Compositions and methods for silencing MYOC expression

Abstract

The present disclosure relates to double-stranded ribonucleic acid (dsRNA) compositions that target MYOC and methods of using such dsRNA compositions to alter (e.g., inhibit) the expression of MYOC.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 215,804, filed June 28, 2021, the benefit of priority to U.S. Provisional Patent Application No. 63 / 287,404, filed December 8, 2021, and the benefit of priority to U.S. Provisional Patent Application No. 63 / 351,033, filed June 10, 2022. The entire contents of the foregoing applications are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on June 22, 2022, is named A108868_1490WO_SL.txt and is 596,467 bytes in size.

[0003] The present disclosure relates to the specific inhibition of expression of MYOC. [Background technology]

[0004] Glaucoma (e.g., primary open-angle glaucoma (POAG)) is the leading cause of irreversible vision loss in today's elderly population. Misfolding of the MYOC protein blocks its secretion from trabecular meshwork cells, leading to increased intraocular pressure, which in turn compresses and damages the optic nerve, reducing its ability to transmit visual information to the brain and resulting in vision loss. New treatments for glaucoma are needed. Summary of the Invention

[0005] The present disclosure describes the method and iRNA composition for regulating the expression of MYOC.In certain embodiments, the expression of MYOC is reduced or inhibited using MYOC-specific iRNA.This inhibition can be useful for treating disorders related to MYOC expression, such as eye disorders (for example, glaucoma, for example, primary open-angle glaucoma (POAG)).

[0006] Thus, described herein are compositions and methods that result in RNA-induced silencing complex (RISC)-mediated cleavage of MYOC RNA transcripts, such as in a cell or a subject (e.g., a mammal, such as a human subject). Also described are compositions and methods for treating disorders associated with MYOC expression, such as glaucoma (e.g., primary open-angle glaucoma (POAG)).

[0007] The iRNA (e.g., dsRNA) included in the compositions featured herein comprises an RNA strand (antisense strand) having a region, e.g., a region of 30 nucleotides or less, generally 19-24 nucleotides in length, that is substantially complementary to at least a portion of an mRNA transcript of MYOC (e.g., human MYOC) (also referred to herein as a "MYOC-specific iRNA"). In some embodiments, the MYOC mRNA transcript is a human MYOC mRNA transcript, such as SEQ ID NO: 1 described herein.

[0008] In some embodiments, an iRNA (e.g., a dsRNA) described herein comprises an antisense strand having a region that is substantially complementary to a region of human MYOC mRNA. In some embodiments, the human MYOC mRNA has the sequence NM_000261.2 (SEQ ID NO: 1). The sequence of NM_000261.2 is also incorporated herein by reference in its entirety. The reverse complement of SEQ ID NO: 1 is provided herein as SEQ ID NO: 2.

[0009] In some aspects, the disclosure provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of myocilin (MYOC), the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides with 0, 1, 2, or 3 mismatches of a portion of the coding strand of human MYOC, and the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides with 0, 1, 2, or 3 mismatches of a corresponding portion of the non-coding strand of human MYOC, such that the sense strand is complementary to at least 15 contiguous nucleotides in the antisense strand.

[0010] In some aspects, the disclosure provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of MYOC, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides with 0, 1, 2, or 3 mismatches of a portion of the nucleotide sequence of SEQ ID NO:2, such that the sense strand is complementary to at least 15 contiguous nucleotides in the antisense strand.

[0011] In some aspects, the disclosure provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of MYOC, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that has 0, 1, 2, or 3 mismatches with the antisense strand nucleotide sequence of duplex AD-1565804, and the sense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that has 0, 1, 2, or 3 mismatches with the sense strand nucleotide sequence of duplex AD-1565804.

[0012] In some aspects, the disclosure provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of MYOC, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that has 0, 1, 2, or 3 mismatches with the antisense strand nucleotide sequence of duplex AD-1565837, and the sense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that has 0, 1, 2, or 3 mismatches with the sense strand nucleotide sequence of duplex AD-1565837.

[0013] In some aspects, the present disclosure provides human cells or tissues that comprise reduced levels of MYOC mRNA or MYOC protein compared to otherwise similar untreated cells or tissues, optionally where the cells or tissues are not genetically engineered (e.g., the cells or tissues comprise one or more naturally occurring mutations, e.g., MYOC mutations), and optionally the levels are reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the human cells or tissues are trabecular meshwork tissue, ciliary bodies, retinal pigment epithelium (RPE), retinal tissue, astrocytes, pericytes, Müller cells, ganglion cells, endothelial cells, photoreceptor cells, retinal blood vessels (e.g., comprising endothelial cells and vascular smooth muscle cells), or choroidal tissue, e.g., choroidal blood vessels.

[0014] The present disclosure also provides, in some aspects, cells containing a dsRNA agent described herein.

[0015] In another aspect, provided herein are human ocular cells, e.g., trabecular meshwork cells, ciliary body-like cells, RPE cells, retinal cells, astrocytes, pericytes, Müller cells, ganglion cells, endothelial cells, or photoreceptor cells, that comprise reduced levels of MYOC mRNA or MYOC protein compared to otherwise similar untreated cells. In some embodiments, the levels are reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0016] In some aspects, the present disclosure also provides a pharmaceutical composition for inhibiting expression of a gene encoding MYOC, comprising a dsRNA agent described herein.

[0017] The disclosure also provides, in some aspects, a method of inhibiting expression of MYOC in a cell, the method comprising: (a) contacting a cell with a dsRNA agent described herein, or a pharmaceutical composition described herein; (b) maintaining the cells produced in step (a) for a time sufficient to allow degradation of MYOC mRNA transcripts, thereby inhibiting expression of MYOC in the cells.

[0018] The disclosure also provides, in some aspects, a method of inhibiting expression of MYOC in a cell, the method comprising: (a) contacting a cell with a dsRNA agent described herein, or a pharmaceutical composition described herein; (b) maintaining the cells produced in step (a) for a time sufficient to reduce the levels of MYOC mRNA, MYOC protein, or both MYOC mRNA and protein, thereby inhibiting expression of MYOC in the cells.

[0019] The disclosure also provides, in some aspects, a method for inhibiting expression of MYOC in an ocular cell or tissue, the method comprising: (a) contacting a cell or tissue with a dsRNA agent that binds to MYOC; (b) maintaining the cells or tissues produced in step (a) for a time sufficient to reduce the levels of MYOC mRNA, MYOC protein, or both MYOC mRNA and protein, thereby inhibiting expression of MYOC in the cells or tissues.

[0020] The present disclosure also provides, in some aspects, a method of treating a subject diagnosed with a MYOC-associated disorder, comprising administering to the subject a therapeutically effective amount of a dsRNA agent described herein or a pharmaceutical composition described herein, thereby treating the disorder.

[0021] Any of the aspects herein, eg, the compositions and methods described above, any of the embodiments herein (eg, below) may be applied.

[0022] In some embodiments, the coding strand of human MYOC has the sequence of SEQ ID NO: 1. In some embodiments, the non-coding strand of human MYOC has the sequence of SEQ ID NO: 2.

[0023] In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides with 0, 1, 2, or 3 mismatches to the corresponding portion of the nucleotide sequence of SEQ ID NO:1.

[0024] In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, and the antisense strand comprises a nucleotide sequence comprising at least 17 consecutive nucleotides with 0, 1, 2, or 3 mismatches of a portion of the nucleotide sequence of SEQ ID NO: 2, such that the sense strand is complementary to at least 17 consecutive nucleotides in the antisense strand. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 17 consecutive nucleotides with 0, 1, 2, or 3 mismatches of a corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

[0025] In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, and the antisense strand comprises a nucleotide sequence comprising at least 19 consecutive nucleotides with 0, 1, 2, or 3 mismatches of a portion of the nucleotide sequence of SEQ ID NO: 2, such that the sense strand is complementary to at least 19 consecutive nucleotides in the antisense strand. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 19 consecutive nucleotides with 0, 1, 2, or 3 mismatches of a corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

[0026] In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, and the antisense strand comprises a nucleotide sequence comprising at least 21 consecutive nucleotides with 0, 1, 2, or 3 mismatches of a portion of the nucleotide sequence of SEQ ID NO: 2, such that the sense strand is complementary to at least 21 consecutive nucleotides in the antisense strand. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 21 consecutive nucleotides with 0, 1, 2, or 3 mismatches of a corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

[0027] In some embodiments, the portion of the sense strand is a portion within the sense strand of any one of Tables 2A and 2B.

[0028] In some embodiments, the portion of the antisense strand is a portion within the antisense strand of any one of Tables 2A and 2B.

[0029] In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides that have 0, 1, 2, or 3 mismatches with one of the antisense sequences listed in any one of Tables 2A and 2B. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides that have 0, 1, 2, or 3 mismatches with the sense sequence listed in any one of Tables 2A and 2B that corresponds to the antisense sequence.

[0030] In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides that have 0, 1, 2, or 3 mismatches with one of the antisense sequences listed in any one of Tables 2A and 2B. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides that have 0, 1, 2, or 3 mismatches with the sense sequence listed in any one of Tables 2A and 2B that corresponds to the antisense sequence.

[0031] In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides that have 0, 1, 2, or 3 mismatches with one of the antisense sequences listed in any one of Tables 2A and 2B. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides that have 0, 1, 2, or 3 mismatches with the sense sequence listed in any one of Tables 2A and 2B that corresponds to the antisense sequence.

[0032] In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides with 0, 1, 2, or 3 mismatches with one of the antisense sequences listed in any one of Tables 2A and 2B. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides with 0, 1, 2, or 3 mismatches with the sense sequence listed in any one of Tables 2A and 2B that corresponds to the antisense sequence.

[0033] In some embodiments, the sense strand of a dsRNA agent is at least 23 nucleotides in length, eg, 23-30 nucleotides in length.

[0034] In some embodiments, at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties.In some embodiments, the lipophilic moiety is conjugated to one or more positions in the double-stranded region of the dsRNA agent.In some embodiments, the lipophilic moiety is conjugated via a linker or carrier.In some embodiments, the lipophilicity of the lipophilic moiety is expressed by logK ow is measured by and exceeds 0.

[0035] In some embodiments, the hydrophobicity of the double-stranded RNAi agent is greater than 0.2, as measured by the unbound fraction in a plasma protein binding assay of the double-stranded RNAi agent. In some embodiments, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

[0036] In some embodiments, dsRNA agent comprises at least one modified nucleotide.In some embodiments, five or less nucleotides of sense strand and five or less nucleotides of antisense strand are unmodified nucleotide.In some embodiments, all nucleotides of sense strand and all nucleotides of antisense strand comprise modification.

[0037] In some embodiments, at least one of the modified nucleotides is selected from the group consisting of deoxy-nucleotides, 3'-terminal deoxy-thymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, unnatural base containing nucleotides, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimetics, glycol modified nucleotides, and 2-O-(N-methylacetamido) modified nucleotides, and combinations thereof. In some embodiments, no more than five of the nucleotides in the sense strand and no more than five of the nucleotides in the antisense strand comprise a modification other than a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, an unlocked nucleic acid (UNA), or a glycerol nucleic acid (GNA).

[0038] In some embodiments, the dsRNA comprises a non-nucleotide spacer (optionally, the non-nucleotide spacer comprises a C3-C6 alkyl) between two consecutive nucleotides in the sense strand or between two consecutive nucleotides in the antisense strand.

[0039] In some embodiments, each strand is 30 nucleotides or less in length. In some embodiments, at least one strand comprises a 3' overhang of at least 1 nucleotide. In some embodiments, at least one strand comprises a 3' overhang of at least 2 nucleotides. In some embodiments, at least one strand comprises a 3' overhang of 2 nucleotides.

[0040] In some embodiments, the double-stranded region is 15-30 nucleotide pairs in length. In some embodiments, the double-stranded region is 17-23 nucleotide pairs in length. In some embodiments, the double-stranded region is 17-25 nucleotide pairs in length. In some embodiments, the double-stranded region is 23-27 nucleotide pairs in length. In some embodiments, the double-stranded region is 19-21 nucleotide pairs in length. In some embodiments, the double-stranded region is 21-23 nucleotide pairs in length. In some embodiments, each strand has 19-30 nucleotides. In some embodiments, each strand has 19-23 nucleotides. In some embodiments, each strand has 21-23 nucleotides.

[0041] In some embodiments, the agent comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. In some embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3'-end of one strand. In some embodiments, the strand is the antisense strand. In some embodiments, the strand is the sense strand.

[0042] In some embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5'-end of one strand. In some embodiments, the strand is the antisense strand. In some embodiments, the strand is the sense strand.

[0043] In some embodiments, the 5'-end and 3'-end of one strand each comprise a phosphorothioate or methylphosphonate internucleotide linkage, hi some embodiments, the strand is the antisense strand.

[0044] In some embodiments, the base pair at position 1 of the 5'-end of the antisense strand of the duplex is an AU base pair.

[0045] In some embodiments, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0046] In some embodiments, one or more lipophilic moieties are conjugated to one or more interior positions on at least one of the chains, hi some embodiments, one or more lipophilic moieties are conjugated to one or more interior positions on at least one of the chains via a linker or carrier.

[0047] In some embodiments, internal positions include all positions except the two most distant positions on at least one strand. In some embodiments, internal positions include all positions except the three most distant positions on at least one strand. In some embodiments, internal positions exclude the cleavage site region of the sense strand. In some embodiments, internal positions include all positions except positions 9-12, counting from the 5' end of the sense strand. In some embodiments, internal positions include all positions except positions 11-13, counting from the 3' end of the sense strand. In some embodiments, internal positions exclude the cleavage site region of the antisense strand. In some embodiments, internal positions include all positions except positions 12-14, counting from the 5' end of the antisense strand. In some embodiments, internal positions include all positions except positions 11-13 on the sense strand, counting from the 3' end, and positions 12-14 on the antisense strand, counting from the 5' end.

[0048] In some embodiments, the 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. In some embodiments, the 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 on the sense strand and positions 15 and 17 on the antisense strand, counting from the 5' end of each strand.

[0049] In some embodiments, the positions in the double-stranded region exclude the cleavage site region of the sense strand.

[0050] In some embodiments, the sense strand is 21 nucleotides in length, the antisense strand is 23 nucleotides in length, and the lipophilic moiety is conjugated to position 21, 20, 15, 1, 7, 6, or 2 of the sense strand or position 16 of the antisense strand. In some embodiments, the lipophilic moiety is conjugated to position 21, 20, 15, 1, or 7 of the sense strand. In some embodiments, the lipophilic moiety is conjugated to position 21, 20, or 15 of the sense strand. In some embodiments, the lipophilic moiety is conjugated to position 20 or 15 of the sense strand. In some embodiments, the lipophilic moiety is conjugated to position 16 of the antisense strand. In some embodiments, the lipophilic moiety is conjugated to position 6, counting from the 5' end of the sense strand.

[0051] In some embodiments, the lipophilic moiety is an aliphatic compound, an alicyclic compound, or a polyalicyclic compound. In some embodiments, the lipophilic moiety is selected from the group consisting of a lipid, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, a heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. In some embodiments, the lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. In some embodiments, the lipophilic moiety contains a saturated or unsaturated C6-C18 hydrocarbon chain. In some embodiments, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.

[0052] In some embodiments, the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotides at an internal position or in the double-stranded region. In some embodiments, the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl, or an acyclic moiety of a serinol backbone or a diethanolamine backbone system.

[0053] In some embodiments, the lipophilic moiety is conjugated to the double-stranded iRNAi agent via a linker containing an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide linkage, a product of a click reaction, or a carbamate.

[0054] In some embodiments, the lipophilic moiety is conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage.

[0055] In some embodiments, the lipophilic moiety or targeting ligand is conjugated via a biocleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, and functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0056] In some embodiments, the 3' end of the sense strand is protected via an end cap that is an amine-bearing cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

[0057] In some embodiments, the dsRNA agent further comprises a targeting ligand, for example, for targeting ocular tissue or liver tissue.In some embodiments, the ocular tissue is trabecular meshwork tissue, ciliary body, retinal tissue, retinal pigment epithelium (RPE), or choroidal tissue, for example, choroidal blood vessels.

[0058] In some embodiments, the ligand is conjugated to the sense strand. In some embodiments, the ligand is conjugated to the 3' or 5' end of the sense strand. In some embodiments, the ligand is conjugated to the 3' end of the sense strand.

[0059] In some embodiments, the ligand comprises N-acetylgalactosamine (GalNAc). In some embodiments, the targeting ligand comprises one or more GalNAc conjugates or one or more GalNAc derivatives. In some embodiments, the ligand is one or more GalNAc complexes or one or more GalNAc derivatives linked via a monovalent, or bivalent, trivalent, or tetravalent branched linker. In some embodiments, the ligand is: [ka]

[0060] In some embodiments, the dsRNA agent is conjugated to a ligand as shown in the diagram below: [ka] wherein X is O or S. In some embodiments, X is O.

[0061] In some embodiments, the dsRNA agent further includes a terminal chiral modification occurring at a first internucleotide linkage at the 3'-end of the antisense strand having a linking phosphorus atom in the Sp configuration, a terminal chiral modification occurring at a first internucleotide linkage at the 5'-end of the antisense strand having a linking phosphorus atom in the Rp configuration, and a terminal chiral modification occurring at a first internucleotide linkage at the 5'-end of the sense strand having a linking phosphorus atom in either the Rp or Sp configuration.

[0062] In some embodiments, the dsRNA agent further includes a terminal chiral modification occurring at the first and second internucleotide linkages at the 3'-end of the antisense strand having a linking phosphorus atom in the Sp configuration, a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand having a linking phosphorus atom in the Rp configuration, and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand having a linking phosphorus atom in either the Rp or Sp configuration.

[0063] In some embodiments, the dsRNA agent further includes a terminal chiral modification occurring at the first, second, and third internucleotide linkages at the 3'-end of the antisense strand having a linking phosphorus atom in the Sp configuration; a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand having a linking phosphorus atom in the Rp configuration; and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand having a linking phosphorus atom in either the Rp or Sp configuration.

[0064] In some embodiments, the dsRNA agent further includes a terminal chiral modification occurring at the first and second internucleotide linkages at the 3'-end of the antisense strand having a linking phosphorus atom in the Sp configuration, a terminal chiral modification occurring at the third internucleotide linkage at the 3'-end of the antisense strand having a linking phosphorus atom in the Rp configuration, a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand having a linking phosphorus atom in the Rp configuration, and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand having a linking phosphorus atom in either the Rp or Sp configuration.

