G protein-coupled receptor 75 (GPR75) iRNA composition and method of use thereof

Abstract

This invention provides RNAi agents, such as dsRNA agents, that target the G protein-coupled receptor 75 (GPR75) gene. It also provides methods for using such RNAi agents to inhibit the expression of the GPR75 gene in a subject, and methods for treating or preventing GPR75-related diseases such as weight disorders, e.g., obesity. [Solution] A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of the GPR75 gene in cells is provided, comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises a region complementary to a portion of the mRNA encoding the GPR75 gene, each strand is independently 14 to 30 nucleotides long, and the sense strand or antisense strand is conjugated to one or more lipophilic portions.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Application No. 63 / 087,342, filed on 5 October 2020, and U.S. Provisional Application No. 63 / 216,629, filed on 30 June 2021. The entire contents of each of the aforementioned applications are incorporated herein by reference. [Background technology]

[0002] G protein-coupled receptor 75 (GPR75) is a member of the G protein-coupled receptor family. It contains many of the characteristics of GPCRs, namely, a seven-transmembrane domain, an N-terminal N-glycosylation site, and several serine and threonine phosphorylation sites at the C-terminus. Amino acid sequence analysis has shown that GPR75 is most closely related to the putative Caenorhabditis elegans neuropeptide Y receptor (24% homology), rat galanin receptor type 3 (25% homology), and porcine growth hormone secretagogue receptor type 1b (25% homology) [Tarttelin et al. (1999) Biochem Biophys Res Commun. 260:174-180]. GPR75 is classified as a Gq-coupled class A orphan receptor, whose activation is associated with increased intracellular calcium and IP-1 accumulation. GPR75 is expressed in many tissues, and in the brain, it is expressed in the neocortex, entorhinal cortex, hippocampus, thalamus, and hypothalamus.

[0003] 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450-derived eicosanoid, has been shown to bind to and activate the GPR75 receptor. 20-HETE is an omega-hydroxylated metabolite of arachidonic acid produced by enzymes of the cytochrome P450 (CYP) 4A and 4F families. Clinical trials have shown that urinary and / or plasma levels of 20-HETE are elevated in obese and diabetic individuals, demonstrating that 20-HETE stimulates adipogenesis, contributes to the development of diabetes, induces hyperglycemia, and interferes with the cellular action of insulin. Furthermore, mice overexpressing Cyp4a12-20-HETE synthase rapidly develop obesity, hyperglycemia, hyperinsulinemia, and impaired glucose tolerance when fed a high-fat diet. These animals also exhibited insulin resistance in skeletal muscle, liver, and adipose tissue, evident in decreased tyrosine phosphorylation of insulin receptors and insulin receptor substrates. Furthermore, 20-HETE has been shown to inhibit insulin signaling in a GPR75-dependent manner [Gilani, et al. (2019) FASEB J .33(S1): 514.8; Gilani, et al. (2018) Am J Physiol Regul Integr Comp Physiol 315: R934-R944].

[0004] Weight disorders, such as obesity, are a growing health problem in many countries. Weight disorders, including obesity, increase the risk of health problems such as insulin resistance, type 2 diabetes, heart disease, osteoarthritis, sleep apnea, and certain forms of cancer. Losing excess weight can significantly reduce the risk of these health problems. The main treatments for weight disorders, including obesity, are diet and exercise, followed by weight-loss drugs and surgery. Several FDA-approved weight-loss drugs, such as orlistat [Alli®] and sibutramine [Meridia®], are on the market, but none have met the weight-loss targets set by the FDA. Furthermore, several weight-loss drug candidates, also known as appetite suppressants, have been discontinued or withdrawn at various stages of development due to their serious side effects. In addition, while many methods exist to reduce initial weight, maintaining that weight loss in the long term is difficult. Many people who successfully achieve initial weight loss subsequently gain weight again. Furthermore, morbidly obese individuals may require medication to maintain a healthy weight in the long term, even after successful weight-loss surgery. However, currently, there are no weight loss / maintenance drugs on the market.

[0005] Therefore, there is an unmet need for effective treatments for obesity, such as drugs that possess high biological activity and in vivo stability, can effectively inhibit the expression of the target GPR75 gene, and can selectively and efficiently silence the GPR75 gene using the cell's own RNAi mechanism. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] This disclosure provides RNAi compositions that perform RNA-induced silencing complex (RISC) mediated cleavage of the RNA transcript of the gene encoding G protein-coupled receptor 75 (GPR75). The GPR75 gene may be located inside cells, for example, in cells within subjects such as humans. This disclosure also provides methods of using the RNAi compositions of this disclosure to inhibit the expression of the GPR75 gene, or to treat subjects who would benefit from inhibiting or reducing the expression of the GPR75 gene, for example, subjects with GPR75-related disorders, for example, subjects with weight disorders, for example, subjects with obesity, or subjects at risk of developing weight disorders. [Means for solving the problem]

[0007] Accordingly, in one embodiment, the present disclosure provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of G protein-coupled receptor 75 (GPR75), comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence containing at least 15 consecutive nucleotides, including 0, 1, 2, or 3 mismatches in a portion of any one of the nucleotide sequences of SEQ ID NOs: 1 to 4, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of any one of the nucleotide sequences of SEQ ID NOs: 1 to 4, and the antisense strand comprises a nucleotide sequence containing at least 15 consecutive nucleotides, including 0, 1, 2, or 3 mismatches in a corresponding portion of any one of the nucleotide sequences of SEQ ID NOs: 5 to 8, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of any one of the nucleotide sequences of SEQ ID NOs: 5 to 8, and the sense strand or antisense strand is conjugated to one or more lipophilic portions.

[0008] In one embodiment, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of the GPR75 gene in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a region complementary to a portion of the mRNA encoding the GPR75 gene (any one of SEQ ID NOs: 1 to 4), each strand independently being 14 to 30 nucleotides long, and the sense strand or antisense strand being conjugated to one or more lipophilic portions.

[0009] In yet another aspect, the present invention provides a double-stranded RNAi agent for inhibiting the expression of the GPR75 gene in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises at least 15 consecutive nucleotides that differ by 3 nucleotides or less from any one of the antisense nucleotide sequences in any one of Tables 2, 3, 5, and 6, each strand independently being 14 to 30 nucleotides long, and the sense strand or antisense strand being conjugated to one or more lipophilic moieties.

[0010] In one embodiment, the sense chain or antisense chain is a sense chain or antisense chain selected from the group consisting of any one of the sense chains and antisense chains in Tables 2, 3, 5, and 6.

[0011] In another aspect, the present invention relates to a double-stranded RNAi agent for inhibiting the expression of G protein-coupled receptor 75 (GPR75) in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotides of SEQ ID NO: 1, 38-60; 50-72; 148-181; 153-181; 153-175; 159-181; 228-250; 240-262; 341-363; 341-368; 346-368; 369-396; 369-391; 374-396; 388-410; 414-436; 424-461; 424-446; 42 4~451;434~456;439~461;429~451;457~504;462~504;462~491;482~504;469~491;457~479;462~584;475~497;469~491;509~537;509~531;515~537 ;544~576;544~566;549~571;580~607;580~602;585~607;595~617;615~647;615~637;620~642;620~647;625~647;773~806;773~795;773~795;778~ 800;784~806;837~872;837~859;843~872;843~865;850~872;860~882;889~911;900~936;900~922;908~936;908~930;914~936;938~990;938~960;9 43~965;968~990;1060~1101;1060~1082;1066~1088;1073~1095;1079~1101;1097~1119;1238~1260;1268~1290;1284~1393;1284~1306;1292~1393; 1292~1314;1292~1383;1292~1314;1301~1323;1307~1383;1307~1342;1307~1329;1313~1335;1371~1393;1351~1373;1320~1342;1336~1358;1345~ 1367;1351~1373;1361~1383;1366~1388;1393~1415;1422~1463;1422~1444;1441~1463;1487~1526;1487~1509;1493~1526;1493~1515;1498~1520;1504~1526;1515~1571;1515~1557;1515~1543;1515~1537;1521~1543;1530~1552;1535~1557;1540~1562;1549~1571;1559~1586;1559~1581;1564~1586;1583~1629;1583~1605;1588~1610;1595~1617;1600~1629;1600~1622;1607~1629;1624~1646;1635~1657;1672~1721;1672~1710;1677~1699;1699~1721;1672~1699;1688~1710;1672~1694;1683~1705;1693~1714;1732~1754;1744~1798;1751~1773;1758~1780;1767~1789;1776~1798;1790~1818;1790~1812;1796~1818;1808~1856;1808~1848;1808~1836;1808~1830;1826~1848;1814~1836;1819~1841;1834~1856;1877~2082;1877~1899;1882~2082;1882~1925;1882~1963;1882~1904;1887~1693;1887~1909;1898~1920;1903~1925;1908~1930;1913~1935;1913~1950;1921~1950;1921~1943;1928~1950;1933~1955;1941~1963;1946~1968;1953~1985;1953~2082;1953~1975;1938~1985;1958~1980;1963~1985;1968~1990;1974~1996;1974~2065;1974~2082;1974~2002;1980~2002;1985~2007;1990~2012;1990~2033;1999~2021;2005~2033;2005~2027;2011~2033;2017~2039;2025~2055;2025~2047;2033~2055;2038~2060;2043~2065;2033~2055;2048~2070;2054~2082;2054~2076;The present invention provides a double-stranded RNAi agent comprising at least 15 consecutive nucleotides that differ by 3 nucleotides or less from any one of the nucleotide sequences 2060-2082, an antisense strand comprising at least 15 consecutive nucleotides derived from the corresponding nucleotide sequence of SEQ ID NO: 2, and the sense strand or antisense strand being conjugated to one or more lipophilic moieties.

[0012] In one embodiment, both the sense chain and the antisense chain are conjugated to one or more lipophilic moieties.

[0013] In one embodiment, the lipophilicity of the lipophilic portion, as measured by logKow, is greater than 0.

[0014] In one embodiment, the hydrophobicity of a double-stranded RNAi agent, as measured by the unbound fraction in a plasma protein binding assay of the double-stranded RNAi agent, is greater than 0.2.

[0015] In one embodiment, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

[0016] In one embodiment, the dsRNA agent comprises at least one modified nucleotide.

[0017] In some embodiments, substantially all nucleotides in the antisense strand are modified nucleotides.

[0018] In another embodiment, all nucleotides of the sense strand and all nucleotides of the antisense strand include modifications.

[0019] In one embodiment, at least one of the modified nucleotides is a deoxy-nucleotide, a 3'-terminal deoxythymidine (dT) nucleotide, a 2'-O-methyl-modified nucleotide, a 2'-fluoro-modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformation-restricted nucleotide, a restricted ethyl nucleotide, a debasalized nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-hydroxyl-modified nucleotide, a 2'-methoxyethyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramide, a nucleotide containing a non-natural base, a tetrahydropyran-modified nucleotide, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing a 5'-phosphorothioate group, nucleotides containing a 5'-methylphosphonate group, nucleotides containing a 5'-phosphate or 5'-phosphate mimetic, nucleotides containing vinylphosphonate, nucleotides containing adenosine-glycol nucleic acid (GNA), nucleotides containing thymidine-glycol nucleic acid (GNA) S isomers, nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3'-phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, 2'-O-hexadecyl nucleotides, nucleotides containing 2'-phosphate, cytidine-2'-phosphate nucleotides, guanosine-2' -Selected from the group consisting of phosphate nucleotides, 2'-O-hexadecyl-cytidine-3'-phosphate nucleotides, 2'-O-hexadecyl-adenosine-3'-phosphate nucleotides, 2'-O-hexadecyl-guanosine-3'-phosphate nucleotides, 2'-O-hexadecyl-uridine-3'-phosphate nucleotides, 5'-vinyl phosphonates (VP), 2'-deoxyadenosine-3'-phosphate nucleotides, 2'-deoxycytidine-3'-phosphate nucleotides, 2'-deoxyguanosine-3'-phosphate nucleotides, 2'-deoxythymidine-3'-phosphate nucleotides, 2'-deoxyuridine nucleotides, cholesteryl derivatives, and terminal nucleotides linked to a bisdecylamide dodecanoate group, as well as combinations thereof.

[0020] In another embodiment, the modified nucleotide is selected from the group consisting of 2'-deoxy-2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, 3'-terminal deoxythymidine nucleotides (dT), locked nucleotides, debasalized nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-O-methyl-modified nucleotides, nucleotides containing glycol nucleic acids (GNA), morpholino nucleotides, phosphoramides, and nucleotides containing non-natural bases.

[0021] In another embodiment, the modified nucleotide includes a short sequence of a 3'-terminal deoxythymidine nucleotide (dT).

[0022] In yet another embodiment, the modifications on the nucleotide include 2'-O-methyl modification, 2'-deoxy- modification, 2'-fluoro modification, 5'-vinylphosphonate (VP) modification, and 2'-O-hexadecyl nucleotide modification.

[0023] In certain embodiments, the double-stranded RNAi agent does not contain inverted debasalized nucleotides.

[0024] In one embodiment, the dsRNA agent further comprises at least one phosphorothioate nucleotide linkage.

[0025] In one embodiment, the dsRNA agent contains 6 to 8 phosphorothioate nucleotide linkages.

[0026] In one embodiment, each chain is 30 nucleotides or less in length.

[0027] In one embodiment, at least one strand includes a 3' overhang of at least one nucleotide.

[0028] In another embodiment, at least one strand includes a 3' overhang of at least two nucleotides.

[0029] The double-stranded region may be 15–30 nucleotide pairs long, 17–23 nucleotide pairs long, 17–25 nucleotide pairs long, 23–27 nucleotide pairs long, 19–21 nucleotide pairs long, or 21–23 nucleotide pairs long.

[0030] Each strand of the dsRNA agent may be 15–30, 17–20, 19–30 nucleotides long; 19–23 nucleotides long; or 21–23 nucleotides long, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long.

[0031] In certain embodiments, the double-stranded RNAi agent further comprises a lipophilic ligand, such as a C16 ligand, conjugated to the 3' end of the sense strand via a monovalent, branched divalent, or trivalent linker.

[0032] In one embodiment, the ligand is conjugated to the 2' position of a nucleotide or modified nucleotide in a sense or antisense chain. For example, the C16 ligand has the following structure:

[0033] [ka] (In the formula, * (where B indicates binding to an adjacent nucleotide, and B is a nucleic acid base or nucleic acid base analog, and B may be adenine, guanine, cytosine, thymine, or uracil.) It can be conjugated as shown.

[0034] In other embodiments, the agent further comprises a targeted ligand that targets liver tissue, such as one or more GalNAc derivatives, conjugated to a double-stranded RNAi agent via a linker or carrier.

[0035] In yet another embodiment, the agent further comprises a lipophilic ligand, e.g., a C16 ligand, conjugated to the 3' end of the sense chain via a monovalent or branched divalent or trivalent linker, and a targeted ligand that targets liver tissue, e.g., one or more GalNAc derivatives, conjugated to the 3' end of the sense chain via a monovalent or branched divalent or trivalent linker.

[0036] In one embodiment, one or more lipophilic portions are conjugated at one or more internal positions in at least one chain.

[0037] In one embodiment, one or more lipophilic portions are conjugated via a linker or carrier to one or more internal positions in at least one chain.

[0038] In certain embodiments, the lipophilic portion is not the cholesterol portion.

[0039] In certain embodiments, the drug may further comprise a targeted ligand that targets liver tissue, such as one or more GalNAc derivatives, which may be conjugated to a double-stranded RNAi agent via a linker or carrier.

[0040] In yet another embodiment, the agent may further include one or more lipophilic moieties conjugated to one or more internal nucleotide positions via a linker or carrier, and a targeting ligand that targets liver tissue, such as one or more GalNAc derivatives, which may be conjugated to a double-stranded RNAi agent via a linker or carrier.

[0041] In one embodiment, the internal positions include all positions except the two terminal positions from each end of at least one chain.

[0042] In another embodiment, the internal positions include all positions except the three terminal positions from each end of at least one chain.

[0043] In another embodiment, the internal location excludes the region of the sense chain's cleavage site.

[0044] In yet another embodiment, the internal position includes all positions except positions 9–12, counting from the 5' end of the sense strand. In a particular embodiment, the sense strand is 21 nucleotides long.

[0045] In one embodiment, the internal position includes all positions except positions 11-13, counting from the 3' end of the sense strand. The internal position may also exclude the cleavage region of the antisense strand. In a particular embodiment, the sense strand is 21 nucleotides long.

[0046] In one embodiment, the internal position excludes the region of the antisense chain's cleavage site.

[0047] In one embodiment, the internal position includes all positions except positions 12–14, counting from the 5' end of the antisense strand. In a particular embodiment, the antisense strand is 23 nucleotides long.

[0048] In one embodiment, the internal positions include all positions except positions 11-13 counting from the 3' end of the sense strand and positions 12-14 counting from the 5' end of the antisense strand. In a particular embodiment, the sense strand is 21 nucleotides long and the antisense strand is 23 nucleotides long.

[0049] In one embodiment, one or more lipophilic moieties are conjugated to one or more internal positions selected from the group consisting of positions 4-8 and 13-18 in the sense chain and positions 6-10 and 15-18 in the antisense chain, counting from the 5' end of each chain.

[0050] In one embodiment, one or more lipophilic moieties are conjugated to one or more internal positions selected from the group consisting of positions 5, 6, 7, 15, and 17 in the sense strand and positions 15 and 17 in the antisense strand, counting from the 5' end of each strand. In a particular embodiment, the sense strand is 21 nucleotides long and the antisense strand is 23 nucleotides long.

[0051] In one embodiment, the location within the double-stranded region excludes the cleavage region of the sense strand.

[0052] In one embodiment, the sense chain is 21 nucleotides long, the antisense chain is 23 nucleotides long, and the lipophilic portion is conjugated at positions 21, 20, 15, 1, 7, 6, or 2 on the sense chain or at position 16 on the antisense chain.

[0053] In one embodiment, the lipophilic portion is conjugated to position 21, position 20, position 15, position 1, or position 7 of the sense chain.

[0054] In one embodiment, the lipophilic portion is conjugated to position 21, position 20, or position 15 of the sense chain.

[0055] In one embodiment, the lipophilic portion is conjugated to position 20 or position 15 of the sense chain.

[0056] In one embodiment, the lipophilic portion is conjugated to position 16 of the antisense chain.

[0057] In one embodiment, the lipophilic portion is an aliphatic compound, an alicyclic compound, or a polyalicyclic compound.

[0058] In one embodiment, the lipophilic portion is selected from the group consisting of lipids, cholesterol, retinoic acid, cholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. In a particular embodiment, the lipophilic portion is not cholesterol.

[0059] In one embodiment, the lipophilic portion contains a saturated or unsaturated C4-C30 hydrocarbon chain and a suitable functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0060] In one embodiment, the lipophilic portion contains saturated or unsaturated C6-C18 hydrocarbon chains.

[0061] In one embodiment, the lipophilic portion contains saturated or unsaturated C16 hydrocarbon chains.

[0062] In some embodiments, a saturated or unsaturated C16 hydrocarbon chain is conjugated at position 6, counting from the 5' end of the chain.

[0063] In one embodiment, the lipophilic portion is conjugated via a carrier that replaces one or more nucleotides in an internal position or double-stranded region.

[0064] In one embodiment, the support is a cyclic group 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; or an acyclic moiety based on a selinol skeleton or a diethanolamine skeleton.

[0065] In one embodiment, the lipophilic portion is conjugated to a double-stranded iRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfamide linkage, click reaction product, or carbamate.

[0066] In one embodiment, the lipophilic portion is conjugated to a nucleic acid base, a sugar portion, or an internucleoside linkage.

[0067] In one embodiment, the double-stranded RNAi agent further comprises a phosphate or phosphate mimetic at the 5' end of the antisense strand. The phosphate mimetic may be a 5'-vinylphosphonate (VP).

[0068] In certain embodiments, the RNAi agent does not contain inverted debasalized nucleotides.

[0069] In certain embodiments, the double-stranded RNAi agent does not contain a targeted ligand.

[0070] In certain embodiments, the double-stranded RNAi agent further comprises a targeted ligand, such as a hydrophilic ligand, that targets a receptor that mediates delivery to liver tissue. In certain embodiments, the targeted ligand is a C16 ligand. In certain embodiments, the lipophilic ligand is not a cholesterol moiety.

[0071] In one embodiment, the lipophilic moiety or targeted ligand is conjugated via a biocleavable linker selected from the group consisting of DNA, RNA, disulfides, amides, and functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, and mannose, as well as combinations thereof.

[0072] In one embodiment, the 3' end of the sense chain is protected via an end cap which is a cyclic group having an amine, and the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanil, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuranil, and dekalinyl.

[0073] In one embodiment, the dsRNA agent further comprises a targeted ligand that targets liver tissue.

[0074] In one embodiment, the targeted ligand is a GalNAc conjugate.

[0075] In one embodiment, the dsRNA agent further includes a terminal chiral modification occurring at the first nucleotide linkage at the 3' end of the antisense strand, having a linked phosphorus atom in the Sp configuration; a terminal chiral modification occurring at the first nucleotide linkage at the 5' end of the antisense strand, having a linked phosphorus atom in the Rp configuration; and a terminal chiral modification occurring at the first nucleotide linkage at the 5' end of the sense strand, having a linked phosphorus atom in either the Rp configuration or the Sp configuration.

[0076] In one embodiment, the dsRNA agent further includes a terminal chiral modification occurring at the first and second nucleotide linkages at the 3' end of the antisense strand, having a linked phosphorus atom in the Sp configuration; a terminal chiral modification occurring at the first nucleotide linkage at the 5' end of the antisense strand, having a linked phosphorus atom in the Rp configuration; and a terminal chiral modification occurring at the first nucleotide linkage at the 5' end of the sense strand, having a linked phosphorus atom in either the Rp configuration or the Sp configuration.

[0077] In one embodiment, the dsRNA agent further includes terminal chiral modifications occurring at the first, second, and third nucleotide linkages at the 3' end of the antisense strand, having a linked phosphorus atom in the Sp configuration; terminal chiral modifications occurring at the first nucleotide linkage at the 5' end of the antisense strand, having a linked phosphorus atom in the Rp configuration; and terminal chiral modifications occurring at the first nucleotide linkage at the 5' end of the sense strand, having a linked phosphorus atom in either the Rp or Sp configuration.

[0078] In one embodiment, the dsRNA agent further includes terminal chiral modifications occurring at the first and second nucleotide linkages at the 3' end of the antisense strand, having a linked phosphorus atom in the Sp configuration; terminal chiral modifications occurring at the third nucleotide linkage at the 3' end of the antisense strand, having a linked phosphorus atom in the Rp configuration; terminal chiral modifications occurring at the first nucleotide linkage at the 5' end of the antisense strand, having a linked phosphorus atom in the Rp configuration; and terminal chiral modifications occurring at the first nucleotide linkage at the 5' end of the sense strand, having a linked phosphorus atom in either the Rp or Sp configuration.

[0079] In one embodiment, the dsRNA agent further includes a terminal chiral modification occurring at the first and second nucleotide linkages at the 3' end of the antisense strand, having a linked phosphorus atom in the Sp configuration; a terminal chiral modification occurring at the first and second nucleotide linkages at the 5' end of the antisense strand, having a linked phosphorus atom in the Rp configuration; and a terminal chiral modification occurring at the first nucleotide linkage at the 5' end of the sense strand, having a linked phosphorus atom in either the Rp or Sp configuration.

[0080] In one embodiment, the dsRNA agent further comprises a phosphate or phosphate mimetic at the 5' end of the antisense strand.

[0081] In one embodiment, the phosphate mimetic is 5'-vinylphosphonate (VP). When the phosphate mimetic is 5'-vinylphosphonate (VP), the 5' terminal nucleotide has the following structure.

[0082] [ka] [In the formula, * This indicates the position of binding to the 5' position of the adjacent nucleotide; R is hydrogen, hydroxy, methoxy, fluoro, or another 2'-modification as described herein (e.g., hydroxy or methoxy); B is a nucleic acid base or a modified nucleic acid base, and B may be adenine, guanine, cytosine, thymine, or uracil. It may have.

[0083] In one embodiment, the base pair at one position of the 5' end of the antisense strand of the double helix is ​​an AU base pair.

[0084] In one embodiment, the sense strand has a total of 21 nucleotides, and the antisense strand has a total of 23 nucleotides.

[0085] The present invention further provides cells for inhibiting the expression of the GPR75 gene, a pharmaceutical composition, and a pharmaceutical composition comprising a lipid preparation containing the dsRNA agent of the present invention.

[0086] In one embodiment, the present invention provides a method for inhibiting the expression of the GPR75 gene in cells. The method comprises contacting cells with the dsRNA agent of the present invention or the pharmaceutical composition of the present invention; and maintaining the cells produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the GPR75 gene, thereby inhibiting the expression of the GPR75 gene in cells.

[0087] In one embodiment, the cells are located within the object.

[0088] In one embodiment, the subject is a human being.

[0089] In one embodiment, the expression of the GPR75 gene is inhibited by at least 50%.

[0090] In one embodiment, the present invention provides a method for treating subjects at risk of developing weight disorders, such as subjects with GPR75-related disorders, such as obesity, or subjects at risk of becoming obese, such as subjects who are overweight or are overweight or obese and have lost weight but failed to maintain it. The method comprises administering a therapeutically effective amount of the dsRNA agent or pharmaceutical composition of the present invention to the subject, thereby treating the subject.

[0091] In one embodiment, the subject is a human being.

[0092] In one embodiment, the treatment includes improvement of at least one sign or symptom of the disease. In some embodiments, administration of the dsRNA agent results in a decrease in the subject's BMI. In some embodiments, administration of the dsRNA agent results in a decrease in the subject's blood glucose levels. In other embodiments, administration of the dsRNA agent results in a decrease in the subject's blood lipid levels.

[0093] In one embodiment, the dsRNA agent is administered to the subject in doses ranging from approximately 0.01 mg / kg to approximately 50 mg / kg.

[0094] In some embodiments, the double-stranded RNAi agent is administered intrathecally to the subject.

[0095] In some embodiments, the double-stranded RNAi agent is administered subcutaneously to the subject.

[0096] In one embodiment, the method further includes administering an additional agent or therapy suitable for the treatment or prevention of GPR75-related disorders.

[0097] In one embodiment, the additional therapeutic agent is selected from the group consisting of antidiabetic agents, antidiabetic complication agents, cardiovascular disease agents, anti-dyslipidemia agents, antihypertensive or antihypertensive agents, anti-obesity agents, non-alcoholic steatohepatitis (NASH) agents, chemotherapeutic agents, immunotherapeutic agents, immunosuppressants, anti-inflammatory agents, anti-steatosis agents, anti-fibrotic agents, immunomodulators, tyrosine kinase inhibitors, anti-fibrotic agents, and any combination thereof.

[0098] The present invention is further illustrated by the following detailed description. [Brief explanation of the drawing]

[0099] [Figure 1] Figure 1 is a graph depicting the relative quantification of Gpr75 mRNA levels normalized to ActB and Gapdh in the brains of diet-induced obese mice 21 days after a single 150 μg dose of the double-stranded siRNA or intracerebroventricular injection of a control. * indicates P<0.05 compared to the control siRNA. [Modes for carrying out the invention]

[0100] The present invention provides iRNA compositions that perform RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the GPR75 gene. The GPR75 gene may be present in cells, for example, in cells within subjects such as humans. The use of these iRNAs enables targeted degradation of the mRNA of the corresponding gene (GPR75 gene) in mammals. The disclosure also provides methods of using the RNAi compositions of the disclosure to inhibit the expression of the GPR75 gene in order to treat subjects with GPR75-related disorders, such as weight disorders, e.g., obesity, or subjects at risk of developing weight disorders, e.g., overweight subjects or subjects who are overweight or obese and have lost weight but failed to maintain the weight loss.

[0101] The iRNA of the present invention can be up to approximately 30 nucleotides in length, for example, 15-30, 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- The RNAi agent of the Disclosure comprises an RNA strand (antisense strand) having a region of 24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, wherein the region is substantially complementary to at least a portion of the mRNA transcript of the GPR75 gene. In certain embodiments, the RNAi agent of the Disclosure comprises an RNA strand (antisense strand) having a region of about 21-23 nucleotides in length, which is substantially complementary to at least a portion of the mRNA transcript of the GPR75 gene.

[0102] In certain embodiments, one or both strands of the double-stranded RNAi agent of the present invention have a region of at least 19 consecutive nucleotides that is substantially complementary to at least a portion of the mRNA transcript of the GPR75 gene, and is up to 66 nucleotides long, for example, 36-66, 26-36, 25-36, 31-60, 22-43, or 27-53 nucleotides long. In some embodiments, such an iRNA agent having a longer antisense strand may include, for example, a second RNA strand (sense strand) of 20-60 nucleotides long, in which case the sense strand and antisense strand form a double helix of 18-30 consecutive nucleotides.

