have the S configuration, and vice versa). The tethering connection point is preferably nitrogen. Preferred examples of carrier D include the following:

[0466] [ka]

[0467] In certain embodiments, the support may be based on a piperidine ring system (E), for example, X is N(CO)R 7 or NR 7 And Y is CR 9 R 10 And Z is CR 11 R 12 That is the case.

[0468] [ka]

[0469] OFG 1Preferably, it is attached to a primary carbon, for example, an extra-ring alkylene group, for example, a methylene group (n=1) or ethylene group (n=2) attached to one of the carbons of a 6-membered ring [E is -(CH2)] n OFG 1 ] Connected. OFG 2 Preferably, one of the carbon atoms of the 6-membered ring (E is -OFG) 2 It is directly connected to (CH2). n OFG 1 and OFG 2 The groups may be arranged in a geminal manner in the ring, meaning both groups may be attached to the same carbon, for example, C-2, C-3, or C-4. Alternatively, -(CH2) n OFG 1 and OFG 2 The groups may be arranged in a vicinal manner on the ring, that is, both groups may be connected to carbon atoms of adjacent rings, for example, -(CH2) n OFG 1 It may also be connected to C-2, OFG 2 It may also be connected to C-3;-(CH2) n OFG 1 It may also be connected to C-3, OFG 2 It may also be connected to C-2;-(CH2) n OFG 1 It may also be connected to C-3, OFG 2 It may also be connected to C-4; or -(CH2) n OFG 1 It may also be connected to C-4, OFG 2 It may be connected to C-3. The piperidine-based monomer may therefore contain a linkage (e.g., a carbon-carbon bond), in which case the bond rotation is restricted with respect to that particular linkage, for example, the restriction arises from the presence of a ring. Thus, -(CH2) n OFG 1 and OFG 2The isomers may be cis or trans relative to each other in any of the pairings detailed above. Therefore, all cis / trans isomers are explicitly included. Monomers may also contain one or more chiral centers and therefore may exist as racemic compounds and racemic mixtures, single enantiomers, individual diastereomers and diastereomer mixtures. All such isomeric forms of monomers are explicitly included (e.g., CH2OFG 1 and OFG 2 Both centers having the configuration may have the R configuration; or both may have the S configuration; or one center may have the R configuration and the other center may have the S configuration, and vice versa. The tethering connection point is preferably nitrogen.

[0470] In certain embodiments, the support may be based on a piperazine ring system (F), for example, X is N(CO)R 7 Or NR 7 And Y is NR 8 And Z is CR 11 R 12 It may be or may be based on a morpholine ring system (G), for example, X is N(CO)R 7 Or NR 7 And Y is O, and Z is CR 11 R 12 That is the case.

[0471] [ka] OFG 1 Preferably, it is attached to a primary carbon, for example, an extra-ring alkylene group, for example, a methylene group attached to one of the carbons of a six-membered ring (F or G is -CH2OFG). 1 ) Connected to OFG 2 Preferably, one of the carbon atoms of the 6-membered ring (F or G, in the case of -OFG) 2 ) is directly connected. For both F and G, -CH2OFG 1 It may also be connected to C-2, OFG2 It may be connected to C-3; or vice versa. In certain embodiments, CH2OFG 1 and OFG 2 The carbon atoms may be geminally substituted on one of the carbon atoms mentioned above. Piperazine and morpholine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds), in which case the bond rotation is restricted with respect to that particular linkage, for example, by the presence of a ring. Thus, CH2OFG 1 and OFG 2 In any of the pairings detailed above, the atoms may be cis or trans relative to each other. Therefore, all cis / trans isomers are explicitly included. Monomers may also contain one or more chiral centers and therefore may exist as racemic compounds and racemic mixtures, single enantiomers, individual diastereomers and diastereomer mixtures. All such isomeric forms of monomers are explicitly included (e.g., CH2OFG 1 and OFG 2 Both centers having R configurations may have R configurations; or both may have S configurations; or one center may have an R configuration and the other center may have an S configuration, and vice versa. R''' may be, for example, a C1-C6 alkyl group, preferably CH3. The tethering connection point is preferably nitrogen at both F and G.

[0472] In certain embodiments, the carrier may be based on a decalin ring system, for example, X is CH2; Y is CR 9 R 10 And; Z is CR 11 R 12 And R 5 and R 11 The two groups together are C6 cycloalkyl (H, z=2), or the support may be based on an indan ring system, for example, X is CH2; Y is CR 9 R 10 And; Z is CR 11 R12 And R 5 and R 11 These elements combine to form a C5 cycloalkyl group (H, z=1).

[0473] [ka] OFG 1 Preferably, it is connected to a primary carbon, for example, one of C-2, C-3, C-4, or C-5 [H is -(CH2)] n OFG 1 It is connected to an extra-ring methylene group (n=1) or ethylene group (n=2) linked to ]. OFG 2 Preferably, one of C-2, C-3, C-4, or C-5 (H-OFG) 2 It is directly connected to (CH2). n OFG 1 and OFG 2 The groups may be arranged in a geminal manner in the ring, meaning both groups may be attached to the same carbon, for example, C-2, C-3, C-4, or C-5. Alternatively, -(CH2) n OFG 1 and OFG 2 The groups may be arranged in a vicinal manner on the ring, that is, both groups may be connected to carbon atoms of adjacent rings, for example, -(CH2) n OFG 1 It may also be connected to C-2, OFG 2 It may also be connected to C-3;-(CH2) n OFG 1 It may also be connected to C-3, OFG 2 It may also be connected to C-2;-(CH2) n OFG 1 It may also be connected to C-3, OFG 2 It may also be connected to C-4; or -(CH2) n OFG 1 It may also be connected to C-4, OFG 2It may also be connected to C-3;-(CH2) n OFG 1 It may also be connected to C-4, OFG 2 It may also be connected to C-5; or -(CH2) n OFG 1 It may also be connected to C-5, OFG 2 It may be connected to C-4. Decalin or indan-based monomers may therefore contain linkages (e.g., carbon-carbon bonds), in which case bond rotation is restricted with respect to that particular linkage, for example, the restriction arises from the presence of a ring. Thus, -(CH2) n OFG 1 and OFG 2 In any of the pairings detailed above, the atoms may be cis or trans relative to each other. Therefore, all cis / trans isomers are explicitly included. Monomers may also contain one or more chiral centers and therefore may exist as racemic compounds and racemic mixtures, single enantiomers, individual diastereomers and diastereomer mixtures. All such isomeric forms of monomers are explicitly included (e.g., CH2OFG 1 and OFG 2 Both centers having C-1 and C-6 may have the R configuration; or both may have the S configuration; or one center may have the R configuration and the other center may have the S configuration, and vice versa. In a preferred embodiment, the substituents at C-1 and C-6 are trans relative to each other. The tethering connection point is preferably C-6 or C-7.

[0474] Other carriers include those based on 3-hydroxyproline (J).