[0065] In some embodiments, the dsRNA agent further includes a terminal chiral modification occurring at the first and second internucleotide linkages at the 3'-end of the antisense strand, which have a linking phosphorus atom in the Sp configuration; a terminal chiral modification occurring at the first and second internucleotide linkages at the 5'-end of the antisense strand, which have a linking phosphorus atom in the Rp configuration; and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, which has a linking phosphorus atom in either the Rp or Sp configuration.

[0066] In some embodiments, the dsRNA agent further comprises a phosphate or phosphate mimic at the 5'-end of the antisense strand. In some embodiments, the phosphate mimic is 5'-vinylphosphonate (VP).

[0067] In various embodiments of the foregoing dsRNA agents, the dsRNA agents target hotspot regions of the mRNA encoding MYOC.

[0068] In another aspect, the invention provides dsRNA agents that target hotspot regions of myocilin (MYOC) mRNA.

[0069] In some embodiments, the cells described herein, e.g., human cells, are produced by a process comprising contacting a human cell with a dsRNA agent described herein.

[0070] In some embodiments, a pharmaceutical composition described herein comprises a dsRNA agent and a lipid formulation.

[0071] In some embodiments (e.g., embodiments of the methods described herein), the cell is in a subject. In some embodiments, the subject is human. In some embodiments, MYOC mRNA levels are inhibited by at least 50%. In some embodiments, MYOC protein levels are inhibited by at least 50%. In some embodiments, MYOC expression is inhibited by at least 50%. In some embodiments, inhibiting MYOC expression reduces MYOC protein levels in a biological sample (e.g., an aqueous ocular fluid sample) from the subject by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In some embodiments, inhibiting MYOC gene expression reduces MYOC mRNA levels in a biological sample (e.g., an aqueous ocular fluid sample) from the subject by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0072] In some embodiments, the subject has been diagnosed with a MYOC-related disorder. In some embodiments, the subject meets at least one diagnostic criterion for a MYOC-related disorder. In some embodiments, the MYOC-related disorder is glaucoma. In some embodiments, the MYOC-related disorder is primary open-angle glaucoma (POAG).

[0073] In some embodiments, the ocular cell or tissue is trabecular meshwork tissue, ciliary bodies, RPE, retinal tissue, astrocytes, pericytes, Müller cells, ganglion cells, endothelial cells, photoreceptor cells, retinal blood vessels (e.g., including endothelial cells and vascular smooth muscle cells), or choroidal tissue, such as, for example, choroidal blood vessels.

[0074] In some embodiments, the MYOC-related disorder is glaucoma. In some embodiments, the glaucoma is caused by or associated with elevated intraocular pressure. In some embodiments, the glaucoma is primary open-angle glaucoma (POAG).

[0075] In some embodiments, treating includes ameliorating at least one sign or symptom of the disorder, in some embodiments, the at least one sign or symptom includes optic nerve damage, vision loss, tunnel vision, blurred vision, ocular pain, or one or more measures of the presence, level, or MYOC activity (e.g., MYOC gene, MYOC mRNA, or MYOC protein).

[0076] In some embodiments, a level of MYOC higher than the baseline level indicates that the subject has glaucoma. In some embodiments, treatment includes preventing progression of the disorder. In some embodiments, treatment includes one or more of: (a) inhibiting or reducing MYOC expression or activity, (b) reducing levels of misfolded MYOC protein, (c) reducing trabecular meshwork cell death, (d) reducing intraocular pressure, or (e) increasing visual acuity.

[0077] In some embodiments, the treatment results in an average reduction of at least 30% from baseline in MYOC mRNA in trabecular meshwork tissue, ciliary bodies, retina, RPE, retinal blood vessels (e.g., comprising endothelial cells and vascular smooth muscle cells), or choroidal tissue, e.g., choroidal blood vessels. In some embodiments, the treatment results in an average reduction of at least 60% from baseline in MYOC mRNA in trabecular meshwork tissue, ciliary bodies, retina, RPE, retinal blood vessels (e.g., comprising endothelial cells and vascular smooth muscle cells), or choroidal tissue, e.g., choroidal blood vessels. In some embodiments, the treatment results in an average reduction of at least 90% from baseline in MYOC mRNA in trabecular meshwork tissue, ciliary bodies, retina, RPE, retinal blood vessels (e.g., comprising endothelial cells and vascular smooth muscle cells), or choroidal tissue, e.g., choroidal blood vessels.

[0078] In some embodiments, after treatment, subject experiences a knockdown period of at least 8 weeks after a single administration of dsRNA, as assessed by MYOC protein in retina.In some embodiments, treatment results in a knockdown period of at least 12 weeks after a single administration of dsRNA, as assessed by MYOC protein in retina.In some embodiments, treatment results in a knockdown period of at least 16 weeks after a single administration of dsRNA, as assessed by MYOC protein in retina.

[0079] In some embodiments, the subject is a human.

[0080] In some embodiments, a dsRNA agent is administered to a subject at a dose of about 0.01 mg / kg to about 50 mg / kg.

[0081] In some embodiments, dsRNA agent is administered to subject intraocularly.In some embodiments, intraocular administration comprises intravitreal administration, for example, intravitreal injection, intrascleral administration, for example, intrascleral injection, subconjunctival administration, for example, subconjunctival injection, retrobulbar administration, for example, retrobulbar injection, intracameral administration, for example, intracameral injection, or subretinal administration, for example, subretinal injection.

[0082] In some embodiments, the dsRNA agent is administered to the subject intravenously. In some embodiments, the dsRNA agent is administered to the subject locally.

[0083] In some embodiments, the methods described herein further comprise measuring the level of MYOC (e.g., MYOC gene, MYOC mRNA, or MYOC protein) in the subject. In some embodiments, measuring the level of MYOC in the subject comprises measuring the level of MYOC protein in a biological sample (e.g., an aqueous ocular fluid sample) from the subject. In some embodiments, the methods described herein further comprise performing a blood test, an imaging test, or an aqueous ocular fluid biopsy (e.g., an aqueous humor tap).

[0084] In some embodiments, the method of further measuring the level of MYOC (e.g., MYOC gene, MYOC mRNA, or MYOC protein) in a subject described herein is performed before treatment with a dsRNA agent or pharmaceutical composition. In some embodiments, upon determining that the subject has a MYOC level greater than the reference level, a dsRNA agent or pharmaceutical composition is administered to the subject. In some embodiments, measuring the level of MYOC in the subject is performed after treatment with a dsRNA agent or pharmaceutical composition.

[0085] In some embodiments, the methods described herein further include treating the subject with a therapy suitable for treating or preventing a MYOC-associated disorder, e.g., the therapy includes laser trabeculoplasty, trabeculectomy, minimally invasive glaucoma surgery, or placement of a drainage tube in the eye. In some embodiments, the methods described herein further include administering to the subject an additional agent suitable for treating or preventing a MYOC-associated disorder. In some embodiments, the additional agent includes a carbonic anhydrase inhibitor, a prostaglandin, a beta-blocker, an alpha-adrenergic agonist, a carbonic anhydrase inhibitor, a Rho kinase inhibitor, or a cholinergic agonist, or any combination thereof. In some embodiments, the additional agent includes an oral medication or an eye drop.

[0086] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. [Brief explanation of the drawings]

[0087] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain features of the present disclosure.

[0088] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

[0089] [Figure 1A] Figure 1A shows the experimental setup for testing the effect of human MYOC siRNA AD-822899 on intraocular pressure (IOP) in SAM mice containing a humanized MYOC locus containing the Y437H mutation (SAM-MYOC mice, homozygous for each allele) treated with AAV2.Y3F-SAM-g4. [Figure 1B] Figure 1B shows intraocular pressure (IOP) in SAM mice containing a humanized MYOC locus containing the Y437H mutation (SAM-MYOC mice, homozygous for each allele) treated with AAV2.Y3F-SAM-g4 or without any treatment (untreated). Mice treated with AAV2.Y3F-SAM-g4 were then treated with either human MYOC siRNA or control luciferase siRNA. [Figure 2A] Figure 2A shows the experimental setup for testing the effects of human MYOC siRNAs AD-822899, AD-1565804, AD-1565837, AD-1193175, and AD-1565503 on intraocular pressure (IOP) in SAM mice containing a humanized MYOC locus containing the Y437H mutation (SAM-MYOC mice, homozygous for each allele) treated with LV-SAM-g4. [Figure 2B] Figure 2B shows intraocular pressure (IOP) in SAM mice containing a humanized MYOC locus containing the Y437H mutation (SAM-MYOC mice, homozygous for each allele) treated with LV-SAM-g4 or PBS, and LV-SAM-g4-treated mice were subsequently treated with human MYOC siRNA AD-822899, AD-1565804, AD-1565837, AD-1193175, or AD-1565503. [Figure 2C]Figure 2C shows qPCR results showing the ratio of human MYOC mRNA expression to Gapdh in SAM mice containing a humanized MYOC locus containing the Y437H mutation (SAM-MYOC mice, homozygous for each allele) treated with LV-SAM-g4 or PBS, and LV-SAM-g4-treated mice were subsequently treated with human MYOC siRNA AD-822899, AD-1565804, AD-1565837, AD-1193175, or AD-1565503. [Figure 2D] Figure 2D shows RNASCOPE® analysis showing the percentage of human MYOC mRNA expression in eyes from SAM mice containing a humanized MYOC locus containing the Y437H mutation (SAM-MYOC mice, homozygous for each allele) treated with LV-SAM-g4 or PBS, and the LV-SAM-g4-treated mice were subsequently treated with human MYOC siRNA AD-1565804 or AD-1565837. [Figure 3A] Figure 3A shows the experimental setup for testing the effect of human MYOC siRNA AD-1565804 or AD-1565837 on intraocular pressure (IOP) in SAM mice containing a humanized MYOC locus containing the Y437H mutation (SAM-MYOC mice, homozygous for each allele) treated with LV-SAM-g4. [Figure 3B] Figure 3B shows intraocular pressure (IOP) in SAM mice containing a humanized MYOC locus containing the Y437H mutation (SAM-MYOC mice, homozygous for each allele) treated with LV-SAM-g4 or PBS, and the LV-SAM-g4-treated mice were subsequently treated with human MYOC siRNA AD-1565804 or AD-1565837. [Figure 3C]Figure 3C shows qPCR results showing the ratio of human MYOC mRNA expression to Gapdh in SAM mice containing a humanized MYOC locus containing the Y437H mutation (SAM-MYOC mice, homozygous for each allele) treated with LV-SAM-g4 or PBS, and LV-SAM-g4-treated mice were subsequently treated with human MYOC siRNA AD-1565804 or AD-1565837. [Figure 4] Figure 4 shows the percentage of MYOC protein remaining in TM relative to PBS at 85 days after administration of either the AD-1565837 or AD-1565804 duplex in a non-human primate (NHP) model. Results are shown for two different MYOC antibodies, R&D and Abnova. [Figure 5] FIG. 5 shows the percentage of MYOC protein remaining in the aqueous humor before, and after administration of either the AD-1565837 duplex or the AD-1565804 duplex on days −35, 22, 50, and 85 in a non-human primate (NHP) model. [Figure 6A] FIG. 6A shows the percentage of MYOC protein remaining in the vitreous and ciliary humor relative to PBS at 85 days after administration of either the AD-1565837 duplex or the AD-1565804 duplex in a non-human primate (NHP) model. [Figure 6B] FIG. 6B shows the percentage of MYOC protein remaining in the iris and sclera relative to PBS at 85 days after administration of either the AD-1565837 or AD-1565804 duplex in a non-human primate (NHP) model. [Figure 7] FIG. 7 shows the percentage of MYOC mRNA remaining in aqueous humor relative to PBS at 85 days after administration of either the AD-1565837 duplex or the AD-1565804 duplex in a non-human primate (NHP) model. DETAILED DESCRIPTION OF THE INVENTION

[0090] Details of various embodiments of the disclosure are set forth in the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and drawings, and from the claims.

[0091] iRNA is responsible for the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). Described herein are iRNAs and methods for using them to regulate (e.g., inhibit) the expression of MYOC. Also described are compositions and methods for treating disorders associated with the expression of MYOC, such as glaucoma (e.g., primary open-angle glaucoma (POAG)).

[0092] Human MYOC is a secretory glycoprotein of approximately 57 kDa that regulates the activation of several signaling pathways in neighboring cells to control different processes, including cell adhesion, cell-matrix adhesion, cytoskeleton organization, and cell migration. MYOC is typically expressed and secreted by various tissues, including the retina and structures involved in aqueous humor regulation, such as trabecular meshwork tissue and ciliary body. Abnormal MYOC is associated with glaucoma, such as primary open-angle glaucoma (POAG). Without wishing to be bound by theory, abnormal MYOC may exacerbate the pathogenesis of glaucoma, for example, by impeding the drainage of aqueous humor, which results in increased intraocular pressure.

[0093] The following description discloses how to make and use compositions containing iRNA to modulate (e.g., inhibit) the expression of MYOC, as well as compositions and methods for treating disorders associated with the expression of MYOC.

[0094] In some aspects, featured herein are pharmaceutical compositions containing a MYOC iRNA and a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of MYOC, and methods of using the pharmaceutical compositions to treat disorders associated with expression of MYOC (e.g., glaucoma, e.g., primary open-angle glaucoma (POAG)).

[0095] I. definition For convenience, the meanings of certain terms and phrases used in the specification, examples, and appended claims are provided below. If there is an apparent inconsistency between the use of a term in other parts of this specification and the definition provided in this section, the definition in this section shall control.

[0096] When referring to a numerical value or numerical range, the term "approximately" means that the referenced number or numerical range is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary, for example, by 1% to 15% of the stated number or numerical range.

[0097] The term "more than" or "at least" preceding a number or a series of numbers is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that may be logically included, if this is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 17 nucleotides of a 20-nucleotide nucleic acid molecule" means that 17, 18, 19, or 20 nucleotides have the specified property. When "at least" precedes a series of numbers or ranges, it is understood that "at least" can modify each of the series of numbers or ranges.

[0098] As used herein, "less than or equal to" and "less than or equal to" are understood to include the value adjacent to the phrase and logically smaller values ​​or integers up to zero from the context.For example, a duplex with a mismatch of "2 nucleotides or less" to a target site has 2, 1, or 0 mismatches.When "less than or equal to" is used before a series of numbers or ranges, it is understood that "less than or equal to" can modify each of the series of numbers or ranges.

[0099] As used herein, "less than" does not include the value adjacent to the phrase, and is understood to include smaller values ​​or integers up to zero that are logical from the context.For example, a duplex with a mismatch of "less than 3 nucleotides" to a target site has 2, 1 or 0 mismatches.When "less than" is before a series of numbers or ranges, it is understood that "less than" can modify each of the series of numbers or ranges.

[0100] As used herein, "more than" does not include the value adjacent to the word, but is understood to include larger values ​​or integers up to logical infinity from the context. For example, a duplex with a mismatch to a target site of "more than 3 nucleotides" has 4, 5, 6 or more mismatches. When "more than" is before a series of numbers or ranges, it is understood that "more than" can modify each of the series of numbers or ranges.

[0101] As used herein, the "up to" in "up to 10" is understood as up to and including 10, i.e., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0102] Ranges provided herein are understood to include all individual integer values ​​and all subranges within the ranges.

[0103] The terms "activate," "promote," "upregulate expression," "increase expression," and the like, insofar as they refer to the MYOC gene, refer herein to at least partial activation of expression of the MYOC gene, as indicated by an increase in the amount of MYOC mRNA that can be isolated from or detected in a first cell or group of cells in which the MYOC gene is transcribed and that have been treated to increase expression of the MYOC gene, compared to a second cell or group of cells that are substantially identical to the first cell or group of cells but have not been so treated (control cells).

[0104] In some embodiments, expression of the MYOC gene is activated by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of an iRNA described herein. In some embodiments, expression of the MYOC gene is activated by at least about 60%, 70%, or 80% by administration of an iRNA described herein. In some embodiments, expression of the MYOC gene is activated by at least about 85%, 90%, or 95% or more by administration of an iRNA described herein. In some embodiments, MYOC gene expression is increased by at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or more in cells treated with an iRNA described herein compared to expression in untreated cells. Activation of expression by small dsRNA is described, for example, in Li et al., 2006 Proc. Natl. Acad. Sci. USA 103:17337-42, and U.S. Patent Application Publication Nos. 2007 / 0111963 and 2005 / 226848, each of which is incorporated herein by reference.

[0105] The terms "silence," "inhibit expression," "downregulate expression," "suppress expression," and the like, when referring to MYOC, refer herein to at least partial suppression of MYOC expression, as assessed, for example, based on MYOC mRNA expression, MYOC protein expression, or another parameter functionally linked to MYOC expression. For example, inhibition of MYOC expression may be manifested by a reduction in the amount of MYOC mRNA that can be isolated or detected in a first cell or group of cells treated such that MYOC is transcribed and expression of MYOC is inhibited, compared to a control. The control may be a second cell or group of cells substantially identical to the first cell or group of cells, except that the second cell or group of cells has not been so treated (control cells). The degree of inhibition is usually expressed as a percentage of the control level, e.g.,

number

[0106] Alternatively, the degree of inhibition can be given in terms of the reduction of a parameter functionally linked to MYOC expression, such as the amount of protein encoded by the MYOC gene.The reduction of a parameter functionally linked to MYOC expression can also be expressed as a percentage of control level.In principle, MYOC silencing can be determined in any cell that expresses MYOC, either constitutively or by genome engineering, and by any suitable assay.

[0107] For example, in certain instances, expression of MYOC is suppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of an iRNA disclosed herein. In some embodiments, MYOC is suppressed by at least about 60%, 65%, 70%, 75%, or 80% by administration of an iRNA disclosed herein. In some embodiments, MYOC is suppressed by at least about 85%, 90%, 95%, 98%, 99%, or more by administration of an iRNA described herein.

[0108] The term "antisense strand" or "guide strand" refers to the strand of an iRNA, e.g., a dsRNA, that includes a region that is substantially complementary to a target sequence.

[0109] As used herein, the term " complementary region " refers to the region on the antisense strand that is substantially complementary to the sequence defined herein, for example, target sequence.If complementary region is not completely complementary to target sequence, its mismatch can be in the inner region or terminal region of the molecule.In some embodiments, complementary region comprises 0, 1 or 2 mismatches.