[0103] The use of the iRNAs of the present invention enables targeted degradation of the corresponding GPR75 mRNA in mammals. Thus, methods and compositions comprising these iRNAs are useful for treating subjects with weight disorders, such as GPR75-related disorders like obesity, or subjects at risk of developing weight disorders such as obesity, such as overweight subjects or subjects who are overweight or obese and have lost weight but failed to maintain it.

[0104] The following detailed description discloses methods for preparing and using compositions containing iRNA to inhibit the expression of the GPR75 gene, as well as compositions, uses, and methods for treating subjects who would benefit from the inhibition and / or reduction of GPR75 gene expression, e.g., subjects suspected of or diagnosed with a GPR75-related disorder.

[0105] I. Definition To make the present invention more easily understandable, certain terms are first defined. In addition, whenever parameter values ​​or ranges of values ​​are listed, intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

[0106] The articles "a" and "an" are used herein to mean one or more (i.e., at least one) grammatical objects of the article. For example, "an element" means one or more elements, e.g., multiple elements.

[0107] The term "including" is used herein to mean "including but not limited to" and is interchangeable with the phrase. The term "or" is used herein to mean "and / or" unless explicitly indicated in the context and is interchangeable with the phrase.

[0108] The term “approximately” is used herein to mean within a typical range of crossover in the art. For example, “approximately” can be understood as approximately 2 standard deviations from the mean. In certain embodiments, “approximately” means ±10%. In certain embodiments, “approximately” means ±5%. It will be understood that when “approximately” precedes a series of numbers or ranges, it can modify each of the numbers or ranges in that series.

[0109] The terms “at least,” “greater than,” or “more than,” preceding a number or a range of numbers are understood to include, where evident from the context, the number adjacent to the term “at least,” and all subsequent numbers or integers that may logically be included. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 18 nucleotides in a 21-nucleotide nucleic acid molecule” means that 18, 19, 20, or 21 nucleotides have the stated characteristic. It will be understood that when the term “at least” precedes a range of numbers or a range, “at least” can modify each of the numbers and ranges in the range.

[0110] As used herein, “less than or equal to” or “or less than or equal to” is understood to mean from the value adjacent to the phrase and any value or integer logically smaller than that value, down to zero, where logically from the context. For example, a double helix with an overhang of “less than or equal to 2 nucleotides” has an overhang of 2, 1, or 0 nucleotides. If “less than or equal to” precedes a series of numbers or ranges, it will be understood that “less than or equal to” can modify each of the numbers or ranges in that series. As used herein, a range includes both an upper and lower limit.

[0111] As used herein, the detection method may include determining whether the amount of analyte present is below the detection level of the method.

[0112] If the indicated target site does not match the nucleotide sequence on the sense or antisense strand, the indicated sequence takes precedence.

[0113] In the event of any inconsistency between a given sequence and its indicated site on a transcript or other sequence, the nucleotide sequence described herein shall prevail.

[0114] As used herein, the term “G protein-coupled receptor 75” (“GPR75”) refers to the well-known gene and polypeptide also known in the industry as “Prosecutor’s G protein-coupled receptor 75,” “WI-31133,” “GPRchr2,” and “WI31133.” GPR75 binds to 20-HETE, inhibiting insulin signaling and leading to obesity.

[0115] The term "GPR75" includes human GPR75, e.g., the amino acid and nucleotide sequences found in GenBank accession number NM_006794.4 (SEQ ID NO: 1); mouse GPR75, e.g., the amino acid and nucleotide sequences found in GenBank accession number NM_175490.4 (SEQ ID NO: 2); and rat GPR75, e.g., the amino acid and nucleotide sequences found in GenBank accession number NM_001109096.1 (SEQ ID NO: 3).

[0116] The term "GPR75" also includes the amino acid and nucleotide sequences found in the rhesus macaque (Macaca mulatta) GPR75, for example, in GenBank accession number NM_001204509.2 (SEQ ID NO: 4).

[0117] Further examples of GPR75 mRNA sequences are readily available, for example, through the websites of GenBank, UniProt, OMIM, and the Macac Genome Project.

[0118] Exemplary GPR75 nucleotide sequences can also be found in SEQ ID NOs: 1-4. SEQ ID NOs: 5-8 are the reverse complement sequences of SEQ ID NOs: 1-4, respectively.

[0119] Further information on GPR75 is available, for example, in the NCBI Gene database at www.ncbi.nlm.nih.gov / gene / 10936.

[0120] 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.

[0121] As used herein, the terms “G protein-coupled receptor 75” and “GPR75” also refer to naturally occurring DNA sequence variations of the GPR75 gene. Numerous sequence variations within the GPR75 gene have been identified and can be found, for example, in NCBI dbSNP and UniProt (see, for example, https: / / www.ncbi.nlm.nih.gov / snp / ?term=GPR75, whose entire contents are incorporated herein by reference as of the filing date of this application).

[0122] As used herein, “target sequence” refers to a contiguous portion of a nucleotide sequence in an mRNA molecule formed during the transcription of the GPR75 gene, such as mRNA, which is the product of RNA processing of the primary transcript. In one embodiment, the target portion of the sequence will be at least sufficiently long to function as a substrate for RNAi-dependent cleavage in or near a portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the GPR75 gene. In one embodiment, the target sequence is located within the protein-coding region of the GPR75 gene. In another embodiment, the target sequence is located within the 3'UTR of the GPR75 gene.

[0123] The target sequence can be approximately 9-36 nucleotides long, for example, approximately 15-30 nucleotides long. For example, the target sequence can be approximately 15-30 nucleotides long, 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, 1 The target sequence may be 9-28, 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 long. In some embodiments, the target sequence is about 19-30 nucleotides long. In other embodiments, the target sequence is about 19-25 nucleotides long. In yet another embodiment, the target sequence is about 19-23 nucleotides long. In some embodiments, the target sequence is about 21-23 nucleotides long. It is conceivable that intermediate ranges and lengths between those listed above are also part of the present invention.

[0124] As used herein, the term “sequence-containing chain” means an oligonucleotide containing a chain of nucleotides described by a sequence as referred to using the standard nucleotide terminology.

[0125] "G," "C," "A," "T," and "U" generally represent nucleotides containing guanine, cytosine, adenine, thymidine, and uracil as bases, respectively. However, it will be understood that the term "ribonucleotide" or "nucleotide" can also mean modified nucleotides or substituted portions (see, for example, Table 1), as will be described in more detail below. Those skilled in the art will be well aware that guanine, cytosine, adenine, and uracil can be replaced by other portions without substantially altering the base-pairing properties of oligonucleotides containing such substituted portions. When a cDNA sequence is provided, it will be understood that the corresponding mRNA or RNAi agent contains U instead of T. 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 substituted, for example, with nucleotides containing inosine in the nucleotide sequences of the dsRNAs characterized in the present invention. In another example, adenine and cytosine in either oligonucleotide can be substituted with guanine and uracil, respectively, to form a G-U Wobble base pair with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods characterized in the present invention. Furthermore, it will be understood by those skilled in the art that T in the target gene sequence or its reverse complement is often substituted with U in the RNAi agents of the present invention.

[0126] The terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interfering agent,” as used interchangeably herein, mean agents containing RNA as defined herein, which mediate targeted cleavage in RNA transcription via the RNA-induced silencing complex (RISC) pathway. RNA interference (RNAi) is a process that directs sequence-specific degradation of mRNA. RNAi modulates, for example, inhibits the expression of the GPR75 gene in cells, such as in mammalian subjects.

[0127] In one embodiment, the RNAi agent of this disclosure comprises a single-stranded RNAi that interacts with a target RNA sequence, such as a GPR75 mRNA sequence, to instruct the cleavage of the target RNA. While we do not wish to be bound by theory, it is thought that long double-stranded RNA introduced into a cell is degraded into double-stranded small interfering RNA (siRNA) containing sense and antisense strands by a type III endonuclease known as Dicer [Sharp et al. (2001) Genes Dev. 15:485]. Dicer, a ribonuclease III-like enzyme, processes these dsRNAs into 19-23 base pair small interfering RNAs with characteristic two base 3' overhangs [Bernstein, et al., (2001) Nature 409:363]. These siRNAs are then introduced into an RNA-induced silencing complex (RISC), in which one or more helicases unwind the siRNA double helix, allowing the complementary antisense strand to induce target recognition [Nykanen, et al., (2001) Cell 107:309]. Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing [Elbashir, et al., (2001) Genes Dev. 15:188]. Thus, in one embodiment, this disclosure relates to single-stranded RNA (ssRNA) (the antisense strand of the siRNA double helix) that is generated in a cell and facilitates the formation of the RISC complex, thereby silencing a target gene. Accordingly, the term "siRNA" is used herein to also mean the RNAi described above.

[0128] In another embodiment, the RNAi agent may be a single-stranded RNA 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 are generally 15–30 nucleotides long and are chemically modified. Designs and tests of single-stranded RNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883–894, the entire contents of which are incorporated herein by reference. Any antisense nucleotide sequences described herein may be used as single-stranded siRNAs described herein, or as single-stranded siRNAs chemically modified by the methods described in Lima et al., (2012) Cell 150:883–894.

[0129] In another embodiment, the “RNAi agent” for use in the compositions and methods of this disclosure is double-stranded RNA, and is referred to herein as “double-stranded RNAi agent,” “double-stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA.” The term “dsRNA” means a complex of ribonucleic acid molecules having a double-stranded structure containing two antiparallel and substantially complementary nucleic acid strands, which are said to have “sense” and “antisense” orientations with respect to the target RNA, i.e., the GPR75 mRNA sequence. In some embodiments of this disclosure, double-stranded RNA (dsRNA) induces the degradation of target RNA, e.g., mRNA, by a post-transcriptional gene silencing mechanism referred herein as RNA interference or RNAi.

[0130] Generally, dsRNA molecules may contain ribonucleotides, but as will be described in detail herein, each or both strands may also contain one or more ribonucleotides, such as deoxyribonucleotides, modified nucleotides, etc. In addition, as used herein, “RNAi agent” may include chemically modified ribonucleotides; RNAi agent may include substantial modifications in multiple nucleotides.

[0131] As used herein, the term “modified nucleotide” means a nucleotide having independently a modified sugar moiety, a modified nucleotide linkage, or a modified nucleic acid base. Therefore, the term “modified nucleotide” encompasses substitution, addition, or removal of, for example, a functional group or atom, to the nucleoside linkage, sugar moiety, or nucleic acid base. Modifications suitable for use in the agents of this disclosure encompass all types of modifications disclosed herein or known in the art. Any such modification used in an siRNA-type molecule is encompassed by “RNAi agent” for the purposes of this specification and the claims.

[0132] In certain embodiments of this disclosure, the presence of deoxyribonucleotides, which are recognized as naturally occurring forms of nucleotides when present in an RNAi agent, can be considered to constitute modified nucleotides.

[0133] The double-stranded region can be of any length that allows for the specific degradation of the desired target RNA by the RISC pathway, as well as lengths of approximately 9 to 36 base pairs, e.g., approximately 15 to 30 base pairs, e.g., 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, e.g., approximately 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 1 7, 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- The base pair lengths can range from 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. Intermediate ranges and lengths between those listed above are also conceivable to be part of the present invention.

[0134] The two strands forming a double helix structure may be different parts of one larger RNA molecule, or they may be separate RNA molecules. If the two strands are part of one larger molecule and are therefore connected by an unpaired nucleotide chain between the 3' end of one strand and the 5' end of the other strand forming the double helix structure, then the connecting 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 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides or nucleotides not targeting the dsRNA site. 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 unpaired nucleotides.

[0135] In certain embodiments, two chains of a double-stranded oligomeric compound can be linked together. The two chains can 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. When 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 can be linked together by an oligonucleotide linker containing, but not limited to, (N)n (wherein 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 (wherein 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 chains may also be linked together by a non-nucleoside linker, for example, the linker described herein. It will be understood by those skilled in the art that any oligonucleotide chemical modification or mutation described herein can be used in the oligonucleotide linker.

[0136] Hairpin and dumbbell-shaped oligomeric compounds will 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 be equal to or less than 200, 100, or 50 in length. In some embodiments, the double-stranded region ranges in length from 15 to 30, 17 to 23, 19 to 23, and 19 to 21 nucleotide pairs.

[0137] In some embodiments, the hairpin oligomer compound may have a single-stranded overhang or terminal unpaired region at 3', and in some embodiments, on the antisense side of the hairpin. In some embodiments, the overhang is 1-4 nucleotides long, more commonly 2-3 nucleotides long. The hairpin oligomer compound capable of inducing RNA interference is also referred to herein as “shRNA”.

[0138] The two substantially complementary strands of dsRNA are contained within separate RNA molecules, and these molecules can be covalently linked, although this is not always necessary. The two strands are covalently linked between the 3' end of one strand and the 5' end of the other strand, forming a double-stranded structure, by means other than an uninterrupted chain of nucleotides; this connecting structure is called a "linker." RNA strands can have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA minus all the overhangs present in the double-stranded structure. In addition to the double-stranded structure, RNAi can contain one or more nucleotide overhangs.

[0139] In one embodiment, the RNAi agent of this disclosure is a dsRNA, each strand being 24-30 nucleotides long, which interacts with a target RNA sequence, e.g., a GPR75 mRNA sequence, to induce cleavage of the target RNA. Although not intended to be bound by theory, the long double-stranded RNA introduced into the cell is degraded into siRNA by a type III endonuclease known as Dicer [Sharp et al. (2001) Genes Dev. 15:485]. Dicer, a ribonuclease III-like enzyme, processes the dsRNA into small interfering RNAs of 19-23 base pairs with characteristic two-base 3' overhangs [Bernstein, et al., (2001) Nature 409:363]. Next, the siRNA is incorporated into the RNA-induced silencing complex (RISC), in which one or more helicases unwind the siRNA double helix, allowing the complementary antisense strand to induce target recognition [Nykanen, et al., (2001) Cell 107:309]. Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing [Elbashir, et al., (2001) Genes Dev. 15:188].

[0140] In one embodiment, the RNAi agent of the present invention is a dsRNA agent, each strand containing 19-23 nucleotides that interact with the GPR75 mRNA sequence to induce cleavage of the target RNA. Although we do not wish to be bound by theory, the long double-stranded RNA introduced into the cell is degraded into siRNA by a type III endonuclease known as Dicer [Sharp et al. (2001) Genes Dev. 15:485]. Dicer, a ribonuclease III-like enzyme, processes the dsRNA into small interfering RNAs of 19-23 base pairs with characteristic two base 3' overhangs [Bernstein, et al., (2001) Nature 409:363]. Next, the siRNA is incorporated into an RNA-induced silencing complex (RISC), in which one or more helicases unwind the siRNA double helix, allowing the complementary antisense strand to induce target recognition [Nykanen, et al., (2001) Cell 107:309]. Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing [Elbashir, et al., (2001) Genes Dev. 15:188]. In one embodiment, the RNAi agent of the present invention is a 24-30 nucleotide dsRNA that interacts with the GPR75 mRNA sequence to induce cleavage of the target RNA.

[0141] As used herein, the term “nucleotide overhang” means at least one unpaired nucleotide protruding from the double-stranded structure of an RNAi agent, such as a dsRNA. For example, a nucleotide overhang exists if the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. A dsRNA may contain an overhang of at least one nucleotide; or the overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang may contain or consist of nucleotide / nucleoside analogs such as deoxynucleotides / nucleosides. 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 of either the antisense strand or the sense strand of the dsRNA.

[0142] In one embodiment of dsRNA, 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, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet another embodiment, at least one strand of the RNAi agent includes a 5' overhang of at least one nucleotide. In a particular embodiment, at least one strand includes a 5' overhang of at least two nucleotides, e.g., 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.

[0143] 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, 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more nucleotides in the overhang are replaced with a nucleoside thiophosphate.

[0144] In certain embodiments, the overhang in the sense strand, the antisense strand, or both may include an extended length longer than 10 nucleotides, for example, 1–30 nucleotides, 2–30 nucleotides, 10–30 nucleotides, or 10–15 nucleotides. In certain embodiments, the extended overhang is located in the sense strand of the double helix. In certain embodiments, the extended overhang is located at the 3' end of the sense strand of the double helix. In certain embodiments, the extended overhang is located at the 5' end of the sense strand of the double helix. In certain embodiments, the extended overhang is located in the antisense strand of the double helix. 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 overhang are replaced with a nucleoside thiophosphate. In certain embodiments, the overhang includes a self-complementary portion such that the overhang can form a stable hairpin structure under physiological conditions.

[0145] The terms “blunt” or “blunt-ended,” as used herein in relation to dsRNA, mean that there are no unpaired nucleotides or nucleotide analogs at any given end of the dsRNA; that is, there are no nucleotide overhangs. One or both ends of a dsRNA can be blunt. If both ends of a dsRNA are blunt, it is said to be blunt-ended. For clarity, a “blunt-ended” dsRNA is a dsRNA that is blunt at both ends, i.e., a dsRNA in which there are no nucleotide overhangs at either end of the molecule. In most cases, such a molecule will be double-stranded over its entire length.

[0146] The terms "antisense strand" or "guide strand" refer to a strand of iRNA, such as a dsRNA, that contains a region substantially complementary to the target sequence, such as the GPR75 mRNA sequence.

[0147] As used herein, the term “complementary region” refers to a region on an antisense strand that is substantially complementary to a sequence, e.g., a target sequence, e.g., a GPR75 nucleotide sequence, as defined herein. If the complementary region is not perfectly complementary to the target sequence, the mismatch may be in an internal or terminal region of the molecule. Generally, the most acceptable mismatch is in a terminal region, e.g., within 5, 4, 3, or 2 nucleotides of the 5' or 3' end of an RNAi agent.

[0148] In some embodiments, the double-stranded RNA agent of the present invention includes nucleotide mismatches in the antisense strand. In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention includes four or fewer mismatches with the target mRNA, for example, the antisense strand includes four, three, two, one, or zero mismatches with the target mRNA. In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention includes four or fewer mismatches with the sense strand, for example, the antisense strand includes four, three, two, one, or zero mismatches with the sense strand. In some embodiments, the double-stranded RNA agent of the present invention includes nucleotide mismatches in the sense strand. In some embodiments, the sense strand of the double-stranded RNA agent of the present invention includes four or fewer mismatches with the antisense strand, for example, the sense strand includes four, three, two, one, or zero 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 mismatch is, for example, at the 3' terminal nucleotide of the iRNA agent. In some embodiments, the mismatch is not present in the seed region.

[0149] 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., three, two, one, or zero mismatches). In one embodiment, the RNAi agents described herein contain two or fewer mismatches. In one embodiment, the RNAi agents described herein contain one or fewer mismatches. In one embodiment, the RNAi agents described herein contain zero mismatches. In certain embodiments, if the antisense strand of the RNAi agent contains a mismatch to the target sequence, the mismatch may, as appropriate, be limited to within the last five nucleotides from the 5' or 3' end of the complementary region. For example, in such embodiments, for a 23-nucleotide RNAi agent, the strand complementary to the GPR75 gene region generally does not contain any mismatches within the central 13 nucleotides. By using the methods described herein or methods known in the art, it is possible to determine whether an RNAi agent containing a mismatch to the target sequence is effective in inhibiting the expression of the GPR75 gene. In particular, if specific complementary regions in the GPR75 gene are known to be altered, it is important to consider the effectiveness of RNAi agents with mismatches that inhibit GPR75 gene expression.

[0150] When used herein, the terms “sense strand” or “passenger strand” mean a strand of an RNAi agent that contains a region substantially complementary to the antisense strand region as defined herein.

[0151] As used herein, “substantially all nucleotides are modified” means that most of the nucleotides are modified, but not entirely, and may contain 5, 4, 3, 2, or 1 or fewer unmodified nucleotides.

[0152] As used herein, the term “cleavage region” means a region located directly adjacent to a cleavage site. A cleavage site is a site on the target where a cleavage occurs. In some embodiments, a cleavage region includes three bases directly adjacent to either end of a cleavage site. In some embodiments, a cleavage region includes two bases directly adjacent to either end of a cleavage site. In some embodiments, in detail, a cleavage site occurs at a site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region includes nucleotides 11, 12, and 13.

[0153] Where used herein, unless otherwise specified, the term “complementary” means, as 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 to form a double helix under certain conditions, when used to describe a first nucleotide sequence in relation to a second nucleotide sequence. Such conditions may be, for example, “stringent conditions,” which may include 400 mM NaCl, 40 mM PIPES, pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12–16 hours, followed by washing [see, for example, Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press]. Other conditions, such as physiologically relevant conditions that may be encountered within living organisms, may also be applicable. Those skilled in the art will be able to determine the most appropriate set of conditions for testing the complementarity of the two sequences by the final application of the hybridized nucleotides.

[0154] In RNAi agents, for example, in dsRNA as described herein, complementary sequences include base pairings of an oligonucleotide or polynucleotide containing a first nucleotide sequence with an oligonucleotide or polynucleotide containing a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as “fully complementary” with respect to each other. However, where herein it is said that the first sequence is “substantially complementary” with respect to the second sequence, the two sequences may be fully complementary, or they may form one or more, but generally five, four, three, or two or fewer, mismatched base pairs during hybridization, while maintaining their ability to hybridize under conditions best suited to their final use, e.g., inhibition of gene expression in vitro or in vivo, in the case of double helixes of up to 30 base pairs. However, if two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, such overhangs are not considered mismatches with respect to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide of 21 nucleotides and another oligonucleotide of 23 nucleotides, wherein the longer oligonucleotide contains a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, can still be considered "perfectly complementary" for the purposes described herein.

[0155] When used herein, “complementary” sequences may include, or may be entirely formed from, non-Watson-Crick base pairs or base pairs formed from non-naturally modified nucleotides, provided that the above requirements regarding their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogsteen base pairings.

[0156] The terms “complementary,” “fully complementary,” and “substantially complementary” as used herein can be used in relation to base matching between two oligonucleotides or polynucleotides, such as between the sense strand and antisense strand of a dsRNA, or between the antisense strand and target sequence of an RNAi agent, as can be understood in context with their use.

[0157] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" a messenger RNA (mRNA) or target sequence means a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of the sequence of interest or target sequence (e.g., mRNA encoding GPR75). For example, a polynucleotide is complementary to at least a portion of GPR75 RNA if its sequence is substantially complementary to an uninterrupted portion of mRNA encoding GPR75.

[0158] Therefore, in some embodiments, the antisense strand polynucleotides disclosed herein are perfectly complementary to the target GPR75 sequence.

[0159] In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to the target GPR75 sequence, and GPR75 comprises a sequence of nucleotides that is at least about 80% complementary over its entire length to the nucleotide sequence of SEQ ID NOs. 1-4, or an equivalent region of the fragment of SEQ ID NOs. 1-4, for example, about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary.

[0160] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target GPR75 sequence and comprise a sequence of nucleotides that is at least about 80% over its entire length, for example, about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary to any one sense strand nucleotide sequence in any one of Tables 2, 3, 5, and 6, or a fragment of any one sense strand nucleotide sequence in any one of Tables 2, 3, 5, and 6.

[0161] In one embodiment, the RNAi agent of the present disclosure comprises a sense strand substantially complementary to an antisense polynucleotide which is identical to the target GPR75 sequence, wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence which is at least about 80%, for example, about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary over its entire length to the nucleotide sequence of SEQ ID NOs. 5-8 or an equivalent region of any fragment of SEQ ID NOs. 5-8.

[0162] In some embodiments, the iRNA of the present invention comprises a sense strand substantially complementary to an antisense polynucleotide, and then complementary to a target GPR75 sequence, wherein the sense strand polynucleotide comprises a sequence of nucleotides that is at least about 80% complementary over its entire length to any one antisense strand nucleotide sequence in any one of Tables 2, 3, 5, and 6, or any fragment of any one antisense strand nucleotide sequence in any one of Tables 2, 3, 5, and 6, e.g., about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary.

[0163] In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the fragments of the target GPR75 sequence, with nucleotides 38-60; 50-72; 148-181; 153-181; 153-175; 159-181; 228-250; 240-262; 341-363; 341-368; 346-368; 369-396; 369-391; 374-396; 388-410; 414-436; 424-461; 424-446; 424-451; 434-456; 439-461; 429-451; 45 7~504;462~504;462~491;482~504;469~491;457~479;462~584;475~497;469~491;509~537;509~531;515~537;544~576;544~566;549~571;580~60 7;580~602;585~607;595~617;615~647;615~637;620~642;620~647;625~647;773~806;773~795;773~795;778~800;784~806;837~872;837~859;843 ~872;843~865;850~872;860~882;889~911;900~936;900~922;908~936;908~930;914~936;938~990;938~960;943~965;968~990;1060~1101;1060~ 1082;1066~1088;1073~1095;1079~1101;1097~1119;1238~1260;1268~1290;1284~1393;1284~1306;1292~1393;1292~1314;1292~1383;1292~1314; 1301~1323;1307~1383;1307~1342;1307~1329;1313~1335;1371~1393;1351~1373;1320~1342;1336~1358;1345~1367;1351~1373;1361~1383;1366~ 1388;1393~1415;1422~1463;1422~1444;1441~1463;1487~1526;1487~1509;1493~1526;1493~1515;1498~1520;1504~1526;1515~1571;1515~1557;1515~1543;1515~1537;1521~1543;1530~1552;1535~1557;1540~1562;1549~1571;1559~1586;1559~1581;1564~1586;1583~1629;1583~1605;1588~1610;1595~1617;1600~1629;1600~1622;1607~1629;1624~1646;1635~1657;1672~1721;1672~1710;1677~1699;1699~1721;1672~1699;1688~1710;1672~1694;1683~1705;1693~1714;1732~1754;1744~1798;1751~1773;1758~1780;1767~1789;1776~1798;1790~1818;1790~1812;1796~1818;1808~1856;1808~1848;1808~1836;1808~1830;1826~1848;1814~1836;1819~1841;1834~1856;1877~2082;1877~1899;1882~2082;1882~1925;1882~1963;1882~1904;1887~1693;1887~1909;1898~1920;1903~1925;1908~1930;1913~1935;1913~1950;1921~1950;1921~1943;1928~1950;1933~1955;1941~1963;1946~1968;1953~1985;1953~2082;1953~1975;1938~1985;1958~1980;1963~1985;1968~1990;1974~1996;1974~2065;1974~2082;1974~2002;1980~2002;1985~2007;1990~2012;1990~2033;1999~2021;2005~2033;2005~2027;2011~2033;2017~2039;2025~2055;2025~2047;2033~2055;2038~2060;2043~2065;2033~2055;2048~2070;2054~2082;2054~2076;and includes a sequence of nucleotides that is at least 80% complementary over its entire length to the SEQ ID NO: 1 fragment selected from the group 2060-2082, e.g., approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary;

[0164] In some embodiments, the double-stranded region of the double-stranded iRNA agent is equal to the length of 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.

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

[0166] In some embodiments, the sense strand of the double-stranded iRNA agent is equal to 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 in length.

[0167] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are independently 15 to 30 nucleotides long.

[0168] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are independently 19 to 25 nucleotides long.

[0169] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are independently 21-23 nucleotides long.

[0170] In one embodiment, the sense strand of the iRNA agent is 21 nucleotides long, and the antisense strand is 23 nucleotides long, and the strands form a double-stranded region of 21 consecutive base pairs with a single-stranded overhang of 2 nucleotides long at the 3' end.

[0171] In one aspect of the present invention, the agent for use in the methods and compositions of the present invention is a single-stranded antisense nucleic acid molecule that inhibits target mRNA by an antisense inhibition mechanism. The single-stranded antisense RNA molecule is complementary to a sequence in the target mRNA. Single-stranded antisense oligonucleotides can inhibit translation in a stoichiometric manner by forming base pairs with mRNA and physically blocking the translation mechanism. See Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense RNA molecule may be about 15 to about 30 nucleotides long and have a sequence complementary to the target sequence. For example, the single-stranded antisense RNA molecule may include a sequence that is at least about 15, 16, 17, 18, 19, 20 or more consecutive nucleotides derived from any one of the antisense sequences described herein.

[0172] In one embodiment, at least partial repression of GPR75 gene expression is assessed by a reduction in the amount of GPR75 mRNA that can be isolated from or detected in the first treated cells or group of cells, compared to a second group of cells or cells (control cells) that are substantially identical to the first group of cells but not treated in the same way as the first, and in which GPR75 gene expression is inhibited. The degree of inhibition can be expressed in the following ways:

[0173]

number

[0174] In one embodiment, inhibition of expression is determined by a double luciferase assay in which the RNAi agent is present at 10 nM.

[0175] The phrase "contacting cells with an RNAi agent," such as dsRNA, as used herein, encompasses contacting cells by any possible means. Contacting cells with an RNAi agent includes contacting cells with an RNAi agent in vitro or in vivo. Contact may be direct or indirect. For example, an RNAi agent may be brought into physical contact with cells by performing the method individually, or an RNAi agent may be placed in a situation that allows or causes subsequent contact with cells.