[0475] [ka] Therefore, -(CH2) n OFG 1 and OFG 2These may be cis or trans relative to each other. Therefore, all cis / trans isomers are explicitly included. Monomers may also contain one or more chiral centers and therefore may exist as racemic compounds and racemic mixtures, single enantiomers, individual diastereomers and diastereomer mixtures. All such isomeric forms of monomers are explicitly included (e.g., CH2OFG 1 and OFG 2 Both centers having the configuration may have the R configuration; or both may have the S configuration; or one center may have the R configuration and the other center may have the S configuration, and vice versa. The tethering connection point is preferably nitrogen.

[0476] Further details regarding representative cyclic sugar substitution-based carriers can be found in U.S. Patents 7,745,608 and 8,017,762, which are incorporated herein by reference in their entirety.

[0477] b. Monomers (acyclic) based on sugar substitution Monomers based on acyclic sugar substitution, such as monomers conjugated with a sugar substitution-based ligand, are also referred to herein as ribose-substituted monomer subunit (RRMS) monomer compounds. Preferred acyclic carriers may have formula LCM-3 or LCM-4.

[0478] [ka]

[0479] In some embodiments, x, y, and z may be 0, 1, 2, or 3 independently of each other. In formula LCM-3, if y and z are different, the tertiary carbon may have either an R or S configuration. In preferred embodiments, x is zero, and in formula LCM-3, y and z are each 1 (e.g., selinol-based), and in formula LCM-3, y and z are each 1. Each of the following formulas LCM-3 or LCM-4 may be substituted with, for example, hydroxy, alkoxy, or perhaloalkyl.

[0480] Further details regarding representative acyclic sugar substitution-based carriers can be found in U.S. Patents 7,745,608 and 8,017,762, which are incorporated herein by reference in their entirety.

[0481] One or more C 22 A hydrocarbon chain is conjugated at one or more internal positions in at least one of the chains. Internal positions refer to any nucleotide at any position in the chain except for terminal positions from the 3' and 5' ends (for example, positions 2:1 from the 3' end and position 1 from the 5' end are excluded).

[0482] In one embodiment, one or more C 22 A hydrocarbon chain is conjugated at one or more internal positions in at least one of the chains, and the internal positions include all positions except the two terminal positions from each end of the chain (for example, positions 4:1 and 2 counting from the 3' end and positions 1 and 2 counting from the 5' end are excluded). In one embodiment, one or more C 22 A hydrocarbon chain is conjugated at one or more internal positions in at least one of the chains, where internal positions include all positions except the three terminal positions from each end of the chain (for example, positions 6 counting from the 3' end: positions 1, 2, and 3, and positions 1, 2, and 3 counting from the 5' end are excluded).

[0483] In one embodiment, one or more C 22 The hydrocarbon chain is conjugated at one or more internal positions in at least one of the chains, excluding the region of the sense chain cleavage site, for example, one or more C 22 The hydrocarbon chain is not conjugated at positions 9-12 counting from the 5' end of the sense chain, for example, one or more C 22 The hydrocarbon chain is not conjugated at positions 9–11, counting from the 5' end of the sense chain. Alternatively, internal positions exclude positions 11–13, counting from the 3' end of the sense chain.

[0484] In one embodiment, one or more C 22 The hydrocarbon chain is conjugated at one or more internal positions on at least one of the chains, excluding the region of the antisense chain's cleavage site. For example, the internal positions exclude positions 12–14, counting from the 5' end of the antisense chain.

[0485] In one embodiment, one or more C 22 The hydrocarbon chain is conjugated at one or more internal positions in at least one of the chains, excluding positions 11–13 in the sense chain (counting from the 3' end) and positions 12–14 in the antisense chain (counting from the 5' end).

[0486] In one embodiment, one or more C 22 The hydrocarbon chains are conjugated to one or more of the following internal positions, counting from the 5' end of each chain: positions 4–8 and 13–18 in the sense chain, and positions 6–10 and 15–18 in the antisense chain.

[0487] In one embodiment, one or more C 22The hydrocarbon chain is conjugated to one or more of the following internal positions: positions 5, 6, 7, 15, 16, and 17 in the sense chain, and positions 15 and 17 in the antisense chain, counting from the 5' end of each chain. In one embodiment, one or more C 22 The hydrocarbon chain is conjugated at the 6th position of the sense chain, counting from the 5' end of each chain. In one embodiment, one or more C 22 The hydrocarbon chain is conjugated at the 16th position in the sense chain, counting from the 5' end of each chain. In some embodiments, one or more C 22 The hydrocarbon chain is conjugated to the phosphate linkage between the nucleic acid bases, sugar moieties, or nucleosides of the dsRNA agent.

[0488] VI. Synthesis of the RNAi agents of this disclosure The nucleic acids described herein may be synthesized or modified by methods well established in the industry, for example, by those described in “Current protocols in nucleic acid chemistry,” Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which are incorporated herein by reference.

[0489] siRNA may be produced by various methods, for example, in bulk. Exemplary methods include organic synthesis and RNA cleavage, such as in vitro cleavage.

[0490] A.Organic synthesis siRNA may be prepared by separately synthesizing single-stranded RNA molecules or individual double-stranded RNA molecules, and then the component strands may be annealed. Large bioreactors, such as OligoPilot II from Pharmacia Biotec AB (Uppsala Sweden), can be used to produce large quantities of specific RNA strands of a given siRNA. The OligoPilot II reactor can efficiently couple nucleotides using only 1.5-fold molar excess phosphoramidite nucleotides. Ribonucleotide amidites are used to construct the RNA strands. A standard monomer addition cycle can be used to synthesize 21-23 nucleotide strands of siRNA. Typically, two complementary strands are prepared separately and then annealed, for example, after release from a solid support and deprotection.

[0491] Organic synthesis can be used to produce distinct siRNA species. The species complementarity to a specific target gene can be precisely determined. For example, a species may be complementary to a region containing polymorphisms, such as single-nucleotide polymorphisms. Furthermore, the location of the polymorphisms can be precisely defined. In some embodiments, the polymorphisms are located within an internal region, for example, within at least 4, 5, 7, or 9 nucleotides from one or both ends.

[0492] B.dsiRNA cleavage siRNA may also be produced by cleaving larger siRNAs. Cleavage can be mediated in vitro or in vivo. For example, the following method can be used to produce iRNA by in vitro cleavage.

[0493] 1. In vitro transcription dsiRNA is produced by transcribing a nucleic acid (DNA) segment in both directions. For example, the HiScribe® RNAi transcription kit (New England Biolabs) provides a vector and method for producing dsiRNA of a nucleic acid segment cloned into a vector at a position flanked on both sides by a T7 promoter. Separate templates are generated for the T7 transcription of the two complementary strands of the dsiRNA. The templates are transcribed in vitro by the addition of T7 RNA polymerase to produce dsiRNA. Similar methods using PCR and / or other RNA polymerases (e.g., T3 or SP6 polymerase) may contain endotoxins that could contaminate recombinant preparations. In one embodiment, the RNA produced by this method is carefully purified to remove endotoxins that could contaminate recombinant preparations.