[0110] The term "sense strand" or "passenger strand," as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand, as those terms are defined herein.

[0111] The term "blunt" or "blunt-end" when used herein in relation to dsRNA means that there is no unpaired nucleotide or nucleotide analogue at the predetermined end of dsRNA, i.e., there is no nucleotide overhang.One end or both ends of dsRNA can be blunt.When both ends of dsRNA are blunt, dsRNA is said to be blunt-ended.For clarity, "blunt-end" dsRNA is the dsRNA that has both ends blunt, i.e., there is no nucleotide overhang at either end of the molecule.In most cases, this molecule will be double-stranded throughout its entire length.

[0112] As used herein, unless otherwise indicated, the term "complementary," when used to describe a first nucleotide sequence in the context of a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize to form a double-stranded structure with an oligonucleotide or polynucleotide comprising a second nucleotide sequence under specified conditions, as would be understood by one of skill in the art. Such conditions may be, for example, stringent conditions, such as 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12-16 hours, followed by washing. Other conditions, such as physiologically relevant conditions that may be encountered in an organism, may be applied. Those skilled in the art can determine the most appropriate set of conditions for testing the complementarity of two sequences depending on the ultimate use of the hybridized nucleotides.

[0113] A complementary sequence within an iRNA, such as a dsRNA described herein, involves base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence with an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as being "fully complementary" to each other. However, when a first sequence is referred to herein as being "substantially complementary" to a second sequence, the two sequences may be perfectly complementary, or they may form one or more, but generally no more than 5, 4, 3, or 2, mismatched base pairs upon hybridization of a duplex of up to 30 base pairs, while maintaining the ability to hybridize under conditions most relevant to its ultimate use, such as inhibiting gene expression via the RISC pathway. However, if two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs shall not be considered mismatches in determining complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, where the longer oligonucleotide comprises a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, may still be referred to as "fully complementary" for purposes described herein.

[0114] Complementary sequences, as used herein, may also include or be formed entirely of non-Watson-Crick base pairs and / or base pairs formed from non-natural modified nucleotides, including, but not limited to, G:U wobble base pairs or Hoogsteen base pairs, so long as they meet the above requirements regarding their ability to hybridize.

[0115] The terms "complementary," "fully complementary," and "substantially complementary" herein may be used in reference to base matching between the sense and antisense strands of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context of their use.

[0116] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding a MYOC protein). For example, a polynucleotide is complementary to at least a portion of a MYOC mRNA if the sequence is substantially complementary to an uninterrupted portion of the mRNA encoding MYOC. The term "complementarity" refers to the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.

[0117] As used herein, the term "complementary region" refers to a region of another sequence, for example, the sense sequence of a dsRNA and the corresponding antisense sequence, or the antisense strand of an iRNA and a target sequence, for example, the MYOC nucleotide sequence defined herein, that is substantially complementary to one nucleotide sequence agent. If the complementary region is not completely complementary to the target sequence, the mismatch can be within the internal region or terminal region of the antisense strand of the iRNA. Generally, the most tolerable mismatch is within the terminal region, for example, within 5, 4, 3, or 2 nucleotides of the 5' or 3' end of the iRNAi agent.

[0118] As used herein, "contacting" includes direct contact with a cell as well as indirect contact with a cell. For example, when a composition containing an iRNA is administered to a subject (e.g., intraocularly, topically, or intravenously), it may come into contact with a cell in the subject.

[0119] When referring to iRNA, "introducing into a cell" means facilitating or resulting in uptake or absorption into the cell. Absorption or uptake of iRNA can occur through spontaneous diffusive or active intracellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; iRNA can also be "introduced into a cell," where the cell is part of an organism. In such instances, introduction into a cell includes delivery to an organism. For example, for in vivo delivery, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be via a β-glucan delivery system, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677, and U.S. Patent Application Publication No. 2005 / 0281781, which are incorporated herein by reference in their entireties. In vitro introduction into cells can include methods known in the art, such as electroporation and lipofection. Further approaches are described herein below or are known in the art. As used herein, "disorder associated with MYOC expression," "disease associated with MYOC expression," "pathological process associated with MYOC expression," "MYOC-associated disorder," "MYOC-associated disease," and the like include any condition, disorder, or disease in which MYOC expression is altered (e.g., decreased or increased compared to a baseline level, e.g., a level characteristic of a non-diseased subject). In some embodiments, MYOC expression is decreased. In some embodiments, MYOC expression is increased. In some embodiments, the decrease or increase in MYOC expression is detectable in a tissue sample (e.g., an aqueous ocular fluid sample) from the subject. The decrease or increase may be assessed relative to levels observed in the same individual before the onset of the disorder or relative to other individuals without the disorder. The decrease or increase may be limited to a particular organ, tissue, or region of the body (e.g., the eye). MYOC-associated disorders include, but are not limited to, glaucoma (e.g., primary open-angle glaucoma (POAG)).

[0120] The term "glaucoma," as used herein, refers to any disease of the eye caused by or associated with damage to the optic nerve. In some embodiments, glaucoma is associated with elevated intraocular pressure. In some embodiments, glaucoma is asymptomatic. In other embodiments, glaucoma has one or more symptoms, such as loss of peripheral vision, tunnel vision, or blind spots. A non-limiting example of glaucoma treatable using the methods provided herein is primary open-angle glaucoma (POAG).

[0121] As used herein, the terms "double-stranded RNA," "dsRNA," or "siRNA" refer to an iRNA comprising an RNA molecule or complex of molecules having a hybridized double-stranded region containing two antiparallel and substantially complementary nucleic acid strands, referred to as having "sense" and "antisense" orientations relative to a target RNA. The double-stranded region can be of any length that allows for specific degradation of the desired target RNA, for example, via the RISC pathway, but typically ranges from 9 to 36 base pairs in length, e.g., 15 to 30 base pairs in length. Considering a duplex of 9 to 36 base pairs, the duplex can be any length within this range, for example, 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, including any subrange therebetween, including 15 to 30 base pairs, 15 to 26 base pairs, 15 to 23 base pairs, 15 to 22 base pairs, 15 to 21 base pairs, 15 to 20 base pairs, 15 to 19 base pairs, 15 to 18 base pairs, 15 to 17 base pairs, 18 to 30 base pairs, dsRNAs produced in cells by treatment with Dicer and similar enzymes generally fall within the 19-22 base pair length range, including, but not limited to, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs. One strand of the double-stranded region of the dsDNA contains a sequence that is substantially complementary to a region of the target RNA. The two strands that form the double-stranded structure may be derived from a single RNA molecule with at least one self-complementary region, or may be formed from two or more separate RNA molecules.When the double-stranded region is formed from two strands of a single molecule, the molecule may have a double-stranded region (referred to herein as a "hairpin loop") separated by a single nucleotide between the 3' end of one strand and the 5' end of the other strand that form the duplex structure. The hairpin loop can contain at least one unpaired nucleotide, and in some embodiments, the hairpin loop can contain at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23, or more unpaired nucleotides. When the two substantially complementary strands of a dsRNA are composed of separate RNA molecules, the molecules need not be, but can be, covalently linked. In some embodiments, the two strands are covalently linked by means other than a hairpin loop, and the connecting structure is a linker.

[0122] In some embodiments, an iRNA agent can be a "single-stranded siRNA" introduced into a cell or organism to inhibit a target mRNA. In some embodiments, a single-stranded RNAi agent can bind to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides long and are optionally chemically modified. The design and testing of single-stranded siRNAs is described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein (e.g., sequences provided in Table 2A or 2B) can be used as the single-stranded siRNAs described herein and, optionally, chemically modified as described herein, e.g., by the methods described in Lima et al., (2012) Cell 150:883-894.

[0123] In some embodiments, RNA interference agents comprise single-stranded RNAs that interact with target RNA sequences and mediate cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNAs introduced into cells are degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer, a ribonuclease III-like enzyme, processes this dsRNA into 19-23 base pair small interfering RNAs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to 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, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in some embodiments, the present disclosure relates to single-stranded RNAs that promote the formation of a RISC complex to effect silencing of a target gene. Each "G", "C", "A", "T" and "U" generally represent the nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as base, respectively.However, it should be understood that the term "deoxyribonucleotide" or "ribonucleotide" or "nucleotide" can also refer to modified nucleotide or alternative replacement part, as will be described in more detail below.Those skilled in the art are well aware that guanine, cytosine, adenine and uracil can be replaced with other parts without substantially changing the base pairing properties of the oligonucleotide that contains the nucleotide with such replacement part.For example, but not limited to, the nucleotide that contains inosine as its base can base pair with the nucleotide that contains adenine, cytosine or uracil.Therefore, the nucleotide that contains uracil, guanine or adenine can be replaced with the nucleotide that contains inosine, for example, in the nucleotide sequence of the dsRNA that is mentioned in the present disclosure. In another example, adenine and cytosine anywhere within the oligonucleotide can be substituted with guanine and uracil, respectively, to form a GU wobble base pair with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods featured in this disclosure.

[0124] As used herein, the term "iRNA," "RNAi," "iRNA agent," or "RNAi agent" or "RNAi molecule" refers to an agent that contains RNA, as that term is defined herein, and mediates targeted cleavage of RNA transcripts, for example, via the RNA-induced silencing complex (RISC) pathway. In some embodiments, the iRNA described herein results in the inhibition of MYOC expression, for example, in cells or mammals. Inhibition of MYOC expression can be assessed based on a reduction in MYOC mRNA levels or a reduction in MYOC protein levels.

[0125] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound, e.g., covalently bonds the two parts of a compound.

[0126] The terms "lipophilic" or "lipophilic moiety" refer broadly 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, log K ow In this case, K ow is the ratio of the concentration of a chemical in the octanol phase to the concentration of the chemical in the aqueous phase of a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it can also be predicted by using coefficients attributable to the structural components of a chemical calculated using first principles or empirical methods (see, e.g., Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), incorporated herein by reference in its entirety). It provides a thermodynamic measure of a substance's tendency to prefer a non-aqueous or oily environment rather than water (i.e., its hydrophilic / lipophilic balance). In principle, a chemical can be expressed as its logK ow is greater than 0, the molecule is lipophilic in nature. Typically, a lipophilic moiety has a logK ow For example, the log K of 6-aminohexanol ow For example, the log K of cholesteryl N-(hexan-6-ol)carbamate is predicted to be approximately 0.7. ow is predicted to be 10.7.

[0127] The lipophilicity of a molecule can be changed depending on the functional groups it contains. For example, adding a hydroxyl or amine group to the end of the lipophilic moiety can increase the partition coefficient (e.g., logK ow ) may increase or decrease in value.

[0128] Alternatively, the hydrophobicity of the double-stranded RNAi agent conjugated with one or more lipophilic moieties can be measured by its protein binding properties.For example, in certain embodiments, the unbound fraction of the plasma protein binding assay of double-stranded RNAi agent can be determined to be positively correlated with the relative hydrophobicity of double-stranded RNAi agent, which can be positively correlated with the silencing activity of double-stranded RNAi agent.

[0129] In some embodiments, the plasma protein binding assay that is determined is electrophoretic mobility shift assay (EMSA) using human serum albumin protein.The exemplary protocol of this binding assay is described in detail, for example, in International Application PCT / US2019 / 031170.The hydrophobicity of double-stranded RNAi agent measured by the unbound fraction of siRNA in binding assay is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45 or greater than 0.5 in the case of enhanced siRNA in vivo delivery.

[0130] Thus, conjugating a lipophilic moiety to an internal position of a double-stranded RNAi agent provides optimal hydrophobicity in siRNA for enhanced in vivo delivery.

[0131] The term "lipid nanoparticle" or "LNP" refers to a vesicle comprising a lipid layer that encapsulates a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an RNAi agent or a plasmid from which an RNAi agent is transcribed. LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0132] As used herein, the term "modulate expression" refers to at least partial "inhibition" or partial "activation" of gene (e.g., MYOC gene) expression in cells treated with an iRNA composition described herein compared to the expression of the corresponding gene in control cells. Control cells include untreated cells or cells treated with a non-targeting control iRNA.

[0133] Those skilled in the art will recognize that the term "RNA molecule" or "ribonucleic acid molecule" encompasses not only RNA molecules expressed or found in nature, but also RNA analogs and derivatives containing one or more ribonucleotide / ribonucleoside analogs or derivatives described herein or known in the art. Strictly speaking, a "ribonucleoside" contains a nucleoside base and a ribose sugar, and a "ribonucleotide" is a ribonucleoside having one, two, or three phosphate moieties or analogs thereof (e.g., phosphorothioates). However, the terms "ribonucleoside" and "ribonucleotide" can be considered equivalent when used herein. RNA can be modified, for example, in the nucleobase structure, ribose structure, or ribose-phosphate backbone structure, as described below. However, molecules containing ribonucleoside analogs or derivatives must retain the ability to form double strands. As non-limiting examples, the RNA molecule may also include at least one modified ribonucleoside, including, but not limited to, a 2'-O-methyl modified nucleoside, a nucleoside containing a 5' phosphorothioate group, a terminal nucleoside linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, an acyclic nucleoside, a glycol nucleotide, a 2'-deoxy-2'-fluoro modified nucleoside, a 2'-amino modified nucleoside, a 2'-alkyl modified nucleoside, a morpholino nucleoside, a phosphoramidate or unnatural base containing nucleoside, or any combination thereof. Alternatively, or in combination, the RNA molecule can include at least two modified ribonucleosides, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least twenty, or more, up to the entire length of the dsRNA molecule. The modification need not be the same for each of such multiple modified ribonucleosides in the RNA molecule.In some embodiments, modified RNAs contemplated for use in the methods and compositions described herein are peptide nucleic acids (PNAs) that have the ability to form the required double-stranded structure and allow or mediate the specific degradation of target RNAs, e.g., via the RISC pathway. For clarity, it is understood that the term "iRNA" does not encompass naturally occurring double-stranded DNA molecules or DNA molecules containing 100% deoxynucleosides.

[0134] In some aspects, modified ribonucleosides include deoxyribonucleosides. In such examples, the iRNA agent can include, for example, one or more deoxynucleosides, including deoxynucleoside overhangs, or one or more deoxynucleosides within the double-stranded portion of the dsRNA. In certain embodiments, the RNA molecule includes, for example, at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more (but not 100%) deoxyribonucleosides, in one or both strands.

[0135] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of an iRNA, such as a dsRNA. For example, a nucleotide overhang exists when the 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa. A dsRNA can contain at least one nucleotide overhang, or the overhang can contain at least two nucleotides, at least three nucleotides, at least four nucleotides, or at least five nucleotides, or more. A nucleotide overhang can comprise or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides, etc. An overhang can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, a certain overhanging nucleotide can be present on the 5'-end, 3'-end, or both ends of either the antisense strand or the sense strand of a dsRNA.

[0136] In some embodiments, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' and / or 5' end. In some embodiments, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' and / or 5' end. In some embodiments, one or more of the nucleotides in the overhang are substituted with a nucleoside thiophosphate.

[0137] As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of a therapeutic agent (e.g., an iRNA) and a pharmaceutically acceptable carrier. As used herein, a "pharmacologically effective amount," "therapeutically effective amount," or simply "effective amount" refers to an amount of an agent (e.g., an iRNA) effective to produce an intended pharmacological, therapeutic, or preventative result. For example, in a method for treating a disorder associated with MYOC expression (e.g., glaucoma, e.g., primary open-angle glaucoma (POAG)), an effective amount includes an amount effective to reduce one or more symptoms associated with the disorder (e.g., an amount effective to (a) inhibit or reduce MYOC expression or activity, (b) reduce levels of misfolded MYOC protein, (c) reduce trabecular meshwork cell death, (d) reduce intraocular pressure, or (e) increase visual acuity). For example, a measurable parameter associated with a disease or disorder may be reduced or eliminated. If a given clinical treatment is considered effective when there is at least a 10% reduction in a parameter, then a therapeutically effective amount of an agent for treating that disease or disorder is the amount necessary to obtain at least a 10% reduction in that parameter. For example, a therapeutically effective amount of an iRNA targeting MYOC can reduce MYOC mRNA levels or MYOC protein levels by any measurable amount, e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0138] The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, glucose, water, glycerol, ethanol, and combinations thereof. This term specifically excludes cell culture media. For orally administered drugs, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable carriers such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavoring agents, coloring agents, and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrants. Binders may include starch and gelatin, while lubricants, if present, are generally magnesium stearate, stearic acid, or talc. If necessary, tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract. The agents contained in the drug formulation are further described herein below.

[0139] As used herein, the term "SNALP" refers to stable nucleic acid-lipid particles.SNALP refers to lipid vesicles that coat a reduced aqueous interior and contain nucleic acids such as iRNA or the plasmid into which iRNA is transcribed.SNALP is described, for example, in U.S. Patent Application Publication Nos. 2006 / 0240093, 2007 / 0135372, and WO 2009 / 082817.These applications are incorporated herein by reference in their entirety.In some embodiments, the SNALP is SPLP.As used herein, the term "SPLP" refers to nucleic acid-lipid particles that contain plasmid DNA encapsulated in lipid vesicles.

[0140] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by a sequence referenced using standard nucleotide nomenclature.

[0141] As used herein, a "subject" to be treated according to the methods described herein includes a human or non-human animal, e.g., a mammal. A mammal may be, for example, a rodent (e.g., a rat or a mouse) or a primate (e.g., a monkey). In some embodiments, the subject is a human.

[0142] A "subject in need thereof" includes a subject who has, is suspected of having, or is at risk of developing a disorder associated with MYOC expression, e.g., overexpression (e.g., glaucoma). In some embodiments, the subject has or is suspected of having a disorder associated with MYOC expression or overexpression. In some embodiments, the subject is at risk of developing a disorder associated with MYOC expression or overexpression.

[0143] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of a gene, such as MYOC, including mRNAs that are the product of RNA processing of a primary transcript. The target portion of the sequence will be at least long enough to serve as a substrate for iRNA-guided cleavage at or near that portion. For example, target sequences are generally 9-36 nucleotides in length, e.g., 15-30 nucleotides in length, including all subranges therebetween. By way of non-limiting example, target sequences may be 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26 ...30 nucleotides, 19-30 nucleotides, 19-30 The amino acid sequence may be 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22 nucleotides.