[0176] Cell contact in vitro can be achieved, for example, by incubating cells with an RNAi agent. Cell contact in vivo can be achieved, for example, by injecting an RNAi agent into or near the tissue in which the cells are located, or by injecting an RNAi agent into another region, for example, the central nervous system (CNS), by intra-shelter injection, intravitreous injection, or other injection, as appropriate, or by injecting the agent into the bloodstream or subcutaneous space so that the agent subsequently reaches the tissue in which the cells to be contacted are located. For example, an RNAi agent may include, or be coupled to, a ligand that directs the RNAi agent to a site of interest, for example, the CNS, or otherwise stabilizes it, for example, a lipophilic moiety as described below and further detailed in, for example, PCT / US2019 / 031170, which is incorporated herein by reference. In some embodiments, an RNAi agent may include, or be coupled to, a ligand that directs the RNAi agent to a site of interest, for example, the liver, or otherwise stabilizes it, for example, one or more GalNAc derivatives as described below. In other embodiments, the RNAi agent may comprise a lipophilic moiety and one or more GalNAc derivatives, or may be coupled thereto. A combination of in vitro and in vivo methods for contact is also possible. For example, cells may be contacted with the RNAi agent in vitro and then transferred to the target.

[0177] In one embodiment, contacting cells with an RNAi agent includes “introducing” or “delivering the RNAi agent into cells” by promoting or carrying out uptake or absorption into the cells. Absorption or uptake of the RNAi agent may occur by spontaneously diffusive or active cellular processes, or by adjuvants or devices. Introducing the RNAi agent into cells may be in vitro or in vivo. For example, in the case of in vivo introduction, the RNAi agent 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 in this specification or are known in the art.

[0178] The term "lipophilic" or "lipophilic moiety" broadly refers to any compound or chemical moiety that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is by the octanol-water partition coefficient logK. ow This is by which, in this case, K ow The octanol-water partition coefficient is the ratio of the concentration of a chemical in the octanol phase to the concentration of a chemical in the aqueous phase in a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it can also be predicted by using a coefficient derived from the structural components of the chemical, calculated using first-principles or empirical methods [see, for example, Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), whose entirety is incorporated herein by reference]. It provides a thermodynamic measure of a substance's tendency to prefer non-aqueous or oily environments rather than water (i.e., the hydrophilic / lipophilic balance). In principle, a chemical is logK ow If logK is greater than 0, it is lipophilic. 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. owis expected to be approximately 0.7. By using the same method, the logK of cholesteryl N-(hexan-6-ol) carbamate ow is expected to be 10.7.

[0179] The lipophilicity of the molecule can be altered with respect to the functional groups the molecule has. For example, by adding a hydroxyl group or an amine group to the end of the lipophilic moiety, the partition coefficient (e.g., logK ow ) value of the lipophilic moiety can be increased or decreased.

[0180] Alternatively, the hydrophobicity of a double-stranded RNAi agent conjugated to one or more lipophilic moieties can be measured by its protein binding properties. For example, in certain embodiments, it can be determined that the unbound fraction of a plasma protein binding assay of a double-stranded RNAi agent is positively correlated with the relative hydrophobicity of the double-stranded RNAi agent, which can be positively correlated with the silencing activity of the double-stranded RNAi agent.

[0181] In one embodiment, the plasma protein binding assay to be determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. An exemplary protocol for this binding assay is described in detail, for example, in PCT Publication No. WO2019 / 217459. The hydrophobicity of the double-stranded RNAi agent measured by the fraction of unbound siRNA in the binding assay is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45, or greater than 0.5 for enhanced in vivo delivery of the siRNA.

[0182] Thus, conjugating the lipophilic moiety to an internal position of the double-stranded RNAi agent provides an optimal hydrophobicity for enhanced in vivo delivery in siRNA.

[0183] The term “lipid nanoparticle” or “LNP” refers to a vesicle containing a lipid layer that encapsulates a pharmaceutically active molecule, such as a nucleic acid molecule, such as an RNAi agent or a plasmid from which an RNAi agent 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.

[0184] As used herein, “Subject” refers to animals such as primates (humans, non-human primates, e.g., monkeys, and chimpanzees), mammals (including cattle, pigs, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, or mice), or birds that express the target gene endogenously or heterologously. In one embodiment, the subject is a human being treated or evaluated for a disease, disorder, or condition that would benefit from reduced GPR75 expression; a human being at risk for a disease, disorder, or condition that would benefit from reduced GPR75 expression; a human being having a disease, disorder, or condition that would benefit from reduced GPR75 expression; or a human being treated for a disease, disorder, or condition that would benefit from reduced GPR75 expression as described herein. In some embodiments, the subject is a human female. In other embodiments, the subject is a human male. In one embodiment, the subject is a human adult. In another embodiment, the subject is a child subject.

[0185] As used herein, the terms “to treat” or “treatment” mean a beneficial or desired outcome, for example, the alleviation or improvement of one or more signs or symptoms associated with GPR75 expression or GPR75 protein production, for example, GPR75-related disorders, for example, obesity, or symptoms associated with undesirable GPR75 expression; a reduction in the degree of undesirable GPR75 activation or stabilization; or improvement or mitigation of undesirable GPR75 activation or stabilization. “Treatment” may also mean an extension of survival compared to the survival expected if no treatment is performed.

[0186] The term “lower” in relation to GPR75 levels or disease markers or symptoms in a subject means a statistically significant decrease in such levels. A decrease could be, for example, at least 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In certain embodiments, the decrease is at least 20%. In certain embodiments, the decrease is at least 50% of a disease marker, e.g., a protein level or gene expression level. Where relating to GPR75 levels in a subject, “lower” means reducing to a level acceptable as within the normal range for an individual without such disorder. In certain embodiments, the expression of a target is normalized, i.e., reduced to a level acceptable as within the normal range for an individual without such disorder, e.g., BMI, blood glucose level, blood lipid level, blood oxygen level, white blood cell count, renal function, spleen function, liver function. As used herein, “reduce” in a subject means a reduction in gene expression or protein production in cells within the subject, and does not require a reduction in expression in all cells or tissues of the subject. For example, as used herein, “reduce” in a subject may include a reduction in gene expression or protein production within the subject.

[0187] The term “reduce” can also be used in relation to normalizing the symptoms of a disease or condition, i.e., reducing the difference between the level in a subject with a GPR75-related disease and the level in a normal subject without a GPR75-related disease toward or to the level of a normal subject without a GPR75-related disease. As used herein, “normal” is considered the upper limit of normal when the disease is associated with an elevated level of symptoms. “Normal” is considered the lower limit of normal when the disease is associated with a reduced level of symptoms.

[0188] As used herein, "prevention" or "preventing" when used in reference to a disease, disorder, or condition that would benefit from a decrease in the expression of the GPR75 gene or the production of the GPR75 protein, means that the subject has a reduced likelihood of developing symptoms associated with such disease, disorder, or condition, such as symptoms of a GPR75-related disease, such as a weight disorder such as obesity, such as diabetes, or lipid metabolism disorders. Not developing a disease, disorder, or condition, or a decrease in the development of symptoms associated with such disease, disorder, or condition (e.g., at least about a 10% decrease on a clinically acceptable scale for the disease or disorder), or a delay in the presentation of symptoms (e.g., days, weeks, months, or years) is considered effective prevention.

[0189] As used herein, the term "GPR75-related disease" is a disease or disorder that would benefit from a decrease in the expression or activity of GPR75. The term "GPR75-related disease" is a disease or disorder caused by or associated with GPR75 expression or GPR75 protein production. The term "GPR75-related disease" includes diseases, disorders, or conditions that would benefit from a decrease in the expression or GPR75 protein activity of GPR75. Non-limiting examples of GPR75-related diseases include weight disorders such as obesity.

[0190] As used herein, "weight disorder" is a disorder associated with abnormal or excessive fat accumulation and body weight. Such disorders may include obesity, independent components of metabolic syndrome (e.g., central obesity, FBG / pre-diabetes / diabetes, hypercholesterolemia, hypertriglyceridemia, and hypertension), hypometabolic states, hypothyroidism, uremia, and other conditions associated with the risk of weight gain (including rapid weight gain), weight maintenance after weight loss, or weight regain after weight loss, including metabolic syndrome.

[0191] Weight can be evaluated by the body mass index (BMI), which is the individual's weight in kilograms divided by the square of his or her height in meters. A BMI less than about 18.5 indicates that the subject is underweight; a BMI from about 18.5 to less than about 25 indicates that the subject is of normal weight; a BMI of about 30.0 or higher indicates that the subject is obese.

[0192] Additional diseases or conditions associated with weight disorders will be apparent to those skilled in the art and are within the scope of this disclosure.

[0193] Symptoms related to GPR75-related diseases, such as weight disorders such as obesity, include, for example, excessive fat mass, a BMI of about 25 or higher, an increased body mass index, a lower metabolic rate, central obesity, FBG / pre-diabetes / diabetes, hypercholesterolemia, hypertriglyceridemia, and hypertension, insulin resistance, lack of ability to regulate blood sugar, high blood glucose levels, diabetes, and / or excessive weight gain. Further details regarding the signs and symptoms of various diseases or conditions are provided herein and are known in the art.

[0194] "Therapeutically effective amount", as used herein, when administered to a subject having a GPR75-related disease, is intended to include an amount of an RNAi agent sufficient to effect the treatment of the disease (eg, by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of the disease). A "therapeutically effective amount" can vary depending on the RNAi agent, how the agent is administered, the disease and its severity, as well as the medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, and other individual characteristics of the subject being treated, if any.

[0195] When used herein, “Prophylactic effective dose” is intended to include an amount of RNAi agent sufficient to prevent or improve one or more symptoms of a disease or disease when administered to a subject with a GPR75-related disorder, e.g., weight disorder, e.g., obesity. Improvement of disease includes slowing the course of the disease or reducing the severity of the disease if it develops later. The “Prophylactic effective dose” may vary depending on the RNAi agent, how the drug is administered, the degree of risk of the disease, and the patient’s medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, and any other individual characteristics of the treated patient.

[0196] The “therapeutic dose” or “preventive dose” also includes the amount of RNAi agent that produces several desired local or systemic effects in a reasonable benefit / risk ratio applicable to any treatment. The RNAi agent used in the method of this disclosure may be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0197] The term "pharmaceutically acceptable" is used herein to mean a compound, material, composition, or dosage form that is suitable for use in contact with the tissues of human and animal subjects within the bounds of sound medical judgment, at a reasonable benefit / risk ratio, without excessive toxicity, irritation, allergic reaction, or other problems or complications.

[0198] When used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material, that is involved in the transport or delivery of the compound of interest from one organ or part of the body to another organ, e.g., another part of the body. Each carrier must be “acceptable” in the sense that it is compatible with the other raw materials of the formulation and must not be harmful to the subject being treated. Some examples of materials that can function as pharmaceutically acceptable carriers include: (1) sugars, e.g., lactose, glucose, and sucrose; (2) starches, e.g., corn starch and potato starch; (3) cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) lubricants, e.g., magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, e.g., cocoa butter and suppository waxes; (9) oils, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil. (10) Glycols, e.g., propylene glycol; (11) Polyols, e.g., glycerin, sorbitol, mannitol, and polyethylene glycol; (12) Esters, e.g., ethyl oleate and ethyl laurate; (13) Agar; (14) Buffers, e.g., magnesium hydroxide and aluminum hydroxide; (15) Alginic acid; (16) Phenothermally hydrated; (17) Isotonic saline; (18) Ringer's solution; (19) Ethyl alcohol; (20) pH buffer solution; (21) Polyesters, polycarbonates, or polyanhydrides; (22) Bulking agents, e.g., polypeptides and amino acids; (23) Serum components, e.g., serum albumin, HDL, and LDL; and (22) other non-toxic affinity substances used in pharmaceutical formulations.Pharmacochemically acceptable carriers for pulmonary delivery are known in the art and will vary depending on the desired location for drug deposition, e.g., the upper or lower respiratory tract, and the type of device used for delivery, e.g., nebulizers, dry powder inhalers.

[0199] The term “sample,” as used herein, encompasses similar bodily fluids, cells, or tissues isolated from a subject, as well as collections of bodily fluids, cells, or tissues present within the subject. Examples of bodily fluids include blood, serum and serous fluid, plasma, bronchial fluid, sputum, cerebrospinal fluid, ocular fluid, lymph, urine, saliva, and sputum. Tissue samples may include samples from tissues, organs, or local areas. For example, a sample may be obtained from a specific organ, a part of an organ, or bodily fluids or cells within those organs. In certain embodiments, a sample may originate from the brain [e.g., the entire brain or a specific segment of the brain, e.g., the striatum, or a specific type of cell in the brain, e.g., neurons and glial cells (astrocytes, oligodendrocytes, microglia)]. In other embodiments, “sample derived from subject” means liver tissue (or its subcomponents) derived from the subject. In some embodiments, “sample obtained from subject” means blood obtained from the subject or plasma or serum obtained therefrom. In further embodiments, “subject-derived sample” means brain tissue (or its minor components) or retinal tissue (or its minor components) derived from the subject.

[0200] II. RNAi agents of the present disclosure This specification describes RNAi agents that inhibit the expression of the GPR75 gene. In one embodiment, the RNAi agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of the GPR75 gene in cells of a subject, such as a mammal such as a human, such as a subject with a GPR75-related disorder, such as a weight disorder, such as obesity, or a subject at risk of a GPR75-related disease.

[0201] The dsRNA contains an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed in the expression of the target RNA, e.g., the GPR75 gene. The complementary region is approximately 15 to 30 nucleotides in length or less. In contact with cells expressing the GPR75 gene, the RNAi agent inhibits the expression of the GPR75 gene (e.g., human gene, primate gene, non-primate gene) by at least 50% when assayed by, for example, PCR or branched DNA (bDNA) based methods, or protein-based methods, such as immunofluorescence analysis using Western blotting or flow cytometry techniques. In certain embodiments, the inhibition of expression is at least 50% when assayed by the Dual-Glo luciferase assay in Example 1, where the siRNA is at a concentration of 10 nM.

[0202] dsRNA comprises two RNA strands that are complementary and hybridize to form a double-stranded structure under the conditions in which the dsRNA is used. One strand of the dsRNA (the antisense strand) contains a complementary region that is substantially complementary and generally perfectly complementary to the target sequence. For example, the target sequence can be obtained from the sequence of mRNA formed during the expression of the GPR75 gene. The other strand (the sense strand) contains a region complementary to the antisense strand, thereby the two strands hybridize to form a double-stranded structure when combined under suitable conditions. As described elsewhere in this specification and known in the art, the complementary sequence of the dsRNA can also be included as a self-complementary region of a single nucleic acid molecule so as to be relative on separate oligonucleotides.

[0203] Generally, double-stranded structures are 15 to 30 base pairs long, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-2 The lengths are 9, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. In certain embodiments, the double-stranded structure is 18 to 25 base pairs long, for example, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24, 20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24, or 24-25 base pairs long, for example, 19-21 base pairs long. It is conceivable that intermediate ranges and lengths between those listed above are also part of this disclosure.

[0204] Similarly, the complementary region to the target sequence is 15 to 30 nucleotides long, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19- 27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotide lengths, for example, 19-23 nucleotide lengths or 21-23 nucleotide lengths. It is conceivable that intermediate ranges and lengths between the above-listed ranges and lengths are also part of this disclosure.

[0205] In some embodiments, dsRNAs are 15 to 23 nucleotides long, or 25 to 30 nucleotides long. Generally, dsRNAs are long enough to function as substrates for Dicer enzymes. For example, it is well known in the art that dsRNAs longer than about 21–23 nucleotides can function as substrates for Dicer. As those skilled in the art will also recognize, the RNA region targeted for cleavage is in most cases a longer RNA molecule, often a portion of an mRNA molecule. Where applicable, the “portion” of the mRNA target is a sequence of mRNA targets long enough to allow it to be a substrate for RNAi-dependent cleavage (i.e., cleavage via the RISC pathway).

[0206] Those skilled in the art will know that the double-stranded region is the primary functional portion of dsRNA, for example, 15 to 36 base pairs, for example, 15-36, 15-35, 15-34, 15-33, 15-32, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 1 You will also recognize that these are 8-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs, for example, a double-stranded region of 19-21 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 is a dsRNA, insofar as it is processed into a functional double helix of, for example, 15-30 base pairs, which targets the desired RNA for cleavage. Thus, those skilled in the art will recognize that in one embodiment, miRNA is a dsRNA. In another embodiment, dsRNA is not a naturally occurring miRNA. In another embodiment, an RNAi agent useful for targeting GPR75 is not generated in the target cell by cleavage of a larger dsRNA.

[0207] The dsRNA described herein may further include one or more single-stranded nucleotide overhangs, for example, 1, 2, 3, or 4 nucleotides. The nucleotide overhang may include or consist of nucleotide / nucleoside analogs such as deoxynucleotides / nucleosides. The overhang may be on the sense strand, on the antisense strand, or any combination thereof. Furthermore, the nucleotides of the overhang may be located on the 5' end, the 3' end, or both of either the antisense strand or the sense strand of the dsRNA. In certain embodiments, longer, elongated overhangs are possible.

[0208] dsRNA can be synthesized by standard methods known in the art, as further discussed below, for example, by using automated DNA synthesizers, such as those commercially available from Biosearch, Applied Biosystems, Inc.

[0209] The iRNA compounds of the present invention can be prepared using a two-step method. First, the individual strands of a double-stranded RNA molecule are prepared separately. Then, the strands of the component are annealed. Individual strands of an siRNA compound can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that oligonucleotide chains containing non-natural or modified nucleotides can be easily prepared. The single-stranded oligonucleotides of the present invention can be prepared using solution-phase or solid-phase organic synthesis or both.

[0210] siRNA can be produced in large quantities by various methods. Exemplary methods include organic synthesis and RNA cleavage, such as in vitro cleavage.

[0211] siRNA can be prepared by separately synthesizing the corresponding strands of single-stranded RNA molecules or double-stranded RNA molecules, and then the component strands can be annealed.

[0212] Large bioreactors, such as the OligoPilot II from Pharmacia Biotec AB (Uppsala, Sweden), can be used to generate large quantities of specific RNA strands for a given siRNA. The OligoPilot II reactor can efficiently couple nucleotides using only a 1.5 molar excess of phosphoramidite nucleotides. Ribonucleotide amidites are used to create the RNA strands. Standard cycles of monomer addition can be used to synthesize 21 - 23 nucleotide strands for siRNA. Typically, two complementary strands are generated separately and then annealed, for example, after release from a solid support and deprotection.

[0213] Individual siRNA species can be generated using organic synthesis. The complementarity of those species to the GPR75 gene can be accurately identified. For example, those species can be complementary to regions containing polymorphisms, such as single nucleotide polymorphisms. Furthermore, the position of the polymorphism can be accurately defined. In some embodiments, the polymorphism is located in an internal region, for example, at least 4, 5, 7, or 9 nucleotides from one or both ends.

[0214] In one embodiment, the generated RNA is carefully purified and removed, and for example, end-siRNA is cleaved to siRNA in vitro using Dicer or an equivalent RNaseIII-based activity. For example, dsiRNA can be incubated in an in vitro extract from Drosophila or using purified components, such as purified RNase or the 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.

[0215] dsiRNA cleavage typically generates multiple siRNA species, each consisting of a specific 21-23 nucleotide 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.

[0216] Regardless of the synthesis method, siRNA preparations can be prepared in a solution suitable for formulation (e.g., aqueous or organic solution). For example, the siRNA preparation can be precipitated, redissolved in pure double-distilled water, and lyophilized. The dried siRNA can then be resuspended in a solution suitable for the intended formulation process.

[0217] In one embodiment, the dsRNA of the present disclosure comprises at least two nucleotide sequences, namely a sense sequence and an antisense sequence. The sense strand sequence for GPR75 may be selected from the group of sequences provided in any one of Tables 2, 3, 5, and 6, and the corresponding nucleotide sequence of the sense strand and the antisense strand may be selected from the group of sequences in any one of Tables 2, 3, 5, and 6. In this embodiment, one of the two sequences is complementary to the other of the two sequences, and one sequence is substantially complementary to the sequence of mRNA produced in the expression of the GPR75 gene. Thus, in this embodiment, the dsRNA would comprise two oligonucleotides with respect to GPR75, one oligonucleotide described as the sense strand (passenger strand) in any one of Tables 2, 3, 5, and 6, and a second oligonucleotide described as the corresponding antisense strand (guide strand) of the sense strand in any one of Tables 2, 3, 5, and 6.

[0218] In one embodiment, a substantially complementary sequence to the dsRNA is contained in separate oligonucleotides. In another embodiment, a substantially complementary sequence to the dsRNA is contained in a single oligonucleotide.

[0219] While the sequences provided herein are described as modified or conjugated sequences, it will be understood that the RNA of the RNAi agent disclosed herein, for example, the dsRNA disclosed herein, may include any one of the sequences in Tables 2, 3, 5, and 6, which may be unmodified, unconjugated, or modified or conjugated in a manner different from those described herein. One or more lipophilic ligands or one or more GalNAc ligands may be included at any of the positions of the RNAi agent provided herein.

[0220] Those skilled in the art are well aware that dsRNAs having double-stranded structures of about 20 to 23 base pairs, for example, 21 base pairs, have been welcomed as particularly effective in introducing RNA interference [Elbashir et al., (2001) EMBO J., 20:6877-6888]. However, others have found that shorter or longer RNA double-stranded structures may also be effective [Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226]. In the embodiments described above, due to the nature of the oligonucleotide sequences provided herein, the dsRNAs described herein may include at least one strand of a minimum length of 21 nucleotides. It can be reasonably expected that shorter double-stranded structures, with some nucleotides subtracted from one or both ends, may be equally effective compared to the dsRNAs described above. Accordingly, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more consecutive nucleotides obtained from one of the sequences provided herein, and which differ in their ability to inhibit the expression of the GPR75 gene by 10, 15, 20, 25, or 30% or less inhibition from dsRNAs containing the complete sequence using in vitro assays with Cos7 and 10 nM concentration RNA agents and PCR assays provided in the examples herein, are conceivable to be within the scope of this disclosure.

[0221] In addition, the RNAs described herein identify sites in the GPR75 transcript that are susceptible to RISC-mediated cleavage. Therefore, this disclosure further features RNAi agents that target these sites. As used herein, an RNAi agent is said to target a specific site within an RNA transcript if it facilitates cleavage of the transcript at any of these specific sites. Such an RNAi agent would generally consist of at least about 15 consecutive nucleotides, such as at least 19 nucleotides, from one of the sequences provided herein coupled to an additional nucleotide sequence taken from a region adjacent to a selected sequence in the GPR75 gene.

[0222] The RNAi agents described herein may contain one or more mismatches with respect to the target sequence. In one embodiment, the RNAi agents described herein contain three or fewer mismatches (i.e., three, two, one, or zero mismatches). In one embodiment, the RNAi agents described herein contain two or fewer mismatches. In one embodiment, the RNAi agents described herein contain one or fewer mismatches. In one embodiment, the RNAi agents described herein contain zero mismatches. In certain embodiments, if the antisense strand of the RNAi agent contains mismatches with respect to the target sequence, the mismatches may, as appropriate, be limited to the last five nucleotides from the 5' or 3' end of the complementary region. For example, in such embodiments, for a 23-nucleotide RNAi agent, the strand complementary to the GPR75 gene region generally does not contain any mismatches within the central 13 nucleotides. Using the methods described herein or methods known in the art, it can be determined whether an RNAi agent containing mismatches with respect to the target sequence is effective in inhibiting the expression of the GPR75 gene. In particular, if specific complementary regions in the GPR75 gene are known to mutate, it is important to consider the effectiveness of RNAi agents with mismatches that inhibit GPR75 gene expression.

[0223] III. Modified RNAi agents of this disclosure In one embodiment, the RNA of the RNAi agent of the Disclosure, e.g., dsRNA, is unmodified and does not contain, for example, chemical modifications or conjugations known in the Art and described herein. In some embodiments, the RNA of the RNAi agent 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 RNAi agent of the Disclosure are modified. In other embodiments of the Disclosure, all of the nucleotides of the RNAi agent of the Disclosure are modified. The RNAi agent of the Disclosure that is "substantially all of its nucleotides modified" is modified, but not entirely, and may contain 5, 4, 3, 2, or 1 or fewer unmodified nucleotides. In yet another embodiment of the Disclosure, the RNAi agent of the Disclosure may contain 5, 4, 3, 2, or 1 or fewer modified nucleotides.

[0224] The nucleic acids featured in this disclosure can be synthesized or modified by methods well established in the art, for example, those described in "Current protocols in nucleic acid chemistry," Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, reverse linking) or 3'-end modifications (conjugation, DNA nucleotides, reverse linking, etc.), base modifications, such as replacement of stabilizing bases, destabilizing bases or bases that form base pairs with partners in an expanded repertoire, base removal (debasing nucleotides) or conjugated bases, sugar modifications (e.g., at the 2' or 4' position) or sugar replacement, or skeletal modifications including modification or replacement of phosphodiester bonds. Specific examples of RNAi agents useful in the embodiments described herein, but not limited to, RNA containing a modified skeleton or lacking natural nucleoside linkages, include RNA containing a modified skeleton or lacking natural nucleoside linkages. Among RNAs having a modified skeleton, those lacking a phosphorus atom in their skeleton are particularly noteworthy. For the purposes of this specification, as sometimes mentioned in the art, modified RNAs lacking a phosphorus atom in their internucleoside skeleton can also be considered oligonucleosides. In some embodiments, the modified RNAi agent has a phosphorus atom in its internucleoside skeleton.

[0225] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methylphosphonates, and other alkylphosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates having the usual 3'-5' linkage, their analogues with 2'-5' linkages, and those with reverse polarity where adjacent pairs of nucleoside units are linked from 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, such as sodium salts, mixed salts, and free acid forms are also included.

[0226] Representative U.S. patents teaching the preparation of the phosphorus-containing linkages described above include, but are not limited to, U.S. Patents 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, and 5,286,71 No. 7, No. 5,321,131, No. 5,399,676, No. 5,405,939, No. 5,453,496, No. 5,455,233, No. 5,466,677, No. 5,476 , No. 925, No. 5,519,126, No. 5,536,821, No. 5,541,316, No. 5,550,111, No. 5,563,253, No. 5,571,799, No. 5,5 87,361, 5,625,050, 6,028,188, 6,124,445, 6,160,109, 6,169,170, 6,172,209, No. 6,239,265, No. 6,277,603, No. 6,326,199, No. 6,346,614, No. 6,444,423, No. 6,531,590, No. 6,534,639 Examples include U.S. Patent Nos. 6,608,035, 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029 and U.S. Reissue Patent No. RE39464, the entirety of each of these is incorporated herein by reference.

[0227] Modified RNA skeletons that do not contain phosphorus atoms have skeletons formed by short alkyl or cycloalkyl nucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl nucleoside linkages, or one or more short heteroatoms or heterocyclic nucleoside linkages. These include morpholino linkages (some formed from the sugar moiety of nucleosides), siloxane skeletons, sulfide, sulfoxide and sulfone skeletons, formacetyl and thioformacetyl skeletons, methyleneformacetyl and thioformacetyl skeletons, alkene-containing skeletons, sulfamate skeletons, methyleneimino and methylenehydrazino skeletons, sulfonate and sulfonamide skeletons, amide skeletons, and others having mixed N, O, S and CH2 component moieties.

[0228] Representative U.S. patents teaching the preparation of the above-mentioned oligonucleotides include, but are not limited to, U.S. Patents 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,64,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, and the same. Nos. 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439 are examples, the entire contents of each of these are incorporated herein by reference.

[0229] In other embodiments, RNA mimetics suitable for use in RNAi agents are envisioned in which both sugar and nucleoside linkages, i.e., the nucleotide unit backbone, are replaced with novel groups. The base units are maintained for hybridization with suitable nucleic acid target compounds. One such oligomeric compound, an RNA mimetic known to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, in particular an aminoethylglycine backbone. The nucleic acid bases are retained and directly or indirectly bonded to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds, but not limited to, U.S. Patents 5,539,082, 5,714,331, and 5,719,262, the entire contents of each of which are incorporated herein by reference. Further PNA compounds suitable for use in the RNAi agents of this disclosure are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0230] Some embodiments featured in this disclosure include RNAs and heteroatom skeletons having a phosphorothioate backbone, in particular the --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2--[known as the methylene(methylimino) or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2-- and --N(CH3)--CH2--CH2-- and oligonucleosides having an amide backbone as referenced in U.S. Patent No. 5,602,240. In some embodiments, the RNAs featured herein have a morpholino backbone structure as referenced in U.S. Patent No. 5,034,506. Natural phosphodiester backbones may be represented as OP(O)(OH)-OCH2-.