[0494] 2. In vitro dissection dsRNA is cleaved into siRNA in vitro using, for example, Dicer or an equivalent RNase III-based activity. For example, dsiRNA may be incubated in vitro in an extract from Drosophila, or it may be incubated with purified components, such as purified RNase or RISC complex (RNA-induced silencing complex). See, for example, Ketting et al. Genes Dev 2001 Oct 15;15(20):2654-9 and Hammond Science 2001 Aug 10;293(5532):1146-50.

[0495] dsiRNA cleavage typically produces multiple siRNA species, each consisting of a specific 21-23nt fragment of the source dsiRNA molecule. For example, there may be siRNAs containing sequences complementary to the overlapping and adjacent regions of the source dsiRNA molecule.

[0496] Regardless of the synthesis method, siRNA preparations may be prepared in a solution suitable for the formulation (e.g., aqueous and / or organic solutions). For example, the siRNA preparation may be precipitated, redissolved in pure redistilled water, and freeze-dried. The dried siRNA may then be resuspended in a solution suitable for the intended formulation process.

[0497] C.1 or C 22 Preparation of dsRNA agents conjugated to hydrocarbon chains In some embodiments, one or more C 22 The hydrocarbon chain is conjugated to the dsRNA agent via nucleic acid bases, sugar moieties, or nucleoside linkages.

[0498] The conjugation to the purine nucleic acid base or its derivative may be present at any position, including atoms inside and outside the ring. In some embodiments, the 2, 67, or 8 position of the purine nucleic acid base is C 22 It is connected to a hydrocarbon chain. Conjugations to pyrimidine nucleic acid bases or their derivatives may also be present at any position. In some embodiments, positions 2, 5 and 6 of the pyrimidine nucleic acid base are C 22 It may be substituted with hydrocarbon chains. One or more C 22 When a hydrocarbon chain is conjugated to a nucleic acid base, the preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bonding interaction necessary for base pairing. In one embodiment, one or more C 22 The hydrocarbon chain may be conjugated to a nucleic acid base via a linker containing alkyl, alkenyl, or amide linkages.

[0499] The conjugation to the sugar portion of the nucleoside may be present on any carbon atom. 22 Exemplary carbon atoms in the sugar moiety, which may have a hydrocarbon chain attached, include 2', 3', and 5' carbon atoms. 22The hydrocarbon chain may also be attached, for example, to the 1' position of a debasic residue. In one embodiment, one or more C 22 The hydrocarbon chain may be conjugated to the sugar moiety via a 2'-O modification, with or without a linker.

[0500] Nucleoside linkages also include one or more C 22 It may have a hydrocarbon chain. In the case of a phosphorus-containing linkage (e.g., phosphodiester, phosphorothioate, phosphoramidate, etc.), one or more C 22 The hydrocarbon chain may be directly linked to a phosphorus atom or an O, N, or S atom bonded to a phosphorus atom. In the case of internucleoside links containing amines or amides (e.g., PNA), one or more C 22 The hydrocarbon chain may be connected to the nitrogen atom or adjacent carbon atom of the amine or amide.

[0501] There are many methods for preparing oligonucleotide (oligonuclotides) conjugates. Generally, oligonucleotides are conjugated to a conjugate by contacting the reactive group in the oligonucleotide (e.g., OH, SH, amine, carboxyl, aldehyde, etc.) with the reactive group in the conjugate. In some embodiments, one reactive group is electrophilic and the other is nucleophilic.

[0502] For example, the electrophilic group may be a carbonyl-containing functional group, and the nucleophilic group may be an amine or a thiol. Methods for the conjugation of nucleic acids and related oligomeric compounds with and without linking groups are well described in literature such as, for example, Manoharan in Antisense Research and Applications, Crooke and LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, which is incorporated herein by reference in its entirety.

[0503] In one embodiment, the first (complementary) RNA strand and the second (sense) RNA strand may be synthesized separately, in which case one of the RNA strands is pendant C 22 The first and second RNA strands, comprising a hydrocarbon chain, may be mixed to form dsRNA. The step of synthesizing the RNA strand preferably involves solid-phase synthesis, in which individual nucleotides are joined together at their ends in a successive synthetic cycle via the formation of 3'-5' phosphodiester bonds between nucleotides.

[0504] In one embodiment, C having a phosphoramidite group 22 The hydrocarbon chain is coupled to either the 3' or 5' end of either the first (complementary) or second (sense) RNA chain in the final synthetic cycle. In solid-phase RNA synthesis, nucleotides are initially in the form of nucleoside phosphoramidites. In each synthetic cycle, additional nucleoside phosphoramidites are ligated to the -OH groups of the nucleotides incorporated up to that point. One or more C 22 If the hydrocarbon chain has a phosphoramidite group, it may be coupled to the free OH end of RNA previously synthesized by solid-phase synthesis in a manner similar to that of nucleoside phosphoramidites. Synthesis can be carried out in an automated and standardized manner using conventional RNA synthesizers. Synthesis of molecules having a phosphoramidite group may involve phosphytylation of the free hydroxyl to generate the phosphoramidite group.

[0505] One or more C 22 The synthesis procedure for hydrocarbon chain conjugate phosphoramidites is illustrated in Example 1.

[0506] Generally, oligonucleotides may be synthesized using protocols known in the art, for example, as described in Caruthers et al., Methods in Enzymology (1992) 211:3-19;WO99 / 54459; Wincott et al., Nucl. Acids Res. (1995) 23:2677-2684; Wincott et al., Methods Mol. Bio., (1997) 74:59; Brennan et al., Biotechnol. Bioeng. (1998) 61:33-45; and U.S. Patent No. 6,001,311, each of which is incorporated herein by reference as a whole. Generally, the synthesis of oligonucleotides involves conventional nucleic acid protecting and coupling groups, for example, dimethoxytrityl at the 5' end and phosphoramidite at the 3' end. In non-limiting examples, small-scale synthesis is performed using the Expedite 8909 RNA synthesizer, sold by Applied Biosystems, Inc. (Weiterstadt, Germany), with ribonucleoside phosphoramidites, sold by ChemGenes Corporation (Ashland, Mass.). Alternatively, the synthesis may be carried out in a 96-well plate synthesizer, for example, in an apparatus produced by Protogene (Palo Alto, Calif.), or by methods such as those described in Usman et al., J. Am. Chem. Soc. (1987) 109:7845; Scaringe, et al., Nucl. Acids Res. (1990) 18:5433; Wincott, et al., Nucl. Acids Res. (1990) 23:2677-2684; and Wincott, et al., Methods Mol. Bio. (1997) 74:59, each of which is incorporated herein by reference as a whole.