[0144] As used herein, phrases such as "therapeutically effective amount" and "prophylactically effective" refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of any disorder or pathological process associated with MYOC expression (e.g., glaucoma, e.g., primary open-angle glaucoma (POAG)). The particular amount that is therapeutically effective may vary depending on factors known in the art, such as, for example, the type of disorder or pathological process, the patient's medical history and age, the stage of the disorder or pathological process, and the administration of other therapies.

[0145] In the context of this disclosure, the terms "treat," "treatment," and the like refer to preventing, delaying, alleviating, or alleviating at least one symptom associated with a disorder associated with MYOC expression, or slowing or reversing the progression or expected progression of such a disorder. For example, when employed to treat glaucoma, the methods featured herein may function to reduce or prevent one or more symptoms of glaucoma, or to reduce the risk or severity of an associated condition, as described herein. Thus, unless the context clearly indicates otherwise, the terms "treat," "treatment," and the like are intended to encompass prevention, e.g., prevention of a disorder and / or symptoms of a disorder associated with MYOC expression. Treatment can also mean prolonging survival compared to expected survival if no treatment is administered.

[0146] "Lowering," in the context of a disease marker or symptom, means any decrease, e.g., a statistically or clinically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. The decrease can be to a level that is accepted as within the normal range for individuals without such disorder.

[0147] As used herein, "MYOC" refers to the "myocilin" corresponding to the mRNA ("MYOC mRNA"), or the corresponding protein ("MYOC protein"). The sequence of the human MYOC mRNA transcript can be found in SEQ ID NO:1.

[0148] II. iRNA Agents Described herein are iRNA agents that modulate (eg, inhibit) the expression of MYOC.

[0149] In some embodiments, the iRNA agent activates expression of MYOC in a cell or mammal.

[0150] In some embodiments, the iRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting expression of MYOC in a cell or subject (e.g., in a mammal, e.g., in a human), the dsRNA comprising an antisense strand having a complementary region that is complementary to at least a portion of an mRNA formed in the expression of MYOC, the complementary region being 30 nucleotides or less in length, generally 19-24 nucleotides in length, and the dsRNA, upon contact with a cell that expresses MYOC, inhibits expression of MYOC, e.g., by at least 10%, 20%, 30%, 40%, or 50%.

[0151] Modulation (e.g., inhibition) of MYOC expression can be assayed, for example, by PCR or branched DNA (bDNA)-based methods, or by protein-based methods such as Western blot. Expression of MYOC in cell cultures such as COS cells, ARPE-19 cells, hTERT RPE-1 cells, HeLa cells, primary hepatocytes, HepG2 cells, primary cultured cells, or biological samples from subjects can be assayed by measuring MYOC mRNA levels, for example, by bDNA or TaqMan assays, or by measuring protein levels, for example, by immunofluorescence analysis using Western blot or flow cytometry techniques.

[0152] dsRNA typically contains two RNA strands that are sufficiently complementary to hybridize to form a double-stranded structure under the conditions in which the dsRNA will be used. One strand of the dsRNA (the antisense strand) typically contains a region of complementarity that is substantially complementary, and generally completely complementary, to a target sequence derived from the sequence of an mRNA formed during MYOC expression. The other strand (the sense strand) typically contains a region that is complementary to the antisense strand, such that the two strands hybridize to form a double-stranded structure when combined under suitable conditions. Typically, the double-stranded structure is 15 to 30 base pairs in length, more commonly 18 to 25 base pairs, even more commonly 19 to 24 base pairs, and most commonly 19 to 21 base pairs in length. Similarly, the region of complementarity to the target sequence is 15 to 30, more usually 18 to 25, even more usually 19 to 24, and most usually 19 to 21 nucleotides in length.

[0153] In some embodiments, the dsRNA is 15-20 nucleotides in length, and in other embodiments, the dsRNA is 25-30 nucleotides in length. As those skilled in the art will recognize, the target region of an RNA targeted for cleavage will most often be a portion of a longer RNA molecule, often an mRNA molecule. Where relevant, a "portion" of an mRNA target is a contiguous sequence of the mRNA target long enough to serve as a substrate for RNAi-dependent cleavage (i.e., cleavage via the RISC pathway). Even double-stranded dsRNA as little as 9 base pairs in length can, under some circumstances, mediate RNA cleavage by RNAi. In most cases, the target will be at least 15 nucleotides in length, e.g., 15-30 nucleotides in length.

[0154] Those skilled in the art will also recognize that the double-stranded region is the primary functional portion of a dsRNA, e.g., a double-stranded region of about 9-36 base pairs, e.g., 15-30 base pairs. That is, in some embodiments, an RNA molecule or complex of RNA molecules having a double-stranded region of more than 30 base pairs is a dsRNA, as long as it is processed into a functional duplex of, e.g., 15-30 base pairs, that targets the desired RNA for cleavage. Thus, those skilled in the art will recognize that, in some embodiments, an miRNA is therefore a dsRNA. In some embodiments, the dsRNA is not a naturally occurring miRNA. In some embodiments, an iRNA agent useful for targeting MYOC expression is not generated in a target cell by cleavage of a larger dsRNA.

[0155] The dsRNA described herein can further comprise one or more single-stranded nucleotide overhangs.DsRNA can be synthesized by standard methods known in the art, for example, by using automatic DNA synthesizer, for example, commercially available from Biosearch, Applied Biosystems, Inc., as will be further discussed below.

[0156] In some embodiments, the MYOC is human MYOC.

[0157] In certain embodiments, the dsRNA comprises a sense strand that comprises or consists of a sense sequence selected from the sense sequences provided in Table 2A or 2B, and an antisense strand that comprises or consists of an antisense sequence selected from the antisense sequences provided in Table 2A or 2B.

[0158] In some embodiments, the dsRNA comprises at least a sense and an antisense nucleotide sequence, whereby the sense strand is selected from the sequences provided in Table 2A or 2B and the corresponding antisense strand is selected from the sequences provided in Table 2A or 2B.

[0159] In these embodiments, one of the two sequences is complementary to the other of the two sequences, where one of the sequences is substantially complementary to the sequence of the mRNA produced by expression of MYOC. Thus, the dsRNA will comprise two oligonucleotides, one of which will be described as the sense strand and the second oligonucleotide as the corresponding antisense strand. As described elsewhere herein and known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being present on separate oligonucleotides.

[0160] Those skilled in the art are well aware that dsRNA with a double-stranded structure of about 20-23 base pairs, for example, 21 base pairs in particular, has been hailed as being particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888).However, others have found that shorter or longer RNA double-stranded structures can also be effective.

[0161] In the embodiments described above, due to the nature of the oligonucleotide sequences provided in Table 2A or 2B, the dsRNA described herein can comprise at least one strand that is a minimum of 19 nucleotides in length. It can be reasonably expected that shorter duplexes having one of the sequences in Table 2A or 2B, minus only a few nucleotides on one or both ends, will be similarly effective compared to the dsRNAs described above.

[0162] In some embodiments, the dsRNA has a subsequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences in Table 2A or 2B.

[0163] In some embodiments, the dsRNA has an antisense sequence comprising at least 15, 16, 17, 18, or 19 contiguous nucleotides of the antisense sequence provided in Table 2A or 2B, and a sense sequence comprising at least 15, 16, 17, 18, or 19 contiguous nucleotides of the corresponding sense sequence provided in Table 2A or 2B.

[0164] In some embodiments, the dsRNA comprises an antisense sequence comprising at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of the antisense sequence provided in Table 2A or 2B, and a sense sequence comprising at least 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides of the corresponding sense sequence provided in Table 2A or 2B.

[0165] In some such embodiments, a dsRNA includes only a portion of a sequence provided in Table 2A or 2B, but is equally effective at inhibiting the level of MYOC expression as a dsRNA including the full-length sequence provided in Table 2A or 2B. In some embodiments, the dsRNA differs in its inhibition of the expression level of MYOC by no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% inhibition compared to a dsRNA including the entire sequence disclosed herein.

[0166] In some embodiments, the iRNAs described herein comprise an antisense strand that comprises at least 15 contiguous nucleotides with 0, 1, 2, or 3 mismatches to a portion of the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the iRNAs described herein comprise a sense strand that comprises at least 15 contiguous nucleotides with 0, 1, 2, or 3 mismatches to the corresponding portion of the nucleotide sequence of SEQ ID NO: 1. Human MYOC mRNA can have the sequence of SEQ ID NO: 1 provided herein.

[0167] Homo sapiens myocilin (MYOC), mRNA

[0168] The reverse complement of SEQ ID NO:1 is provided herein as SEQ ID NO:2.

[0169] In some embodiments, the iRNAs described herein comprise at least 15 contiguous nucleotides from one of the sequences provided in Tables 2A and 2B, and can optionally be linked to additional nucleotide sequences taken from regions contiguous to the selected sequence in MYOC.

[0170] Target sequences are generally 15-30 nucleotides in length, although there is wide variation in the suitability of specific sequences within this range for directing cleavage of any given target RNA. While the various software packages and guidelines described herein provide guidance for identifying optimal target sequences for any given gene target, an empirical approach can also be undertaken in which a "window" or "mask" of a given size (21 nucleotides, as a non-limiting example) is placed literally or figuratively (including, for example, in silico) over the target RNA sequence to identify sequences within a size range that can function as target sequences. By incrementally shifting the sequence "window" one nucleotide upstream or downstream from the initial target sequence position, the next potential target sequence can be identified until a complete set of possible sequences has been identified for any given target size selected. This process, combined with systematic synthesis and testing of identified sequences (using assays described herein or known in the art) to identify optimally performing sequences, can identify RNA sequences that mediate the best inhibition of target gene expression when targeted with an iRNA agent. It is therefore contemplated that further optimization of inhibitory efficiency may be achieved by progressively "window walking" by one nucleotide upstream or downstream of a given sequence to identify sequences with equivalent or better inhibitory properties.

[0171] Furthermore, it is contemplated that further optimization can be achieved, for example, for any sequence identified in Tables 2A and 2B, by systematically adding or removing nucleotides to generate longer or shorter sequences, and then testing the resulting sequences by walking up and down the target RNA through longer or shorter windows. Again, combining this approach with generating new candidate targets to test the effectiveness of iRNAs based on their target sequences in inhibition assays known in the art or described herein can lead to further improvements in the efficiency of inhibition. Furthermore, such optimized sequences can be adjusted, for example, by introducing modified nucleotides described herein or known in the art, adding or changing overhangs, or other modifications known in the art and / or discussed herein, to further optimize the molecule as an expression inhibitor (e.g., increasing serum stability or circulating half-life, increasing thermostability, enhancing transmembrane delivery, targeting specific locations or cell types, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.).

[0172] In some embodiments, the present disclosure provides an iRNA of Table 2B that is unmodified or unconjugated. In some embodiments, the RNAi agent of the present disclosure has a nucleotide sequence provided in Table 2A, but lacks one or more ligands or moieties shown in the table. A ligand or moiety (e.g., a lipophilic ligand or moiety) can be included at any of the positions provided herein.

[0173] The iRNAs described herein may contain one or more mismatches to the target sequence. In some embodiments, the iRNAs described herein contain three or fewer mismatches. In some embodiments, when the antisense strand of an iRNA contains a mismatch to the target sequence, the area of ​​the mismatch is not located in the center of the complementary region. In some embodiments, when the antisense strand of an iRNA contains a mismatch to the target sequence, the mismatch can be limited to within the last five nucleotides from either the 5' or 3' end of the complementary region. For example, in the case of a 23-nucleotide iRNA agent, the RNA strand complementary to a region of MYOC generally does not contain any mismatches within the central 13 nucleotides. Using methods described herein or known in the art, it is possible to determine whether an iRNA containing mismatches to the target sequence is effective in inhibiting MYOC expression. It is important to consider the effectiveness of an iRNA with mismatches in inhibiting MYOC expression, especially when a particular complementary region in the MYOC gene is known to have polymorphic sequence variation within the population.

[0174] In some embodiments, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, typically 1 or 2, nucleotides. In some embodiments, dsRNAs with at least one nucleotide overhang have superior inhibitory properties compared to their blunt-ended counterparts. In some embodiments, the RNA (e.g., dsRNA) of the iRNA is chemically modified to enhance stability or other beneficial characteristics. Nucleic acids featured in this disclosure can be synthesized or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S. Lett. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, incorporated herein by reference. Modifications include, for example, (a) terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, inverted linkage, etc.) and 3'-end modifications (conjugation, DNA nucleotides, inverted linkage, etc.); (b) base modifications, such as substitution with a stabilizing base, a destabilizing base, or a base that base-pairs with an extended repertoire partner, base removal (abasic nucleotide), or a conjugated base; (c) sugar modifications (e.g., at the 2'- or 4'-position, or with an acyclic sugar) or sugar substitution; and (d) backbone modifications, including modifications or replacement of phosphodiester bonds. Specific examples of useful RNA compounds in the present disclosure include, but are not limited to, RNAs containing modified backbones or containing non-natural internucleoside linkages. RNAs with modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For purposes herein, and as sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered oligonucleosides. In certain embodiments, a modified RNA will have a phosphorus atom in its internucleoside backbone.

[0175] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and those with reversed polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.Various salts, mixed salts, and free acid forms are also included.

[0176] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Patent Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,7 No. 17, No. 5,321,131, No. 5,399,676, No. 5,405,939, No. 5,453,496, No. 5,455,233, No. 5,466,677, No. 5,47 No. 6,925, No. 5,519,126, No. 5,536,821, No. 5,541,316, No. 5,550,111, No. 5,563,253, No. 5,571,799, No. 5 , 587,361, 5,625,050, 6,028,188, 6,124,445, 6,160,109, 6,169,170, 6,172,209 No. 6,239,265, No. 6,277,603, No. 6,326,199, No. 6,346,614, No. 6,444,423, No. 6,531,590, No. 6,534, Nos. 6,639, 6,608,035, 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029, and U.S. Patent No. RE39464, the contents of each of which are incorporated herein by reference in their entirety.

[0177] Modified RNA backbones that do not contain a phosphorus atom in the backbone have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages, including those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S and CH2 constituent moieties.

[0178] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,64,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, Nos. 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439, the entire contents of each of which are incorporated herein by reference.

[0179] In other RNA mimics suitable for or contemplated for use in iRNA, both the sugar and internucleoside linkages of the nucleotide units, i.e., their backbones, are replaced with novel groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimic known to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are linked directly or indirectly to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082, 5,714,331, and 5,719,262, each of which is incorporated herein by reference. Further teaching of PNA compounds can be found, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0180] Some embodiments featured in this disclosure include dsRNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, particularly those of the above-referenced U.S. Pat. No. 5,489,677, such as --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [(known as the methylene(methylimino) or MMI backbone)], --CH2--O--N(CH3)--CH2--, -- These include -CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- (the natural phosphodiester backbone is represented as --O--P--O--CH2--), as well as the amide backbone of the above-referenced U.S. Patent No. 5,602,240. In some embodiments, the RNAs featured herein have the morpholino backbone structure of the above-referenced U.S. Patent No. 5,034,506.

[0181] Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can 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 are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 It can be alkenyl or alkynyl. Exemplary suitable modifications include O[(CH) 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, where n and m are from 1 to about 10. In other embodiments, the dsRNA includes one of the following at the 2' position: C1 to C 10lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH, OCN, Cl, Br, CN, CF, OCF, SOCH, SOCH, ONO, NO, N, NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group for improving the pharmacokinetic properties of iRNA, or group for improving the pharmacodynamic properties of iRNA, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-CHCHOCH, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is the group O(CH2)2ON(CH3)2, also known as 2'-dimethylaminooxyethoxy, i.e., 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH2)2.

[0182] In other embodiments, an iRNA agent includes one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) acyclic nucleotides (or nucleosides). In certain embodiments, the sense strand or the antisense strand, or both the sense and antisense strands, include fewer than five acyclic nucleotides per strand (e.g., four, three, two, or one acyclic nucleotide per strand). The one or more acyclic nucleotides can be found, for example, in the double-stranded region of the sense strand or the antisense strand, or both strands of the iRNA agent; at the 5'-end, the 3'-end, or both the 5'-end and the 3'-end of the sense strand or the antisense strand, or both strands. In some embodiments, the one or more acyclic nucleotides are present in positions 1-8 of the sense strand or the antisense strand, or both. In some embodiments, the one or more acyclic nucleotides are found in the antisense strand at positions 4-10 (e.g., positions 6-8) from the 5'-end of the antisense strand. In some embodiments, the one or more acyclic nucleotides are found in one or both 3' overhangs of the iRNA agent.

[0183] As used herein, the term "acyclic nucleotide" or "acyclic nucleoside" refers to any nucleotide or nucleoside having an acyclic sugar, e.g., an acyclic ribose. Exemplary acyclic nucleotides or nucleosides can include a nucleobase, e.g., a natural nucleobase or a modified nucleobase (e.g., a nucleobase described herein). In certain embodiments, the bond between any of the ribose carbons (C1, C2, C3, C4, or C5), independently or in combination, is absent in a nucleotide. In some embodiments, the bond between the C2-C3 carbons of the ribose ring is absent, e.g., an acyclic 2'-3'-seco-nucleotide monomer. In other embodiments, the bond between C1-C2, C3-C4, or C4-C5 is absent (e.g., a 1'-2', 3'-4', or 4'-5'-seco-nucleotide monomer). Exemplary acyclic nucleotides are disclosed in U.S. Pat. No. 8,314,227, the entire contents of which are incorporated herein by reference. For example, the acyclic nucleotide can include any of monomers D-J in Figures 1-2 of U.S. Patent No. 8,314,227. In some embodiments, the acyclic nucleotide includes the following monomers: [ka] The base is a nucleobase, eg, a natural nucleobase or a modified nucleobase (eg, a nucleobase described herein).

[0184] In certain embodiments, acyclic nucleotides can be modified or derivatized, for example, by conjugating the acyclic nucleotide to another moiety, such as a ligand (e.g., GalNAc, cholesterol ligand), alkyl, polyamine, sugar, polypeptide, etc.