[0231] Modified RNA may also contain one or more substituted sugar moieties. RNAi agents characterized herein, such as dsRNA, may contain one of the following at the 2' position: OH;F;O-, S- or N-alkyl;O-, S- or N-alkenyl;O-, S- or N-alkynyl or O-alkyl-O-alkyl, where alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1-C 10 Alkyl or C2-C 10 It can be an alkenyl or alkinyl. An exemplary suitable modification is O[(CH2) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n CH3)2 is an example, where n and m are approximately 1 to 10. In other embodiments, the dsRNA is located at the 2' position as follows: C1~C 10The modifications include one of the following: lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, interfering substance, group for improving the pharmacokinetic properties of RNAi agents or group for improving the pharmacokinetic properties of RNAi agents, and other substituents having similar properties. In some embodiments, the modifications include 2'-methoxyethoxy (2'-O-(2-methoxyethyl) or 2'-MOE, also known as 2'-O--CH2CH2OCH3) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Other exemplary modifications include 2'-dimethylaminooxyethoxy, i.e., the O(CH2)2ON(CH3)2 group also known as 2'-DMAOE, as described below in the examples herein, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH3)2. Further exemplary modifications include 5'-Me-2'-F nucleotide, 5'-Me-2'-OMe nucleotide, 5'-Me-2'-deoxynucleotide (both R and S isomers in these three families), 2'-alkoxyalkyl, and 2'-NMA (N-methylacetamide).

[0232] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), 2'-O-hexadecyl, and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of the RNAi agent, particularly on the 3' terminal nucleotide or at the 3' position and 5' position of the sugar in the 2'-5' ligated dsRNA. The RNAi agent may also have sugar mimetic moieties, such as a cyclobutyl moiety instead of a pentofuranosyl sugar. Representative U.S. patents teaching the preparation of such modified sugar structures include, but are not limited to, U.S. Patents 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, and 5,56 Examples include patents 7,811, 5,576,427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, and 5,700,920, some of which are jointly owned with this application. The entire content of each of the aforementioned is incorporated herein by reference.

[0233] The RNAi agents of this disclosure may also include modifications or substitutions of nucleic acid bases (often simply referred to as “bases” in the art). As used herein, “unmodified” or “natural” nucleic acid bases include the purine bases adenine (A) and guanine (G), the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include other synthetic and natural nucleic acid bases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, and 5-uracil. This includes (pseudracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and (anal) other 8-substituted adenines and guanines, 5-halo, in particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine (daazaadenine), as well as 3-deazaguanine and 3-deazaadenine.Further nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808, Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008, The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleic acid bases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in this disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitution has been shown to increase nucleic acid double-strand stability by 0.6–1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276–278), and more specifically, is an exemplary base substitution when combined with 2'-O-methoxyethyl sugar modification.

[0234] Representative U.S. patents teaching the preparation of the above-mentioned modified nucleic acid bases and certain other modified nucleic acid bases include, but are not limited to, U.S. Patents 3,687,808, 4,845,205, 5,130,30, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, and 5,587,469, Reference numbers 5,594,121, 5,596,091, 5,614,617, 5,681,941, 5,750,692, 6,015,886, 6,147,200, 6,166,197, 6,222,025, 6,235,887, 6,380,368, 6,528,640, 6,639,062, 6,617,438, 7,045,610, 7,427,672, and 7,495,088 are examples, the entire contents of each of these are incorporated herein by reference.

[0235] The RNAi agents of this disclosure can also be modified to include one or more bicyclic sugar moieties. A “bicyclic sugar” is a furanosyl ring modified by a ring formed by a bridge between two carbon atoms, whether adjacent or non-adjacent. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety that includes a ring formed by a bridge that connects two carbon atoms of the sugar ring, whether adjacent or non-adjacent, thereby forming a bicyclic ring system. In certain embodiments, the bridge optionally connects the 4'-carbon and 2'-carbon of the sugar ring via a 2'-acyclic oxygen atom. Thus, in some embodiments, the agents of this disclosure may include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety, wherein the ribose moiety includes an additional bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide having a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge. This structure efficiently "locks" ribose into the 3'-end conformation. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects [Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447, Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843, Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193]. Examples of bicyclic nucleosides for use in the polynucleotides of this disclosure include, but are not limited to, nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, one or more bicyclic nucleosides containing a 4'-to-2' bridge may be used as antisense polynucleotide agents of this disclosure.

[0236] Locked nucleosides have a structure (stereochemistry is omitted).

[0237] [ka] (In the formula, B is a nucleic acid base or a modified nucleic acid base, and L is a linking group that bonds the 2'-carbon of the ribose ring to the 4'-carbon.) It can be represented by [this].

[0238] Examples of such 4'-to-2' crosslinked bicyclic nucleosides include, but are not limited to, 4'-(CH2)-O-2'(LNA), 4'-(CH2)-S-2', 4'-(CH2)2-O-2'(ENA), 4'-CH(CH3)-O-2' (also known as "restricted ethyl" or "cEt") and 4'-CH(CH2OCH3)-O-2' (and its analogues, see, e.g., U.S. Patent No. 7,399,845), 4'-C(CH3)(CH3)-O-2' (and its analogues, see, e.g., U.S. Patent No. 8,2 See Patent No. 78,283), 4'-CH2-N(OCH3)-2' (and its analogues, e.g., see U.S. Patent No. 8,278,425), 4'-CH2-ON(CH3)-2' (e.g., see U.S. Patent Publication No. 2004 / 0171570), 4'-CH2-N(R)-O-2' (wherein R is H, C1-C12 alkyl or nitrogen protecting group) (e.g., see U.S. Patent No. 7,427,672), 4'-CH2-C(H)(CH3)-2' (e.g., Chattopadhyaya Examples include 4'-CH2-C(-CH2)-2' (and its analogues, see, for example, U.S. Patent No. 8,278,426), and 4'-CH2-C(-CH2)-2' (see, et al., J. Org. Chem., 2009, 74, 118-134). The entire contents of each of the foregoing are incorporated herein by reference.

[0239] Further representative U.S. patents and U.S. patent publications teaching the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490, 6,525,191, 6,670,461, 6,770,748, 6,794,499, 6,998,484, 7,053,207, 7,034,133, 7,084,125, and the same. Examples include patent numbers 7,399,845, 7,427,672, 7,569,686, 7,741,457, 8,022,193, 8,030,467, 8,278,425, 8,278,426, 8,278,283, US2008 / 0039618, and US2009 / 0012281, the entire contents of each of these are incorporated herein by reference.

[0240] For example, any of the aforementioned bicyclic nucleosides having one or more stereochemical sugar configurations, including α-L-ribofuranose and β-D-ribofuranose, can be prepared (see WO99 / 14226).

[0241] The RNAi agents of this disclosure can also be modified to include one or more restricted ethyl nucleotides. As used herein, “restricted ethyl nucleotide” or “cEt” is a locked nucleic acid containing a bicyclic sugar moiety including a 4'-CH(CH3)-O~2' bridge (i.e., L in the structure). In one embodiment, the restricted ethyl nucleotide is in the S conformation and is referred to herein as “S-cEt”.

[0242] The RNAi agents of this disclosure may also comprise one or more “conformation-restricted nucleotides” (“CRNs”). CRNs are nucleotide analogs having a linker connecting the C2' and C4' carbons of ribose, or the -C3' and -C5' carbons of ribose. CRNs lock the ribose ring into a stable conformation and increase hybridization affinity to mRNA. The linker is long enough to position the oxygen in an optimal position for stability and affinity, resulting in less ribose ring puckering.

[0243] Representative publications that instruct the preparation of certain CRNs mentioned above include, but are not limited to, US2013 / 0190383 and WO2013 / 036868, the entire contents of which are incorporated herein by reference.

[0244] In some embodiments, the RNAi agents of this disclosure comprise one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA are unlocked acyclic nucleic acids in which any sugar bond has been removed, forming an unlocked "sugar" residue. In one example, UNA also encompass monomers in which the bond between C1'-C4' (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 Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, incorporated herein by reference].

[0245] Representative U.S. publications teaching the preparation of UNAs include, but are not limited to, U.S. 8,314,227 and U.S. Patent Publications 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020, the entire contents of each of which are incorporated herein by reference.

[0246] Potentially stabilizing modifications to the ends of RNA molecules include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyluridine-3'-phosphate, reverse base dT (idT), and others. The disclosure of these modifications can be found in WO2011 / 005861.

[0247] Other modifications of the RNAi agents of this disclosure include 5' phosphates or 5' phosphate mimics, for example, a 5' terminal phosphate or phosphate mimic on the antisense strand of the RNAi agent. Suitable phosphate mimics are disclosed, for example, in US2012 / 0157511, the entirety of which is incorporated herein by reference.

[0248] A. Modified RNAi agents containing motifs of the present disclosure In certain embodiments of this disclosure, the double-stranded RNAi agents of this disclosure include agents having chemical modifications such as those disclosed in WO2013 / 075035, the entirety of which is incorporated herein by reference. As shown herein and in WO2013 / 075035, one or more motifs of three identical modifications on a triple nucleotide can 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 otherwise be fully modified. The introduction of these motifs disrupts the modification pattern of the sense or antisense strand, if present. The RNAi agent may be conjugated with a lipophilic ligand, for example, a C16 ligand on the sense strand. The RNAi agent may be modified, for example, with (S)-glycol nucleic acid (GNA) modification at one or more residues on the antisense strand.

[0249] Accordingly, this disclosure provides a double-stranded RNAi agent capable of inhibiting the expression of a target genome or gene (i.e., the GPR75 gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may be 15 to 30 nucleotides long. For example, each strand may be 16 to 30 nucleotides long, 17 to 30 nucleotides long, 25 to 30 nucleotides long, 27 to 30 nucleotides long, 17 to 23 nucleotides long, 17 to 21 nucleotides long, 17 to 19 nucleotides long, 19 to 25 nucleotides long, 19 to 23 nucleotides long, 19 to 21 nucleotides long, 21 to 25 nucleotides long, or 21 to 23 nucleotides long. In a particular embodiment, each strand is 19 to 23 nucleotides long.

[0250] The sense strand and antisense strand typically form a double-stranded RNA ("dsRNA"), also referred herein as the "RNAi agent." The double-stranded region of the RNAi agent may be 15–30 nucleotide pairs long. For example, the double-stranded region may be 16–30 nucleotide pairs long, 17–30 nucleotide pairs long, 27–30 nucleotide pairs long, 17–23 nucleotide pairs long, 17–21 nucleotide pairs long, 17–19 nucleotide pairs long, 19–25 nucleotide pairs long, 19–23 nucleotide pairs long, 19–21 nucleotide pairs long, 21–25 nucleotide pairs long, or 21–23 nucleotide pairs long. In another example, the double-stranded region is selected from lengths of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides. In some embodiments, the double-stranded region is 19–21 nucleotide pairs long.

[0251] In one embodiment, the RNAi agent may contain one or more overhang regions or capping groups at the 3' end, 5' end, or both ends of one or both strands. The overhangs may be 1 to 6 nucleotides long, e.g., 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides. In some embodiments, the nucleotide overhang region is 2 nucleotides long. The overhangs may result from one strand being longer than the other or from two strands of equal length being twisted. The overhangs may form a mismatch with the target mRNA, or may be complementary to the targeted gene sequence, or may be a different sequence. The first and second strands may also be joined, for example, by additional bases forming a hairpin, or by other non-base linkers.

[0252] In one embodiment, each nucleotide in the overhang region of the RNAi agent may independently be a modified or unmodified nucleotide, including, but not limited to, 2'-sugar-modified nucleotides such as 2-F, 2'-O-methyl, thymidine (T), and any combination thereof.

[0253] For example, TT could be an overhang sequence at any end of either strand. The overhang could form a mismatch with the target mRNA, be complementary to the targeted gene sequence, or be a different sequence altogether.

[0254] The sense strand, antisense strand, or 5'- or 3'-overhangs of both strands of an RNAi agent can be phosphorylated. In some embodiments, the overhang region(s) contains two nucleotides with a phosphorothioate between them, and the two nucleotides may be identical or different. In one embodiment, the overhang is located at the 3' end of the sense strand, antisense strand, or both strands. In one embodiment, this 3'-overhang is located in the antisense strand. In one embodiment, this 3'-overhang is located in the sense strand.

[0255] RNAi agents may contain only a single overhang that can enhance RNAi interference activity without affecting their overall stability. For example, a single-stranded overhang may be located at the 3' end of the sense strand or the 3' end of the antisense strand. RNAi may also have a blunt end located at the 5' end of the antisense strand (i.e., the 3' end of the sense strand), or vice versa. Generally, the antisense strand of RNAi has a nucleotide overhang at the 3' end and a blunt end at the 5' end. Without getting bogged down in theory, the blunt end at the 5' end of an asymmetric antisense strand and the 3' end overhang of the antisense strand are advantageous for guide strand loading into RISC processes.

[0256] In one embodiment, the RNAi agent is 19 nucleotides long with blunt ends at both ends, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 7, 8, and 9 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0257] In another embodiment, the RNAi agent is 20 nucleotides long with blunt ends at both ends, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0258] In yet another embodiment, the RNAi agent is 21 nucleotides long with blunt ends at both ends, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0259] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, with one end of the RNAi agent being blunt and the other end containing a 2-nucleotide overhang. In one embodiment, the 2-nucleotide overhang is at the 3' end of the antisense strand. When the 2-nucleotide overhang is at the 3' end of the antisense strand, there may be two phosphorothioate nucleotide linkages between the three terminal nucleotides, where two of the three nucleotides are the overhang nucleotides and the third nucleotide is the nucleotide that follows the overhang nucleotide. In one embodiment, the RNAi agent further has two phosphorothioate nucleotide linkages between the three terminal nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand. In one embodiment, any nucleotide in the sense strand and antisense strand of the RNAi agent, including a nucleotide that is part of a motif, is a modified nucleotide. In one embodiment, each residue is independently modified, for example, with 2'-O-methyl or 2'-fluoro in an alternating motif. The RNAi agent may further contain a ligand (e.g., a lipophilic ligand, optionally a C16 ligand).

[0260] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, the sense strand being 25-30 nucleotides long and starting from the 5' terminal nucleotide (position 1), positions 1-23 of the first strand containing at least 8 ribonucleotides; the antisense strand being 36-66 nucleotides long and starting from the 3' terminal nucleotide, containing at least 8 ribonucleotides at positions 1-23 of the sense strand to form a double helix, at least 3' terminal nucleotides of the antisense strand not pairing with the sense strand, up to 6 consecutive 3' terminal nucleotides not pairing with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides, and the 5' end of the antisense strand containing 10-30 consecutive nucleotides not pairing with the sense strand The sense strand contains nucleotides, thereby forming a single-stranded 5' overhang of 10–30 nucleotides, and at least the 5' and 3' terminal nucleotides of the sense strand are bases that pair with the nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands, the antisense strand being sufficiently complementary to the target RNA along at least 19 ribonucleotides of the length of the antisense strand, and reducing target gene expression when the double-stranded nucleic acid is introduced into mammalian cells, the sense strand containing at least one motif of three 2'-F modifications on three consecutive nucleotides, at least one of which occurs at or near the cleavage site, and the antisense strand containing at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0261] In one embodiment, the RNAi agent comprises sense and antisense strands, the RNAi agent comprising a first strand having a length of at least 25 and at most 29 nucleotides, and a second strand having a length of at most 30 nucleotides and having at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, the 3' end of the first strand and the 5' end of the second strand form a blunt end, the second strand is 1 to 4 nucleotides longer at its 3' end than the first strand, the double-stranded region is a region of at least 25 nucleotides, the second strand is sufficiently complementary to the target mRNA along the length of the second strand of at least 19 nucleotides, and the RNAi agent reduces the expression of the target gene when introduced into mammalian cells, the dicer cleavage of the RNAi agent yields an siRNA containing the 3' end of the second strand, thereby reducing the expression of the target gene in mammals. Optionally, the RNAi agent may further comprise a ligand.

[0262] In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on a triple nucleotide sequence, one of which occurs at a cleavage site in the sense strand.

[0263] In one embodiment, the antisense strand of the RNAi agent may also contain at least one motif of three identical modifications on a triple nucleotide, one of which occurs at or near a cleavage site in the antisense strand.

[0264] For RNAi agents having a double-stranded region of 17–23 nucleotides in length, the cleavage sites on the antisense strand are typically at positions 10, 11, and 12 from the 5' end. Therefore, three identical modification motifs may occur at positions 9, 10, and 11, 10, 11, and 12, 11, 12, and 13, 12, 13, and 14, or 13, 14, and 15 of the antisense strand, with the number starting from the first nucleotide from the 5' end of the antisense strand, or the number starting from the first pair-formed nucleotide within the double-stranded region from the 5' end of the antisense strand. The cleavage sites in the antisense strand may also vary depending on the length of the double-stranded region of the RNAi from the 5' end.

[0265] The sense strand of an RNAi agent may contain at least one motif of three identical modifications on a triple nucleotide at the cleavage site of the strand, and the antisense strand may have at least one motif of three identical modifications on a triple nucleotide at or near the cleavage site of the strand. When the sense strand and antisense strand form a dsRNA double helix, the sense strand and antisense strand can be sequenced such that one motif of three nucleotides on the sense strand and one motif of three nucleotides on the antisense strand have at least one nucleotide duplication, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may be duplicated, or all three nucleotides may be duplicated.

[0266] In one embodiment, the sense strand of an RNAi agent may contain two or more motifs of three identical modifications on a triple nucleotide sequence. The first motif may occur at or near a cleavage site on the strand, and the other motifs may be wing modifications. In this specification, the term “wing modification” refers to a motif occurring on a different part of the strand, away from the motif at or near the cleavage site on the same strand. Wing modifications are either adjacent to the first motif or at least one or more nucleotides away. If the motifs are immediately adjacent to each other, their chemistry is distinct from each other; if the motifs are one or more nucleotides away, their chemistry may be identical or different. There may be two or more wing modifications. For example, if there are two wing modifications, each wing modification may occur at one end relative to the first motif at or near the cleavage site, or on either side of the read motif.

[0267] Similar to the sense strand, the antisense strand of an RNAi agent may contain two or more motifs of three identical modifications on a triple nucleotide sequence, with at least one motif occurring at or near a cleavage site on the strand. This antisense strand may also contain one or more wing modifications in a sequence similar to those present on the sense strand.

[0268] In one embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0269] In another embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two pairs of nucleotides forming the double-stranded region at the 3', 5', or both ends of the strand.

[0270] If the sense strand and antisense strand of an RNAi agent each contain at least one wing modification, the wing modification may be located at the same end of the double-stranded region and may have one, two, or three nucleotide duplicates.

[0271] If the sense strand or antisense strand of the RNAi agent each contains at least two wing modifications, the sense strand and antisense strand can be arranged such that two modifications from one strand each enter one end of the double-stranded region with 1, 2, or 3 nucleotide duplicates, and two modifications from one strand each enter the other end of the double-stranded region with 1, 2, or 3 nucleotide duplicates, and one strand of the two modifications enters each side of the read motif with 1, 2, or 3 nucleotide duplicates in the double-stranded region.

[0272] In one embodiment, the RNAi agent includes a double-strand mismatch(s) or combination thereof with the target. Mismatches may occur in overhang regions or double-strand regions. Base pairs can be ranked based on their tendency to promote dissociation or dissolution (e.g., by the free energy of association or dissociation of a particular pairing, the simplest approach being to examine pairs on a basis of individual pairs, although the following adjacent analysis or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C, G:U is preferred over G:C, and I:C is preferred over G:C (I = inosine). Mismatches, e.g., non-canonical pairing or non-canonical pairing (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairing, and pairing involving universal bases is preferred over canonical pairing.

[0273] In one embodiment, the RNAi agent includes 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, independently selected from the group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical pairing, non-canonical pairing, or pairings involving universal bases, in order to facilitate the dissociation of the antisense strand at the 5' end of the double helix.

[0274] In one embodiment, the nucleotide at position 1 in the double-strand 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-strand region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the double-strand region from the 5' end of the antisense strand is an AU base pair.

[0275] In another embodiment, the nucleotide at the 3' end of the sense strand is deoxythymidine (dT). In yet another embodiment, the nucleotide at the 3' end of the antisense strand is deoxythymidine (dT). In one embodiment, there is a short sequence of deoxythymine nucleotides, e.g., two dT nucleotides at the 3' end of the sense or antisense strand.

[0276] In one embodiment, the sense strand sequence is given by formula (I): 5'n p -N a -(XXX) i -N b -YYY -N b -(ZZZ) j -N a -n q 3' (I) [In the formula, i and j are independently either 0 or 1. p and q are each independently between 0 and 6. each N a Each independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two differently modified nucleotides. each N bEach independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides. each n p and n q These independently represent overhang nucleotides, Nb and Y do not have the same modifications, and XXX, YYY, and ZZZ each independently represent one motif of three identical modifications on a sequence of three nucleotides. This can be represented by [formula]. In one embodiment, YYY are all 2'-F modified nucleotides.

[0277] In one embodiment, N a or N b This includes alternating modification patterns.

[0278] In one embodiment, the YYY motif occurs at or near the sense strand cleavage site. For example, if the RNAi agent has a double-stranded region of 17-23 nucleotides in length, the YYY motif may occur at or near the sense strand cleavage site (e.g., at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13), the number may start from the first nucleotide from the 5' end, or, as appropriate, the number may start from the first pair-formed nucleotide in the double-stranded region from the 5' end.

[0279] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Therefore, the sense chain is given by the following equation: 5' n p -N a -YYY-N b -ZZZ-N a -n q 3' (Ib), 5' n p -N a -XXX-N b -YYY-N a -n q 3' (Ic), or 5' n p -N a-XXX-N b -YYY-N b -ZZZ-N a -n q 3' (Id) It can be represented by [this].

[0280] If the sense chain is represented by formula (Ib), then N b This represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-5, 0-4, 0-2, or 0.

[0281] each N a These can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0282] If the sense chain is expressed as equation (Ic), then N b This represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. a These may also independently represent oligonucleotide sequences containing 2–20, 2–15, or 2–10 modified nucleotides.

[0283] When the sense chain is expressed as formula (Id), each N b Independently, represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-5, 0-4, 0-2, or 0. In some embodiments, N b is 0, 1, 2, 3, 4, 5, or 6. a These can also independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0284] Each of X, Y, and Z may be the same as or different from one another.

[0285] In other embodiments, i is 0, j is 0, and the sense chain is given by: 5' n p -N a-YYY-N a -n q 3' (Ia) It can be represented by [this].

[0286] If the sense chain is represented by equation (Ia), then each N a These may independently contain oligonucleotide sequences comprising 2-20, 2-15, or 2-10 modified nucleotides.

[0287] In one embodiment, the antisense strand sequence of RNAi is given by formula (II): 5' n q’ -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p ' 3' (II) [In the formula, k and l are independently either 0 or 1. p' and q' are each independently between 0 and 6. each N a ' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two differently modified nucleotides. each N b 'Independently, this represents an oligonucleotide sequence containing 0 to 10 modified nucleotides, each n p 'and n q ' independently represents an overhang nucleotide, N b 'and Y' do not have the same modifier, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif of three identical modifications on a triple nucleotide chain. It can be represented by [this].

[0288] In one embodiment, N a 'or N b ' includes alternating modification patterns.

[0289] The Y’Y’Y’ motif occurs at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a double-stranded region 17 to 23 nucleotides in length, the Y’Y’Y’ motif may occur at positions 9, 10, 11; positions 10, 11, 12; positions 11, 12, 13; positions 12, 13, 14; or positions 13, 14, 15 of the antisense strand, where the numbering starts from the first nucleotide from the 5’ end, or optionally, the numbering may start from the first paired nucleotide within the double-stranded region from the 5’ end. In some embodiments, the Y’Y’Y’ motif occurs at positions 11, 12, 13.

[0290] In one embodiment, the Y’Y’Y’ motif consists of nucleotides that are all 2’-OMe modified.

[0291] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

[0292] Thus, the antisense strand has the following formula: 5’ n q’ -N a ’-Z’Z’Z’-N b ’-Y’Y’Y’-N a ’-n p’ 3’ (IIb), 5’ n q’ -N a ’-Y’Y’Y’-N b ’-X’X’X’-n p’ 3’ (IIc), or 5’ n q’ -N a ’- Z’Z’Z’-N[[ID=4​​​​​​​​​​​​​​’ represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0. Each N a ’ independently represents an oligonucleotide sequence containing modified nucleotides of 2-20, 2-15 or 2-10.

[0294] When the antisense strand is represented by formula (IIc), N b ’ represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0. Each N a ’ independently represents an oligonucleotide sequence containing modified nucleotides of 2-20, 2-15 or 2-10.

[0295] When the antisense strand is represented by formula (IId), each N b ’ independently represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0. Each N a ’ independently represents an oligonucleotide sequence containing modified nucleotides of 2-20, 2-15 or 2-10. In some embodiments, N b is 0, 1, 2, 3, 4, 5 or 6.

[0296] In other embodiments, k is 0, l is 0, and the antisense strand has the following formula: 5’ n p’ -N a’ -Y’Y’Y’- N a’ -n q’ 3’ (Ia) can be represented by.

[0297] When the antisense strand is represented by formula (IIa), each N a ’ independently represents an oligonucleotide sequence containing modified nucleotides of 2-20, 2-15 or 2-10.

[0298] Each of X', Y', and Z' may be identical or different from the others.

[0299] Each nucleotide in the sense and antisense strands can be independently modified with LNA, glycol nucleic acid (GNA), hexitol nucleic acid (HNA), 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide in the sense and antisense strands can be independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y', and Z' may, in particular, represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0300] In one embodiment, the sense strand of the RNAi agent may contain a YYY motif occurring at positions 9, 10, and 11 of the strand when the double-stranded region is 21nt, the number starting from the first nucleotide from the 5' end, or optionally starting from the first pair-formed nucleotide in the double-stranded region from the 5' end, where Y represents a 2'-F modification. The sense strand may further contain an XXX motif or a ZZZ motif as a wing modification at the opposite end of the double-stranded region, where XXX and ZZZ independently represent a 2'-OMe modification or a 2'-F modification.

[0301] In one embodiment, the antisense strand may contain a Y'Y'Y' motif occurring at positions 11, 12, and 13 of the strand, the number starting from the first nucleotide from the 5' end, or optionally starting from the first pair-formed nucleotide in the double-stranded region from the 5' end, where Y' represents a 2'-O-methyl modification. The antisense strand may further contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the double-stranded region, where X'X'X' and Z'Z'Z' independently represent a 2'-OMe modification or a 2'-F modification.

[0302] A sense strand represented by any one of the above equations (Ia), (Ib), (Ic), and (Id) forms a double helix with an antisense strand represented by any one of the above equations (IIa), (IIb), (IIc), and (IId).

[0303] Therefore, the RNAi agent for use in the method of this disclosure may include a sense strand and an antisense strand, each having 14 to 30 nucleotides, and the RNAi double helix is ​​given by formula (III): Sense: 5' n p -N a -(XXX) i -N b - YYY -N b -(ZZZ) j -N a -n q 3' Antisense: 3' n p ’ -N a ’ -(X'X'X') k -N b ’ -Y'Y'Y'-N b ’ -(Z'Z'Z') l -N a ’ -n q ’ 5' (III) [In the formula, i, j, k, and l are each independently either 0 or 1. p, p', q, and q' are each independently between 0 and 6. each N a and N a ’ Each independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two differently modified nucleotides. each N b and N b ’ Each independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides. each np ',n p , n q 'and n q Each of these may or may not be present, but they independently represent an overhang nucleotide. XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif of three identical modifications on three consecutive nucleotides. It is represented by [this].

[0304] In one embodiment, i is 0 and j is 0, or i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 0, or both i and j are 1. In another embodiment, k is 0 and l is 0, or k is 1 and l is 0, k is 0 and l is 1, or both k and l are 0, or both k and l are 1.

[0305] An exemplary combination of sense and antisense strands that form an RNAi double helix is ​​given by the following formula: 5' n p - N a -YYY -N a -n q 3' 3' n p ’ -N a ’ -Y'Y'Y' -N a ’ n q ’ 5' (IIIa) 5' n p -N a -YYY -N b -ZZZ -N a -n q 3' 3' n p ’ -N a ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a’ n q ’ 5' (IIIb) 5' n p -N a - XXX -N b -YYY - N a -n q 3' 3' n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N a ’ -n q ’ 5' (IIIc) 5' n p -N a -XXX -N b -YYY -N b - ZZZ -N a -n q 3' 3' n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a -n q ’ 5' (IIId) Includes.

[0306] When an RNAi agent is represented by formula (IIIa), each N a Each of these independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0307] When an RNAi agent is represented by formula (IIIb), each N b Each N independently represents an oligonucleotide sequence containing modified nucleotides 1-10, 1-7, 1-5, or 1-4. aEach of these independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0308] When an RNAi agent is represented by formula (IIIc), each N b , N b ' independently represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a Each of these independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0309] When an RNAi agent is represented by formula (IIId), each N b , N b ' independently represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a , N a ’ N independently represents oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides. a , N a ', N b and N b ’ Each of these independently includes alternating modification patterns.