[0507] The nucleic acid molecules disclosed in this invention may be synthesized separately and then combined after synthesis, for example, by ligation (Moore et al., Science (1992) 256:9923; WO93 / 23569; Shabarova et al., Nucl. Acids Res. (1991) 19:4247; Bellon et al., Nucleosides & Nucleotides (1997) 16:951; Bellon et al., Bioconjugate Chem. (1997) 8:204; or by hybridization after synthesis and / or deprotection. The nucleic acid molecules may be purified by gel electrophoresis using conventional methods, or by high-pressure liquid chromatography (HPLC; Wincott et al., the whole of which is incorporated herein by reference, see above) and resuspended in water.

[0508] VII. Ligand In certain embodiments, the dsRNA agents of the Disclosure may be further modified by covalent linkage of one or more conjugate groups. Generally, but not limited to, conjugate groups modify one or more properties of the linked dsRNA agents of the Disclosure, such as pharmacokinetics, pharmacokinetics, binding, absorption, cellular distribution, intracellular uptake, charge, and clearance. Conjugate groups are conventionally used in the chemical field and may be directly linked to a parent compound, such as an oligomeric compound, or linked via a linkage moiety or linkage group as appropriate. A preferred list of conjugate groups, but not limited to, includes intercalators, reporter molecules, polyamines, polyamides, polyethylene glycol, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, foletes, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, and dyes.

[0509] In some embodiments, the dsRNA agent further comprises a targeted ligand that targets a receptor that mediates delivery to a specific tissue, such as liver tissue. These targeted ligands include one or more C receptors to enable specific systemic delivery. 22 It may be combined with a hydrocarbon chain and conjugated. In one embodiment, the targeting ligand, for example, one or more GalNAc derivatives, is one or more C 22 It is combined with a hydrocarbon chain and conjugated to the dsRNA agent of the present disclosure. In another embodiment, a targeting ligand, for example, one or more GalNAc derivatives, is one or more C 22 It is not combined with a hydrocarbon chain and conjugated to the dsRNA agent of this disclosure.

[0510] Exemplary targeting ligands that target receptor-mediated delivery to adipose tissue include peptide ligands, e.g., angiopeptide-2, lipoprotein receptor-associated protein (LRP) ligands, bEnd.3 cell-binding ligand; transferrin receptor (TfR) ligand (which can utilize the iron transport system in the brain and cargo transport to the brain parenchyma); mannose receptor ligand (which targets olfactory nerve sheath cells and glial cells); glucose transporter proteins; and LDL receptor ligands.

[0511] Suitable conjugate groups for the disclosure of the present invention include lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053); thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765); thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533); and fatty acid chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49); phospholipids, e.g., dihexadecyl-rac-glycerol or triethylammonium-1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777); polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969); adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651); palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229); or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther.(Examples include 1996, 277, 923).

[0512] In general, various entities, such as ligands, may be coupled to the oligomeric compounds described herein. Ligands may be naturally occurring molecules or recombinant or synthetic molecules. Exemplary ligands, but not limited to these, include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolid) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG, e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG]2, polyvinyl alcohol (PVA), polyurethane, and poly(2-ethylacrylic acid) acid)), N-isopropylacrylamide polymer, polyphosphazine, polyethyleneimine, cationic group, spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary compound salts of polyamines, tyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin, glycosylated polyamino acids, transferrin, bisphosphonate, polyglutamate, polyaspartate , aptamers, asialofetin, hyaluronan, procollagen, immunoglobulins (e.g., antibodies), insulin, transferrin, albumin, sugar-albumin conjugate, intercalating agents (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (e.g., TPPC4, texaphylline, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., steroids, bile acids, cholesterol, cholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), peptides (e.g., alpha-helical peptides, amphiphilic peptides, RGD peptides, cell-permeable peptides, endosomal soluble / fusion peptides), alkylating agents, phosphates, amino acids, mercaptos, polyamino acids, alkyls, substituted alkyls, radiolabeled markers, enzymes Haptens (e.g., biotin), transport / absorption enhancers (e.g., naproxen, aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraaza macrocyclic compounds), dinitrophenyl, HRP, AP, antibodies, hormones and hormone receptors, lectins, carbohydrates, polyunsaturated carbohydrates, vitamins (e.g., vitamin A, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin and pyridoxal), vitamin cofactors, lipopolysaccharides, p38 Examples include MAP kinase activators, NF-κB activators, taxanes, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latruncrine A, phalloidin, swinholide A, indanosine, myoservin, tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta, gamma interferon, natural or recombinant low-density lipoprotein (LDL), natural or recombinant high-density lipoprotein (HDL), and cell permeabilizers (e.g., a. helical cell permeabilizers).

[0513] Examples of peptides and peptidomimetic ligands include naturally occurring or modified peptides, e.g., D or L peptides; α, β, or γ peptides; N-methyl peptides; azapeptides; peptides having one or more amides, i.e., peptides, urea-substituted linkages, thiourea, carbamate, or sulfonylurea linkages; or cyclic peptides. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules capable of folding into a defined three-dimensional structure similar to that of natural peptides. Peptides or peptidomimetic ligands may be about 5 to 50 amino acids in length, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids.

[0514] Examples of amphiphilic peptides include, but are not limited to, secropine, lycotoxin, paradaxin, buforin, CPF, bombinin-like peptide (BLP), cathelicidine, ceratotoxin, S. clava peptide, hagfish intestinal antimicrobial peptide (HFIAP), magainin, brevinin-2, dermaceptin, melittin, pleurocidin, H2A peptide, African clawed frog peptide, esculentinis-1, and caerin.

[0515] The term “endosomal lytic ligand,” as used herein, refers to a molecule having endosomal lytic properties. Endosomal lytic ligands facilitate the lysis of the compositions or components thereof of this disclosure, and / or their transport from cellular compartments such as endosomes, lysosomes, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other intracellular vesicles into the cytoplasm of a cell. Some exemplary endosomal soluble ligands includ...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of activin A receptor type 1C (ACVR1C) in cells, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2 by three or fewer nucleotides, and one or more C ２２ A dsRNA agent in which a hydrocarbon chain is conjugated to a sense chain.

2. The dsRNA agent according to claim 1, wherein the antisense strand comprises at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the antisense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12.

3. The dsRNA agent according to claim 1, wherein the antisense strand comprises at least 15 consecutive nucleotides that differ by two or fewer nucleotides from any one of the nucleotide sequences of the antisense strand in any one of Tables 2-5, 7, 8, 11, and 12.

4. The dsRNA agent according to claim 1, wherein the antisense strand comprises at least 15 consecutive nucleotides that differ by 11 or fewer nucleotides from any one of the antisense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12.

5. The dsRNA agent according to claim 1, wherein the antisense strand comprises at least 15 consecutive nucleotides from any one of the nucleotide sequences of the antisense strand in any one of Tables 2-5, 7, 8, 11, and 12.

6. The dsRNA agent according to claim 1, wherein the sense strand comprises at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the sense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12, and the antisense strand comprises at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the antisense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12.

7. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of activin A receptor type 1C (ACVR1C) in cells, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprises a region complementary to the mRNA encoding ACVR1C, and the complementary region comprises at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the antisense nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12.

8. The dsRNA agent according to claim 7, wherein the antisense strand comprises at least 15 consecutive nucleotides that differ by two or fewer nucleotides from any one of the nucleotide sequences of the antisense strand in any one of Tables 2-5, 7, 8, 11, and 12.

9. The dsRNA agent according to claim 7, wherein the antisense strand comprises at least 15 consecutive nucleotides that differ by 11 or fewer nucleotides from any one of the nucleotide sequences of the antisense strand in any one of Tables 2-5, 7, 8, 11, and 12.

10. The dsRNA agent according to claim 7, wherein the antisense strand comprises a nucleotide sequence selected from the group consisting of one of the nucleotide sequences of antisense strands in any one of Tables 2-5, 7, 8, 11, and 12.

11. The dsRNA agent according to claim 7, wherein the sense strand comprises at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the sense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12, and the antisense strand comprises at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the antisense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12.

12. The dsRNA agent according to claim 7, wherein the antisense strand comprises at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the nucleotide sequences of a double-stranded antisense strand selected from the group consisting of AD-2640052, AD-2640053, AD-2640060, AD-2509191, and AD-2508176.

13. The dsRNA agent according to claim 7, wherein the sense strand and antisense strand each contain at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the nucleotide sequences of a double-stranded antisense strand selected from the group consisting of AD-2640052, AD-2640053, AD-2640060, AD-2509191, or AD-2508176.

14. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of activin A receptor type 1C (ACVR1C) in cells, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, and the antisense strand is (a) Sequence ID 14921 5'-UUAATUAUCAUCAUUAGCUUUG-3'; (b) Sequence ID 14922 5'-UUUGACACAAAGUUGAGAUAUAG-3'; (c) Sequence ID 14925 5'-UAACAUAUUAUCAUCAUUAGGC-3'; (d) 5'-UAGGACUCAAAAGAUAUUCACAUU-3' of SEQ ID NO: 9291; and (e) Sequence ID 8956 5'-UCACACAAAAAGACAUCACACUU-3' A dsRNA agent comprising at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the nucleotide sequences of an antisense strand selected from the group consisting of the above.

15. The antisense chain, (a) Sequence ID 14921 5'-UUAATUAUCAUCAUUAGCUUUG-3'; (b) Sequence ID 14922 5'-UUUGACACAAAGUUGAGAUAUAG-3'; (c) Sequence ID 14925 5'-UAACAUAUUAUCAUCAUUAGGC-3'; (d) 5'-UAGGACUCAAAAGAUAUUCACAUU-3' of SEQ ID NO: 9291; and (e) Sequence ID 8956 5'-UCACACAAAAAGACAUCACACUU-3' The dsRNA agent according to claim 14, comprising at least 15 consecutive nucleotides that differ by two or fewer nucleotides from any one of the nucleotide sequences of an antisense strand selected from the group consisting of the above.

16. The antisense chain, (a) Sequence ID 14921 5'-UUAATUAUCAUCAUUAGCUUUG-3'; (b) Sequence ID 14922 5'-UUUGACACAAAGUUGAGAUAUAG-3'; (c) Sequence ID 14925 5'-UAACAUAUUAUCAUCAUUAGGC-3'; (d) 5'-UAGGACUCAAAAGAUAUUCACAUU-3' of SEQ ID NO: 9291; and (e) Sequence ID 8956 5'-UCACACAAAAAGACAUCACACUU-3' The dsRNA agent according to claim 14, comprising at least 15 consecutive nucleotides that differ by one or less nucleotides from any one of the nucleotide sequences of an antisense strand selected from the group consisting of the above.

17. The antisense chain, (a) Sequence ID 14921 5'-UUAATUAUCAUCAUUAGCUUUG-3'; (b) Sequence ID 14922 5'-UUUGACACAAAGUUGAGAUAUAG-3'; (c) Sequence ID 14925 5'-UAACAUAUUAUCAUCAUUAGGC-3'; (d) 5'-UAGGACUCAAAAGAUAUUCACAUU-3' of SEQ ID NO: 9291; and (e) Sequence ID 8956 5'-UCACACAAAAAGACAUCACACUU-3' The dsRNA agent according to claim 14, comprising at least 15 consecutive nucleotides from any one of the nucleotide sequences of an antisense strand selected from the group consisting of the following.

18. The dsRNA agent according to claim 14, wherein the sense strand comprises at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the nucleotide sequences of the sense and antisense strands selected from the group consisting of (a) to (e) below, and the antisense strand comprises at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the nucleotide sequences of the sense and antisense strands selected from the group consisting of (a) to (e) below: (a) 5'-AAGCCUAAUGAUGAUAAUUAA-3' of SEQ ID NO: 14909 and 5'-UUATUAUCAUCAUUAGCUUUG-3' of SEQ ID NO: 14921; (b) 5'-AUAUCUCAACUUUGUGUCAAA-3' of SEQ ID NO: 14910 and 5'-UUUGACACAAAGUUGAGAUAUAG-3' of SEQ ID NO: 14922; (c) 5'-CUAAUGAUGAUAAUUAUGUUA-3' of Sequence ID No. 14913 and 5'-UACAUAAUUAUCAUCAUAGGC-3' of Sequence ID No. 14925; (d) 5'-UGUGAAUAUUCUUGAGUCCUA-3' of SEQ ID NO: 7805 and 5'-UAGGACUCAAAAGAAUUCACAUU-3' of SEQ ID NO: 9291; and (e) 5'-GUGUGUAUGUCUUUUGUGUGA-3' of sequence number 7470 and 5'-UCACACAAAGACAUACACACU-3' of sequence number 8956.

19. The dsRNA agent according to claim 14, wherein the sense strand comprises at least 15 consecutive nucleotides that differ by two or fewer nucleotides from any one of the nucleotide sequences of the sense and antisense strands selected from the group consisting of (a) to (e) below, and the antisense strand comprises at least 15 consecutive nucleotides that differ by two or fewer nucleotides from any one of the nucleotide sequences of the sense and antisense strands selected from the group consisting of (a) to (e) below: (a) 5'-AAGCCUAAUGAUGAUAAUUAA-3' of SEQ ID NO: 14909 and 5'-UUATUAUCAUCAUUAGCUUUG-3' of SEQ ID NO: 14921; (b) 5'-AUAUCUCAACUUUGUGUCAAA-3' of SEQ ID NO: 14910 and 5'-UUUGACACAAAGUUGAGAUAUAG-3' of SEQ ID NO: 14922; (c) 5'-CUAAUGAUGAUAAUUAUGUUA-3' of Sequence ID No. 14913 and 5'-UACAUAAUUAUCAUCAUAGGC-3' of Sequence ID No. 14925; (d) 5'-UGUGAAUAUUCUUGAGUCCUA-3' of SEQ ID NO: 7805 and 5'-UAGGACUCAAAAGAAUUCACAUU-3' of SEQ ID NO: 9291; and (e) 5'-GUGUGUAUGUCUUUUGUGUGA-3' of sequence number 7470 and 5'-UCACACAAAGACAUACACACU-3' of sequence number 8956.