[0185] In other embodiments, the iRNA agent includes one or more acyclic nucleotides and one or more LNAs (e.g., LNAs described herein). For example, the one or more acyclic nucleotides and / or one or more LNAs can be present in the sense strand, the antisense strand, or both. The number of acyclic nucleotides in one strand can be the same or different from the number of LNAs in the opposite strand. In certain embodiments, the sense strand and / or antisense strand include fewer than five LNAs (e.g., four, three, two, or one LNA) located in the double-stranded region or 3' overhang. In other embodiments, one or two LNAs are located in the double-stranded region or 3' overhang of the sense strand. Alternatively, or in combination, the sense strand and / or antisense strand include fewer than five acyclic nucleotides (e.g., four, three, two, or one acyclic nucleotide) in the double-stranded region or 3' overhang. In some embodiments, the sense strand of an iRNA agent includes one or two LNAs in the 3' overhang of the sense strand and one or two acyclic nucleotides in the double-stranded region of the antisense strand of the iRNA agent (e.g., positions 4-10 (e.g., positions 6-8) from the 5' end of the antisense strand).

[0186] In other embodiments, inclusion of one or more acyclic nucleotides (alone or in addition to one or more LNAs) in an iRNA agent results in one or more (or all) of the following: (i) reduced off-target effects, (ii) reduced passenger strand participation in RNAi, (iii) increased specificity of the guide strand for its target mRNA, (iv) reduced microRNA off-target effects, (v) increased stability, or (vi) increased resistance to degradation of the iRNA molecule.

[0187] Other modifications include 2'-methoxy (2'-OCH), 2'-5 aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3' position of the sugar on the 3'-terminal nucleotide or in 2'-5'-linked dsRNA, and the 5' position of the 5'-terminal nucleotide. iRNAs can also have sugar mimetics, such as cyclobutyl moieties, in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, 5,567,811, 5,576, Nos. 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 commonly owned with the present application, and each of which is incorporated herein by reference in its entirety.

[0188] iRNAs may also contain modified or substituted nucleobases (often simply referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, 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, These include 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine.

[0189] Additional nucleobases include those disclosed in United States Patent No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P.ed.Wiley-VCH, 2008, those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, JL, ed.John Wiley&Sons, 1990, those disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed in Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993.Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present 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 substitutions have been shown to increase nucleic acid duplex 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 are a further exemplary base substitution, especially when combined with a 2'-O-methoxyethyl sugar modification.

[0190] Representative United States patents that teach the preparation of some of the above-mentioned modified nucleobases, as well as other modified nucleobases, include, but are not limited to, the above-mentioned U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205, 5,130,300, 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, 5, Nos. 587,469, 5,594,121, 5,596,091, 5,614,617, 5,681,941, 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, each of which is incorporated herein by reference. No. 5,750,692 is also incorporated herein by reference.

[0191] The RNA of an iRNA can also be modified to contain one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by bridging two atoms. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety containing a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Thus, in some embodiments, agents of the present disclosure can contain one or more locked nucleic acids (LNAs) (also referred to herein as "locked nucleotides"). In some embodiments, locked nucleic acids are nucleotides having a modified ribose moiety, where the ribose moiety contains, for example, an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo conformation. The addition of a locked nucleic acid to siRNA has been shown to increase siRNA stability in serum, increase thermal stability, 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).

[0192] Examples of bicyclic nucleosides for use in polynucleotides of the present disclosure include, but are not limited to, nucleosides comprising a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, antisense polynucleotide agents of the present disclosure include one or more bicyclic nucleosides comprising a 4' to 2' bridge. Examples of such 4' to 2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (also referred to as "constrained ethyl" or "cEt") and 4'-CH(CHOCH3)-O-2' (and analogs thereof, see, e.g., U.S. Pat. No. 7,399,845), 4'-C(CH3)(CH3)-O-2' (and analogs thereof, see, e.g., U.S. Pat. No. 7,399,845), and 4'-C(CH3)(CH3)-O-2' (and analogs thereof, see, e.g., U.S. Pat. No. 7,399,845). No. 8,278,283), 4'-CH2-N(OCH3)-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,425), 4'-CH2-ON(CH3)-2' (see, e.g., U.S. Patent Publication No. 2004 / 0171570), 4'-CH2-N(R)-O-2', where R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S. Pat. No. 7,427,672), 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134), and 4'-CH2-C(═CH2)-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,426). The contents of each of the foregoing are incorporated herein by reference for the methods provided herein. Representative United States patents that teach the preparation of locked nucleic acids include, but are not limited to, U.S. Patent Nos. 6,268,490, 6,670,461, 6,794,499, 6,998,484, 7,053,207, 7,084,125, 7,399,845, and 8,314,227, each of which is incorporated herein by reference in its entirety.Exemplary LNAs include, but are not limited to, 2',4'-C methylene bicyclonucleotides (see, for example, Wengel et al., PCT Publication Nos. WO 00 / 66604 and WO 99 / 14226).

[0193] Any of the foregoing bicyclic nucleosides can be prepared with one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0194] The RNAi agents of the present disclosure can also be modified to include one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid that includes a bicyclic sugar moiety containing a 4'-CH(CH3)-O-2' bridge. In some embodiments, the constrained ethyl nucleotide is in the S conformation and is referred to herein as an "S-cEt."

[0195] The RNAi agent of the present disclosure may also contain one or more "conformation-restricting nucleotides" ("CRNs"). A CRN is a nucleotide analogue with a linker connecting the C2' and C4' carbons of ribose or the C3' and C5' carbons of ribose. The CRN locks the ribose ring into a stable conformation, increasing hybridization affinity to mRNA. The linker is long enough to position the oxygen at an optimal position for stability and affinity, thereby reducing puckering of the ribose ring.

[0196] Representative publications that teach the preparation of certain of the above CRNs include, but are not limited to, U.S. Patent Application Publication No. 2013 / 0190383 and WO 2013 / 036868, the contents of each of which are incorporated herein by reference for the methods provided herein.

[0197] In some embodiments, the RNAi agent of the present disclosure includes one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNAs are unlocked acyclic nucleic acids in which any of their sugar bonds have been removed to form an unlocked "sugar" residue. In one example, UNAs also encompass monomers in which the C1'-C4' bond has been removed (i.e., a covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another example, the C2'-C3' bond of the sugar (i.e., a carbon-carbon 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).

[0198] Representative U.S. publications that teach the preparation of UNAs include, but are not limited to, U.S. Patent No. 8,314,227, and U.S. Patent Application Publication Nos. 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020, the contents of each of which are incorporated herein by reference for the methods provided herein.

[0199] In other embodiments, an iRNA agent includes one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides. G-clamp nucleotides are modified cytosine analogs, the modification conferring the ability to hydrogen bond with both the Watson-Crick and Hoogsteen faces of complementary guanines within a duplex; see, e.g., Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. A single G-clamp analog substitution within an oligonucleotide can result in substantially enhanced helical thermal stability and mismatch discrimination when hybridized to a complementary oligonucleotide. Inclusion of such nucleotides in an iRNA molecule can enhance affinity and specificity for a nucleic acid target, complementary sequence, or template strand.

[0200] Potential stabilizing modifications to the ends of RNA molecules can 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-docosanoyl-uridine-3''-phosphate, inverted base dT (idT), and others. Disclosure of this modification can be found in WO 2011 / 005861.

[0201] Other modifications of the RNAi agent of the present disclosure include a 5' phosphate or 5' phosphate mimic, such as a 5'-terminal phosphate or phosphate mimic on the antisense strand of the RNAi agent. Suitable phosphate mimics are disclosed, for example, in U.S. Patent Application Publication No. 2012 / 0157511, the contents of which are incorporated herein by reference for the purposes provided herein. In one embodiment, a double-stranded RNAi agent of the present invention further comprises a 5'-phosphate or 5'-phosphate mimic of the 5' nucleotide of the antisense strand. In another embodiment, a double-stranded RNAi agent further comprises a 5'-phosphate mimic of the 5' nucleotide of the antisense strand. In a specific embodiment, the 5' phosphate mimic is 5'-vinylphosphonate (5'-VP). In one embodiment, the phosphate mimic is 5'-cyclopropylphosphonate (VP). In some embodiments, the 5'-end of the antisense strand of a double-stranded iRNA agent does not contain a 5'-vinylphosphonate (VP).

[0202] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of deoxy-nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, glycol-modified nucleotides (GNAs), such as Ggn, Cgn, Tgn, or Agn, nucleotides having a 2' phosphate, such as G2p, C2p, A2p, U2p, and vinyl-phosphonate nucleotides, and combinations thereof. In other embodiments, each of the duplexes of Tables 5 and 6 can be specifically modified to provide alternative double-stranded iRNA agents of the present disclosure. In one example, the 3' end of each sense duplex can be modified by removing the 3'-terminal L96 ligand and replacing two phosphodiester internucleotide linkages with phosphorothioate internucleotide linkages between the three 3'-terminal nucleotides, i.e., 5'-N1-... -Nn-2Nn-1NnL96 3' The three 3' terminal nucleotides (N) of the sense sequence of 5'-N1-... -Nn-2sNn-1sNn 3'.

[0203] RNA targets may have regions or spans of the nucleotide sequence of target RNA that are relatively more susceptible or receptive than other regions of the RNA target to mediating RNA target cleavage through RNA interference induced by the binding of RNAi agents to those regions.The increased receptivity to RNA interference in these "hotspot regions" (or simply "hotspots") means that iRNA agents that target those regions are likely to be more effective in inducing iRNA interference than iRNA agents that target other regions of the target RNA.For example, without being bound by theory, the accessibility of the target region of target RNA can affect the effectiveness of iRNA agents that target that region, and some hotspot regions have increased accessibility.For example, secondary structures formed within an RNA target (e.g., within or near hotspot regions) can affect the ability of iRNA agents to bind to target regions and induce RNA interference.

[0204] According to certain embodiments of the invention, iRNA agents can be designed to target hotspot regions of any of the target RNAs described herein, including any specified portion of the target RNA (e.g., a particular exon). As used herein, a hotspot region can refer to a region of about 19-200, 19-150, 19-100, 19-75, 19-50, 21-200, 21-150, 21-100, 21-75, 21-50, 50-200, 50-150, 50-100, 50-75, 75-200, 75-150, 75-100, 100-20, or 100-150 nucleotides of a target RNA sequence where targeting with an RNAi agent provides a significantly higher likelihood of effective silencing compared to targeting other regions of the same target RNA. According to certain embodiments of the present invention, hotspot regions can comprise a limited region of target RNA, and in some cases, a substantially limited region of target, for example, comprising less than half of the length of target RNA, for example, about 5%, 10%, 15%, 20%, 25%, or 30% of the length of target RNA. Conversely, other regions to which hotspots are compared can cumulatively comprise at least half of the length of target RNA. For example, other regions can cumulatively comprise at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95% of the length of target RNA.

[0205] Comparative regions of the target RNA can be empirically evaluated to identify hotspots using efficacy data obtained from in vitro or in vivo screening assays. For example, RNAi agents targeting various regions spanning the target RNA can be compared for the frequency of effective iRNA agents binding to each region (e.g., the amount of target gene expression inhibited, as measured by mRNA expression or protein expression). Generally, hotspots can be recognized by observing the clustering of multiple effective RNAi agents binding to a limited region of the RNA target. A hotspot can be fully characterized by observing the efficacy of iRNA agents that cumulatively cover at least about 60% of the target region identified as a hotspot, for example, about 70%, about 80%, about 90%, or about 95% or more of the length of the region, including both ends of the region (i.e., at least about 60%, 70%, 80%, 90%, or 95% or more of the nucleotides within the region, including the nucleotides at each end of the region, are targeted by the iRNA agent). According to some embodiments of the invention, an iRNA agent that exhibits at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% inhibition across the region (e.g., about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less of mRNA remaining) can be identified as effective.

[0206] The suitability of RNA regions for targeting can also be evaluated using a quantitative comparison of inhibition measurements across different regions of defined size (e.g., 25, 30, 40, 50, 60, 70, 80, 90, or 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nts). For example, the average level of inhibition can be determined for each region, and the averages for each region can be compared. The average level of inhibition within a hotspot region can be substantially higher than the average of all regions evaluated. According to some embodiments, the average level of inhibition in a hotspot region can be at least about 10%, 20%, 30%, 40%, or 50% higher. According to some embodiments, the average level of inhibition in a hotspot region can be at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 standard deviations higher than the mean of the average. The average level of inhibition may be higher by a statistically significant amount (e.g., p<0.05). According to some embodiments, each inhibition measurement within a hotspot region may exceed a threshold amount (e.g., equal to or less than a threshold amount of residual mRNA). According to some embodiments, each inhibition measurement within a region may be substantially higher than the average of all inhibition measurements across all measured regions. For example, each inhibition measurement in a hotspot region may be at least about 10%, 20%, 30%, 40%, or 50% higher than the average of all inhibition measurements. According to some embodiments, each inhibition measurement may be at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 standard deviations higher than the average of all inhibition measurements. Each inhibition measurement may be higher than the average of all inhibition measurements by a statistically significant amount (e.g., p<0.05). Criteria for assessing hotspots may include various combinations of the above standards being met (e.g., an average level of inhibition of at least about a first amount, with no inhibition measurements below a threshold level of a second amount less than the first amount).

[0207] Therefore, it is expressly contemplated that any iRNA agent, including the specific exemplary iRNA agents described herein, that targets a hotspot region of a target RNA can be selected to induce RNA interference of a target mRNA, preferably because targeting such a hotspot region is more likely to exhibit a robust inhibitory response than targeting a region that is not a hotspot region. An RNAi agent that targets a target sequence that substantially overlaps (e.g., at least about 70%, 75%, 80%, 85%, 90%, 95% of the target sequence length), or preferably exists entirely within a hotspot region, can be considered to target a hotspot region. The hotspot region of the RNA target of the present invention can include any region that shows a higher frequency of targeting by an effective RNAi agent, including any of the criteria described elsewhere herein, regardless of whether the scope of such a hotspot region is explicitly specified.

[0208] In various embodiments, the dsRNA agents of the invention target hotspot regions of the mRNA encoding MYOC.

[0209] III. iRNA motifs In certain aspects of the present disclosure, the double-stranded RNAi agent of the present disclosure includes an agent having chemical modifications, such as those disclosed in International Publication No. 2013 / 075035, the contents of which are incorporated herein by reference for the methods provided herein. As shown herein and in International Publication No. 2013 / 075035, excellent results can be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense or antisense strand of the RNAi agent, particularly at or near the cleavage site. In some embodiments, the sense and antisense strands of the RNAi agent can be otherwise completely modified. The introduction of these motifs, if present, interrupts the modification pattern of the sense or antisense strand. The RNAi agent can optionally be conjugated with a lipophilic moiety or ligand, for example, a C16 moiety or ligand, for example, on the sense strand. The RNAi agent can optionally be modified with an (S)-glycol nucleic acid (GNA) modification, for example, at one or more residues of the antisense strand. The resulting RNAi agent exhibits excellent gene silencing activity.

[0210] In some embodiments, the sense strand sequence has formula (I): 5'np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq3'(I) It can be expressed as During the ceremony, i and j each independently represent 0 or 1; p and q each independently represent 0 to 6; each Na independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each Nb independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each np and nq independently represents an overhanging nucleotide; wherein Nb and Y do not have the same modification, and XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. In some embodiments, YYY are all 2'-F modified nucleotides.

[0211] In some embodiments, Na and / or Nb comprise an alternating pattern of modifications.

[0212] In some embodiments, YYY motif occurs at or near the cleavage site of the sense strand.For example, when the RNAi agent has a double-stranded region of 17-23 nucleotides in length, YYY motif can occur at or near the cleavage site of the sense strand (for example, can occur at position 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13), and this number starts from the first nucleotide from the 5' end, or optionally this number starts from the first paired nucleotide in the double-stranded region from the 5' end.

[0213] In some embodiments, i is 1 and j is 0, or i is 0 and j is 1, or i and j are both 1. Thus, the sense strand has the following formula: 5'np-Na-YYY-Nb-ZZZ-Na-nq3'(Ib), 5'np-Na-XXX-Nb-YYY-Na-nq3'(Ic), or 5'np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq3'(Id), It can be expressed as:

[0214] When the sense strand is represented by Formula (Ib), Nb represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each Na can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0215] When the sense strand is represented as Formula (Ic), Nb represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each Na can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0216] When the sense strand is represented by Formula (Id), each Nb independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. In some embodiments, Nb is 0, 1, 2, 3, 4, 5, or 6. Each Na independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0217] Each of X, Y and Z may be the same or different from one another.

[0218] In other embodiments, i is 0 and j is 0, and the sense strand has the formula 5'n p -N a -YYY-N a -n q 3'(Ia), It can be expressed as:

[0219] When the sense strand is represented by formula (Ia), each N a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0220] In some embodiments, the antisense strand sequence of the RNAi is represented by formula (Ie): 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'(Ie), It can be expressed as During the ceremony, k and l each independently represent 0 or 1; p' and q' each independently represent 0 to 6; each Nb' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each Nb' independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p ' and n q ' independently represent an overhanging nucleotide; N b ' and Y' do not have the same modification, and X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one of three identical modifications on three consecutive nucleotides.

[0221] In some embodiments, N a ' and / or N b ' includes alternating pattern modifications.

[0222] Y'Y'Y' motif occurs at or near the cleavage site of antisense strand.For example, when RNAi agent has a double-stranded region of 17-23 nucleotides in length, Y'Y'Y' motif can occur at position 9, 10, 11, 10, 11, 12, 11, 12, 13, 12, 13, 14, or 13, 14, 15 of antisense strand, this number starting from the first nucleotide from the 5' end, or optionally this number starting from the first paired nucleotide in double-stranded region from the 5' end.In some embodiments, Y'Y'Y' motif occurs at position 11, 12, 13.

[0223] In some embodiments, the Y'Y'Y' motif is all 2'-Ome modified nucleotides.

[0224] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or k and l are both 1.

[0225] 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'(Ig), 5'n q '-N a '-Y'Y'Y'-N b '-X'X'X'-n p '3' (Ih), or 5'n q '-N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'-N a '-n p '3'(Ii), It can be expressed as:

[0226] When the antisense strand is represented by formula (Ig), Nb' represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each Na' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0227] When the antisense strand is represented by Formula (Ii), each Nb' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each Na' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. In some embodiments, Nb is 0, 1, 2, 3, 4, 5, or 6.

[0228] In other embodiments, k is 0 and l is 0, and the antisense strand has the formula: It can be represented as 5'np'-Na'-Y'Y'Y'-Na'-nq'3'(If).

[0229] When the antisense strand is represented as formula (If), each Na' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0230] Each of X', Y' and Z' may be the same as or different from one another.