[0310] In one embodiment, if the RNAi agent is represented by formula (IIId), then N a The modifications are 2'-O-methyl or 2'-fluoro modifications. In another embodiment, if the RNAi agent is represented by formula (IIId), then N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p ' is linked to an adjacent nucleotide via phosphorothioate linkage. In yet another embodiment, if the RNAi agent is represented by formula (IIId), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p'>0 and at least one n p ' is linked to an adjacent nucleotide via a phosphorothioate linkage, and the sense strand is conjugated to one or more C16 (or related) moieties attached by a divalent or trivalent branched linker (described below). In another embodiment, if the RNAi agent is represented by formula (IIId), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p The sense strand is linked to an adjacent nucleotide via a phosphorothioate linkage, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic, for example, C16 (or related) moieties, which may be attached by a divalent or trivalent branched linker.

[0311] In one embodiment, when the RNAi agent is represented by formula (IIIa), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p The sense strand is linked to an adjacent nucleotide via a phosphorothioate linkage, and the sense strand contains at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic, e.g., C16 (or related) moieties attached by a divalent or trivalent branched linker.

[0312] In one embodiment, the RNAi agent is a multimer containing at least two double helixes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), the double helixes being linked by a linker. The linker may or may not be cleavable. The multimer may further contain ligands. Each double helix may target the same gene, or two different genes, or each double helix may target the same gene at two different target sites.

[0313] In one embodiment, the RNAi agent is a multimer containing 3, 4, 5, 6 or more double helixes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), where the double helixes are linked by linkers. The linkers may or may not be cleavable. The multimer may further contain ligands. Each double helix may target the same gene, or two different genes, or each double helix may target the same gene at two different target sites.

[0314] In one embodiment, two RNAi agents represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) may be ligated together at their 5' ends, with one or both of their 3' ends conjugated to a ligand. Each agent may target the same gene, each may target two different genes, or each agent may target the same gene at two different target sites.

[0315] Various publications describe multimeric RNAi agents that may be used in the methods of this disclosure. Such publications include WO2007 / 091269, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887, and WO2011 / 031520, as well as US7858769, the entire contents of each of these publications being incorporated herein by reference.

[0316] In certain embodiments, the compositions and methods of the Disclosure include vinyl phosphonate (VP) modification of an RNAi agent as described herein. In exemplary embodiments, the 5'-vinyl phosphonate modified nucleotide of the Disclosure has the following structure:

[0317] [ka] [In the formula, X is either O or S; R is hydrogen, hydroxyl, fluoro, or C 1~20It 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. It has.

[0318] The vinyl phosphonates of the Disclosure may be attached to either the antisense or sense strand of the dsRNA of the Disclosure. In certain embodiments, the vinyl phosphonates of the Disclosure may be attached to the antisense strand of the dsRNA at its 5' end, as appropriate.

[0319] Vinylphosphonate modifications are also intended for the compositions and methods of the present disclosure. Exemplary vinylphosphonate structures include the structure in which 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).

[0320] E. Thermal destabilization modification In certain embodiments, a dsRNA molecule can be optimized for RNA interference by incorporating a thermal destabilization modification into the seed region of the antisense strand. As used herein, “seed region” refers to positions 2–9 at the 5’ end of the referenced strand. For example, a thermal destabilization modification can be incorporated into the seed region of the antisense strand to reduce or inhibit off-target gene silencing.

[0321] The term "thermally destabilizing modification(s)" includes modifications(s) that would result in dsRNA having an overall Tm lower than that of dsRNA without such modifications(s). For example, thermally destabilizing modifications(s) can lower the Tm of dsRNA by 1–4°C, e.g., 1, 2, 3, or 4°C. The term "thermally destabilized nucleotide" refers to a nucleotide containing one or more thermally destabilizing modifications.

[0322] It has been found that dsRNAs having an antisense strand containing at least one double-strand thermal destabilization modification within the first 9 nucleotide positions counting from the 5' end of the antisense strand exhibit reduced off-target gene silencing activity. Therefore, in some embodiments, the antisense strand contains at least one (e.g., 1, 2, 3, 4, 5 or more) double-strand thermal destabilization modification within the first 9 nucleotide positions of the 5' region of the antisense strand. In some embodiments, one or more double-strand thermal destabilization modifications are located in positions 2-9, such as positions 4-8, from the 5' end of the antisense strand. In some further embodiments, the double-strand thermal destabilization modifications are located at positions 6, 7, or 8 from the 5' end of the antisense strand. In some even further embodiments, the double-strand thermal destabilization modifications are located at position 7 from the 5' end of the antisense strand. In some embodiments, the double-strand thermal destabilization modifications are located at positions 2, 3, 4, 5, or 9 from the 5' end of the antisense strand.

[0323] Examples of thermal destabilization modifications, though not limited to these, include debasing modifications, mismatches with opposing nucleotides on opposing chains, and sugar modifications, such as 2'-deoxy modifications or acyclic nucleotides, such as unlocked nucleic acids (UNAs) or glycol nucleic acids (GNAs).

[0324] Examples of debase modification include, but are not limited to, the following:

[0325] [ka] [In the formula, R = H, Me, Et or OMe; R' = H, Me, Et or OMe; R” = H, Me, Et or OMe]

[0326] [ka] [In the formula, B is a modified or unmodified nucleic acid base.] These are some examples.

[0327] Examples of sugar modifications include, but are not limited to, the following:

[0328] [ka] [In the formula, B is a modified or unmodified nucleic acid base.] These are some examples.

[0329] In some embodiments, the thermal destabilization modification of the double chain is as follows:

[0330] [ka] [In the formula, B is a modified or unmodified nucleic acid base, and each asterisk in the structure represents either R, S, or racemic.] It is selected from the group consisting of the following.

[0331] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar in which, for example, one of the bonds between ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') is absent, or at least one of the ribose carbons or oxygen atoms (e.g., C1', C2', C3', C4', or O4') is absent independently or in combination in the nucleotide. In some embodiments, an acyclic nucleotide is,

[0332] [ka] [In the formula, B is a modified or unmodified nucleic acid base, and R 1 and R 2 R3 is independently H, halogen, OR3, or alkyl; and 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 encompasses monomers in which the bond between C1'-C4' has been removed (i.e., the carbon-oxygen-carbon bond of the covalent bond between the C1' and C4' carbons). 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), whose entirety is incorporated herein by reference]. Acyclic derivatives offer greater skeletal flexibility without affecting Watson-Crick pair formation. Acyclic nucleotides can be linked via 2'-5' or 3'-5' ligatures.

[0333] 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.

[0334] [ka]

[0335] Double-strand thermal destabilization modifications can be a mismatch (i.e., a non-complementary base pair) between a thermally destabilized nucleotide and an opposing nucleotide in the opposing strand within the dsRNA double-strand. Exemplary mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or combinations thereof. Other mismatch base pair formations known in the art are also suitable for the present invention. Mismatches can occur between nucleotides that are either naturally occurring or modified nucleotides; that is, mismatch base pair formation can occur between nucleic acid bases derived from each nucleotide independently of modifications on the ribose sugar of the nucleotides. In certain embodiments, the dsRNA molecule contains at least one nucleic acid base in mismatch pair formation, for example, a 2'-deoxynucleotide, which is located in the sense strand.

[0336] In some embodiments, thermal destabilization modification of the double helix in the seed region of the antisense strand results in a nucleotide in which the Watson-Crick hydrogen bond (WCH bond) with the complementary base on the target mRNA is impaired, e.g., modified nucleic acid base:

[0337] [ka] Includes.

[0338] More examples of debasalized nucleotides, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications are described in detail in WO2011 / 133876, which is incorporated herein by reference in its entirety.

[0339] Thermal destabilization modifications may also include universal base and phosphate modifications in which the ability to form hydrogen bonds with opposing bases is reduced or lost.

[0340] In some embodiments, thermal destabilization modifications of the double helix include nucleotides with non-canonical bases, for example, but not limited to, nucleic acid base modifications in which the ability to form hydrogen bonds with bases in the opposing strand is impaired or completely lost. These nucleic acid base modifications have been evaluated for destabilization of the central region of the dsRNA double helix, as described in WO2010 / 0011895, which is incorporated herein by reference in its entirety. Exemplary nucleic acid base modifications include:

[0341] [ka]

[0342] In some embodiments, the thermal destabilization modification of the double helix in the seed region of the antisense strand involves one or more α-nucleotides complementary to the base on the target mRNA, for example:

[0343] [ka] [In the formula, R is H, OH, OCH3, F, NH2, NHMe, NMe2, or O-alkyl] It includes.

[0344] As an example of phosphate modifications known to reduce the thermal stability of dsRNA double helix compared to natural phosphodiester bonds:

[0345] [ka]

[0346] The alkyl group of the R group can be C1-C6 alkyl. Specific examples of alkyl groups of the R group, though not limited to these, include methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

[0347] As those skilled in the art will recognize, given that the functional roles of nucleic acid bases define the specificity of the RNAi agents of this disclosure, nucleic acid base modifications can be carried out in various ways as described herein, for example, to enhance on-target effects against off-target effects, or to introduce destabilizing modifications into the RNAi agents of this disclosure. However, the range of modifications available and generally present on the RNAi agents of this disclosure tends to be greater with respect to non-nucleonucleotide modifications, such as modifications to the sugar groups or phosphate backbone of polyribonucleotides. Such modifications are described in more detail in other sections of this disclosure and are explicitly intended for the RNAi agents of this disclosure having either natural nucleic acid bases or modified nucleic acid bases, as described above or elsewhere herein.

[0348] In addition to the antisense strand containing thermal destabilization modifications, the dsRNA may also contain one or more stabilization modifications. For example, the dsRNA may contain at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilization modifications. While not limiting, all stabilization modifications may be present on one of the strands. In some embodiments, both the sense and antisense strands contain at least two stabilization modifications. Stabilization modifications can occur on any nucleotide of the sense or antisense strand. For example, a stabilization modification may occur on any nucleotide on the sense or antisense strand, each stabilization modification may occur in an alternating pattern on the sense or antisense strand, or both the sense or antisense strand may contain stabilization modifications in an alternating pattern. The alternating pattern of stabilization modifications on the sense strand may be identical or different to that on the antisense strand, and the alternating pattern of stabilization modifications on the sense strand may have a shift compared to the alternating pattern of stabilization modifications on the antisense strand.

[0349] In some embodiments, the antisense chain includes at least two stabilization modifications (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). Stabilization modifications in the antisense chain may be located at any position, but are not limited. In some embodiments, the antisense includes stabilization modifications at positions 2, 6, 8, 9, 14, and 16 from the 5' end. In some other embodiments, the antisense includes stabilization modifications at positions 2, 6, 14, and 16 from the 5' end. In yet another embodiment, the antisense includes stabilization modifications at positions 2, 14, and 16 from the 5' end.

[0350] In some embodiments, the antisense strand includes at least one stabilizing modification adjacent to the destabilizing modification. For example, the stabilizing modification may be a nucleotide at the 5' or 3' end of the destabilizing modification, i.e., at position -1 or +1 from the position of the destabilizing modification. In some embodiments, the antisense strand includes stabilizing modifications at each of the 5' and 3' ends of the destabilizing modification, i.e., at positions -1 and +1 from the position of the destabilizing modification.

[0351] In some embodiments, the antisense chain includes at least two stabilizing modifications at the 3' end of the destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification.

[0352] In some embodiments, the sense chain includes at least two stabilization modifications (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). Stabilization modifications in the sense chain may be located at any position, but are not limited to these. In some embodiments, the sense chain includes stabilization modifications at positions 7, 10, and 11 from the 5' end. In some other embodiments, the sense chain includes stabilization modifications at positions 7, 9, 10, and 11 from the 5' end. In some embodiments, the sense chain includes stabilization modifications at positions opposite or complementary to positions 11, 12, and 15 of the antisense chain, counting from the 5' end of the antisense chain. In some other embodiments, the sense chain includes stabilization modifications at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense chain, counting from the 5' end of the antisense chain. In some embodiments, the sense chain includes blocks of two, three, or four stabilization modifications.

[0353] In some embodiments, the sense chain does not contain stabilizing modifications in positions that counteract or complement the thermal destabilizing modifications of the double chain in the antisense chain.

[0354] Examples of thermal stabilization modifications include, but are not limited to, 2'-fluoro modifications. Other examples of thermal stabilization modifications include, but are not limited to, LNA.

[0355] In some embodiments, the dsRNA of this disclosure contains at least four (e.g., 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoronucleotides. Not limited to, all 2'-fluoronucleotides may be present in one of the strands. In some embodiments, both the sense and antisense strands contain at least two 2'-fluoronucleotides. 2'-fluoro modifications may occur on any nucleotide of the sense or antisense strand. For example, a 2'-fluoro modification may occur on any nucleotide on the sense or antisense strand, each 2'-fluoro modification may occur in an alternating pattern on the sense or antisense strand, or both the sense or antisense strand may contain 2'-fluoro modifications in an alternating pattern. The alternating pattern of 2'-fluoro modifications on the sense strand may be identical or different to that on the antisense strand, and the alternating pattern of 2'-fluoro modifications on the sense strand may have a shift compared to the alternating pattern of 2'-fluoro modifications on the antisense strand.

[0356] In some embodiments, the antisense chain contains at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoronucleotides. While not limiting, 2'-fluoro modifications in the antisense chain can be located at any position. In some embodiments, the antisense contains 2'-fluoronucleotides at positions 2, 6, 8, 9, 14, and 16 from the 5' end. In some other embodiments, the antisense contains 2'-fluoronucleotides at positions 2, 6, 14, and 16 from the 5' end. In yet another embodiment, the antisense contains 2'-fluoronucleotides at positions 2, 14, and 16 from the 5' end.

[0357] In some embodiments, the antisense strand includes at least one 2'-fluoronucleotide adjacent to the destabilization modification. For example, the 2'-fluoronucleotide may be at the 5' or 3' end of the destabilization modification, i.e., at position -1 or +1 from the position of the destabilization modification. In some embodiments, the antisense strand includes 2'-fluoronucleotides at each of the 5' and 3' ends of the destabilization modification, i.e., at positions -1 and +1 from the position of the destabilization modification.

[0358] In some embodiments, the antisense strand includes at least two 2'-fluoronucleotides at the 3' end of the destabilization modification, i.e., at positions +1 and +2 from the position of the destabilization modification.

[0359] In some embodiments, the sense strand contains at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoronucleotides. While not limiting, 2'-fluoro modifications in the sense strand can be present at any position. In some embodiments, the antisense strand contains 2'-fluoronucleotides at positions 7, 10, and 11 from the 5' end. In some other embodiments, the sense strand contains 2'-fluoronucleotides at positions 7, 9, 10, and 11 from the 5' end. In some embodiments, the sense strand contains 2'-fluoronucleotides at positions opposite or complementary to positions 11, 12, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some other embodiments, the sense strand contains 2'-fluoronucleotides at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some embodiments, the sense strand contains blocks of 2, 3, or 4 2'-fluoronucleotides.

[0360] In some embodiments, the sense strand does not contain a 2'-fluoronucleotide in a position that counteracts or complements the thermal destabilization modification of the double helix in the antisense strand.

[0361] In some embodiments, the dsRNA molecule of the present disclosure comprises a sense strand of 21 nucleotides (nt) and an antisense strand of 23 nucleotides (nt), wherein the antisense strand contains at least one thermally destabilized nucleotide, the at least one thermally destabilized nucleotide occurring in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand), one end of the dsRNA is blunt, the other end contains two overhangs, and the dsRNA may further have at least one of the following features (e.g., 1, 2, 3, 4, 5, 6, or all of 7): Good: (i) the antisense strand contains 2, 3, 4, 5 or 6 2'-fluoro modifications, (ii) the antisense strand contains 1, 2, 3, 4 or 5 phosphorothioate nucleotide linkages, (iii) the sense strand is conjugated with a ligand, (iv) the sense strand contains 2, 3, 4 or 5 2'-fluoro modifications, (v) the sense strand contains 1, 2, 3, 4 or 5 phosphorothioate nucleotide linkages, (vi) the dsRNA contains at least 4 2'-fluoro modifications, and (vii) the dsRNA has a blunt end at the 5' end of the antisense strand. In one embodiment, two nucleotide overhangs are at the 3' end of the antisense.

[0362] In some embodiments, the dsRNA molecule of the present disclosure comprises a sense and an antisense strand, the sense strand being 25–30 nucleotides long and starting from the 5' terminal nucleotide (position 1), positions 1–23 of the sense strand containing at least 8 ribonucleotides; the antisense strand being 36–66 nucleotides long and starting from the 3' terminal nucleotide, at least 8 ribonucleotides in positions that pair with positions 1–23 of the sense strand form a double helix; at least 3' terminal nucleotides of the antisense strand do not pair with the sense strand, up to 6 consecutive 3' terminal nucleotides do not pair with the sense strand, thereby forming a 3' single-stranded overhang of 1–6 nucleotides; and the 5' end of the antisense strand contains 10–30 consecutive nucleotides that do not pair with the sense strand. The sense strand contains, thereby forming a single-stranded 5' overhang of 10–30 nucleotides, and at least the 5' and 3' terminal nucleotides of the sense strand are bases that pair with the nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands, the antisense strand being sufficiently complementary to the target RNA along at least 19 ribonucleotides of the length of the antisense strand, reducing target gene expression when the double-stranded nucleic acid is introduced into mammalian cells, the antisense strand containing at least one thermally destabilized nucleotide, the at least one thermally destabilized nucleotide located in the seed region of the antisense strand (i.e., at positions 2–9 at the 5' end of the antisense strand).For example, thermally destabilized nucleotides occur between positions 14-17 at the 5' end of the sense strand and complementary positions, and the dsRNA may further have at least one of the following features (e.g., 1, 2, 3, 4, 5, 6, or all 7): (i) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense strand contains 1, 2, 3, 4, or 5 phosphorothioate nucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate nucleotide linkages; (vi) the dsRNA contains at least 4 2'-fluoro modifications; and (vii) the dsRNA contains a double-stranded region 12-30 nucleotide pairs long.

[0363] In some embodiments, the dsRNA molecule of the present disclosure comprises a sense strand and an antisense strand, the dsRNA molecule comprising a sense strand having a length of at least 25 and at most 29 nucleotides, and an antisense strand having a length of at most 30 nucleotides, wherein the sense strand comprises a modified nucleotide sensitive to enzymatic degradation at position 11 from its 5' end, the 3' end of the sense strand and the 5' end of the antisense strand form a blunt end, the antisense strand is 1 to 4 nucleotides longer at its 3' end than the sense strand, the double-stranded region is at least 25 nucleotides long, the antisense strand is sufficiently complementary to the target mRNA along the length of the antisense strand by at least 19 nucleotides, the dsRNA molecule reduces target gene expression when introduced into mammalian cells, and dicer cleavage of the dsRNA yields an siRNA containing the 3' end of the antisense strand, thereby reducing target gene expression in mammals The antisense strand contains at least one thermally destabilized nucleotide, the at least one thermally destabilized nucleotide is located in the seed region of the antisense strand (i.e., at position 2-9 of the 5' end of the antisense strand), and the dsRNA may further have at least one of the following features (e.g., all of 1, 2, 3, 4, 5, 6, or 7): (i) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense strand contains 1, 2, 3, 4, or 5 phosphorothioate nucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate nucleotide linkages; (vi) the dsRNA contains at least 4 2'-fluoro modifications; and (vii) the dsRNA has a double-stranded region of 12-29 nucleotide pairs in length.

[0364] In some embodiments, any nucleotide in the sense and antisense strands of a dsRNA molecule may be modified. Each nucleotide may be modified with the same or different modifications, which may include alterations of one or more unbound phosphate oxygens, or one or more bound phosphate oxygens, alterations of the 2' hydroxyl group on the ribose sugar components, large-scale substitution of the phosphate moiety with a "dephospho" linker, modifications or substitutions of naturally occurring bases, and substitutions or modifications of the ribose-phosphate backbone.

[0365] Since nucleic acids are polymers of subunits, many modifications occur at repeating positions within the nucleic acid, for example, modifications of bases or phosphate moieties or unbound oxygen atoms of phosphate moieties. In some cases, modifications occur at all target positions in the nucleic acid, but often they do not. For example, modifications may occur only at the 3' or 5' end, or only in the terminal region, for example, at the terminal nucleotides of the strand, or at the last 2, 3, 4, 5, or 10 nucleotides. Modifications may occur in double-stranded regions, single-stranded regions, or both. Modifications may occur only in the double-stranded regions of RNA, or only in the single-stranded regions of RNA. For example, phosphorothioate modifications at unbound oxygen atoms may occur only at one or both ends, or only in the terminal region, for example, at the terminal nucleotides of the strand, or at the last 2, 3, 4, 5, or 10 nucleotides, or in both double-stranded and single-stranded regions, especially at the ends. The 5' end or both ends may be phosphorylated.

[0366] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in single-stranded overhangs, e.g., in the 5' or 3' overhang, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' or 5' overhang may be modified, for example, with modifications described herein. Modifications may include, for example, the use of 2'-position modification of ribose sugar in modifications known in the art, e.g., the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro(2'-F) or 2'-O-methyl modified in place of ribosaccharides in nucleic acid bases, and modifications at phosphate groups, e.g., phosphorothioate modifications. The overhang does not need to be homologous to the target sequence.

[0367] In some embodiments, each residue in the sense and antisense strands is independently modified with LNA, glycol nucleic acid (GNA), hexitol nucleic acid (HNA), 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, or 2'-fluoro. The strands may contain two or more modifications. In some embodiments, each residue in the sense and antisense strands is independently modified with 2'-O-methyl or 2'-fluoro. It should be understood that these modifications are in addition to at least one thermal destabilization modification of the double helix present in the antisense strand.

[0368] At least two distinct modifications are typically present on the sense and antisense strands. These two modifications may include 2'-deoxy, 2'-O-methyl, or 2'-fluoro modifications, acyclic nucleotides, etc. In some embodiments, the sense and antisense strands each contain two distinctly modified nucleotides selected from 2'-O-methyl or 2'-deoxy. In some embodiments, each residue in the sense and antisense strands is independently modified with 2'-O-methyl nucleotide, 2'-deoxy nucleotide, 2'-deoxy-2'-fluoro nucleotide, 2'-ON-methylacetamide (2'-O-NMA, 2'O-CH2C(O)N(Me)H) nucleotide, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotide, 2'-O-aminopropyl (2'-O-AP) nucleotide, or 2'-ala-F nucleotide. Again, it should be understood that these modifications are in addition to at least one thermal destabilization modification of the double helix present in the antisense strand.

[0369] In some embodiments, the dsRNA molecules of this disclosure include alternating pattern modifications. The terms “alternating motif” or “alternating pattern,” as used herein, refer to a motif having one or more modifications, each modification occurring in alternating nucleotides on a single strand. Alternating nucleotides may refer to one every other nucleotide, one every three nucleotides, or a similar pattern. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif may be “ABABABABABAB…”, “AABBAABBAABB…”, “AABAABAABAAB…”, “AAABAAABAAAB…”, “AAABBBAAABBB…”, or “ABCABCABCABC…”.

[0370] The types of modifications contained within an alternating motif may be identical or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternating pattern, i.e., the modifications on every other nucleotide, may be identical, but each of the sense strand or antisense strand may be selected from several possible modifications within the alternating motif, such as "ABABAB…", "ACACAC…", "BDBDBD…", or "CDCDCD…".

[0371] In some embodiments, the dsRNA molecules of this disclosure include a modification pattern of alternating motifs on the sense strand that is shifted relative to the modification pattern of alternating motifs on the antisense strand. The shift may be such that modified groups of nucleotides on the sense strand correspond to differently modified groups of nucleotides on the antisense strand, and vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA double helix, the alternating motifs on the sense strand may begin with "ABABAB" from 5'-3' of the strand, and the alternating motifs on the antisense strand may begin with "BABABA" from 3'-5' of the strand in the double helix region. As another example, the alternating motifs on the sense strand may begin with "AABBAABB" from 5'-3' of the strand, and the alternating motifs on the antisense strand may begin with "BBAABBAA" at 3'-5' of the strand in the double helix region, resulting in a complete or partial shift of the modification patterns between the sense and antisense strands.

[0372] The dsRNA molecules of this disclosure may further include at least one phosphorothioate or methylphosphonate internucleotide ligation. Phosphothioate or methylphosphonate internucleotide ligation modifications may occur at any position on the chain, on the sense strand, the antisense strand, or on any nucleotide of both. For example, an internucleotide ligation modification may occur on any nucleotide on the sense strand or the antisense strand, each internucleotide ligation modification may occur in an alternating pattern on the sense strand or the antisense strand, or the sense strand or the antisense strand may contain both internucleotide ligation modifications in an alternating pattern. The alternating pattern of internucleotide ligation modifications on the sense strand may be identical or different to that on the antisense strand, and the alternating pattern of internucleotide ligation modifications on the sense strand may have a shift relative to the alternating pattern of internucleotide ligation modifications on the antisense strand.

[0373] In some embodiments, the dsRNA molecule includes phosphorothioate or methylphosphonate internucleotide ligation modifications within the overhang region. For example, the overhang region includes two nucleotides having a phosphorothioate or methylphosphonate internucleotide ligation between the two nucleotides. The internucleotide ligation modifications may also be made to ligate the overhang nucleotides to the terminal pair-forming nucleotides in the double-stranded region. For example, at least two, three, four, or all of the overhang nucleotides may be ligated by phosphorothioate or methylphosphonate internucleotide ligations, and there may be further phosphorothioate or methylphosphonate internucleotide ligations that ligate the overhang nucleotides to the pair-forming nucleotides adjacent to the overhang nucleotides. For example, there may be at least two phosphorothioate internucleotide ligations between three terminal nucleotides, where two of the three nucleotides are overhang nucleotides and the third is the pair-forming nucleotide adjacent to the overhang nucleotide. In one embodiment, these three terminal nucleotides may be the 3' end of the antisense strand.

[0374] In some embodiments, the sense strand of a dsRNA molecule comprises 1 to 10 blocks of 2 to 10 phosphorothioate or methylphosphonate nucleotide links, separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate nucleotide links, one of which is positioned at any position in the oligonucleotide sequence, and the sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide links, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate links.

[0375] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate nucleotide links, separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate nucleotide links, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide links, and an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate links.

[0376] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate internucleotide links, separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate nucleotide interlinks, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide interlinks, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate linkage.

[0377] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate internucleotide links, separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate nucleotide interlinks, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide interlinks, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate linkage.

[0378] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate internucleotide links, separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate nucleotide interlinks, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide interlinks, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate linkage.

[0379] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of six phosphorothioate or methylphosphonate internucleotide links, separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate nucleotide interlinks, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide interlinks, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate linkage.

[0380] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of seven phosphorothioate or methylphosphonate internucleotide links, separated by one, two, three, four, five, six, seven, or eight phosphate nucleotide interlinks, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide interlinks, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate links.

[0381] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide links, separated by one, two, three, four, five, or six phosphate nucleotide interlinks, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide interlinks, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate links.

[0382] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate nucleotide links, separated by one, two, three, or four phosphate nucleotide links, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide links, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate links.

[0383] In some embodiments, the dsRNA molecules of this disclosure further include one or more phosphorothioate or methylphosphonate internucleotide ligation modifications within the terminal positions of 1 to 10 nucleotides of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be ligated by phosphorothioate or methylphosphonate internucleotide ligations at one or both ends of the sense or antisense strand.

[0384] In some embodiments, the dsRNA molecules of this disclosure further include one or more phosphorothioate or methylphosphonate nucleotide ligation modifications within 1 to 10 nucleotides in the internal region of each of the sense or antisense strands of the double helix. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be ligated by phosphorothioate or methylphosphonate ligations at positions 8 to 16 of the double helix region, counting from the 5' end of the sense strand. The dsRNA molecules may further include one or more phosphorothioate or methylphosphonate nucleotide ligation modifications within 1 to 10 terminal positions.

[0385] In some embodiments, the dsRNA molecule of the present disclosure further comprises 1 to 5 phosphorothioate or methylphosphonate nucleotide ligation modifications within positions 1 to 5 of the sense strand and 1 to 5 phosphorothioate or methylphosphonate nucleotide ligation modifications within positions 18 to 23 (counting from the 5' end), as well as 1 to 2 phosphorothioate or methylphosphonate nucleotide ligation modifications within positions 1 and 2 of the antisense strand and 1 to 5 within positions 18 to 23 (counting from the 5' end).

[0386] In some embodiments, the dsRNA molecule of the present disclosure further comprises one phosphorothioate nucleotide ligation modification within positions 1-5 of the sense strand and one phosphorothioate or methylphosphonate nucleotide ligation modification within positions 18-23 (counting from the 5' end), as well as one phosphorothioate nucleotide ligation modification at position 1 or 2 of the antisense strand and two phosphorothioate or methylphosphonate nucleotide ligation modifications within positions 18-23 (counting from the 5' end).