20. The dsRNA agent according to claim 14, wherein the sense strand comprises at least 15 consecutive nucleotides that differ by one or less nucleotides from any one of the nucleotide sequences of the sense and antisense strands selected from the group consisting of (a) to (e) below, and the antisense strand comprises at least 15 consecutive nucleotides that differ by one or less nucleotides from any one of the nucleotide sequences of the sense and antisense strands selected from the group consisting of (a) to (e) below: (a) 5'-AAGCCUAAUGAUGAUAAUUAA-3' of SEQ ID NO: 14909 and 5'-UUATUAUCAUCAUUAGCUUUG-3' of SEQ ID NO: 14921; (b) 5'-AUAUCUCAACUUUGUGUCAAA-3' of SEQ ID NO: 14910 and 5'-UUUGACACAAAGUUGAGAUAUAG-3' of SEQ ID NO: 14922; (c) 5'-CUAAUGAUGAUAAUUAUGUUA-3' of Sequence ID No. 14913 and 5'-UACAUAAUUAUCAUCAUAGGC-3' of Sequence ID No. 14925; (d) 5'-UGUGAAUAUUCUUGAGUCCUA-3' of SEQ ID NO: 7805 and 5'-UAGGACUCAAAAGAAUUCACAUU-3' of SEQ ID NO: 9291; and (e) 5'-GUGUGUAUGUCUUUUGUGUGA-3' of sequence number 7470 and 5'-UCACACAAAGACAUACACACU-3' of sequence number 8956.

21. The dsRNA agent according to claim 14, wherein the sense strand comprises at least 15 consecutive nucleotides from any one of the nucleotide sequences of the sense and antisense strands selected from the group consisting of (a) to (e) below, and the antisense strand comprises at least 15 consecutive nucleotides from any one of the nucleotide sequences of the sense and antisense strands selected from the group consisting of (a) to (e) below: (a) 5'-AAGCCUAAUGAUGAUAAUUAA-3' of SEQ ID NO: 14909 and 5'-UUATUAUCAUCAUUAGCUUUG-3' of SEQ ID NO: 14921; (b) 5'-AUAUCUCAACUUUGUGUCAAA-3' of SEQ ID NO: 14910 and 5'-UUUGACACAAAGUUGAGAUAUAG-3' of SEQ ID NO: 14922; (c) 5'-CUAAUGAUGAUAAUUAUGUUA-3' of Sequence ID No. 14913 and 5'-UACAUAAUUAUCAUCAUAGGC-3' of Sequence ID No. 14925; (d) 5'-UGUGAAUAUUCUUGAGUCCUA-3' of SEQ ID NO: 7805 and 5'-UAGGACUCAAAAGAAUUCACAUU-3' of SEQ ID NO: 9291; and (e) 5'-GUGUGUAUGUCUUUUGUGUGA-3' of sequence number 7470 and 5'-UCACACAAAGACAUACACACU-3' of sequence number 8956.

22. A dsRNA agent according to any one of claims 1 to 21, wherein at least one nucleotide of the dsRNA agent comprises a nucleotide modification.

23. A dsRNA agent according to any one of claims 1 to 22, wherein substantially all of the nucleotides of the sense strand contain nucleotide modifications; substantially all of the nucleotides of the antisense strand contain nucleotide modifications; or substantially all of the nucleotides of both the sense strand and the antisense strand contain nucleotide modifications.

24. A dsRNA agent according to any one of claims 1 to 23, wherein all nucleotides of the sense strand contain nucleotide modifications; all nucleotides of the antisense strand contain nucleotide modifications; or all nucleotides of both the sense strand and the antisense strand contain nucleotide modifications.

25. At least one of the nucleotide modifications is a deoxynucleotide modification, a 3'-terminal deoxythymidine (dT) nucleotide modification, a 2'-O-methyl nucleotide modification, a 2'-fluoronucleotide modification, a 2'-deoxynucleotide modification, a locked nucleotide modification, an unlocked nucleotide modification, a conformation-fixed nucleotide modification, a restricted ethyl nucleotide modification, a debasalized nucleotide modification, a 2'-amino nucleotide modification, a 2'-O-allyl nucleotide modification, a 2'-C-alkyl nucleotide modification, a 2'-hydroxyl nucleotide modification, a 2'-methoxyethyl nucleotide modification, a 2'-O-alkyl nucleotide modification, a morpholino nucleotide modification, or a phosphoramide modification. A dsRNA agent according to any one of claims 22 to 24, selected from the group consisting of modifications, nucleotide modifications including non-natural bases, tetrahydropyran nucleotide modifications, 1,5-anhydrohexitol nucleotide modifications, cyclohexenyl nucleotide modifications, nucleotides including phosphorothioate group modifications, nucleotides including methylphosphonate group modifications, nucleotides including 5'-phosphate modifications, nucleotides including 5'-phosphate mimetic modifications, thermally destabilized nucleotide modifications, glycol-modified nucleotide (GNA) modifications, nucleotides including 2'-phosphate modifications, and 2-O-(N-methylacetamide) nucleotide modifications; and combinations thereof.

26. The dsRNA agent according to any one of claims 22 to 24, wherein at least one of the nucleotide modifications 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.

27. The dsRNA agent according to any one of claims 22 to 24, wherein at least one of the nucleotide modifications is selected from the group consisting of deoxynucleotide modifications, 2'-O-methylnucleotide modifications, 2'-fluoronucleotide modifications, 2'-deoxynucleotide modifications, glycol nucleotide (GNA) modifications, nucleotides comprising 2'-phosphate modifications, nucleotides comprising phosphorothioate group modifications, and vinyl phosphonate nucleotide modifications; and combinations thereof.

28. The dsRNA agent according to any one of claims 22 to 24, wherein at least one of the nucleotide modifications is a nucleotide modified by a thermally destabilizing nucleotide modification.

29. The dsRNA agent according to claim 28, wherein the thermally destabilizing nucleotide modification is selected from the group consisting of debase modification; mismatch with opposing nucleotides in the double helix; destabilizing sugar modification, 2'-deoxy modification, acyclic nucleotide, unlocked nucleic acid (UNA), and glycerol nucleic acid (GNA).

30. A dsRNA agent according to any one of claims 22 to 24, wherein the antisense strand includes 2'-fluoronucleotide modifications at positions 2, 14, and 16 counting from the 5' end.

31. A dsRNA agent according to any one of claims 22 to 24, wherein the antisense strand includes 2'-fluoronucleotide modifications at positions 2, 6, 14, and 16 counting from the 5' end.

32. A dsRNA agent according to any one of claims 22 to 24, wherein the antisense strand contains a 2'-fluoronucleotide modification at positions 9 to 11, counting from the 5' end.