[0231] Each nucleotide of sense strand and antisense strand can be independently modified with LNA, HNA, CeNA, GNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl or 2'-fluoro.For example, each nucleotide of sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.Each X, Y, Z, X', Y' and Z' can specifically represent 2'-O-methyl modification or 2'-fluoro modification.

[0232] In some embodiments, the sense strand of the RNAi agent can contain a YYY motif occurring at positions 9, 10, and 11 of the strand when the double-stranded region is 21 nt, the numbers starting from the first nucleotide from the 5' end, or optionally, the numbers starting from the first paired nucleotide in the double-stranded region from the 5' end, and Y represents a 2'-F modification. The sense strand can further contain a XXX motif or a ZZZ motif as a wing modification at the opposite end of the double-stranded region, and XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0233] In some embodiments, the antisense strand can contain a Y'Y'Y' motif occurring at positions 11, 12, or 13 of the strand, where the numbering starts from the first nucleotide from the 5' end, or optionally, the numbering starts from the first paired nucleotide in the double-stranded region from the 5' end, and Y' represents a 2'-O-methyl modification. The antisense strand can further contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at opposite ends of the double-stranded region, and X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0234] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with the antisense strand represented by any one of the formulas (If), (Ig), (Ih), and (Ii), respectively.

[0235] Thus, certain RNAi agents for use in the methods of the disclosure may include a sense strand and an antisense strand, each strand having 14-30 nucleotides, and the RNAi duplex may have the formula (Ij): Sense:5'n p -N a -(XXX)iN b -YYY-N b -(ZZZ)jN a -n q 3' Antisense: 3'n p '-Na'-(X'X'X')kN b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5', It can be expressed as (Ij) During the ceremony, i, j, k, and l each independently represent 0 or 1; p, p', q and q' each independently represent 0 to 6; each N a and N a each independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b and N b ' independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides; During the ceremony, each n p ',n p , n q ' and n q each of which may or may not be present independently represents an overhanging nucleotide; and XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides.

[0236] In some embodiments, i is 0 and j is 0, or i is 1 and j is 0, or i is 0 and j is 1, or i and j are both 0, or i and j are both 1. In some embodiments, k is 0 and l is 0, or k is 1 and l is 0, or k is 0 and l is 1, or k and l are both 0, or k and l are both 1.

[0237] An exemplary combination of sense and antisense strands that form an RNAi duplex is of 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' (Ik) 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'-Na'-nq'5' (Il) 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'-Na'-n q '5' (Im) 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'-Na'-n q '5' (In), Includes:

[0238] When the RNAi agent is represented by formula (Ik), each N a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0239] When the RNAi agent is represented by formula (II), each N b independently represent an oligonucleotide sequence containing 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. a ' independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0240] When an RNAi agent is represented by formula (Im), each Nb, Nb' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0241] When the RNAi agent is represented by the formula (In), each N b , N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a , N aN' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. a , N a ', N b , and N b ' each independently includes an alternating pattern of modifications.

[0242] Each of X, Y, and Z in formulae (Ij), (Ik), (Il), (Im), and (In) may be the same or different from one another.

[0243] When an RNAi agent is represented by formula (Ij), (Ik), (Il), (Im), and (Im), at least one of the Y nucleotides can be base-paired to one of the Y' nucleotides, alternatively, at least two Y nucleotides are base-paired to the corresponding Y' nucleotide, or all three Y nucleotides are base-paired to the corresponding Y' nucleotide.

[0244] When the RNAi agent is represented by formula (II) or (In), at least one of the Z nucleotides can be base-paired with one of the Z' nucleotides, alternatively, at least two Z nucleotides are base-paired with a corresponding Z' nucleotide, or all three Z nucleotides are base-paired with a corresponding Z' nucleotide.

[0245] When an RNAi agent is represented as formula (Im) or (In), at least one of the X nucleotides can be base-paired to one of the X' nucleotides, alternatively, at least two of the X nucleotides are base-paired to the corresponding X' nucleotide, or all three X nucleotides are base-paired to the corresponding X' nucleotide.

[0246] In some embodiments, the modification on a Y nucleotide is different from the modification on a Y' nucleotide, the modification on a Z nucleotide is different from the modification on a Z' nucleotide, and / or the modification on an X nucleotide is different from the modification on an X' nucleotide.

[0247] In some embodiments, when the RNAi agent has formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification. In some embodiments, when the RNAi agent has formula (In), the Na modification is a 2'-O-methyl or 2'-fluoro modification, and np'>0 and at least one np' are linked to adjacent nucleotides via phosphorothioate linkages. In some embodiments, when the RNAi agent has formula (In), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0 and also at least one np' are linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand is conjugated to one or more moieties or ligands (e.g., one or more lipophilic moieties, optionally one or more C16 moieties, or one or more GalNAc moieties) linked via a bivalent or trivalent branched linker. In some embodiments, when the RNAi agent has formula (In), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0 and also at least one np' is linked to an adjacent nucleotide via a phosphorothioate linkage, and the sense strand is conjugated to one or more moieties or ligands (e.g., one or more lipophilic moieties, optionally one or more C16 moieties, or one or more GalNAc moieties) attached via a divalent or trivalent branched linker.

[0248] In some embodiments, when the RNAi agent has formula (IIIa), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0 and also at least one np' is linked to an adjacent nucleotide via a phosphorothioate linkage, and the sense strand is conjugated to one or more moieties or ligands (e.g., one or more lipophilic moieties, optionally one or more C16 moieties, or one or more GalNAc moieties) attached via a divalent or trivalent branched linker.

[0249] In some embodiments, the RNAi agent is a multimer containing at least two double strands represented by formula (Ij), (Ik), (II), (Im), and (Im), and the double strands are connected by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the double strands can target the same gene or two different genes, or each of the double strands can target the same gene at two different target sites.

[0250] In some embodiments, the RNAi agent is a multimer containing three, four, five, six or more double strands represented by formula (Ij), (Ik), (Il), (Im) and (In), and the double strands are connected by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the double strands can target the same gene or two different genes, or each of the double strands can target the same gene at two different target sites.

[0251] In some embodiments, two RNAi agents represented by formula (Ij), (Ik), (Il), (Im), and (In) are linked to each other at their 5' ends and optionally conjugated to a ligand at one or both of their 3' ends. The agents can each target the same gene or two different genes, or the agents can each target the same gene at two different target sites.

[0252] Various publications describe the multimeric RNAi agent that can be used in the method of the present disclosure.These publications include International Publication No. WO2007 / 091269, International Publication No. WO2010 / 141511, International Publication No. WO2007 / 117686, International Publication No. WO2009 / 014887 and International Publication No. WO2011 / 031520 and US Patent No. 7858769, the contents of each of which are incorporated herein by reference for the method provided herein.In certain embodiments, the RNAi agent of the present disclosure can comprise GalNAc ligand.

[0253] As described in more detail below, RNAi agents containing one or more carbohydrate moieties conjugated to them can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be 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 with another moiety, such as a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is attached. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose-replacement modified subunit (PRMS). The cyclic carrier can be a carbon-cyclic system, i.e., all ring atoms are carbon atoms, or a heterocyclic system, i.e., one or more ring atoms can be heteroatoms, such as nitrogen, oxygen, or sulfur. The cyclic carrier can be a monocyclic ring system or can contain two or more rings, e.g., fused rings. The cyclic carrier can be a fully saturated ring system or can contain one or more double bonds.

[0254] The ligand can be attached to the polynucleotide via a carrier. The carrier comprises (i) at least one "backbone attachment point," preferably two "backbone attachment points," and (ii) at least one "tethering attachment point." As used herein, "backbone attachment point" refers to a functional group, e.g., a hydroxyl group, or generally refers to a bond available and suitable for incorporating the carrier into the backbone of a ribonucleic acid, e.g., a phosphate, or a modified phosphate backbone, e.g., sulfur-containing. In some embodiments, a "tethering attachment point" (TAP) refers to a ring atom, e.g., a carbon atom or heteroatom (other than the atom providing the backbone attachment point), of the cyclic carrier to which the selected moiety is attached. This moiety can be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is attached to the cyclic carrier by an intervening tether. Thus, cyclic carriers often contain functional groups, e.g., amino groups, or generally bonds, that provide a linkage suitable for the incorporation or tethering of another chemical entity, e.g., a ligand, to the constituent ring.

[0255] The RNAi agent may be conjugated to the ligand via a carrier, which may be a cyclic or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is selected from a serinol backbone or a diethanolamine backbone.

[0256] In certain embodiments, the RNAi agent used in the method of the present disclosure is an agent selected from the group of agents listed in any one of Tables 2A and 2B. These agents may further comprise a ligand. The ligand can be attached to the sense strand, the antisense strand, or both strands at the 3'-end, the 5'-end, or both ends. For example, the ligand can be attached to the sense strand, particularly the 3'-end of the sense strand.

[0257] IV. iRNA conjugates The iRNA agent disclosed herein can be in the form of conjugate.Conjugate can be attached to any suitable position of iRNA molecule, for example, the 3'-end or 5'-end of sense strand or antisense strand.Conjugate can be optionally attached via linker.

[0258] In some embodiments, an iRNA agent described herein is chemically linked to one or more ligands, moieties, or conjugates that can confer functionality, for example, by affecting (e.g., enhancing) the activity, cellular distribution, or cellular uptake of the iRNA. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acids Sci. USA, 1989, 86:6553-6556), cholic acid (Manoharan et al., Bior. Med. Chem. Let., 1994, 4:1053-1060), thioethers such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Bior. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), fatty chains such as 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 such as dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0259] In some embodiments, the ligand changes the distribution, targeting, or life span of the iRNA agent into which it is incorporated. In some embodiments, the ligand enhances the affinity of a selected target, for example, a molecule, a cell or cell type, a compartment, such as a cell or organ compartment, a tissue, an organ, or a region of the body, for example, when compared to a species in which such a ligand is not present. Conventional ligands do not participate in double-stranded pairing in double-stranded nucleic acids.

[0260] Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL) or globulins), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid), or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.

[0261] The ligand can also include a targeting group, such as a cell or tissue targeting agent, such as a lectin, a glycoprotein, lipid, or protein, such as an antibody, that binds to a specific cell type, such as a kidney cell. The targeting group can be thyroid-stimulating hormone, melanocyte-stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic.

[0262] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules, such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutanoic acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithophosphate, and the like. Examples of suitable cleavage inhibitors include oleic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0263] Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules with specific affinity for co-ligands, or antibodies, e.g., antibodies that bind to specific cell types, such as ocular cells. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. Ligands can be, for example, lipopolysaccharides, activators of p38 MAP kinase, or activators of NF-κB.

[0264] The ligand can be a substance, e.g., a drug, that can increase the uptake of an iRNA agent into a cell, for example, by disrupting the cytoskeleton of the cell, e.g., by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0265] In some embodiments, the ligands binding to the iRNAs described herein act as pharmacokinetic modulators (PK modulators). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing several phosphorothioate linkages have also been shown to bind to serum proteins, and therefore short oligonucleotides containing multiple phosphorothioate linkages in the backbone, e.g., oligonucleotides of about 5, 10, 15, or 20 bases, are also suitable as ligands (e.g., as PK-modulating ligands) for the present disclosure. Furthermore, aptamers that bind serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0266] Ligand-conjugated oligonucleotides of the present disclosure can be synthesized by using an oligonucleotide bearing a reactive functional pendant side chain, such as that resulting from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide can be reacted directly with a commercially available ligand, a synthesized ligand bearing any of a variety of protecting groups, or a ligand bearing a linking moiety attached thereto.

[0267] The oligonucleotides used in the conjugates of the present disclosure can be conveniently and routinely produced by the well-known technique of solid-phase synthesis.The equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems (Foster City, California).Any other means for such synthesis known in the art can additionally or alternatively be adopted.It is also known to use similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

[0268] In the ligand-conjugated oligonucleotides and sequence-specific linked nucleosides bearing ligand molecules of the present disclosure, the oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors already bearing a linking moiety, ligand-nucleotide or ligand-nucleoside conjugate precursors already bearing a ligand molecule, or non-nucleoside ligand-bearing building blocks.

[0269] When using a nucleotide-conjugate precursor that already possesses a linking moiety, typically, synthesis of the sequence-specifically linked nucleoside is completed, and then a ligand molecule is reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present disclosure are synthesized by automated synthesizers using phosphoramidites derived from ligand-nucleoside conjugates, in addition to commercially available standard and non-standard phosphoramidites routinely used in oligonucleotide synthesis.

[0270] A. Lipophilic part In certain embodiments, the lipophilic moiety is aliphatic, cyclic, e.g., alicyclic, or polycyclic, e.g., polyalicyclic, compounds such as steroids (e.g., sterols) or linear or branched aliphatic hydrocarbons. The lipophilic moiety generally comprises a hydrocarbon chain, which may be cyclic or acyclic. The hydrocarbon chain may contain various substituents or one or more heteroatoms, such as oxygen or nitrogen atoms. Such lipophilic aliphatic moieties include saturated or unsaturated C4-C 30 Hydrocarbons (e.g., C6-C 18 hydrocarbons), saturated or unsaturated fatty acids, waxes (e.g., monohydric alcohol esters of fatty acids and fatty acid diamides), terpenes (e.g., C 10 Terpene, C 15 Sesquiterpene, C 20 Diterpenes, C 30 Triterpenes, and C 40 tetraterpenes), as well as other polyalicyclic hydrocarbons. For example, the lipophilic moiety may be C4-C 30 Hydrocarbon chains (e.g., C4-C 30 In some embodiments, the lipophilic moiety may contain a saturated or unsaturated C-C 18 Hydrocarbon chains (e.g., linear C6-C 18In some embodiments, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain (e.g., a straight-chain C16 alkyl or alkenyl).

[0271] Lipophilic moieties can be attached to RNAi agents by any method known in the art, either through functional groups already present on the lipophilic moiety or through functional groups introduced into the RNAi agent, such as hydroxyl groups (e.g., -CO-CH-OH). Functional groups already present on the lipophilic moiety or introduced into the RNAi agent include, but are not limited to, hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0272] Conjugation of an RNAi agent to a lipophilic moiety can occur, for example, through the formation of an ether, carboxylic acid, or carbamoyl ester bond between a hydroxyl group and an alkyl group R-, an alkanoyl group RCO-, or a substituted carbamoyl group RNHCO-. The alkyl group R can be cyclic (e.g., cyclohexyl) or acyclic (e.g., linear or branched, and saturated or unsaturated). The alkyl group R can be butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, or octadecyl group, etc.

[0273] In some embodiments, the lipophilic moiety is conjugated to the double-stranded RNAi agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, the product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate.

[0274] In another embodiment, the lipophilic moiety is a steroid, such as a sterol.Steroids are polycyclic compounds containing a perhydro-1,2-cyclopentanophenanthrene ring system.Steroids include, but are not limited to, cationic steroids such as bile acids (e.g., cholic acid, deoxycholic acid, and dehydrocholic acid), cortisone, digoxigenin, testosterone, cholesterol, and cortisone."Cholesterol derivative" refers to a compound derived from cholesterol, for example, by substitution, addition, or removal of substituents.

[0275] In another embodiment, the lipophilic moiety is an aromatic moiety. In this context, the term "aromatic" refers broadly to monoaromatic and polyaromatic hydrocarbons. Aromatic groups include C6-C6 groups containing one to three aromatic rings, which may be optionally substituted. 14 These include, but are not limited to, aryl moieties, "aralkyl" or "arylalkyl" groups comprising an aryl group covalently bonded to an alkyl group, either of which independently may be optionally substituted or unsubstituted, and "heteroaryl" groups. As used herein, the term "heteroaryl" refers to groups having 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms, groups having 6, 10, or 14 π electrons shared in a cyclic arrangement, and groups having, in addition to carbon atoms, one to about three heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and sulfur (S).

[0276] As used herein, a "substituted" alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclic group has one to four, preferably one to about three, and more preferably one or two non-hydrogen substituents. Suitable substituents include, but are not limited to, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido groups.

[0277] In some embodiments, the lipophilic moiety is an aralkyl group, e.g., a 2-arylpropanoyl moiety. The structural features of the aralkyl group are selected so that the lipophilic moiety binds to at least one protein in vivo. In certain embodiments, the structural features of the aralkyl group are selected so that the lipophilic moiety binds to serum, vascular, or cellular proteins. In certain embodiments, the structural features of the aralkyl group promote binding to albumin, immunoglobulins, lipoproteins, α-2-macrogululin, or α-1-glycoprotein.

[0278] In certain embodiments, the ligand is naproxen or a structural derivative of naproxen. The synthesis procedure for naproxen can be found in U.S. Patent No. 3,904,682 and U.S. Patent No. 4,009,197, which are incorporated herein by reference in their entireties. Naproxen has the chemical name (S)-6-methoxy-α-methyl-2-naphthaleneacetic acid and the structure: [ka] is.

[0279] In certain embodiments, the ligand is ibuprofen or a structural derivative of ibuprofen. The synthesis procedure for ibuprofen can be found in U.S. Patent No. 3,228,831, which is incorporated herein by reference for the methods provided herein. The structure of ibuprofen is: [ka] is.

[0280] Additional exemplary aralkyl groups are set forth in US Pat. No. 7,626,014, which is incorporated herein by reference for the purposes provided herein.

[0281] In another embodiment, suitable lipophilic moieties include lipids, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl groups, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxytrityl, or phenoxazine.

[0282] In certain embodiments, two or more lipophilic moieties can be incorporated into a double-stranded RNAi agent, especially when the lipophilic moiety has low lipophilicity or hydrophobicity. In some embodiments, two or more lipophilic moieties are incorporated into the same strand of a double-stranded RNAi agent. In some embodiments, the same strand of a double-stranded RNAi agent has one or more incorporated lipophilic moieties. In some embodiments, two or more lipophilic moieties are incorporated into the same position of a double-stranded RNAi agent (i.e., the same nucleobase, the same sugar moiety, or the same internucleotide bond). This can be achieved, for example, by conjugating two or more lipophilic moieties via a carrier, or by conjugating two or more lipophilic moieties via a branched linker, or by conjugating two or more lipophilic moieties via one or more linkers with one or more linkers that sequentially connect the lipophilic moieties.

[0283] The lipophilic moiety can be conjugated to the RNAi agent via direct binding to the ribosugar of the RNAi agent. Alternatively, the lipophilic moiety can be conjugated to the double-stranded RNAi agent via a linker or carrier.