[0387] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications within positions 1–5 of the sense strand and one phosphorothioate nucleotide ligation modification within positions 18–23 (counting from the 5' end), as well as one phosphorothioate nucleotide ligation modification at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide ligation modifications within positions 18–23 (counting from the 5' end).

[0388] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications within positions 1-5 of the sense strand and two phosphorothioate nucleotide ligation modifications within positions 18-23 (counting from the 5' end), as well as one phosphorothioate nucleotide ligation modification at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide ligation modifications within positions 18-23 (counting from the 5' end).

[0389] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications within positions 1–5 of the sense strand and two phosphorothioate nucleotide ligation modifications within positions 18–23 (counting from the 5' end), as well as one phosphorothioate nucleotide ligation modification at positions 1 and 2 of the antisense strand and one phosphorothioate nucleotide ligation modification within positions 18–23 (counting from the 5' end).

[0390] In some embodiments, the dsRNA molecule of the present disclosure further comprises one phosphorothioate nucleotide ligation modification within positions 1-5 of the sense strand and one phosphorothioate nucleotide ligation modification within positions 18-23 (counting from the 5' end), as well as two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide ligation modifications within positions 18-23 (counting from the 5' end).

[0391] In some embodiments, the dsRNA molecule of the present disclosure further comprises one phosphorothioate nucleotide ligation modification within positions 1–5 of the sense strand and one within positions 18–23 (counting from the 5' end), as well as two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand and one phosphorothioate nucleotide ligation modification within positions 18–23 (counting from the 5' end).

[0392] In some embodiments, the dsRNA molecule of the present disclosure further includes one phosphorothioate nucleotide ligation modification (counting from the 5' end) within positions 1–5 of the sense strand, and two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand, and one phosphorothioate nucleotide ligation modification (counting from the 5' end) within positions 18–23.

[0393] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications (counting from the 5' end) within positions 1–5 of the sense strand, one phosphorothioate nucleotide ligation modification at positions 1 and 2 of the antisense strand, and two phosphorothioate nucleotide ligation modifications (counting from the 5' end) within positions 18–23.

[0394] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications within positions 1–5 of the sense strand and one within positions 18–23 (counting from the 5' end), as well as two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand and one within positions 18–23 (counting from the 5' end).

[0395] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications within positions 1-5 of the sense strand and one phosphorothioate nucleotide ligation modification within positions 18-23 (counting from the 5' end), as well as two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide ligation modifications within positions 18-23 (counting from the 5' end).

[0396] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications within positions 1–5 of the sense strand and one phosphorothioate nucleotide ligation modification within positions 18–23 (counting from the 5' end), as well as one phosphorothioate nucleotide ligation modification at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide ligation modifications within positions 18–23 (counting from the 5' end).

[0397] In some embodiments, the dsRNA molecule of the present disclosure further includes two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the sense strand and two phosphorothioate nucleotide ligation modifications at positions 20 and 21 (counting from the 5' end), as well as one phosphorothioate nucleotide ligation modification at position 1 of the antisense strand and one at position 21 (counting from the 5' end).

[0398] In some embodiments, the dsRNA molecule of the present disclosure further comprises one phosphorothioate nucleotide ligation modification at position 1 of the sense strand and one phosphorothioate nucleotide ligation modification at position 21 (counting from the 5' end), and two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide ligation modifications at positions 20 and 21 (counting from the 5' end).

[0399] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the sense strand and two phosphorothioate nucleotide ligation modifications at positions 21 and 22 (counting from the 5' end), as well as one phosphorothioate nucleotide ligation modification at position 1 of the antisense strand and one phosphorothioate nucleotide ligation modification at position 21 (counting from the 5' end).

[0400] In some embodiments, the dsRNA molecule of the present disclosure further comprises one phosphorothioate nucleotide ligation modification at position 1 of the sense strand and one phosphorothioate nucleotide ligation modification at position 21 (counting from the 5' end), and two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide ligation modifications at positions 21 and 22 (counting from the 5' end).

[0401] In some embodiments, the dsRNA molecule of the present disclosure further includes two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the sense strand and two phosphorothioate nucleotide ligation modifications at positions 22 and 23 (counting from the 5' end), as well as one phosphorothioate nucleotide ligation modification at position 1 of the antisense strand and one phosphorothioate nucleotide ligation modification at position 21 (counting from the 5' end).

[0402] In some embodiments, the dsRNA molecule of the present disclosure further includes one phosphorothioate nucleotide ligation modification at position 1 of the sense strand and one phosphorothioate nucleotide ligation modification at position 21 (counting from the 5' end), and two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide ligation modifications at positions 23 and 23 (counting from the 5' end).

[0403] In some embodiments, the compounds of the present disclosure include a pattern of skeletal chiral centers. In some embodiments, the general pattern of skeletal chiral centers includes at least five nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of skeletal chiral centers includes at least six nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of skeletal chiral centers includes at least seven nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of skeletal chiral centers includes at least eight nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of skeletal chiral centers includes at least nine nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of skeletal chiral centers includes at least ten nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of skeletal chiral centers includes at least eleven nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of skeletal chiral centers includes at least twelve nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of skeletal chiral centers includes at least thirteen nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 14 nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 15 nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 16 nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 17 nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 18 nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 19 nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes 8 or fewer nucleotide linkages in the Rp configuration.In some embodiments, the general pattern of the skeletal chiral center includes seven or fewer nucleotide linkages in the Rp configuration. In some embodiments, the general pattern of the skeletal chiral center includes six or fewer nucleotide linkages in the Rp configuration. In some embodiments, the general pattern of the skeletal chiral center includes five or fewer nucleotide linkages in the Rp configuration. In some embodiments, the general pattern of the skeletal chiral center includes four or fewer nucleotide linkages in the Rp configuration. In some embodiments, the general pattern of the skeletal chiral center includes three or fewer nucleotide linkages in the Rp configuration. In some embodiments, the general pattern of the skeletal chiral center includes two or fewer nucleotide linkages in the Rp configuration. In some embodiments, the general pattern of the skeletal chiral center includes one or fewer nucleotide linkages in the Rp configuration. In some embodiments, the general pattern of the skeletal chiral center includes eight or fewer non-chiral nucleotide linkages (phosphodiesters are an example, not limited to this). In some embodiments, the general pattern of the skeletal chiral center includes seven or fewer non-chiral nucleotide linkages. In some embodiments, the general pattern of the skeletal chiral center includes six or fewer non-chiral nucleotide linkages. In some embodiments, the general pattern of the skeletal chiral center includes five or fewer non-chiral nucleotide linkages. In some embodiments, the general pattern of the skeletal chiral center includes four or fewer non-chiral nucleotide linkages. In some embodiments, the general pattern of the skeletal chiral center includes three or fewer non-chiral nucleotide linkages. In some embodiments, the general pattern of the skeletal chiral center includes two or fewer non-chiral nucleotide linkages. In some embodiments, the general pattern of the skeletal chiral center includes one or fewer non-chiral nucleotide linkages. In some embodiments, the general pattern of the skeletal chiral center includes at least 10 nucleotide linkages and eight or fewer non-chiral nucleotide linkages in the Sp configuration.In some embodiments, the general pattern of the skeletal chiral center includes at least 11 internucleotide links and 7 or fewer non-chiral internucleotide links in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 12 internucleotide links and 6 or fewer non-chiral internucleotide links in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 13 internucleotide links and 6 or fewer non-chiral internucleotide links in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 14 internucleotide links and 5 or fewer non-chiral internucleotide links in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 15 internucleotide links and 4 or fewer non-chiral internucleotide links in the Sp configuration. In some embodiments, the internucleotide links in the Sp configuration may or may not be continuous. In some embodiments, the internucleotide links in the Rp configuration may or may not be continuous. In some embodiments, the non-chiral internucleotide links may or may not be continuous.

[0404] In some embodiments, the compounds of the Disclosure include blocks that are stereochemical blocks. In some embodiments, a block is an Rp block in that each nucleotide linkage in the block is Rp. In some embodiments, a 5'-block is an Rp block. In some embodiments, a 3'-block is an Rp block. In some embodiments, a block is an Sp block in that each nucleotide linkage in the block is Sp. In some embodiments, a 5'-block is an Sp block. In some embodiments, a 3'-block is an Sp block. In some embodiments, the oligonucleotides provided include both Rp and Sp blocks. In some embodiments, the oligonucleotides provided include one or more Rp but do not include Sp blocks. In some embodiments, the oligonucleotides provided include one or more Sp but do not include Rp blocks. In some embodiments, the oligonucleotides provided include one or more PO blocks in which each nucleotide linkage is a native phosphate linkage.

[0405] In some embodiments, the compounds of the present disclosure include a 5'-block in which each sugar moiety is an Sp block containing a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each nucleotide linkage is a modified nucleotide linkage and each sugar moiety is an Sp block containing a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each nucleotide linkage is a phosphorothioate linkage and each sugar moiety is an Sp block containing a 2'-F modification. In some embodiments, the 5'-block contains four or more nucleoside units. In some embodiments, the 5'-block contains five or more nucleoside units. In some embodiments, the 5'-block contains six or more nucleoside units. In some embodiments, the 5'-block contains seven or more nucleoside units. In some embodiments, the 3'-block is an Sp block in which each sugar moiety is an Sp block containing a 2'-F modification. In some embodiments, the 3'-block is an Sp block in which each nucleotide linkage is a modified nucleotide linkage and each sugar moiety is an Sp block containing a 2'-F modification. In some embodiments, the 3'-block is an Sp block in which each nucleotide linkage is a phosphorothioate linkage and each sugar moiety contains a 2'-F modification. In some embodiments, the 3'-block contains four or more nucleoside units. In some embodiments, the 3'-block contains five or more nucleoside units. In some embodiments, the 3'-block contains six or more nucleoside units. In some embodiments, the 3'-block contains seven or more nucleoside units.

[0406] In some embodiments, the compounds of the Disclosure comprise a nucleoside of a certain type in the region, or an oligonucleotide followed by a specific type of internucleotide linkage, such as a native phosphate linkage, a modified internucleotide linkage, an Rp chiral internucleotide linkage, an Sp chiral internucleotide linkage, and the like. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by a native phosphate linkage (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by a native phosphate linkage (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by a native phosphate linkage (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by a native phosphate linkage (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by a natural phosphate linkage (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.

[0407] In some embodiments, the antisense strand includes phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23, the antisense strand contains at least one double-stranded thermal destabilization modification located in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand), and the dsRNA may further have at least one of the following features (e.g., all of 1, 2, 3, 4, 5, 6, 7, or 8): (i) the antisense strand includes 2, 3, 4, 5, or 6 2'-fluoro modifications, (ii) (iii) the antisense strand contains 3, 4, or 5 phosphorothioate nucleotide interlinks, (iii) the sense strand is conjugated with a ligand, (iv) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications, (v) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate nucleotide interlinks, (vi) the dsRNA contains at least 4 2'-fluoro modifications, (vii) the dsRNA contains a double-stranded region of 12–40 nucleotide pairs in length, and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0408] In some embodiments, the antisense strand includes phosphorothioate internucleotide links between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, and the antisense strand contains at least one double-stranded thermal destabilization modification located in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand), and the dsRNA may further have at least one of the following features (e.g., all of 1, 2, 3, 4, 5, 6, 7, or 8): (i) the antisense strand has 2, 3, 4, 5, or 6 (ii) the sense strand contains one 2'-fluoro modification, is conjugated with a ligand, (iii) the sense strand contains two, three, four or five 2'-fluoro modifications, (iv) the sense strand contains one, two, three, four or five phosphorothioate nucleotide interlinks, (v) the dsRNA contains at least four 2'-fluoro modifications, (vi) the dsRNA contains a double-stranded region of 12 to 40 nucleotide pairs in length, (vii) the dsRNA contains a double-stranded region of 12 to 40 nucleotide pairs in length, and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0409] In some embodiments, the sense strand includes phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, the antisense strand includes at least one double-stranded thermal destabilization modification located in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand), and the dsRNA may further have at least one of the following features (e.g., all of 1, 2, 3, 4, 5, 6, 7, or 8): (i) the antisense includes 2, 3, 4, 5, or 6 2'-fluoro modifications, (ii) the anti (iii) the sense strand contains 1, 2, 3, 4 or 5 phosphorothioate nucleotide interlinks, (iv) the sense strand contains 2, 3, 4 or 5 2'-fluoro modifications, (v) the sense strand contains 3, 4 or 5 phosphorothioate nucleotide interlinks, (vi) the dsRNA contains at least 4 2'-fluoro modifications, (vii) the dsRNA contains a double-stranded region of 12 to 40 nucleotide pairs in length, and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0410] In some embodiments, the sense strand includes phosphorothioate internucleotide links between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, the antisense strand includes phosphorothioate internucleotide links between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23, the antisense strand contains at least one double-stranded thermal destabilization modification located in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand), and the dsRNA has at least one of the following features (e.g., 1, 2) The dsRNA may further have (3, 4, 5, 6 or 7) all of the following: (i) the antisense strand contains 2, 3, 4, 5 or 6 2'-fluoro modifications; (ii) the sense strand is conjugated with a ligand; (iii) the sense strand contains 2, 3, 4 or 5 2'-fluoro modifications; (iv) the sense strand contains 3, 4 or 5 phosphorothioate internucleotide linkages; (v) the dsRNA contains at least 4 2'-fluoro modifications; (vi) the dsRNA contains a double-stranded region 12–40 nucleotide pairs long; and (vii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0411] In some embodiments, the dsRNA molecules of this disclosure include double-stranded mismatches(s) or combinations thereof with respect to the target. Mismatches may occur in overhang regions or double-stranded regions. Base pairs can be ranked based on their tendency to promote dissociation or fusion (e.g., by the free energy of association or dissociation of a particular pairing, the simplest approach being to examine pairs on a basis of individual pairs, although the following adjacency analysis or similar analysis may also be used). In terms of promoting dissociation: A:U is preferred over G:C, G:U is preferred over G:C, and I:C is preferred over G:C (I = inosine). Mismatches, e.g., non-canonical pairing or non-canonical pairing (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairing, and pairing involving universal bases is preferred over canonical pairing.

[0412] In some embodiments, the dsRNA molecule of the present disclosure includes 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, which can be independently selected from the group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical pairing or pairing other than canonical pairing or pairing including universal bases, in order to facilitate the dissociation of the antisense strand at the 5' end of the double helix.

[0413] In some embodiments, the nucleotide at position 1 in the double-strand 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-strand region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the double-strand region from the 5' end of the antisense strand is an AU base pair.

[0414] It has been found that introducing 4'-modified or 5'-modified nucleotides to the 3' end of a nucleotide phosphodiester (PO), phosphorothioate (PS), or phosphorodithioate (PS2) linkage at any position on a single-stranded or double-stranded oligonucleotide exerts a steric effect on the nucleotide linkage, thereby protecting and stabilizing it from nucleases.

[0415] In some embodiments, a 5'-modified nucleotide is introduced at the 3' end of a dinucleotide at any position in a single-stranded or double-stranded siRNA. For example, a 5'-alkylated nucleotide can be introduced at the 3' end of a dinucleotide at any position in a single-stranded or double-stranded siRNA. The alkyl group at the 5' position of the ribose sugar can be a racemic mixture or a chiralally pure R or S isomer. An exemplary 5'-alkylated nucleotide is 5'-methylnucleotide. 5'-methyl can be either a racemic mixture or a chirally pure R or S isomer.

[0416] In some embodiments, a 4'-modified nucleotide is introduced at the 3' end of a dinucleotide at any position in a single-stranded or double-stranded siRNA. For example, a 4'-alkylated nucleotide can be introduced at the 3' end of a dinucleotide at any position in a single-stranded or double-stranded siRNA. The alkyl group at the 4' position of the ribose sugar can be racemic or a chiralally pure R or S isomer. An exemplary 4'-alkylated nucleotide is 4'-methylnucleotide. The 4'-methyl group can be either racemic or a chirally pure R or S isomer. Alternatively, a 4'-O-alkylated nucleotide can be introduced at the 3' end of a dinucleotide at any position in a single-stranded or double-stranded siRNA. The 4'-O-alkyl group of the ribose sugar can be racemic or a chirally pure R or S isomer. An exemplary 4'-O-alkylated nucleotide is 4'-O-methylnucleotide. 4'-O-methyl can be either a racemic mixture or a chiralally pure R or S isomer.

[0417] In some embodiments, a 5'-alkylated nucleotide is introduced at any position on the sense or antisense strand of the dsRNA, and such modification maintains or improves the potency of the dsRNA. The 5'-alkyl can be either a racemic or a chiralally pure R or S isomer. An exemplary 5'-alkylated nucleotide is 5'-methylnucleotide, which can be either a racemic or a chirally pure R or S isomer.

[0418] In some embodiments, a 4'-alkylated nucleotide is introduced at any position on the sense or antisense strand of the dsRNA, and such modification maintains or improves the potency of the dsRNA. The 4'-alkyl can be either racemic or a chiralally pure R or S isomer. An exemplary 4'-alkylated nucleoside is the 4'-methyl nucleoside. The 4'-methyl can be either racemic or a chirally pure R or S isomer.

[0419] In some embodiments, the 4'-O-alkylated nucleotide is introduced at any position on the sense or antisense strand of the dsRNA, and such modification maintains or improves the potency of the dsRNA. The 5'-alkyl can be either racemic or chiralally pure R or S isomer. An exemplary 4'-O-alkylated nucleod is the 4'-O-methyl nucleotide. The 4'-O-methyl can be either racemic or chiralally pure R or S isomer.

[0420] In some embodiments, the dsRNA molecules of this disclosure may include a 2'-5' ligation (having 2'-H, 2'-OH, and 2'-OMe, and being P=O or P=S). For example, the 2'-5' ligation modification can be used to promote nuclease resistance, to inhibit the binding of sense to the antisense strand, or to avoid sense strand activation by RISC at the 5' end of the sense strand.

[0421] In another embodiment, the dsRNA molecule of this 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 can be used to promote nuclease resistance, to inhibit the binding of sense to the antisense strand, or to avoid sense strand activation by RISC at the 5' end of the sense strand.

[0422] Multimeric siRNAs have been described in various publications, all of which can be used in conjunction with the dsRNAs of this disclosure. Such publications include WO2007 / 091269, US7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887, and WO2011 / 031520, which are incorporated in their entirety herein.

[0423] As described in more detail below, RNAi agents containing the conjugation of one or more carbohydrate moieties can optimize one or more properties of the RNAi agent. Often, the carbohydrate moiety is attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced by another moiety, e.g., a non-carbohydrate (e.g., cyclic) carrier to which a carbohydrate ligand is attached. A ribonucleotide subunit in which the ribose sugar of the subunit is thus replaced is referred herein to as a ribose-replaced modified subunit (RRMS). The cyclic carrier may be a carbocyclic system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring structure, i.e., one or more ring atoms are heteroatoms, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring structure, or may contain two or more rings, e.g., a fused ring. The cyclic carrier may be a fully saturated ring structure, or may contain one or more double bonds.

[0424] Ligands can be attached to polynucleotides via a carrier. The carrier comprises (i) at least one “skeleton attachment site,” e.g., two “skeleton attachment sites,” and (ii) at least one “tethering attachment site.” “Skeleton attachment site,” as used herein, refers to a functional group, e.g., a hydroxyl group, or generally, a bond available and suitable for the incorporation of the carrier into a skeleton, e.g., a phosphate or modified phosphate of ribonucleic acid, e.g., a sulfur-containing skeleton. “Tethering attachment site” (TAP) refers, in some embodiments, to a constituent ring atom of the cyclic carrier connecting a selected moiety, e.g., a carbon atom or heteroatom (separate from the atom providing the skeleton attachment sites). The moiety may be, for example, a carbohydrate, e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, and polysaccharides. The selected moiety may be connected to the cyclic carrier by an intervening tether. Thus, the cyclic carrier will often provide a bond suitable for the incorporation or tethering of another chemical entity, e.g., a ligand, into a constituent ring, e.g., containing a functional group, e.g., an amino group.

[0425] RNAi agents may be conjugated to ligands via a carrier, which may be a cyclic or acyclic group. In some embodiments, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuryl, and decalin. In some embodiments, the acyclic group is selected from a selinol skeleton or a diethanolamine skeleton.

[0426] In certain specific embodiments, the RNAi agent for use in the method of the present disclosure is an agent selected from the group of agents listed in any one of Tables 2, 3, 5, and 6. These agents may further include ligands, such as one or more lipophilic moieties, one or more GalNAc derivatives, or both one or more lipophilic moieties and one or more GalNAc derivatives.

[0427] IV. iRNA conjugated to a ligand Another modification of the iRNA of the present invention involves chemically linking the iRNA with one or more ligands, a moiety or conjugate that enhances the activity, cell distribution or cellular uptake of the iRNA into cells. These include, but are not limited to, lipid parts such as cholesterol (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, for example, beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), fatty acid chains, e.g., dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118, Kabanov et al., FEBS Lett., 1990, 259:327-330, Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654, Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetate (Manoharan et al., Tetrahedron Lett.Examples include the palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237) or the octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0428] In certain embodiments, ligands alter the distribution, targeting, or lifespan of the iRNA agent into which they are incorporated. In some embodiments, ligands provide enhanced affinity to selected targets, such as molecules, cells or cell types, compartments, such as cellular or organ compartments, tissues, organs, or regions of the body, compared to species in which such ligands are absent. Conventional ligands do not participate in double-strand pairing in double-stranded nucleic acids.

[0429] Ligands can be naturally occurring substances, such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, such as synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymer, or polyphosphatidine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helix peptides.

[0430] Ligands may also include targeting groups, such as cell or tissue targeting agents, such as lectins, glycoproteins, lipids or proteins, or antibodies that bind to specific cell types, such as kidney cells. Targeting groups may include thyroid-stimulating hormone, melanocyte-stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, biotin, or RGD peptides or RGD peptide mimetic compounds. In certain embodiments, the ligand is polyvalent galactose, such as N-acetyl-galactosamine.

[0431] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyllin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., 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) lithoglycerol Examples include oenic acid, O3-(oleoyl)colenic acid, dimethoxytrityl or phenoxazine, and peptide conjugates (e.g., Antennapedia peptide, Tat peptide), alkylating agents, phosphoric acid, amino acids, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraaza macrocyclic molecules), dinitrophenyl, HRP, or AP.

[0432] Ligands can be proteins, such as glycoproteins or peptides, molecules or antibodies that have a specific affinity for a co-ligand, such as antibodies that bind to specific cell types, such as cancer cells, endothelial cells, or osteocytes. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, or polyvalent fucose. Ligands can also be, for example, lipopolysaccharides, p38 MAP kinase activators, or NF-κB activators.

[0433] A ligand can be a substance, such as a drug, that can increase the uptake of an iRNA agent into a cell by, for example, disrupting the cytoskeleton of a cell, for example, by disrupting the microtubules, microfibrils, or intermediate fibers of a cell. A drug may be, for example, taxone, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latruncrine A, phalloidin, swinford A, indanosine, or myoserbine.

[0434] In some embodiments, ligands attached to iRNAs, as described herein, act as pharmacokinetic modulators (PK modulators). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEGs, vitamins, and the like. Exemplary PK modulators, but not limited to, include cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, and biotin. Oligonucleotides containing several phosphorothioate linkages are also known to bind to serum proteins; therefore, short oligonucleotides, e.g., oligonucleotides of about 5, 10, 15, or 20 bases containing multiple phosphorothioate linkages in their backbone, are also suitable as ligands (e.g., as PK-modulating ligands) in the present invention. Furthermore, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in embodiments described herein.

[0435] iRNAs conjugated with the ligand of the present invention can be synthesized using oligonucleotides having pendant-reactive functionality, for example, those derived from the attachment of a linking molecule to an oligonucleotide (as described below). These reactive oligonucleotides can be directly reacted with commercially available ligands, synthetic ligands having any of the various protecting groups, or ligands having a linking portion attached thereto.

[0436] The oligonucleotides used in the conjugates of the present invention can be conveniently and routinely prepared by known solid-phase synthesis techniques. Equipment for such synthesis is available from several vendors, including, for example, Applied Biosystems® (Foster City, California). Any other means for such synthesis known in the art may be used further or instead. It is also known that similar techniques can be used to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

[0437] In the ligand-conjugated oligonucleotide and ligand-sequence-specific linked nucleosides of the present invention, the oligonucleotide and oligonucleosides can be assembled in a suitable DNA synthesizer using a standard nucleotide or nucleoside precursor, a nucleotide or nucleoside conjugate precursor already having a linking portion, a ligand-nucleotide or nucleoside conjugate precursor already having a ligand molecule, or a non-nucleoside ligand having a building block.

[0438] When using nucleotide-conjugate precursors that already have a linking region, the synthesis of a sequence-specific linked nucleoside is typically completed, and then the ligand molecule reacts with the linking region to form a ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates, in addition to commercially available and standard and non-standard phosphoramidites routinely used in oligonucleotide synthesis.

[0439] A. Lipid conjugates In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule. Such lipid or lipid-based molecules can typically bind to serum proteins, such as human serum albumin (HSA). HSA-binding ligands enable the distribution of the conjugate to target tissues in the body, such as non-renal target tissues. For example, the target tissue could be the liver, including the parenchymal cells of the liver. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipid or lipid-based ligands can (a) increase the resistance of the conjugate to degradation, (b) increase the targeting or transport into target cells or cell membranes, or (c) modulate binding to serum proteins, such as HSA.

[0440] Lipid-based ligands can be used to modulate, for example, control (e.g., inhibit) the binding of conjugates to target tissues. For instance, lipids or lipid-based ligands that bind more strongly to HSA are less likely to be targeted to the kidneys and therefore less likely to be eliminated from the body. Lipids or lipid-based ligands that do not bind less strongly to HSA can be used to target conjugates to the kidneys.

[0441] In certain embodiments, lipid-based ligands bind to HSA. For example, the ligand can bind to HSA with sufficient affinity, resulting in enhanced distribution of the conjugate to non-renal tissue. However, the affinity is usually not strong enough to reverse the HSA-ligand binding.

[0442] In certain embodiments, the lipid-based ligand may bind weakly to HSA or not bind at all, resulting in enhanced distribution of the conjugate to the kidney. Other moieties that target kidney cells can be used instead of, or in addition to, the lipid-based ligand.

[0443] In another embodiment, the ligand is a portion taken up by target cells, e.g., proliferating cells, e.g., a vitamin. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., malignant or non-malignant species, e.g., cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, e.g., folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low-density lipoprotein (LDL).

[0444] B. Cell permeability agents In another embodiment, the ligand is a cell permeabilizer, such as a helix cell permeabilizer. In certain embodiments, these cell permeabilizers are amphiphilic. Exemplary cell permeabilizers include peptides, such as tat or antennopedia. If the agent is a peptide, it may be modified, including peptidyl mimetic, inverted isomers, non-peptide or pseudopeptide linkages, and the use of D-amino acids. Helix agents are typically α-helix agents and may have lipophilic and oleophobic phases.

[0445] Ligands can be peptides or peptidomimetic molecules. Peptidomimetic molecules (also referred to herein as oligopeptidomimetic molecules) are molecules that can fold into a defined three-dimensional structure similar to that of natural peptides. The attachment of peptides and peptidomimetic molecules to iRNA agents can affect the pharmacokinetic distribution of the iRNA, for example, by enhancing cell recognition and absorption. The peptide or peptidomimetic moiety may be about 5 to 50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0446] Peptides or peptidomimetic molecules can be, for example, cell-penetrating peptides, cationic peptides, amphiphilic peptides, or hydrophobic peptides (e.g., mainly composed of Tyr, Trp, or Phe). The peptide moiety can be a dendrimer peptide, a restrictive peptide, or a cross-linked peptide. Alternatively, the peptide moiety may contain a hydrophobic membrane-transfer sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF with the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 9). RFGF analogues containing hydrophobic MTS (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 10)) can also be targeting moieties. The peptide moiety can be a "delivery" peptide capable of carrying large polar molecules, including peptides, oligonucleotides, and proteins, across the cell membrane. For example, sequences derived from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12)) have been found to be functional as delivery peptides. Peptides or peptidomimetic molecules can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or 1-bead-1-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). Typically, peptides or peptidomimetic molecules tethered to dsRNA agents via integrated monomer units include cell-targeting peptides, such as arginine-glycine-aspartate (RGD) peptides or RGD mimics. The peptide moiety can range in length from approximately 5 to 40 amino acids. The peptide moiety may have structural modifications that increase stability or direct conformational properties, for example. Any of the structural modifications described below are available.

[0447] The RGD peptides for use in the compositions and methods of the present invention may be linear or cyclic, and may be modified to facilitate targeting to specific tissues, for example, by glycosylation or methylation. RGD-containing peptides and peptidiomimemtics may include D-amino acids and synthetic RGD mimics. In addition to RGD, other moieties that target integrin ligands such as PECAM-1 or VEGF may be used.