33. A dsRNA agent according to any one of claims 1 to 32, further comprising a phosphate or phosphate mimetic at the 5' end of the antisense strand.

34. The dsRNA agent according to claim 33, wherein the phosphate mimetic is 5'-vinylphosphonate (VP).

35. The dsRNA agent according to any one of claims 1 to 34, wherein the 3' end of the sense strand is protected by a terminal cap which is a cyclic group having an amine, and the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinil, pyrazolidinyl, imidazolinil, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanil, oxazolidinyl, isoxazolidinyl, morpholinil, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuranil, and dekalinil.

36. A dsRNA agent according to any one of claims 1 to 35, wherein the double-stranded region is a pair of nucleotides with a length of 19 to 30 nucleotides.

37. The dsRNA agent according to claim 36, wherein the double-stranded region is a pair of nucleotides with a length of 19 to 25 nucleotides.

38. The dsRNA agent according to claim 36, wherein the double-stranded region has a length of 19 to 23 nucleotide pairs.

39. The dsRNA agent according to claim 36, wherein the double-stranded region has a length of 23 to 27 nucleotide pairs.

40. The dsRNA agent according to claim 36, wherein the double-stranded region is a pair of nucleotides with a length of 21 to 23 nucleotides.

41. The dsRNA agent according to any one of claims 1 to 40, wherein each chain independently has a length of 30 or less nucleotides.

42. A dsRNA agent according to any one of claims 1 to 41, wherein the sense strand has a length of 21 nucleotides and the antisense strand has a length of 23 nucleotides.

43. The dsRNA agent according to any one of claims 1 to 42, wherein the complementary region has a length of at least 17 nucleotides.

44. The dsRNA agent according to any one of claims 1 to 43, wherein the complementary region has a length of 19 to 23 nucleotides.

45. The dsRNA agent according to any one of claims 1 to 44, wherein the complementary region has a length of 19 nucleotides.

46. A dsRNA agent according to any one of claims 1 to 45, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

47. A dsRNA agent according to any one of claims 1 to 46, wherein at least one strand comprises a 3' overhang of at least two nucleotides.

48. One or more C ２２ A dsRNA agent according to any one of claims 7 to 47, wherein a hydrocarbon chain is conjugated at one or more internal positions in at least one of the chains.

49. C ２２ A dsRNA agent according to any one of claims 1 to 6 and 48, wherein the hydrocarbon chain is saturated or unsaturated.

50. C ２２ The dsRNA agent according to any one of claims 1 to 6, 48, and 49, wherein the hydrocarbon chain is linear or branched.

51. The dsRNA agent according to any one of claims 48 to 50, wherein the internal positions include all positions except two or three terminal positions from each end of at least one strand.

52. The dsRNA agent according to claim 51, wherein the internal position excludes the region of the sense strand cleavage site.

53. The dsRNA agent according to claim 51, wherein the internal position excludes positions 9-12 or 11-13 counting from the 5' end of the sense strand.

54. The dsRNA agent according to claim 51, wherein the internal position excludes the region of the antisense strand cleavage site.

55. The dsRNA agent according to claim 54, wherein the internal position excludes positions 12 to 14, counting from the 5' end of the antisense strand.

56. One or more C ２２ The dsRNA agent according to any one of claims 48 to 55, wherein the hydrocarbon chain is conjugated to one or more of the following internal positions: positions 4–8 and 13–18 in the sense strand and positions 6–10 and 15–18 in the antisense strand, counting from the 5' end of each chain.

57. One or more C ２２ The dsRNA agent according to claim 56, wherein the hydrocarbon chain is conjugated to one or more of the following internal positions: positions 5, 6, 7, 15, 16, and 17 in the sense strand, and positions 15 and 17 in the antisense strand, counting from the 5' end of each chain.

58. One or more C ２２ The dsRNA agent according to claim 57, wherein the hydrocarbon chain is conjugated at the 6th position on the sense strand, counting from the 5' end of the sense strand.

59. One or more C ２２ The dsRNA agent according to claim 57, wherein the hydrocarbon chain is conjugated at the 16th position on the sense strand, counting from the 5' end of the sense strand.

60. one or more C ２２ The dsRNA agent according to any one of claims 1 to 6 and 48 to 59, wherein the hydrocarbon chain is an aliphatic, alicyclic, or polyalicyclic compound.

61. One or more C ２２ The dsRNA agent according to claim 60, wherein the hydrocarbon chain contains a functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

62. One or more C ２２ The hydrocarbon chain is C ２２ A dsRNA agent according to any one of claims 1 to 6 and 48 to 61, wherein the agent is an acid.

63. C ２２ The dsRNA agent according to claim 62, wherein the acid is selected from the group consisting of docosanic 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.

64. One or more C ２２ The hydrocarbon chain is C ２２ A dsRNA agent according to any one of claims 1 to 6 and 48 to 61, wherein the alcohol is used.

65. C ２２ The dsRNA agent according to claim 64, wherein the alcohol is selected from the group consisting of 1-docosanol, 6-octyltetradecane-1-ol, 10-hexylhexadecane-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.

66. One or more C ２２ The hydrocarbon chain is C ２２ A dsRNA agent according to any one of claims 1 to 6 and 48 to 61, wherein the agent is an amide.

67. C ２２ The dsRNA agent according to claim 66, wherein 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-docosenamid, docosa-11-enamide, (4E,13E)-docosa-4,13-dienamide, and (5E,13E)-docosa-5,13-dienamide.

68. One or more C ２２ A dsRNA agent according to any one of claims 1 to 6 and 48 to 67, wherein a hydrocarbon chain is conjugated via a carrier that replaces one or more nucleotides at an internal position.

69. The dsRNA agent according to claim 68, wherein the carrier is a cyclic group selected from the group consisting of pyrrolidinil, pyrazolinil, pyrazolidinil, imidazolinil, imidazolidinil, piperidinil, piperazinil, [1,3]dioxolanil, oxazolidinil, isoxazolidinil, morpholinil, thiazolidinil, isothiazolidinil, quinoxalinil, pyridadinil, tetrahydrofuranil, and dekalinil; or an acyclic portion based on a selinol backbone or a diethanolamine backbone.

70. One or more C ２２ A dsRNA agent according to any one of claims 1 to 6 and 48 to 69, wherein a hydrocarbon chain is conjugated to a dsRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, click reaction product, or carbamate.

71. One or more C ２２ The dsRNA agent according to any one of claims 1 to 6 and 48 to 70, wherein a hydrocarbon chain is conjugated to the dsRNA agent via a linker or carrier, or via phosphate linkage between nucleotides.

72. One or more C ２２ A dsRNA agent according to any one of claims 1 to 6 and 48 to 71, wherein the hydrocarbon chain is conjugated to a phosphate linkage between nucleic acid bases, sugar moieties, or nucleosides.

73. The dsRNA agent according to any one of claims 1 to 72, further comprising an internucleotide linkage of at least one phosphorothioate or methylphosphonate.