[0284] In certain embodiments, the lipophilic moiety may be conjugated to the RNAi agent via one or more linkers (tethers).

[0285] In some embodiments, the lipophilic moiety is conjugated to the double-stranded RNAi agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, the product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate.

[0286] B. Lipid Conjugates In some embodiments, the ligand is a lipid or lipid-based molecule. Such lipid or lipid-based molecule can usually bind serum proteins, for example, human serum albumin (HSA). HSA-binding ligands allow the vascular distribution of the conjugate to target tissue. For example, the target tissue can be the eye. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. The lipid or lipid-based ligand can (a) increase the resistance of the conjugate to degradation, (b) increase targeting or transport into target cells or cell membranes, and / or (c) be used to adjust the binding to serum proteins, for example, HSA.

[0287] Lipid-based ligands can be used to regulate, for example, control (for example, inhibit) the binding of conjugate to target tissue.For example, lipid or lipid-based ligands that bind more strongly to HSA are less likely to target the kidney, and therefore less likely to be eliminated from the body.A lipid or lipid-based ligand that binds less strongly to HSA can be used so that the conjugate targets the kidney.

[0288] In some embodiments, the lipid-based ligand binds HSA. For example, the ligand can bind to HSA with sufficient affinity, thereby enhancing the distribution of the conjugate to non-renal tissues. However, this affinity is usually not so strong that the HSA-ligand binding is irreversible.

[0289] In some embodiments, the lipid-based ligand binds weakly or not at all to HSA, 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.

[0290] In another embodiment, the ligand is a moiety, for example, a vitamin, that is taken up by target cells, for example, proliferating cells.These are particularly useful for treating disorders characterized by unwanted cell proliferation, for example, malignant or non-malignant, for example, cancer cells.Exemplary vitamins include vitamins A, E and K.Other exemplary vitamins include vitamin B, for example, folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by cancer cells.Also included are HSA and low-density lipoprotein (LDL).

[0291] C. Cell-penetrating agents In another aspect, the ligand is a cell-penetrating agent, such as a helical cell-penetrating agent. In some embodiments, the agent is amphipathic. Exemplary agents include peptides, such as tat or antennopedia. If the agent is a peptide, it can be modified, including peptidyl mimetics, invertomers, non-peptide or pseudo-peptide linkages, and the use of D-amino acids. Helical agents are typically α-helical agents and can have a lipophilic phase and a lipophobic phase.

[0292] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules that can fold into defined three-dimensional structures similar to natural peptides. Attachment of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic distribution of iRNAs, such as by enhancing cellular recognition and uptake. The peptide or peptidomimetic portion can be about 5 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0293] The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. Alternatively, the peptide moiety can contain a hydrophobic membrane transport sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 3). An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 4)) can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, capable of transporting large polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 5)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 6)) have been shown to function as delivery peptides. Peptides or peptidomimetics can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). Peptides or peptidomimetics that are typically tethered to dsRNA agents via incorporated monomeric units include cell-targeting peptides, such as arginine-glycine-aspartic acid (RGD)-peptides or RGD mimetics. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications, such as those that increase stability or affect conformational properties. Any of the structural modifications described below can be utilized.

[0294] The RGD peptides for use in the compositions and methods of the present disclosure can be linear or cyclic, and can be modified, for example, glycosylated or methylated, to facilitate targeting to specific tissues.RGD-containing peptides and peptidomimetics can include D-amino acids and synthetic RGD mimics.In addition to RGD, other moieties that target integrin ligands can be used.In some embodiments, the conjugate of this ligand targets PECAM-1 or VEGF.

[0295] RGD peptide moieties can be used to target specific cell types, such as tumor cells, e.g., endothelial tumor cells or breast cancer tumor cells (Zitzmann et al., Cancer Res., 62:5139-43, 2002). RGD peptides can promote targeting of dsRNA agents to tumors in various other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Typically, RGD peptides promote targeting of iRNA agents to the kidney. RGD peptides can be linear or cyclic, and can be modified to promote targeting to specific tissues, e.g., glycosylated or methylated. For example, glycosylated RGD peptides can promote targeting of α V iRNA agents can be delivered to tumor cells that express β3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

[0296] A "cell-penetrating peptide" can penetrate cells, such as microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or Ceropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain a nuclear localization signal (NLS). For example, a cell-penetrating peptide can be a bipartite amphipathic peptide, such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0297] D. Carbohydrate Conjugates and Ligands In some embodiments of the compositions and methods of the present disclosure, the iRNA oligonucleotide further comprises a carbohydrate. As described herein, iRNA conjugated to a carbohydrate is advantageous for in vivo delivery of nucleic acids and is suitable for in vivo therapeutic use. As used herein, "carbohydrate" refers to a compound that is either a carbohydrate itself (which may be linear, branched, or cyclic) composed of one or more monosaccharide units having at least six carbon atoms with an oxygen, nitrogen, or sulfur atom attached to each carbon atom, or a compound having as its part a carbohydrate moiety (which may be linear, branched, or cyclic) composed of one or more monosaccharide units each having at least six carbon atoms with an oxygen, nitrogen, or sulfur atom attached to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide resins. Particular monosaccharides include C5 and above (e.g., C5, C6, C7 or C8) sugars, and disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7 or C8).

[0298] In certain embodiments, the compositions and methods of the present disclosure comprise a C16 ligand. In exemplary embodiments, the C16 ligand of the present disclosure has the following structure (illustrated herein below for uracil base, but the binding of the C16 ligand is contemplated for any base (C, G, A, etc.) present, or for any other nucleotide modification presented herein, provided that the 2' ribonucleotide bond is retained), and is bound at the 2' position of the ribonucleotide in the residue modified in this way. [ka]

[0299] As noted above, the C16 ligand-modified residue presents a linear alkyl at the 2'-ribo position of the exemplary residue so modified (here uracil).

[0300] In some embodiments, the carbohydrate conjugate of an RNAi agent of the present disclosure further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator or a cell-penetrating peptide.

[0301] Additional carbohydrate conjugates (and linkers) suitable for use in the present disclosure include those described in WO 2014 / 179620 and WO 2014 / 179627, the entire contents of each of which are incorporated herein by reference.

[0302] In certain embodiments, the compositions and methods of the present disclosure include vinyl phosphonate (VP) modifications of the RNAi agents described herein. In exemplary embodiments, the vinyl phosphonates of the present disclosure have the following structure: [ka]

[0303] The vinyl phosphonate of the present disclosure can be attached to either the antisense or sense strand of the dsRNA of the present disclosure. In certain preferred embodiments, the vinyl phosphonate of the present disclosure is optionally attached to the antisense strand of the dsRNA at the 5'-end of the antisense strand of the dsRNA. The dsRNAi agent can include a phosphorus-containing group at the 5'-end of the sense strand or the antisense strand. The 5'-terminal phosphorus-containing group can be 5'-terminal phosphate (5'-P), 5'-terminal phosphorothioate (5'-PS), 5'-terminal phosphorodithioate (5'-PS2), 5'-terminal vinyl phosphonate (5'-VP), 5'-terminal methyl phosphonate (MMePhos), or 5'-deoxy-5'-C-malonyl. When the 5'-terminal phosphorus-containing group is 5'-terminal vinyl phosphonate (5'-VP), the 5'-VP can be a 5'-E-VP isomer (i.e., trans-vinyl phosphonate, [ka] 5'-Z-VP isomer (i.e., cis-vinyl phosphonate, [ka] or a mixture thereof.

[0304] Vinyl phosphate modifications are also contemplated for the compositions and methods of the present disclosure. Exemplary vinyl phosphate structures are as follows: [ka]

[0305] In some embodiments, the carbohydrate conjugate comprises a monosaccharide. In some embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). GalNAc conjugates comprising one or more N-acetylgalactosamine (GalNAc) derivatives are described, for example, in U.S. Patent No. 8,106,022, the entire contents of which are incorporated herein by reference. In some embodiments, the GalNAc conjugate functions as a ligand that targets the iRNA to specific cells. In some embodiments, the GalNAc conjugate targets the iRNA to liver cells, for example, by functioning as a ligand for the asialoglycoprotein receptor of liver cells (e.g., hepatocytes).

[0306] In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives can be attached via a linker, e.g., via a bivalent or trivalent branched linker. In some embodiments, the GalNAc conjugate is conjugated to the 3' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 3' end of the sense strand) via a linker, e.g., via a linker as described herein.

[0307] In some embodiments, the GalNAc conjugate is [ka] Formula II.

[0308] In some embodiments, the RNAi agent is attached to the carbohydrate conjugate via a linker as shown in the following schematic diagram, where X is O or S: [ka]

[0309] In some embodiments, the RNAi agent is conjugated to L96, as defined in Table 1 and shown below. [ka]

[0310] In some embodiments, the carbohydrate conjugate for use in the compositions and methods of the present disclosure is selected from the group consisting of: [ka] [ka] [ka] [ka] .

[0311] Other exemplary carbohydrate conjugates for use in the embodiments described herein include, but are not limited to: [ka] (Formula XXIII), and when one of X or Y is an oligonucleotide, the other is hydrogen.

[0312] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and / or a cell-penetrating peptide.

[0313] In some embodiments, the iRNAs of the present disclosure are conjugated to carbohydrates through linkers. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the present disclosure include, but are not limited to: [ka] and [ka] When one of X and Y is an oligonucleotide, the other is hydrogen.

[0314] E. Thermal destabilization modification In certain embodiments, dsRNA molecules can be optimized for RNA interference by incorporating thermally destabilizing modifications into the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand) to reduce or inhibit off-target gene silencing. It has been discovered that dsRNAs having an antisense strand containing at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end of the antisense strand have reduced off-target gene silencing activity. Thus, in some embodiments, the antisense strand contains at least one (e.g., one, two, three, four, five, or more) thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5' region of the antisense strand. In some embodiments, the thermally destabilizing modification of one or more of the duplexes is located at positions 2-9, or preferably 4-8, from the 5' end of the antisense strand. In some further embodiments, the thermally destabilizing modification of the duplex is located at positions 6, 7, or 8 from the 5' end of the antisense strand. In yet some further embodiments, the thermally destabilizing modification of the duplex is located at position 7 from the 5' end of the antisense strand. The term "thermally destabilizing modification" includes a modification that results in a dsRNA having a lower overall melting temperature (Tm) (preferably a Tm that is one, two, three, or four degrees lower) than the Tm of a dsRNA not having such a modification. In some embodiments, the thermally destabilizing modification of the duplex is located at position 2, 3, 4, 5, or 9 from the 5' end of the antisense strand.

[0315] Thermally destabilizing modifications can include, but are not limited to, abasic modifications, mismatches with opposing nucleotides in the opposing strand, and sugar modifications, such as 2'-deoxy modifications or acyclic nucleotides, such as unlocked nucleic acids (UNAs) or glycol nucleic acids (GNAs).

[0316] Exemplary abasic modifications include, but are not limited to, the following: [ka] wherein R=H, Me, Et or OMe, R'=H, Me, Et or OMe, R"=H, Me, Et or OMe [ka] wherein B is a modified or unmodified nucleobase.

[0317] Exemplary sugar modifications include, but are not limited to, the following: [ka] wherein B is a modified or unmodified nucleobase.

[0318] In some embodiments, the thermally destabilizing modification of the duplex is selected from the group consisting of: [ka] where B is a modified or unmodified nucleobase, and the asterisk on each structure represents either R, S, or racemic.

[0319] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, e.g., one in which any of the bonds between the 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 oxygens (e.g., C1', C2', C3', C4', or O4'), independently or in combination, is absent from the nucleotide. In some embodiments, an acyclic nucleotide is [ka] B is a modified or unmodified nucleobase; R 1 and R 2are independently H, halogen, OR3, or alkyl, and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. The term "UNA" refers to an acyclic unlocked nucleic acid in which one of the sugar bonds has been removed to form an unlocked "sugar" residue. In one example, UNA also encompasses monomers in which the C1'-C4' bond has been removed (i.e., a carbon-oxygen-carbon covalent bond between the C1' and C4' carbons). In another example, the C2'-C3' bond of the sugar has been removed (i.e., a carbon-carbon covalent bond between the C2' and C3' carbons) (see Mikhailov et al., Tetrahedron Letters, 26(17):2059 (1985), and Fluiter et al., Mol. Biosyst., 10:1039 (2009), which are incorporated herein by reference in their entireties). Acyclic derivatives offer greater backbone flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be linked via 2'-5' or 3'-5' linkages.

[0320] The term "GNA" refers to glycol nucleic acid, which is a polymer similar to DNA or RNA, but differs in the composition of its "backbone" in that it is made up of repeating glycerol units linked by phosphodiester bonds. [ka]

[0321] The thermally destabilizing modification of the double strand can be a mismatch (i.e., non-complementary base pair) between the thermally destabilizing nucleotide and the opposite nucleotide in the opposite strand of the dsRNA duplex.Exemplary mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or combinations thereof.Other mismatch base pairings known in the art are also contemplated by the present invention.Mismatches can occur between nucleotides that are either naturally occurring or modified nucleotides, i.e., mismatch base pairing can occur between the nucleobases derived from each nucleotide, regardless of the modification on the ribose sugar of the nucleotide.In certain embodiments, the dsRNA molecule contains at least one nucleobase in the mismatch pairing that is a 2'-deoxynucleobase, for example, the 2'-deoxynucleobase is in the sense strand.

[0322] In some embodiments, the thermally destabilizing modifications of the duplex in the seed region of the antisense strand include nucleotides that impair WCH bonding with complementary bases on the target mRNA, such as: [ka]

[0323] Many examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA) and mismatch modifications are described in detail in WO 2011 / 133876, which is incorporated herein by reference in its entirety.

[0324] Thermally destabilizing modifications can also include universal base and phosphate modifications that have reduced or eliminated ability to form hydrogen bonds with opposing bases.

[0325] In some embodiments, the thermal destabilizing modification of double strand comprises the nucleotide with non-standard base, for example but not limited to, the nucleobase modification that the ability to form hydrogen bond with the base in opposite strand is impaired or completely lost.These nucleobase modifications have been evaluated for the destabilization of the central region of dsRNA double strand, as described in International Publication No. 2010 / 0011895, the entire contents of which are incorporated herein by reference.Exemplary nucleobase modifications include: [ka]

[0326] In some embodiments, the thermally destabilizing modifications of the duplex in the seed region of the antisense strand include one or more α-nucleotides that are complementary to bases on the target mRNA, such as: [ka] wherein R is H, OH, OCH3, F, NH2, NHMe, NMe2 or O-alkyl.

[0327] Exemplary phosphate modifications that have been shown to reduce the thermal stability of dsRNA duplexes compared to natural phosphodiester linkages include the following: [ka]

[0328] The alkyl R group can be a C1-C6 alkyl. Specific alkyl R groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

[0329] As those skilled in the art will recognize, considering that the functional role of nucleobase defines the specificity of the RNAi agent of the present disclosure, nucleobase modification can be carried out in various ways as described herein, for example, for the purpose of enhancing on-target effect against off-target effect, for example, introducing destabilizing modification into the RNAi agent of the present disclosure, but generally the range of modification that exists on the RNAi agent of the present disclosure tends to be much larger than the non-nucleobase modification, for example, the modification of the sugar group or phosphate backbone of polyribonucleotide.Such modification will be described in more detail in other sections of the present disclosure, and is expressly intended for the RNAi agent of the present disclosure that has either natural nucleobase or modified nucleobase as described above or elsewhere herein.

[0330] In addition to the antisense strand containing a thermally destabilizing modification, the dsRNA may also contain one or more stabilizing modifications. For example, the dsRNA may contain at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilizing modifications. Without limitation, all of the stabilizing modifications may be present in one strand. In some embodiments, both the sense and antisense strands contain at least two stabilizing modifications. The stabilizing modifications may occur at any nucleotide in either the sense or antisense strand. For example, the stabilizing modifications may occur at any nucleotide in the sense or antisense strand, and each stabilizing modification may occur in an alternating pattern on the sense or antisense strand, or the sense or antisense strand may contain both stabilizing modifications in an alternating pattern. The alternating pattern of stabilizing modifications on the sense strand may be the same as or different from that of the antisense strand, and the alternating pattern of stabilizing modifications on the sense strand may be shifted relative to the alternating pattern of stabilizing modifications on the antisense strand.

[0331] In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilizing modifications. Without limitation, the stabilizing modifications within the antisense strand can be located at any position.

[0332] In some embodiments, the antisense strand comprises stabilizing modifications at positions 2, 6, 8, 9, 14, and 16 from the 5' end. In some other embodiments, the antisense strand comprises stabilizing modifications at positions 2, 6, 14, and 16 from the 5' end. In yet some other embodiments, the antisense strand comprises stabilizing modifications at positions 2, 14, and 16 from the 5' end.

[0333] In some embodiments, the antisense strand comprises at least one stabilizing modification adjacent to a destabilizing modification. For example, the stabilizing modification can be at the 5' or 3' end of the destabilizing modification, i.e., at the -1 or +1 position from the destabilizing modification. In some embodiments, the antisense strand comprises a stabilizing modification at each of the 5' and 3' ends of the destabilizing modification, i.e., at the -1 and +1 positions from the destabilizing modification.

[0334] In some embodiments, the antisense strand comprises at least two stabilizing modifications at the 3' end of the destabilizing modification, ie, at positions +1 and +2 from the position of the destabilizing modification.

[0335] In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilizing modifications. Without limitation, the stabilizing modifications within the sense strand can be located at any position. In some embodiments, the sense strand comprises stabilizing modifications at positions 7, 10, and 11 from the 5' end. In some other embodiments, the sense strand comprises stabilizing modifications at positions 7, 9, 10, and 11 from the 5' end. In some embodiments, the sense strand comprises stabilizing modifications 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 comprises stabilizing modifications 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 comprises blocks of two, three, or four stabilizing modifications.

[0336] In some embodiments, the sense strand does not contain a stabilizing modification at a position opposite or complementary to a thermally destabilizing modification of the duplex in the antisense strand.

[0337] Exemplary thermally stabilizing modifications include, but are not limited to, 2'-fluoro modifications. Other thermally stabilizing modifications include, but are not limited to, LNA.