[0448] The RGD peptide portion can be used to target specific cell types, such as tumor cells, endothelial tumor cells, or breast cancer tumor cells (Zitzmann et al., Cancer Res., 62:5139-43, 2002). RGD peptides can facilitate the targeting of dsRNA agents to tumors in various other tissues, including the lungs, kidneys, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Typically, RGD peptides facilitate the targeting of iRNA agents to the kidneys. RGD peptides can be linear or cyclic and can be modified to facilitate targeting to specific tissues, for example, by glycosylation or methylation. For example, glycosylated RGD peptides can α V It can be delivered to tumor cells that express β3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

[0449] A "cell-permeable peptide" is capable of permeating cells, such as microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. Microbial cell-permeable peptides may be, for example, α-helix linear peptides (e.g., LL-37 or seropin P1), disulfide bond-containing peptides (e.g., α-defensin, β-defensin, or bactenesin), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidine). Cell-permeable peptides may also contain nuclear localization signals (NLS). For example, a cell-permeable peptide may be a bifid amphiphilic peptide such as MPG derived from the fusion peptide domain of the NLS of HIV-1 gp41 and SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0450] C. Carbohydrate Conjugate In some embodiments of the compositions and methods of the present invention, the iRNA further comprises a carbohydrate. Carbohydrate-conjugated iRNA is advantageous for in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” means a compound that is either a carbohydrate itself, or a compound having a carbohydrate moiety composed of one or more monosaccharide units, each having at least six carbon atoms (which may be linear, branched, or cyclic), each having at least six carbon atoms (which may be linear, branched, or cyclic), along with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Typical carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide gum. C5 is an example of a specific monosaccharide, and the above-mentioned (e.g., C5, C6, C7, or C8) sugars, disaccharides, and trisaccharides include sugars (e.g., C5, C6, C7, or C8) that have two or three monosaccharide units.

[0451] In certain embodiments, the carbohydrate conjugate includes a monosaccharide.

[0452] In certain embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). GalNAc conjugates comprising one or more N-acetylgalactosamine (GalNAc) derivatives are described, for example, in US8,106,022, the entire contents of which are incorporated herein by reference. In some embodiments, the GalNAc conjugate acts as a ligand that targets iRNA to specific cells. In some embodiments, the GalNAc conjugate targets iRNA to liver cells, for example, by acting as a ligand for the asialocrycoprotein receptor in liver cells (e.g., hepatocytes).

[0453] In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives can be attached via a linker, for example, a divalent or trivalent branched linker. In some embodiments, the GalNAc conjugate is conjugated to the 3' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (for example, to the 3' end of the sense strand) via a linker, for example, a linker as described herein. In some embodiments, the GalNAc conjugate is conjugated to the 5' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (for example, to the 5' end of the sense strand) via a linker, for example, a linker as described herein.

[0454] In certain embodiments of the present invention, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a divalent linker. In yet another embodiment of the present invention, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a trivalent linker. In yet another embodiment of the present invention, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a tetravalent linker.

[0455] In certain embodiments, the double-stranded RNAi agent of the present invention comprises one GalNAc or GalNAc derivative attached to an iRNA agent. In certain embodiments, the double-stranded RNAi agent of the present invention comprises multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently attached to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers.

[0456] In some embodiments, for example, if the two strands of the iRNA agent of the present invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of each of the other strands, forming a hairpin loop containing a plurality of unpaired nucleotides, each unpaired nucleotide in the hairpin loop may independently contain GalNAc or a GalNAc derivative attached via a monovalent linker. The hairpin loop may also be formed by an extended overhang in one of the two strands.

[0457] In some embodiments, for example, if the two strands of the iRNA agent of the present invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of each of the other strands, forming a hairpin loop containing a plurality of unpaired nucleotides, each unpaired nucleotide in the hairpin loop may independently contain GalNAc or a GalNAc derivative attached via a monovalent linker. The hairpin loop may also be formed by an extended overhang in one of the two strands.

[0458] In some embodiments, the GalNAc conjugate is

[0459] [ka]

[0460] In some embodiments, the RNAi agent is attached to a carbohydrate conjugate via a linker, as shown in the schematic diagram below, where X is O or S.

[0461] [ka]

[0462] In some embodiments, the RNAi agent is defined in Table 1 and conjugated to L96 as shown below:

[0463] [ka]

[0464] In certain embodiments, carbohydrate conjugates for use in the compositions and methods of the present invention are selected from the group consisting of:

[0465] [ka] [ka] [ka] [ka] [ka] [ka] [In the formula, Y is either O or S, and n is between 3 and 6] (Formula XXIV)

[0466] [ka] [In the formula, Y is either O or S, and n is between 3 and 6] (Formula XXV)

[0467] [ka] [In the formula, X is either O or S] (Formula XXVII),

[0468] [ka] [ka]

[0469] In certain embodiments, the carbohydrate conjugate for use in the compositions and methods of the present invention is a monosaccharide. In certain embodiments, the monosaccharide is N-acetylgalactosamine, for example,

[0470] [ka] That is the case.

[0471] Other representative carbohydrate conjugates for use in the embodiments described herein, but not limited to,

[0472] [ka] [In the formula, one of X or Y is an oligonucleotide, and the other is hydrogen.] These are some examples.

[0473] In some embodiments, suitable ligands are ligands disclosed in WO2019 / 055633, the entirety of which is incorporated herein by reference. In one embodiment, the ligand has the following structure:

[0474] [ka] Includes.

[0475] In certain embodiments, the RNAi agents of the Disclosure may include a GalNAc ligand, even if such a GalNAc ligand is currently expected to have limited value for the subarachnoid / CNS delivery pathway(s) of the Disclosure.

[0476] In certain embodiments of the present invention, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a divalent linker. In yet another embodiment of the present invention, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a trivalent linker. In yet another embodiment of the present invention, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a tetravalent linker.

[0477] In certain embodiments, the double-stranded RNAi agent of the present invention comprises one GalNAc or GalNAc derivative attached to the 5' end of the sense strand of an iRNA agent, e.g., a dsRNA agent, or to the 5' end of one or both sense strands of a dual-targeted RNAi agent described herein. In certain embodiments, the double-stranded RNAi agent of the present invention comprises a plurality of (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently attached to a plurality of nucleotides of the double-stranded RNAi agent via a plurality of monovalent linkers.

[0478] In some embodiments, for example, if the two strands of the iRNA agent of the present invention are part of one larger molecule linked by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of the other strand, forming a hairpin loop containing a plurality of unpaired nucleotides, each of the unpaired nucleotides in the hairpin loop may independently contain GalNAc or a GalNAc derivative attached via a monovalent linker.

[0479] In some embodiments, the carbohydrate conjugate further comprises one or more of the above-mentioned ligands, but is not limited to a carbohydrate conjugate, such as a PK modulator or a cell-permeable peptide.

[0480] Further carbohydrate conjugates and linkers suitable for use in the present invention include those described in WO2014 / 179620 and WO2014 / 179627, the entire contents of which are incorporated herein by reference.

[0481] D. Linker In some embodiments, the conjugates or ligands described herein can be attached to iRNA oligonucleotides using a variety of linkers, which may or may not be cleavable.

[0482] The term "linker" or "linking group" refers to an organic part that connects two parts of a compound, for example, by covalent bonding.Linkers are typically directly bonded or composed of atoms such as oxygen or sulfur, units such as NR8, C(O), C(O)NH, SO, SO2, SO2NH, or not, but one or more methylene groups may be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylal Quinnyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylal Kenyl, alkyl heteroarylalkynyl, alkenyl heteroarylalkyl, alkenyl heteroarylalkenyl, alkenyl heteroarylalkynyl, alkenyl heteroarylalkynyl, alkynyl heteroarylalkyl, alkynyl heteroarylalkenyl, alkynyl heteroarylalkynyl, alkyl heterocyclylalkyl, alkyl heterocyclylalkenyl, alkyl heterocyclylalkynyl (alkylhererocyclylalkynyl), alkenyl heterocyclylalkyl, alkenyl heterocyclylalkenyl, alkenyl heterocyclylalkynyl, alkynyl heterocyclylalkynyl, alkynyl heterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkyl heteroaryl, alkenyl heteroaryl, alkynyl heteroaryl (alkynylhereroaryl), R8 is hydrogen, acyl, aliphatic or substituted aliphatic, and includes a chain of atoms such as substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc.In certain embodiments, the linker has approximately 1 to 24 atoms, 2 to 24, 3 to 24, 4 to 24, 5 to 24, 6 to 24, 6 to 18, 7 to 18, 8 to 18 atoms, 7 to 17, 8 to 17, 6 to 16, 7 to 16, or 8 to 16 atoms.

[0483] A cleavable linker is one that is sufficiently stable outside the cell but, upon entering the target cell, is cleaved, releasing the two parts held together by the linker. In one embodiment, the cleavable linker is cleaved at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or more, or at least about 100 times faster in the target cell or under a first reference condition (which may be selected to mimic or represent intracellular conditions, for example) than in the target blood or under a second reference condition (which may be selected to mimic or represent conditions found in blood or serum).

[0484] Cleavable linking groups are susceptible to the influence of cleavage agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleavage agents are more common or found at higher levels or activity inside cells than in serum or blood. Examples of such degrading agents include redox agents selected for specific substrates or those without substrate specificity, including reducing agents such as mercaptans present in cells that can degrade redox-cleavable linking groups by oxidase or reductase or reduction, esterases, endosomes, or agents that can create an acidic environment, such as those that result in a pH of 5 or less, general acids, peptidases (which may be substrate-specific), and enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as phosphatases.

[0485] Cleavable linking groups, such as disulfide bonds, can be susceptible to pH changes. While human serum has a pH of 7.4, the average intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes have a more acidic pH in the range of 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers may have cleavable linking groups that are cleaved at a selected pH, thereby releasing cationic lipids from ligands into the cell or into desired compartments within the cell.

[0486] Linkers may contain cleavable linking groups that can be cleaved by specific enzymes. The type of cleavable linking group incorporated into the linker may vary depending on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid via a linker containing an ester group. Liver cells are rich in esterases, and therefore, linkers are cleaved more efficiently in liver cells than in cell types that are not rich in esterases. Other cell types rich in esterases include lung, renal cortex, and testicular cells.

[0487] When targeting peptidase-rich cell types such as liver cells and synovial cells, linkers containing peptide bonds can be used.

[0488] Generally, the suitability of a candidate cleavable linker can be evaluated by testing the ability of a degrading agent (or condition) to cleave the candidate linker. It would also be desirable to test the candidate cleavable linker for its ability to resist cleavage in blood or in contact with other non-target tissues. Thus, the relative sensitivity to cleavage between the first and second conditions can be determined, with the first being selected to exhibit cleavage in target cells and the second being selected to exhibit cleavage in other tissues or biological fluids, such as blood or serum. Evaluations can be carried out in cell-free systems, in cells, in cell cultures, in organs or tissue cultures, or in whole animals. It may be useful to perform initial evaluations in cell-free or culture conditions and confirm them with further evaluations in whole animals. In certain embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0489] i. Redox-cleavable linking groups In certain embodiments, the cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of a reductively cleavable linking group is a disulfide linking group (-SS-). To determine whether a candidate cleavable linking group is a suitable “reductively cleavable linking group” or whether it is suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, one can turn to the methods described herein. For example, 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 that would be observed in cells, e.g., target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In one embodiment, a candidate compound is cleaved by up to about 10% in blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times 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 rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions selected to mimic intracellular media compared to conditions selected to mimic extracellular media.

[0490] ii. Phosphate-based cleavable linking groups In certain embodiments, the cleavable linker includes a phosphate-based cleavable linking group. The phosphate-based cleavable linking group is cleaved by agents that decompose or hydrolyze the phosphate group. Examples of agents that cleave phosphate groups in cells include enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S- (wherein each instance of Rk may be independently a C1-C20 alkyl, a C1-C20 haloalkyl, a C6-C10 aryl, or a C7-C12 aralkyl). Exemplary embodiments include -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. In one embodiment, the phosphate-based linking group is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0491] iii. Acid-cleavable linking groups In certain embodiments, a cleavable linker includes an acid-cleavable linking group. An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In some embodiments, an acid-cleavable linking group is cleaved in an acidic environment having a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0 or lower), or by a drug such as an enzyme that can act as a general acid. In cells, certain low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for acid-cleavable linking groups. Examples of acid-cleavable linking groups, but not limited to them, include hydrazones, esters, and amino acid esters. Acid-cleavable groups may have the general formula -C=NN-, C(O)O, or -OC(O). An exemplary embodiment is when the carbon bonded to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group, such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0492] iv. Ester-based cleavable linking groups In certain embodiments, the cleavable linker includes an ester-based cleavable linking group. This ester-based linking group is cleaved by enzymes such as esterases and amidases in the cell. Examples of ester-based cleavable linking groups, but not limited to them, include esters of alkylene, alkenylene, and alkynylene groups. Ester-cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0493] v. Peptide-based cleavable linking groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linking group. The peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. The peptide-based cleavable linking group is a peptide bond formed between amino acids, resulting in oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable group does not contain an amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene, or alkylene. A peptide bond is a special type of amide bond formed between amino acids, resulting in peptides and proteins. The peptide-based cleavable group is generally limited to peptide bonds (i.e., amide bonds) formed between amino acids, resulting in peptides and proteins, and does not contain the entire amide functional group. The peptide-based cleavable linking group has the general formula -NHCHRAC(O)NHCHRBC(O)- (wherein RA and RB are the R groups of two adjacent amino acids). These candidates can be evaluated using methods similar to those described above.

[0494] In some embodiments, the iRNA of the present invention is conjugated to a carbohydrate via a linker. Examples of iRNA carbohydrate conjugates having linkers of the compositions and methods of the present invention, but not limited to these, include:

[0495] [ka] [ka] [ka] [If either X or Y is an oligonucleotide, then the other is hydrogen.] These are some examples.

[0496] In certain embodiments of the compositions and methods of the present invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a divalent or trivalent branched linker.

[0497] In certain embodiments, the dsRNA of the present invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures represented by any of the formulas (XLV) to (XLVIII):

[0498] [ka] [In the formula, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C each independently represent a number from 0 to 20 for each occurrence, and the repeating units may be the same or different. P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C Each occurrence is independently of the following: non-existent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O. Q 2A Q 2B Q 3A Q 3B Q 4A Q 4B Q 5A Q 5B Q 5C Each occurrence is independently of whether it is a non-existent alkylene, a substituted alkylene, and one or more methylenes, O, S, S(O), SO2, N(R) N), C(R')=C(R''), C≡C or C(O) may interrupt or terminate by one or more of these. R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C For each occurrence, independently, the non-existent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R) a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO,

[0499] [ka] or heterocycline, L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C R represents a ligand, that is, each instance independently represents a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. a [H is an amino acid side chain]. Trivalent conjugate GalNAc derivatives target genes, e.g., those of formula (XLIX):

[0500] [ka] [In the formula, L 5A , L 5B and L 5C This represents a monosaccharide, for example, a GalNAc derivative. It is particularly useful for use in conjunction with RNAi agents to inhibit the expression of [specific gene / substance].

[0501] Examples of suitable divalent and trivalent branched linker groups for conjugating GalNAc derivatives include, but are not limited to, the structures listed above, such as formulas II, VII, XI, X, and XIII.

[0502] Representative U.S. patents teaching the preparation of RNA conjugates include, but are not limited to, U.S. Patents 4,828,979, 4,948,882, 5,218,105, 5,525,465, 5,541,313, 5,545,730, 5,552,538, 5,578,717, 5,580,731, 5,591,584, 5,109,124, 5,118,802, 5,138,045, 5,414,077, and 5,486,603, 5,512,439, 5,578,718, 5,608,046, 4,587,044, 4,605,735, 4,667,025, 4,762,779, 4,789,737, 4,824,941, 4,835,263, 4,876,335, 4,904,582, 4,958,013, 5,082,830, 5,112,963, 5,214,136, 5,082,830, No. 5,112,963, No. 5,214,136, No. 5,245,022, No. 5,254,469, No. 5,258,506, No. 5,262,536, No. 5,272,250, No. 5,292,873, No. 5,317,098, No. No. 5,371,241, No. 5,391,723, No. 5,416,203, No. 5,451,463, No. 5,510,475, No. 5,512,667, No. 5,514,785, No. 5,565,552, No. 5,567,810, No. Examples include Nos. 5,574,142, 5,585,481, 5,587,371, 5,595,726, 5,597,696, 5,599,923, 5,599,928, 5,688,941, 6,294,664, 6,320,017, 6,576,752, 6,783,931, 6,900,297, 7,037,646, and 8,106,022, the entire contents of each of these are incorporated herein by reference.

[0503] It is not necessary for all positions in a given compound to be uniformly modified; in fact, two or more of the modifications can be incorporated into a single compound, or even into a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0504] In relation to the present invention, "chimeric" iRNA compound or "chimeric" refers to an iRNA compound, e.g., a dsRNA agent, that contains two or more chemically distinct regions, each composed of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region in which the RNA is modified to confer increased resistance to nuclease degradation, increased cellular uptake, or increased binding affinity to a target nucleic acid. Further regions of the iRNA can act as substrates for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA double-stranded DNA. Therefore, activation of RNase H results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, when chimeric dsRNAs are used, it is often possible to obtain results that can be compared with shorter iRNAs compared to phosphorothioate deoxy dsRNAs that hybridize to the same target region. Cleavage of RNA targets can be routinely detected by gel electrophoresis and, if necessary, by relevant nucleic acid hybridization techniques known in the art.

[0505] In certain cases, the RNA of an iRNA can be modified with non-ligand groups. Several non-ligand molecules have been conjugated to iRNAs to enhance their activity, cell distribution, or cell uptake, and procedures for carrying out such conjugations are available in the scientific literature. These non-ligand portions include lipid portions such as cholesterol [Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61, Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553], cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers, for example, 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), fatty acid chains, e.g., 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., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651, Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969) or adamantane acetate (Manoharan et al.These included (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., 1996, 277:923). Representative U.S. patents teaching the preparation of such RNA conjugates are listed above. A typical conjugation protocol involves the synthesis of RNA having aminolinkers at one or more positions in its sequence. The amino group then reacts with the conjugated molecule using an appropriate coupling or activating reagent. The conjugation reaction can be performed while the RNA is still bound to a solid support or after the RNA has been cleaved in solution. Purification of the RNA conjugate by HPLC usually yields a pure conjugate.

[0506] V. Delivery of RNAi agents of this disclosure The delivery of the RNAi agents of this disclosure to cells in a subject, such as a human subject (e.g., a subject that requires it, e.g., a subject with GPR75-related disorder, e.g., weight disorder, e.g., an obese subject, e.g., a subject that has or is at risk of developing or having weight disorder), can be achieved in several different ways. For example, delivery may be carried out by contacting cells with the RNAi agents of this disclosure either in vitro or in vivo. In vivo delivery may be carried out directly by administering the RNAi agent, e.g., a composition comprising dsRNA, to the subject. Alternatively, in vivo delivery may be carried out indirectly by administering one or more vectors that encode the RNAi agent and induce its expression. These alternatives are described further below.

[0507] In general, any method of delivering nucleic acid molecules (in vitro or in vivo) can be adapted to the use of RNAi agents in this disclosure [see, for example, Akhtar S. and Julian RL., (1992) Trends Cell. Biol. 2(5):139-144 and WO94 / 02595, which are incorporated herein in their entirety by reference]. In the case of in vivo delivery, factors to consider for delivering RNAi agents include, for example, the biological stability of the delivered agent, prevention of nonspecific effects, and accumulation of the delivered agent in the target tissue. Nonspecific effects of RNAi agents can be minimized by local administration, e.g., direct injection or transplantation into tissue or local administration of preparations. Local administration to the treatment site maximizes the local concentration of the agent, limits exposure of the agent to systemic tissues that may be harmed or degraded by the agent, and allows for a lower total dose of RNAi agent administered. Several studies have demonstrated successful knockdown of gene products when RNAi agents are administered locally. For example, pulmonary delivery of dsRNAs, such as SOD1, by inhalation, has been shown to efficiently knock down gene and protein expression in lung tissue, and to have excellent uptake of dsRNAs by the bronchioles and alveoli of the lung. Intraocular delivery of VEGF dsRNA by intravitreous injection in cynomolgus monkeys [Tolentino, MJ. et al., (2004) Retina 24:132-138] and subretinal injection in mice [Reich, SJ. et al. (2003) Mol. Vis. 9:210-216] have both been shown to prevent neovascularization in experimental models of age-related macular degeneration. In addition, direct intratumoral injection of dsRNA into mice can reduce tumor volume [Pille, J. et al. (2005) Mol. Ther. 11:267-274] and prolong the survival of mice with tumors [Kim, WJ. et al., (2006) Mol. Ther. 14:343-350; Li, S. et al., (2007) Mol. Ther. 15:515-523].RNA interference can be administered to the CNS by direct injection [Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, PH. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al. (2002) BMC Neurosci. 3:18; Shishkina, GT., et al. (2004) Neuroscience 129:521-528; Thakker, ER., et al. (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594-602] and to the lungs by intranasal administration [Howard, KA. et al., (2006) Mol. Ther. [14:476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55], success has also been demonstrated by local delivery. When RNAi agents are administered systemically to treat a disease, the RNA can be modified, or instead, delivered using a drug delivery system; both methods function to prevent the rapid degradation of dsRNA by endonucleases and exonucleases in vivo. Modification of RNA or drug carriers can also enable the targeting of RNAi agents to target tissues and avoid undesirable off-target effects (for example, although we do not wish to be bound by theory, the use of GNAs described herein has been identified to destabilize the seed region of dsRNAs, and such off-target effects are significantly attenuated by such seed region destabilization, thereby increasing the preference of such dsRNAs for on-target efficacy compared to off-target effects). RNAi agents can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation.For example, when RNAi agents derived to ApoB conjugated to a lipophilic cholesterol portion were systemically injected into mice, it resulted in knockdown of apoB mRNA in both the liver and jejunum [Soutschek, J. et al., (2004) Nature 432:173-178]. Conjugation of RNAi agents to aptamers has been shown to inhibit tumor growth and mediate tumor reduction in a mouse model of prostate cancer [McNamara, JO. et al., (2006) Nat. Biotechnol. 24:1005-1015]. In alternative embodiments, RNAi agents can be delivered using drug delivery systems, such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems promote the binding of molecular RNAi agents (negatively charged) and enhance interactions with negatively charged cell membranes, thereby enabling efficient uptake of RNAi agents by cells. Cationic lipids, dendrimers, or polymers can bind to RNAi agents or be induced to form vesicles or micelles that encapsulate RNAi agents [see, for example, Kim SH. et al., (2008) Journal of Controlled Release 129(2):107-116]. Vesicle or micelle formation further prevents the degradation of RNAi agents when administered systemically. Methods for preparing and administering cationic RNAi agent conjugates are well within the capabilities of those skilled in the art [see, for example, Sorensen, DR., et al. (2003) J. Mol. Biol 327:761-766; Verma, UN. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al. (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety].Some non-limiting examples of drug delivery systems useful for systemic delivery of RNAi agents include DOTAP [Sorensen, DR., et al (2003), supra; Verma, UN. et al., (2003), supra], oligofectamine, "solid nucleic acid lipid particles" [Zimmermann, TS. et al., (2006) Nature 441:111-114], cardiolipin [Chien, PY. et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091], and polyethyleneimine [Bonnet ME. et al., (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol.]. Examples include

[71659] , Arg-Gly-Asp(RGD) peptide [Liu, S. (2006) Mol. Pharm. 3:472-487], and polyamidoamine [Tomalia, DA. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804]. In some embodiments, the RNAi agent forms a complex with cyclodextrin for systemic administration. Methods of administration and pharmaceutical compositions of RNAi agents and cyclodextrin can be found in U.S. Patent No. 7,427,605, which is incorporated herein by reference in whole.

[0508] Certain aspects of the present disclosure relate to a method for reducing the expression of the GPR75 gene in cells, comprising contacting the cells with a double-stranded RNAi agent of the present disclosure. In one embodiment, the cells are liver cells, and optionally hepatocytes. In one embodiment, the cells are nerve cells.

[0509] In certain embodiments, the RNAi agent is taken up by one or more tissues or cell types present in an organ, such as the liver or kidney.

[0510] Another aspect of the present disclosure relates to a method for reducing the expression and / or activity of the GPR75 gene in a subject, comprising administering the subject a double-stranded RNAi agent of the present disclosure.

[0511] Another aspect of the present disclosure relates to a method for treating a subject who has, is at risk of having, or is at risk of developing a GPR75-related disorder, comprising administering to the subject a therapeutically effective dose of the double-stranded RNAi agent of the present disclosure, thereby treating the subject. In some embodiments, the GPR75-related disorder includes weight disorders, such as obesity.

[0512] In one embodiment, the double-stranded RNAi agent is administered subcutaneously.

[0513] In one embodiment, the double-stranded RNAi agent is administered intrathecally. By intrathecal administration of the double-stranded RNAi agent, the method can reduce the expression of GPR75 target genes in brain (e.g., striatum) or spinal tissue, such as the cortex, cerebellum, cervical vertebrae, lumbar vertebrae, and thoracic vertebrae.

[0514] In one embodiment, the double-stranded RNAi agent is administered intravenously.

[0515] For the sake of clarity, the formulations, compositions, and methods described in this section will primarily relate to modified siRNA compounds. However, it should be understood that these formulations, compositions, and methods can be practiced with other siRNA compounds, such as unmodified siRNA compounds, and that such practices are also within this disclosure. Compositions containing RNAi agents can be delivered to the target by a variety of routes. Exemplary routes include intrathecal, pulmonary, intravenous, subcutaneous, intraventricular, oral, topical, rectal, vaginal, nasal, and intraocular administration.

[0516] The RNAi agents of this disclosure can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise one or more RNAi agents and a pharmaceutically acceptable carrier. As used herein, the phrase “pharmaceutically acceptable carrier” is intended to include any solvent, dispersion medium, coating agent, antifungal and antifungal agent, isotonic and absorption retardant, and similar, that is suitable for pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active ingredient, their use in a composition is conceived. Supplementary active ingredients may also be incorporated into a composition.

[0517] The pharmaceutical compositions of this disclosure may be administered in several ways, depending on whether a topical or systemic treatment is desired and the area to be treated. Administration may be intratracheal, intranasal, topical (e.g., ophthalmic, vaginal, rectal, intranasal, percutaneous, etc.), oral, parenteral, or pulmonary by inhalation or insufflation of powders or aerosols, for example, by a nebulizer. Parenteral administration may include intravenous infusion, subcutaneous, intraperitoneal, or intramuscular injection, or intrathecal or intraventricular administration.

[0518] The route and site of administration may be selected to enhance targeting. For example, to target muscle cells, intramuscular injection into the target muscle would be a logical choice. Lung cells may be targeted by administration of RNAi agents in powder or aerosol form. Vascular endothelial cells may be targeted by coating balloon catheters with RNAi agents and mechanical delivery of RNA.

[0519] The composition for pulmonary delivery may include, for example, an aqueous solution for intranasal or oral inhalation administration, a suitable carrier consisting of, for example, lipids (liposomes, niosomes, microemulsions, lipid micelles, solid lipid nanoparticles) or polymers (polymer micelles, dendrimers, polymer nanoparticles, nonogels, nanocapsules), or an adjuvant for oral inhalation administration. The aqueous composition may be sterile and may optionally contain buffers, diluents, absorption enhancers, and other suitable additives. Such administration enables both systemic and local delivery of the double-stranded RNAi agent of the present invention.

[0520] Intranasal administration may include infusing or blowing double-stranded RNA agents into the nasal cavity using a syringe or dispensing device, either in a single dose or by applying several drops by atomization. Suitable dosage forms for intranasal administration include droplets, powders, atomized mists, and sprays. Nasal delivery devices include, but are not limited to, steam inhalers, nasal droppers, spray bottles, metered-dose spray pumps, gas-driven spray nebulizers, nebulizers, mechanical powder nebulizers, respiratory-operated inhalers, and insufflerators. Devices for delivery to the respiratory system, e.g., deep into the lungs, include nebulizers, pressurized metered-dose inhalers, dry powder inhalers, and thermal evaporation aerosol devices. Devices for inhalation delivery are available from commercial suppliers. Devices may be disposable or reusable, fixed or variable dose, single or multiple dose, depending on, for example, the disease or disorder to be prevented or treated, the volume of the drug to be delivered, the frequency of drug delivery, and other considerations in the industry.