74. The dsRNA agent according to claim 73, wherein the internucleotide linkage of phosphorothioate or methylphosphonate is located at the 3' end of one strand.

75. The dsRNA agent according to claim 74, wherein the chain is an antisense chain.

76. The dsRNA agent according to claim 74, wherein the strand is a sense strand.

77. The dsRNA agent according to claim 73, wherein the internucleotide linkage of phosphorothioate or methylphosphonate is located at the 5' end of one strand.

78. The dsRNA agent according to claim 77, wherein the chain is an antisense chain.

79. The dsRNA agent according to claim 77, wherein the strand is a sense strand.

80. The dsRNA agent according to claim 73, wherein the internucleotide linkages of phosphorothioate or methylphosphonate are located at the 5' and 3' ends of one strand.

81. The dsRNA agent according to claim 80, wherein the strand is an antisense strand.

82. A dsRNA agent according to any one of claims 1 to 81, wherein the base pair at position 1 of the 5' end of the double-stranded antisense strand is an AU base pair.

83. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of activin A receptor type 1C (ACVR1C) in cells, wherein the dsRNA agent comprises a sense strand having 18 to 23 nucleotides and at least 3 2'-F modifications, and an antisense strand having 18 to 23 nucleotides and 4 2'-F modifications, and the dsRNA agent has a lipophilic moiety conjugated to the sense strand.

84. The dsRNA agent according to claim 83, wherein the antisense strand contains 2'-F modifications at positions 2, 14, and 16, counting from the 5' end.

85. The dsRNA agent according to claim 83 or 84, wherein the sense strand contains a 2'-F modification at positions 9 to 11, counting from the 5' end.

86. The dsRNA agent according to any one of claims 83 to 85, wherein the lipophilic portion is a C22 hydrocarbon chain connected to the 6th or 16th position of the sense strand, counting from the 5' end.

87. The dsRNA agent according to any one of claims 83 to 86, wherein the dsRNA agent is selected from the group consisting of AD-2509512, AD-2640052, AD-2640053, AD-2640060, AD-2509191, AD-2509494, AD-2509477, and AD-2508176.

88. Cells containing the dsRNA agent according to any one of claims 1 to 87.

89. A pharmaceutical composition for inhibiting the expression of the activin A receptor type 1C (ACVR1C) gene, comprising a dsRNA agent according to any one of claims 1 to 87 and a pharmaceutically acceptable carrier.

90. The pharmaceutical composition according to claim 89, wherein the dsRNA agent is in an unbuffered solution.

91. The pharmaceutical composition according to claim 90, wherein the unbuffered solution is physiological saline or water.

92. The pharmaceutical composition according to claim 89, wherein the dsRNA agent is in a buffer solution.

93. The pharmaceutical composition according to claim 92, wherein the buffer solution comprises an acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof.

94. The pharmaceutical composition according to claim 92, wherein the buffer solution is phosphate-buffered saline (PBS).

95. A method for inhibiting the expression of the activin A receptor type 1C (ACVR1C) gene in cells, comprising contacting cells with a dsRNA agent according to any one of claims 1 to 87 or a pharmaceutical composition according to any one of claims 89 to 94, thereby inhibiting the expression of the ACVR1C gene in cells.

96. The method according to claim 95, wherein the cells are adipocytes.

97. The method according to claim 95 or 96, wherein the cells are within the scope.

98. The method according to claim 97, wherein the subject is a human.

99. The method according to claim 97 or 98, wherein the subject has ACVR1C-related disorder.

100. The method according to claim 99, wherein the ACVR1C-related disorder is a metabolic disorder.

101. The method according to claim 100, wherein the metabolic disorder is a metabolic syndrome.

102. The method according to claim 100, wherein the metabolic disorder is a cardiovascular disease.

103. The method according to claim 100, wherein the metabolic disorder is hypertension.

104. The method according to any one of claims 95 to 103, wherein contact with a dsRNA agent inhibits the expression of the ACVR1C gene by at least 50%, 60%, 70%, 80%, 90%, or 95%.

105. The method according to any one of claims 95 to 104, wherein inhibiting the expression of the ACVR1C gene reduces the ALK7 protein level in the serum of the subject by at least 50%, 60%, 70%, 80%, 90%, or 95%.

106. A method for treating a subject having a disorder in which a reduction in activin A receptor type 1C (ACVR1C) expression is expected to be beneficial, comprising administering a therapeutically effective amount to the subject a dsRNA agent according to any one of claims 1 to 87 or a pharmaceutical composition according to any one of claims 89 to 94, thereby treating the subject having a disorder in which a reduction in ACVR1C expression is expected to be beneficial.

107. A method for preventing at least one symptom in a subject having a disorder in which a reduction in activin A receptor type 1C (ACVR1C) expression is expected to be beneficial, comprising administering a preventive amount to the subject a dsRNA agent according to any one of claims 1 to 87 or a pharmaceutical composition according to any one of claims 89 to 94, thereby preventing at least one symptom in the subject having a disorder in which a reduction in ACVR1C expression is expected to be beneficial.

108. The method according to claim 106 or 107, wherein the disorder is an ACVR1C-related disorder.

109. The method according to claim 108, wherein the ACVR1C-related disorder is a metabolic disorder.

110. The method according to claim 109, wherein the metabolic disorder is a metabolic syndrome.

111. The method according to claim 109, wherein the metabolic disorder is a cardiovascular disease.

112. The method according to claim 109, wherein the metabolic disorder is hypertension.

113. The method according to any one of claims 106 to 112, wherein the subject is a human.

114. The method according to any one of claims 106 to 113, wherein administration of a dsRNA agent to the subject results in a reduction in the accumulation of ALK7 protein in the subject.

115. The method according to any one of claims 106 to 114, wherein the dsRNA agent is administered subcutaneously to the subject.

116. The method according to any one of claims 106 to 115, further comprising determining the level of ACVR1C in a sample from a subject.

117. The method according to any one of claims 106 to 116, further comprising administering an additional therapeutic agent to treat ACVR1C-related disorders.

118. The method according to claim 117, wherein the additional therapeutic agent is selected from the group consisting of insulin, glucagon-like peptide 1 agonists, sulfonylurea, seglycinide, biguanide, thiazolidinedione, alpha-glucosidase inhibitors, SGLT2 inhibitors, DPP-4 inhibitors, HMG-CoA reductase inhibitors, statins, and any combination thereof.

119. A kit comprising a dsRNA agent according to any one of claims 1 to 87 or a pharmaceutical composition according to any one of claims 89 to 94.

120. A vial containing a dsRNA agent according to any one of claims 1 to 87 or a pharmaceutical composition according to any one of claims 89 to 94.

121. A syringe comprising a dsRNA agent according to any one of claims 1 to 87 or a pharmaceutical composition according to any one of claims 89 to 94.

122. An RNA-induced silencing complex (RISC) comprising the antisense strand of any of the dsRNA agents described in claims 1 to 87.

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