[0521] Oral inhalation administration may include the use of devices, such as single-dose / multi-dose dry powder inhalers (DPIs) induced by passive respiration or active force, for delivering double-stranded RNAi agents to the pulmonary system. Suitable dosage forms for oral inhalation administration include powders and liquids. Suitable devices for oral inhalation administration include nebulizers, metered-dose inhalers, and dry powder inhalers. Dry powder inhalers are the most common devices used to deliver drugs, particularly proteins, to the lungs. Exemplary commercially available dry powder inhalers include Spinhaler (Fisons Pharmaceuticals, Rochester, NY) and Rotahaler (GSK, RTP, NC). Several types of nebulizers are available, namely jet nebulizers, ultrasonic nebulizers, and vibrating mesh nebulizers. Jet nebulizers are driven by pressurized air. Ultrasonic nebulizers use piezoelectric transducers to create droplets from an open liquid reservoir. Vibrating mesh nebulizers use a perforated membrane actuated by an annular piezoelectric element to vibrate in a resonant bending mode. The pores in the membrane have a large cross-sectional size on the liquid supply side and a narrow cross-sectional size on the side from which the droplet emerges. The size and number of pores can be adjusted according to the therapeutic application. The selection of a suitable device depends on parameters such as the properties of the drug and its formulation, the site of action, and the pathophysiology of the lung. Aqueous suspensions and solutions are efficiently atomized. Aerosols based on mechanically generated vibrating mesh technology are also successfully used to deliver proteins to the lungs.

[0522] The dosage of RNAi agents for pulmonary administration may vary from one target gene to another, and the appropriate dosage to be applied may need to be determined individually for each target gene. Typically, this dosage ranges from 10 μg to 2 mg, 50 μg to 1500 μg, or 100 μg to 1000 μg.

[0523] Topical formulations may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, solutions, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, and thickeners may also be necessary or desirable. Coated condoms and gloves may also be useful.

[0524] Oral administration compositions include powders or granules, suspensions or solutions in water, syrups, exylates or non-aqueous media, tablets, capsules, drops, or lozenges. For tablets, suitable carriers include salts of lactose, sodium citrate, and phosphoric acid. Various disintegrants, such as starch, and lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycol. When an aqueous suspension is required for oral administration, nucleic acid compositions can be combined with emulsifiers and suspension agents. Certain sweeteners or flavorings may be added if desired. Suitable compositions for oral administration of the agents of the present invention are further described in PCT application number PCT / US20 / 33156, the entire contents of which are incorporated herein by reference.

[0525] Compositions for intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents, and other suitable additives.

[0526] Preparations for parenteral administration may include sterile aqueous solutions that may also contain buffers, diluents, and other suitable additives. Intracerebroventricular injection may be facilitated, for example, by an intracerebroventricular catheter attached to a reservoir. For intravenous use, the total concentration of the solute may be controlled to make the preparation isotonic.

[0527] In one embodiment, the administration of an siRNA compound, such as a double-stranded siRNA compound or ssiRNA compound, composition, is parenteral, for example, intravenous (e.g., as a bolus or diffuse infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracerebral, subcutaneous, transmucosal, buccal, sublingual, endoscopic, transrectal, oral, transvaginal, topical, pulmonary, intranasal, transurethral, ​​or transocular. The administration may be given by the patient or by another person, such as a healthcare provider. The drug may be provided in measured doses or in a dispenser that delivers weighed doses. The selected mode of delivery is described in more detail below.

[0528] Intrathecal administration In one embodiment, double-stranded RNAi agents are delivered by intrathecal injection (i.e., injection into cerebrospinal fluid that immerses brain and spinal cord tissue). Intrathecal injection of RNAi agents into cerebrospinal fluid can be performed as a bolus injection or by a minipump that can be implanted subcutaneously, providing regular and consistent delivery of siRNA into the cerebrospinal fluid. The circulation of cerebrospinal fluid from the choroid plexus, where it is produced, descends around the spinal cord and dorsal root ganglia, then passes through the cerebellum and crosses the cortex to the arachnoid granulations, where the fluid can exit the CNS, and depending on the size, stability, and solubility of the injected compound, molecules delivered intrathecally can attack targets throughout the CNS.

[0529] In some embodiments, intrathecal administration is performed via a pump. The pump may be an osmotic pump surgically implanted. In one embodiment, the osmotic pump is implanted in the subarachnoid space of the spinal canal to facilitate intrathecal administration.

[0530] In some embodiments, intrathecal administration is performed via an intrathecal delivery system for a drug, which includes a reservoir containing a certain amount of the drug and a pump configured to deliver a portion of the drug contained in the reservoir. Further details of this intrathecal delivery system can be found in WO2015 / 116658, which is incorporated herein by reference in its entirety.

[0531] The amount of RNAi agent injected intrathecally may change from one target gene to another, and the appropriate amount to be applied may need to be determined individually for each target gene. Typically, this amount ranges from 10 μg to 2 mg, 50 μg to 1500 μg, or 100 μg to 1000 μg.

[0532] Vector-coding RNAi agents disclosed herein RNAi agents targeting the GPR75 gene can be expressed from transcript units inserted into DNA or RNA vectors [see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; WO 00 / 22113, WO 00 / 22114, and US 6,054,299]. Expression may persist (for several months or more) depending on the specific construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, and they may be integrated or non-integrated vectors. Transgenes can also be constructed to allow passage as extrachromosomal plasmids [Gassmann, et al., (1995) Proc. Natl. Acad. Sci. USA 92:1292].

[0533] Individual strands of an RNAi agent can be transcribed from a promoter on an expression vector. If two separate strands are expressed to produce, for example, dsRNA, two separate expression vectors can be co-introduced into target cells (e.g., by transfection or infection). Alternatively, each individual strand of dsRNA can be transcribed by a promoter both located on the same expression plasmid. In one embodiment, the dsRNA is expressed as a reverse repeat polynucleotide linked by a linker polynucleotide sequence such that the dsRNA has a stem-and-loop structure.

[0534] RNAi agent expression vectors are generally DNA plasmids or viral vectors. By using expression vectors compatible with eukaryotic cells, preferably expression vectors compatible with vertebrate cells, recombinant constructs for the expression of RNAi agents described herein can be generated. The delivery of RNAi agent expression vectors may be systemic, for example, by intravenous or intramuscular administration, by administration to target cells explanted from a patient and subsequent reintroduction into the patient, or by any other means that enable introduction into desired target cells.

[0535] Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, e.g., lentivirus vectors, Moloney's mouse leukemia virus, etc., but are not limited to these; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomevirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) vesicular virus vectors, e.g., orthopox, e.g., varicella virus vector, etc., or avipox, e.g., canarypox or avian diphtheria, etc.; and (j) helper-dependent or gutless adenoviruses. Replication-deficient viruses may also be advantageous. Different vectors may or may not be incorporated into the cell genome. The construct may, if desired, contain a viral sequence for transfection. Alternatively, the construct can be incorporated into an episomal replication-capable vector, suc...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of G protein-coupled receptor 75 (GPR75) in cells, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence containing at least 15 consecutive nucleotides, including 0, 1, 2, or 3 mismatches in a portion of any one of the nucleotide sequences of SEQ ID NOs: 1 to 4, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of any one of the nucleotide sequences of SEQ ID NOs: 1 to 4, and the antisense strand comprises a nucleotide sequence containing at least 15 consecutive nucleotides, including 0, 1, 2, or 3 mismatches in a corresponding portion of any one of the nucleotide sequences of SEQ ID NOs: 5 to 8, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of any one of the nucleotide sequences of SEQ ID NOs: 5 to 8, and the sense strand or antisense strand is conjugated to one or more lipophilic portions.

2. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of G protein-coupled receptor 75 (GPR75) in cells, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a region complementary to a portion of the mRNA encoding the GPR75 gene (any one of SEQ ID NOs: 1 to 4), each strand independently being 14 to 30 nucleotides long, and the sense strand or antisense strand being conjugated to one or more lipophilic regions.

3. A double-stranded RNAi agent for inhibiting the expression of the G protein-coupled receptor 75 (GPR75) gene in cells, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises at least 15 consecutive nucleotides that differ by 3 nucleotides or less from any one of the antisense nucleotide sequences in any one of Tables 2, 3, 5, and 6, each strand independently having a length of 14 to 30 nucleotides, and the sense strand or antisense strand is conjugated to one or more lipophilic moieties.

4. The dsRNA agent according to any one of claims 1 to 3, wherein the sense strand or antisense strand is a sense strand or antisense strand selected from the group consisting of any one of the sense strands and antisense strands in Tables 2, 3, 5, and 6.

5. A double-stranded RNAi agent for inhibiting the expression of G protein-coupled receptor 75 (GPR75) in cells, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises nucleotides 38-60; 50-72; 148-181; 153-181; 153-175; 159-181; 228-250; 240-262; 341-363; 341-368; 346-368; 369-396; 369-391; 374-396; 388-410; 414-436; 424-461; 424-446; 424-451; 434-45 6; 439-461; 429-451; 457-504; 462-504; 462-491; 482-504; 469-491; 457-479; 462-584; 475-497; 469-491; 509-537; 509-531; 515-537; 544-576; 54 4-566; 549-571; 580-607; 580-602; 585-607; 595-617; 615-647; 615-637; 620-642; 620-647; 625-647; 773-806; 773-795; 773-795; 778-800; 784-806 ;837-872;837-859;843-872;843-865;850-872;860-882;889-911;900-936;900-922;908-936;908-930;914-936;938-990;938-960;943-965;968 ~990; 1060~1101; 1060~1082; 1066~1088; 1073~1095; 1079~1101; 1097~1119; 1238~1260; 1268~1290; 1284~1393; 1284~1306; 1292~1393; 1292~1314; 1292-1383; 1292-1314; 1301-1323; 1307-1383; 1307-1342; 1307-1329; 1313-1335; 1371-1393; 1351-1373; 1320-1342; 1336-1358; 1345-1367; 1351- 1373; 1361-1383; 1366-1388; 1393-1415; 1422-1463; 1422-1444; 1441-1463; 1487-1526; 1487-1509; 1493-1526; 1493-1515; 1498-1520; 1504-1526;１５１５～１５７１；１５１５～１５５７；１５１５～１５４３；１５１５～１５３７；１５２１～１５４３；１５３０～１５５２；１５３５～１５５７；１５４０～１５６２；１５４９～１５７１；１５５９～１５８６；１５５９～１５８１；１５６４～１５８６；１５８３～１６２９；１５８３～１６０５；１５８８～１６１０；１５９５～１６１７；１６００～１６２９；１６００～１６２２；１６０７～１６２９；１６２４～１６４６；１６３５～１６５７；１６７２～１７２１；１６７２～１７１０；１６７７～１６９９；１６９９～１７２１；１６７２～１６９９；１６８８～１７１０；１６７２～１６９４；１６８３～１７０５；１６９３～１７１４；１７３２～１７５４；１７４４～１７９８；１７５１～１７７３；１７５８～１７８０；１７６７～１７８９；１７７６～１７９８；１７９０～１８１８；１７９０～１８１２；１７９６～１８１８；１８０８～１８５６；１８０８～１８４８；１８０８～１８３６；１８０８～１８３０；１８２６～１８４８；１８１４～１８３６；１８１９～１８４１；１８３４～１８５６；１８７７～２０８２；１８７７～１８９９；１８８２～２０８２；１８８２～１９２５；１８８２～１９６３；１８８２～１９０４；１８８７～１６９３；１８８７～１９０９；１８９８～１９２０；１９０３～１９２５；１９０８～１９３０；１９１３～１９３５；１９１３～１９５０；１９２１～１９５０；１９２１～１９４３；１９２８～１９５０；１９３３～１９５５；１９４１～１９６３；１９４６～１９６８；１９５３～１９８５；１９５３～２０８２；１９５３～１９７５；１９３８～１９８５；１９５８～１９８０；１９６３～１９８５；１９６８～１９９０；１９７４～１９９６；１９７４～２０６５；１９７４～２０８２；１９７４～２００２；１９８０～２００２；１９８５～２００７；１９９０～２０１２；１９９０～２０３３；１９９９～２０２１；２００５～２０３３；２００５～２０２７；２０１１～２０３３；２０１７～２０３９；２０２５～２０５５；２０２５～２０４７；２０３３～２０５５；２０３８～２０６０；２０４３～２０６５；２０３３～２０５５；２０４８～２０７０；２０５４～２０８２；２０５４～２０７６；A double-stranded RNAi agent comprising at least 15 consecutive nucleotides that differ by 3 nucleotides or less from any one of the nucleotide sequences 2060 to 2082, wherein the antisense strand comprises at least 15 consecutive nucleotides derived from the corresponding nucleotide sequence of SEQ ID NO: 2, and the sense strand or antisense strand is conjugated to one or more lipophilic moieties.

6. The dsRNA agent according to any one of claims 1 to 5, wherein both the sense strand and the antisense strand are conjugated to one or more lipophilic moieties.

7. The dsRNA agent according to any one of claims 1 to 6, wherein the lipophilic portion is conjugated at one or more positions in the double-stranded region of the dsRNA agent.

8. A dsRNA agent according to any one of claims 1 to 7, wherein the lipophilic portion is conjugated via a linker or carrier.

9. A dsRNA agent according to any one of claims 1 to 8, wherein the lipophilicity of the lipophilic portion measured by logKow is greater than 0.

10. A dsRNA agent according to any one of claims 1 to 9, wherein the hydrophobicity of the double-stranded RNAi agent, as measured by the unbound fraction in a plasma protein binding assay of the double-stranded RNAi agent, is greater than 0.

2.

11. The dsRNA agent according to claim 10, wherein the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

12. A dsRNA agent according to any one of claims 1 to 11, comprising at least one modified nucleotide.

13. The dsRNA agent according to claim 12, wherein five or fewer nucleotides of the sense strand and five or fewer nucleotides of the antisense strand are unmodified nucleotides.

14. The dsRNA agent according to claim 12, wherein all nucleotides of the sense strand and all nucleotides of the antisense strand are modified.

15. At least one of the modified nucleotides is a deoxy-nucleotide, a 3'-terminal deoxythymidine (dT) nucleotide, a 2'-O-methyl-modified nucleotide, a 2'-fluoro-modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a restricted ethyl nucleotide, a debasalized nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-methoxyethyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramide, a nucleotide containing a non-natural base, a tetrahydropyran-modified nucleotide, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing a 5'-phosphorothioate group, nucleotides containing a 5'-methylphosphonate group, nucleotides containing a 5'-phosphate or 5'-phosphate mimetic, nucleotides containing vinylphosphonate, nucleotides containing adenosine-glycol nucleic acid (GNA), nucleotides containing thymidine-glycol nucleic acid (GNA) S isomers, nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3'-phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, 2'-O-hexadecyl nucleotides, nucleotides containing 2'-phosphate, cytidine-2'-phosphate nucleotides, guanosine-2'-phosphate nucleotides A dsRNA agent according to any one of claims 12 to 14, selected from the group consisting of 2'-O-hexadecyl-cytidine-3'-phosphate nucleotide, 2'-O-hexadecyl-adenosine-3'-phosphate nucleotide, 2'-O-hexadecyl-guanosine-3'-phosphate nucleotide, 2'-O-hexadecyl-uridine-3'-phosphate nucleotide, 5'-vinyl phosphonate (VP), 2'-deoxyadenosine-3'-phosphate nucleotide, 2'-deoxycytidine-3'-phosphate nucleotide, 2'-deoxyguanosine-3'-phosphate nucleotide, 2'-deoxythymidine-3'-phosphate nucleotide, 2'-deoxyuridine nucleotide, cholesteryl derivatives, terminal nucleotides linked to a bisdecylamide dodecanoate group, and combinations thereof.

16. The dsRNA agent according to claim 15, wherein the modified nucleotide is selected from the group consisting of nucleotides comprising 2'-deoxy-2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, 3'-terminal deoxythymidine nucleotides (dT), locked nucleotides, debasalized nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, and non-natural bases.

17. The dsRNA agent according to claim 15, wherein the modified nucleotide comprises a short sequence of a 3'-terminal deoxythymidine nucleotide (dT).

18. The dsRNA agent according to claim 15, wherein the nucleotide modifications are 2'-O-methyl modification, 2'-deoxy- modification, 2'-fluoro modification, 5'-vinylphosphonate (VP) modification, and 2'-O-hexadecyl nucleotide modification.

19. The dsRNA agent according to claim 15, further comprising at least one phosphorothioate nucleotide linkage.

20. The dsRNA agent according to claim 19, comprising 6 to 8 phosphorothioate nucleotide linkages.

21. A dsRNA agent according to any one of claims 1 to 20, wherein each chain is 30 nucleotides or less in length.

22. A dsRNA agent according to any one of claims 1 to 21, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

23. A dsRNA agent according to any one of claims 1 to 21, wherein at least one strand comprises a 3' overhang of at least two nucleotides.

24. A dsRNA agent according to any one of claims 1 to 23, wherein the double-stranded region is 15 to 30 nucleotide pairs long.

25. The dsRNA agent according to claim 24, wherein the double-stranded region has a length of 17 to 23 nucleotide pairs.

26. The dsRNA agent according to claim 24, wherein the double-stranded region has a length of 17 to 25 nucleotide pairs.

27. The dsRNA agent according to claim 24, wherein the double-stranded region has a length of 23 to 27 nucleotide pairs.

28. The dsRNA agent according to claim 24, wherein the double-stranded region has a length of 19 to 21 nucleotide pairs.

29. The dsRNA agent according to claim 24, wherein the double-stranded region has a length of 21 to 23 nucleotide pairs.

30. A dsRNA agent according to any one of claims 1 to 29, wherein each chain has 19 to 30 nucleotides.

31. A dsRNA agent according to any one of claims 1 to 29, wherein each chain has 19 to 23 nucleotides.

32. A dsRNA agent according to any one of claims 1 to 29, wherein each chain has 21 to 23 nucleotides.

33. A dsRNA agent according to any one of claims 1 to 32, wherein one or more lipophilic moieties are conjugated at one or more internal positions in at least one strand.

34. The dsRNA agent according to claim 33, wherein one or more lipophilic moieties are conjugated to one or more internal positions in at least one strand via a linker or carrier.

35. The dsRNA agent according to claim 34, wherein the internal positions include all positions from each end of at least one strand except for the two terminal positions.

36. The dsRNA agent according to claim 34, wherein the internal positions include all positions from each end of at least one strand except for the three terminal positions.

37. A dsRNA agent according to any one of claims 34 to 36, wherein the internal position excludes the sense strand cleavage site region.

38. The dsRNA agent according to claim 37, wherein the internal position includes all positions except positions 9 to 12, counting from the 5' end of the sense strand.

39. The dsRNA agent according to claim 37, wherein the internal position includes all positions except positions 11 to 13, counting from the 3' end of the sense strand.

40. A dsRNA agent according to any one of claims 34 to 36, wherein the internal position is excluding the antisense strand cleavage site region.

41. The dsRNA agent according to claim 40, wherein the internal position includes all positions except positions 12 to 14, counting from the 5' end of the antisense strand.

42. The dsRNA agent according to any one of claims 34 to 36, wherein the internal position includes all positions except positions 11 to 13 counting from the 3' end of the sense strand and positions 12 to 14 counting from the 5' end of the antisense strand.

43. A dsRNA agent according to any one of claims 1 to 42, wherein one or more lipophilic moieties are conjugated to one or more internal positions selected from the group consisting of positions 4-8 and 13-18 on the sense strand and positions 6-10 and 15-18 on the antisense strand, counting from the 5' end of each strand.

44. The dsRNA agent according to claim 43, wherein one or more lipophilic moieties are conjugated to one or more internal positions selected from the group consisting of positions 5, 6, 7, 15, and 17 in the sense strand and positions 15 and 17 in the antisense strand, counting from the 5' end of each strand.

45. The dsRNA agent according to claim 7, wherein the position in the double-stranded region is excluding the sense strand cleavage site region.

46. A dsRNA agent according to any one of claims 1 to 45, wherein the sense strand is 21 nucleotides long, the antisense strand is 23 nucleotides long, and the lipophilic portion is conjugated at position 21, position 20, position 15, position 1, position 7, position 6, or position 2 of the sense strand or at position 16 of the antisense strand.

47. The dsRNA agent according to claim 46, wherein the lipophilic portion is conjugated to position 21, position 20, position 15, position 1, or position 7 of the sense strand.

48. The dsRNA agent according to claim 46, wherein the lipophilic portion is conjugated to position 21, position 20, or position 15 of the sense strand.

49. The dsRNA agent according to claim 46, wherein the lipophilic portion is conjugated to position 20 or position 15 of the sense strand.

50. The dsRNA agent according to claim 46, wherein the lipophilic portion is conjugated to position 16 of the antisense strand.

51. The dsRNA agent according to any one of claims 1 to 50, wherein the lipophilic portion is an aliphatic compound, an alicyclic compound, or a polyalicyclic compound.

52. The dsRNA agent according to claim 51, wherein the lipophilic portion is selected from the group consisting of lipids, cholesterol, retinoic acid, cholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

53. The dsRNA agent according to claim 52, wherein the lipophilic portion contains a saturated or unsaturated C4-C30 hydrocarbon chain and a suitable functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

54. The dsRNA agent according to claim 53, wherein the lipophilic portion contains a saturated or unsaturated C6-C18 hydrocarbon chain.

55. The dsRNA agent according to claim 53, wherein the lipophilic portion contains a saturated or unsaturated C16 hydrocarbon chain.

56. The dsRNA agent according to claim 55, wherein a saturated or unsaturated C16 hydrocarbon chain is conjugated at position 6, counting from the 5' end of the chain.

57. The dsRNA agent according to any one of claims 1 to 54, wherein the lipophilic portion is conjugated via a carrier that replaces one or more nucleotides in the internal position or double-stranded region.

58. The dsRNA agent according to claim 56, 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 serinol skeleton or a diethanolamine skeleton.

59. The dsRNA agent according to any one of claims 1 to 54, wherein the lipophilic portion is conjugated to a double-stranded iRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfamide linkage, click reaction product, or carbamate.

60. A double-stranded iRNA agent according to any one of claims 1 to 59, wherein the lipophilic portion is conjugated to a nucleic acid base, a sugar portion, or an internucleoside linkage.

61. A dsRNA agent according to any one of claims 1 to 60, wherein the lipophilic moiety or targeting ligand is conjugated via a biocleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, galactosamine, glucosamine, glucose, galactose, mannose-functionalized monosaccharides or oligosaccharides, and combinations thereof.

62. The dsRNA agent according to any one of claims 1 to 61, wherein the 3' end of the sense strand is protected via an end cap which is a cyclic group having an amine, and the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinil, pyrazolidinil, imidazolinil, imidazolidinil, piperidinil, piperazinil, [1,3]dioxolanil, oxazolidinil, isoxazolidinil, morpholinil, thiazolidinil, isothiazolidinil, quinoxalinil, pyridadinil, tetrahydrofuranil, and dekalinil.

63. A dsRNA agent according to any one of claims 1 to 62, further comprising a targeted ligand that targets liver tissue.

64. The dsRNA agent according to claim 63, wherein the targeting ligand is a GalNAc conjugate.

65. A terminal chiral modification occurring at the first nucleotide linkage at the 3' end of an antisense chain, having a linked phosphorus atom in the Sp stereoconfiguration. A terminal chiral modification occurring at the first internucleotide linkage at the 5' end of the antisense chain, having a linked phosphorus atom in the Rp stereoconfiguration, and Terminal chiral modification occurring at the first nucleotide linkage at the 5' end of the sense strand, having a linked phosphorus atom in either the Rp or Sp stereoconfiguration. A dsRNA agent according to any one of claims 1 to 64, further comprising:

66. Terminal chiral modifications occurring in the internucleotide linkage between the first and second nucleotides at the 3' end of an antisense chain, having a linked phosphorus atom in the Sp stereoconfiguration, A terminal chiral modification occurring at the first internucleotide linkage at the 5' end of an antisense chain, having a linked phosphorus atom in the Rp stereoconfiguration, and Terminal chiral modification occurring at the first nucleotide linkage at the 5' end of the sense strand, having a linked phosphorus atom in either the Rp or Sp stereoconfiguration. A dsRNA agent according to any one of claims 1 to 64, further comprising:

67. Terminal chiral modifications occurring in the internucleotide linkages of the first, second, and third nucleotides at the 3' end of an antisense chain, having linked phosphorus atoms in the Sp stereoconfiguration. A terminal chiral modification occurring at the first internucleotide linkage at the 5' end of an antisense chain, having a linked phosphorus atom in the Rp stereoconfiguration, and Terminal chiral modification occurring at the first nucleotide linkage at the 5' end of the sense strand, having a linked phosphorus atom in either the Rp or Sp stereoconfiguration. A dsRNA agent according to any one of claims 1 to 64, further comprising:

68. Terminal chiral modifications occurring in the internucleotide linkage between the first and second nucleotides at the 3' end of an antisense chain, having a linked phosphorus atom in the Sp stereoconfiguration, A terminal chiral modification occurring at the third nucleotide linkage at the 3' end of an antisense chain, having a linked phosphorus atom in the Rp stereoconfiguration. A terminal chiral modification occurring at the first internucleotide linkage at the 5' end of an antisense chain, having a linked phosphorus atom in the Rp stereoconfiguration, and Terminal chiral modification occurring at the first nucleotide linkage at the 5' end of the sense strand, having a linked phosphorus atom in either the Rp or Sp stereoconfiguration. A dsRNA agent according to any one of claims 1 to 64, further comprising:

69. Terminal chiral modifications occurring in the internucleotide linkage between the first and second nucleotides at the 3' end of an antisense chain, having a linked phosphorus atom in the Sp stereoconfiguration, Terminal chiral modifications occurring in the internucleotide linkage between the first and second nucleotides at the 5' end of an antisense chain, which have linked phosphorus atoms in the Rp stereoconfiguration, and Terminal chiral modification occurring at the first nucleotide linkage at the 5' end of the sense strand, having a linked phosphorus atom in either the Rp or Sp stereoconfiguration. A dsRNA agent according to any one of claims 1 to 64, further comprising:

70. A dsRNA agent according to any one of claims 1 to 69, further comprising a phosphate or phosphate mimetic at the 5' end of the antisense strand.

71. The dsRNA agent according to claim 70, wherein the phosphate mimetic is 5'-vinylphosphonate (VP).

72. A dsRNA agent according to any one of claims 1 to 69, wherein the base pair at one position of the 5' end of the double-stranded antisense strand is an AU base pair.

73. A dsRNA agent according to any one of claims 1 to 69, wherein the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

74. Cells containing the dsRNA agent according to any one of claims 1 to 73.

75. A pharmaceutical composition for inhibiting the expression of the GPR75 gene, comprising a dsRNA agent according to any one of claims 1 to 73.

76. A pharmaceutical composition comprising a dsRNA agent and a lipid preparation according to any one of claims 1 to 73.

77. A device for oral inhalation administration comprising a dsRNA agent according to any one of claims 1 to 73.

78. The device according to claim 77, selected from the group consisting of a nebulizer, a metered-dose inhaler, and a dry powder inhaler.

79. A method for inhibiting the expression of the GPR75 gene in cells, (a) Contacting cells with a dsRNA agent according to any one of claims 1 to 73, or a pharmaceutical composition according to claim 75 or 76, or a device according to claim 77 or 78, and (b) Maintain the cells produced in step (a) for a sufficient amount of time to obtain degradation of the GPR75 gene, thereby inhibiting the expression of the GPR75 gene in the cells. A method that includes this.

80. The method according to claim 79, wherein the cells are located within the target.

81. The method according to claim 80, wherein the subject is a human.

82. The method according to any one of claims 77 to 81, wherein the expression of the GPR75 gene is inhibited by at least 50%.

83. A method for treating a subject having a G protein-coupled receptor 75 (GPR75) related disease or a subject at risk of developing a GPR75 related disease, comprising administering to the subject a therapeutically effective amount of a dsRNA agent according to any one of claims 1 to 73, or a pharmaceutical composition according to claim 75 or 76, or a device according to claim 77 or 78, thereby treating the subject.

84. The method according to claim 83, wherein the subject is a human.

85. The method according to claim 84, wherein the GPR75-related disorder is a weight-loss disorder.

86. The method according to claim 85, wherein the weight disorder is obesity.

87. The method according to any one of claims 83 to 86, wherein the treatment comprises improvement of at least one sign or symptom of a disease.

88. The method according to any one of claims 83 to 87, wherein administration of a dsRNA agent results in a decrease in the blood glucose level of the subject.

89. The method according to any one of claims 83 to 88, wherein the dsRNA agent is administered to the subject at a dose of approximately 0.01 mg / kg to approximately 50 mg / kg.

90. The method according to any one of claims 83 to 89, wherein the dsRNA agent is administered to the subject intrathecally.

91. The method according to any one of claims 83 to 89, wherein the dsRNA agent is administered subcutaneously to the subject.

92. The method according to any one of claims 83 to 91, further comprising administering to an additional agent or therapy suitable for the treatment or prevention of GPR75-related disorders.

93. The method according to claim 92, wherein the additional therapeutic agent is selected from the group consisting of diabetic agents, diabetic complication agents, cardiovascular disease agents, anti-dyslipidemia agents, antihypertensive or anti-hypertension agents, anti-obesity agents, non-alcoholic steatohepatitis (NASH) agents, chemotherapy agents, immunotherapy agents, immunosuppressants, anti-inflammatory agents, anti-steatosis agents, and any combination thereof.

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