[0338] In some embodiments, the dsRNA of the present disclosure contains at least four (e.g., four, five, six, seven, eight, nine, ten, or more) 2'-fluoro nucleotides. Without limitation, all of the 2'-fluoro nucleotides can be present in one strand. In some embodiments, both the sense and antisense strands contain at least two 2'-fluoro nucleotides. The 2'-fluoro modification can occur on either the sense strand or the antisense strand's nucleotide. For example, the 2'-fluoro modification can occur on any nucleotide on the sense strand or the antisense strand, and each 2'-fluoro modification can occur in an alternating pattern on the sense strand or the antisense strand, or both the sense strand and the antisense strand contain 2'-fluoro modifications in an alternating pattern. The alternating pattern of 2'-fluoro modifications on the sense strand can be the same as or different from that of the antisense strand, and the alternating pattern of 2'-fluoro modifications on the sense strand can be shifted relative to the alternating pattern of 2'-fluoro modifications on the antisense strand.

[0339] In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modifications in the antisense strand can be present at any position. In some embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 8, 9, 14, and 16 from the 5' end. In some other embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end. In yet some other embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 14, and 16 from the 5' end.

[0340] In some embodiments, the antisense strand comprises at least one 2'-fluoro nucleotide adjacent to the destabilizing modification.For example, the 2'-fluoro nucleotide can be the nucleotide at the 5'-end or 3'-end of the destabilizing modification, i.e., at the -1 or +1 position from the position of the destabilizing modification.In some embodiments, the antisense strand comprises a 2'-fluoro nucleotide at each of the 5'-end and 3'-end of the destabilizing modification, i.e., at the -1 and +1 positions from the position of the destabilizing modification.

[0341] In some embodiments, the antisense strand comprises at least two 2'-fluoro nucleotides at the 3' end of the destabilizing modification, ie, at positions +1 and +2 from the position of the destabilizing modification.

[0342] In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modifications within the sense strand can be present at any position. In some embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 7, 10, and 11 from the 5'-end. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 7, 9, 10, and 11 from the 5'-end. In some embodiments, the sense strand comprises 2'-fluoro nucleotides 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 comprises 2'-fluoro nucleotides 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 comprises two, three, or four blocks of 2'-fluoro nucleotides.

[0343] In some embodiments, the sense strand does not contain a 2'-fluoro nucleotide at a position opposite or complementary to a thermally destabilizing modification of the duplex in the antisense strand.

[0344] In some embodiments, a dsRNA molecule of the disclosure comprises a 21-nucleotide (nt) sense strand and a 23-nucleotide (nt) antisense strand, wherein the antisense strand contains at least one thermally destabilized nucleotide, wherein the at least one thermally destabilized nucleotide occurs within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), one end of the dsRNA is blunt while the other end comprises a 2-nt overhang, and the dsRNA optionally comprises at least one (e.g., one, two, three, four, five, six, or all seven) of the following: Further features include: (i) the antisense strand comprises 2, 3, 4, 5, or 6 2'-fluoro modifications, (ii) the antisense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, (iii) the sense strand is conjugated with a ligand, (iv) the sense strand comprises 2, 3, 4, or 5 2'-fluoro modifications, (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, (vi) the dsRNA comprises at least four 2'-fluoro modifications, and (vii) the dsRNA comprises a blunt end at the 5' end of the antisense strand. Preferably, a 2-nt overhang is at the 3' end of the antisense strand.

[0345] In some embodiments, any nucleotide in the sense strand and antisense strand of dsRNA molecule can be modified.Each nucleotide can be modified with the same or different modifications, and these modifications can include: changing one or both of the non-linked phosphate oxygens, or one or more of the linked phosphate oxygens; changing the ribose sugar component, for example, changing the 2' hydroxyl on the ribose sugar; replacing the phosphate moiety extensively with "dephosphorylation" linker; modifying or replacing naturally occurring bases; and replacing or modifying the ribose phosphate backbone.

[0346] Because nucleic acids are polymers of subunits, many modifications occur at positions that are repeated within nucleic acids, such as modifications of bases or phosphate moieties, or unlinked Os in phosphate moieties.In some cases, modifications occur at all target positions in nucleic acids, but in many cases, they do not occur.For example, modifications can occur only at the 3'-end or 5'-end position, or only in terminal regions, such as at the terminal nucleotide position of the chain or at the last 2, 3, 4, 5, or 10 nucleotides of the chain.Modifications can occur in double-stranded regions, single-stranded regions, or both.Modifications can occur only in the double-stranded region of RNA, or only in the single-stranded region of RNA.For example, phosphorothioate modification at unlinked O positions can occur only at one or both ends, or only in terminal regions, such as at the terminal nucleotide position of the chain or at the last 2, 3, 4, 5, or 10 nucleotides, or in double-stranded and single-stranded regions, especially at the ends. The 5' end can be phosphorylated.

[0347] This may, for example, enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in the single-stranded overhang, for example, in the 5' overhang or 3' overhang, or in both overhangs.For example, it may be desirable to include purine nucleotides in the overhang.In some embodiments, all or part of the bases in the 3' overhang or 5' overhang can be modified, for example, with the modifications described herein.Modifications may include, for example, the use of modifications at the 2' position of the ribose sugar with modifications known in the art, for example, the use of modified deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl, instead of the ribosugar of the nucleobase, and modifications at the phosphate group, for example, phosphorothioate modifications.The overhang does not need to be homologous to the target sequence.

[0348] In some embodiments, each residue of the sense strand and the antisense strand is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, or 2'-fluoro. These strands may contain two or more modifications. In some embodiments, each residue of the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro. It should be understood that these modifications are in addition to at least one thermally destabilizing modification of the duplex present in the antisense strand.

[0349] At least two different modifications are typically present on the sense strand and the antisense strand. These modifications can be 2'-deoxy, 2'-O-methyl, or 2'-fluoro modifications, acyclic nucleotides, etc. In some embodiments, the sense strand and the antisense strand each contain two differently modified nucleotides selected from 2'-O-methyl or 2'-deoxy. In some embodiments, each residue in the sense strand and the antisense strand is independently modified with a 2'-O-methyl nucleotide, a 2'-deoxy nucleotide, a 2'-deoxy-2'-fluoro nucleotide, a 2'-ON-methylacetamide (2'-O-NMA) nucleotide, a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotide, a 2'-O-aminopropyl (2'-O-AP) nucleotide, or a 2'-ara-F nucleotide. Again, it should be understood that these modifications are in addition to at least one thermally destabilizing modification of the duplex present in the antisense strand.

[0350] In some embodiments, the dsRNA molecules of the present disclosure contain an alternating pattern of modifications, particularly in the B1, B2, B3, B1', B2', B3', and B4' regions. The term "alternating motif" or "alternating pattern," as used herein, refers to a motif having one or more modifications, with each modification occurring on alternating nucleotides in a single strand. The alternating nucleotides may refer to one every other nucleotide, one every three nucleotides, or similar patterns. For example, if A, B, and C each represent a type of modification to a nucleotide, the alternating motif may be "ABABABABABAB...," "AABBAABBAABB...," "AABAABAABAAB...," "AAABAAABAAAB...," "AAABBBAAABBB...," or "ABCABCABCABC...," etc.

[0351] The types of modifications contained within an alternating motif can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternation pattern, i.e., the modifications on every other nucleotide, can be the same, but each of the sense or antisense strands can be selected from several possible modifications within the alternating motif, such as "ABABAB...," "ACACAC...," "BDBDBD...," or "CDCDCD...," etc.

[0352] In some embodiments, dsRNA molecules of the present disclosure include an alternating motif modification pattern on the sense strand that is shifted relative to the alternating motif modification pattern on the antisense strand. The shift can be such that modified groups on nucleotides in the sense strand correspond to differently modified groups on nucleotides in the antisense strand, or vice versa. For example, when a sense strand is paired with an antisense strand in a dsRNA duplex, the alternating motif in the sense strand can begin with "ABABAB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand can begin with "BABABA" from the 3'-5' end of the strand in the double-stranded region. As another example, the alternating motif in the sense strand can begin with "AABBAABB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand can begin with "BBAABBAA" from the 3'-5' end of the strand in the double-stranded region, resulting in a complete or partial shift in the modification pattern between the sense and antisense strands.

[0353] The dsRNA molecule of the present disclosure can further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage.Phosphorothioate or methylphosphonate internucleotide linkage modification can occur on the nucleotide of either sense strand or antisense strand or both strands at any position of the strand.For example, internucleotide linkage modification can occur on any nucleotide on sense strand or antisense strand, each internucleotide linkage modification can occur in an alternating pattern on sense strand or antisense strand, or sense strand or antisense strand contains both internucleotide linkage modifications in an alternating pattern.The alternating pattern of internucleotide linkage modification on sense strand can be the same or different from that of antisense strand, and the alternating pattern of internucleotide linkage modification on sense strand can be shifted relative to the alternating pattern of internucleotide linkage modification on antisense strand.

[0354] In some embodiments, the dsRNA molecule comprises phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region.For example, the overhang region comprises two nucleotides, and the two nucleotides comprise two nucleotides with phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides.Internucleotide linkage modification can also be made to link the overhang nucleotide to the terminal paired nucleotide in the double-stranded region.For example, at least 2, 3, 4 or all of the overhang nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there can be an additional phosphorothioate or methylphosphonate internucleotide linkage connecting the overhang nucleotide to the paired nucleotide adjacent to the overhang nucleotide.For example, there can be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, such that two of the three nucleotides are overhang nucleotides and the third is the paired nucleotide adjacent to the overhang nucleotide.Preferably, these terminal three nucleotides can be at the 3'-end of the antisense strand.

[0355] In some embodiments, the sense strand of a dsRNA molecule contains 1 to 10 blocks of two to ten phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the sense strand is paired with an antisense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand containing either phosphorothioate or methylphosphonate or phosphate linkages.

[0356] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0357] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0358] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0359] In some embodiments, the antisense strand of the dsRNA molecule contains two blocks of five phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand containing either phosphorothioate or methylphosphonate or phosphate linkages.

[0360] In some embodiments, the antisense strand of a dsRNA molecule contains two blocks of six phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand containing either phosphorothioate or methylphosphonate or phosphate linkages.

[0361] In some embodiments, the antisense strand of a dsRNA molecule contains two blocks of seven phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, or 8 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand containing either phosphorothioate or methylphosphonate or phosphate linkages.

[0362] In some embodiments, the antisense strand of a dsRNA molecule contains two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, or 6 phosphate internucleotide linkages, one of which is located at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand containing either phosphorothioate or methylphosphonate or phosphate linkages.

[0363] In some embodiments, the antisense strand of a dsRNA molecule contains two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by one, two, three, or four phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand containing either phosphorothioate, methylphosphonate, or phosphate linkages.

[0364] In some embodiments, dsRNA molecules of this disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1 to 10 of the termini of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides can be linked via phosphorothioate or methylphosphonate internucleotide linkages at one or both termini of the sense or antisense strand.

[0365] In some embodiments, dsRNA molecules of the present disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-10 of the interior region of each double strand of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides can be linked via phosphorothioate methylphosphonate internucleotide linkage modifications at positions 8-16 of the double-stranded region, counting from the 5' end of the sense strand, and the dsRNA molecule can optionally further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-10 of that end.

[0366] In some embodiments, dsRNA molecules of this disclosure further comprise one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the sense strand, and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and one to five within positions 18-23 of the antisense strand.

[0367] In some embodiments, dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification within the range of positions 1-5 (counting from the 5' end) and one phosphorothioate or methylphosphonate internucleotide linkage modification within the range of positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate or methylphosphonate internucleotide linkage modifications within the range of positions 18-23 of the antisense strand.

[0368] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18-23 of the antisense strand.

[0369] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1 to 5 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18 to 23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18 to 23 of the antisense strand.

[0370] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1 to 5 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18 to 23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within the range of positions 18 to 23 of the antisense strand (counting from the 5' end).

[0371] In some embodiments, dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification within the range of positions 1 to 5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18 to 23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within the range of positions 18 to 23 of the antisense strand (counting from the 5' end).

[0372] In some embodiments, dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification within the range of positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the antisense strand.

[0373] In some embodiments, dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification within the range of positions 1 to 5 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within the range of positions 18 to 23 (counting from the 5' end) of the antisense strand.

[0374] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end) of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 (counting from the 5' end) of the antisense strand.

[0375] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one within positions 18-23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one within positions 18-23 of the antisense strand (counting from the 5' end).

[0376] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1 to 5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18 to 23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18 to 23 of the antisense strand.

[0377] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18-23 of the antisense strand.

[0378] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one at position 21 (counting from the 5' end) of the antisense strand.

[0379] In some embodiments, dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 (counting from the 5' end) of the antisense strand.

[0380] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0381] In some embodiments, dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 (counting from the 5' end) of the antisense strand.

[0382] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 22 and 23 of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0383] In some embodiments, dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 (counting from the 5' end) of the antisense strand.

[0384] In some embodiments, compounds of the present disclosure comprise a pattern of backbone chiral centers. In some embodiments, the regular pattern of backbone chiral centers comprises at least 5 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 6 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 7 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 8 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 9 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 16 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 17 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 18 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 19 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 8 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 7 or fewer internucleotide linkages in the Rp configuration.In some embodiments, the regular pattern of backbone chiral centers comprises six or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises five or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises four or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises three or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises two or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises one or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises eight or fewer non-chiral internucleotide linkages (phosphodiesters as a non-limiting example). In some embodiments, the regular pattern of backbone chiral centers comprises seven or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises six or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises five or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 4 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 3 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 2 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 1 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration and 8 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration and 7 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration and 6 or fewer non-chiral internucleotide linkages.In some embodiments, the regular pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration and no more than 6 non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration and no more than 5 non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration and no more than 4 non-chiral internucleotide linkages. In some embodiments, the internucleotide linkages in the Sp configuration may be contiguous or non-contiguous. In some embodiments, the internucleotide linkages in the Rp configuration may be contiguous or non-contiguous. In some embodiments, the non-chiral internucleotide linkages may be contiguous or non-contiguous.

[0385] In some embodiments, compounds of the present disclosure include blocks that are stereochemical blocks. In some embodiments, the blocks are Rp blocks, in that each internucleotide linkage of the block is Rp. In some embodiments, the 5'-block is an Rp block. In some embodiments, the 3'-block is an Rp block. In some embodiments, the blocks are Sp blocks, in that each internucleotide linkage of the block is Sp. In some embodiments, the 5'-block is an Sp block. In some embodiments, the 3'-block is an Sp block. In some embodiments, provided oligonucleotides include both Rp and Sp blocks. In some embodiments, provided oligonucleotides include one or more Rp blocks but no Sp blocks. In some embodiments, provided oligonucleotides include one or more Sp blocks but no Rp blocks. In some embodiments, provided oligonucleotides include one or more PO blocks, where each internucleotide linkage is a natural phosphate linkage.

[0386] In some embodiments, compounds of the present disclosure include a 5'-block that is an Sp block in which each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each internucleotide linkage is a modified internucleotide linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each internucleotide linkage is a phosphorothioate linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block comprises four or more nucleoside units. In some embodiments, the 5'-block comprises five or more nucleoside units. In some embodiments, the 5'-block comprises six or more nucleoside units. In some embodiments, the 5'-block comprises seven or more nucleoside units. In some embodiments, the 3'-block is an Sp block in which each sugar moiety comprises a 2'-F modification. In some embodiments, the 3'-block is an Sp block in which each internucleotide linkage is a modified internucleotide linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 3'-block is an Sp block, in which each of the internucleotide linkages is a phosphorothioate linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 3'-block comprises 4 or more nucleoside units. In some embodiments, the 3'-block comprises 5 or more nucleoside units. In some embodiments, the 3'-block comprises 6 or more nucleoside units. In some embodiments, the 3'-block comprises 7 or more nucleoside units.

[0387] In some embodiments, compounds of the disclosure include a region of nucleosides or oligonucleotides followed by a particular type of internucleotide linkage, such as a natural phosphate linkage, a modified internucleotide linkage, an Rp chiral internucleotide linkage, an Sp chiral internucleotide linkage, etc. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by a natural 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 natural 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 natural 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 natural 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.

[0388] In some embodiments, the dsRNA molecule of the present disclosure comprises mismatches in the double strand with the target, or a combination thereof. Mismatches can occur in the overhang region or in the double-stranded region. Base pairs can be ranked based on their tendency to promote dissociation or melting (for example, based on the free energy of association or dissociation of a particular pairing; the simplest approach is to examine each pair individually, but then adjacent or similar analysis can also be used). In terms of promoting dissociation, A:U is more preferable than G:C, G:U is more preferable than G:C, and I:C is more preferable than G:C (I=inosine). Mismatches, such as non-standard pairings or non-standard pairings (described elsewhere herein), are more preferable than standard pairings (A:T, A:U, G:C), and pairings containing universal bases are more preferable than standard pairings.

[0389] In some embodiments, the dsRNA molecules of the present disclosure comprise at least one mismatch pair within the duplex region from the 5' end of the antisense strand, which may be independently selected from the group of A:U, G:U, I:C, and a non-canonical or non-standard or universal base pair, for example, a mismatch pair that promotes dissociation of the antisense strand at the 5' end of the duplex.

[0390] In some embodiments, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first one, two, or three base pairs in the double-stranded region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0391] It has been found that the introduction of a 4'- or 5'-modified nucleotide at the 3' end of a dinucleotide phosphodiester (PO), phosphorothioate (PS), or phosphorodithioate (PS2) linkage at any position in a single- or double-stranded oligonucleotide can exert a steric effect on the internucleotide linkage, thereby protecting or stabilizing it against nucleases.

[0392] In some embodiments, 5'-modified nucleoside is introduced at the 3' end of dinucleotide at any position in single-stranded or double-stranded siRNA.For example, 5'-alkylated nucleoside can be introduced at the 3' end of dinucleotide at any position in single-stranded or double-stranded siRNA.The alkyl group at the 5' position of ribose sugar can be racemic or chirally pure R or S isomer.Exemplary 5'-alkylated nucleoside includes 5'-methyl nucleoside.5'-methyl can be racemic or chirally pure R or S isomer.

[0393] In some embodiments, 4'-modified nucleosides are introduced at the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA. For example, 4'-alkylated nucleosides can be introduced at the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA. The alkyl group at the 4' position of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4'-alkylated nucleoside is 4'-methyl nucleoside. 4'-methyl can be racemic or chirally pure R or S isomer. Alternatively, 4'-O-alkylated nucleosides can be introduced at the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA. The 4'-O-alkyl of the ribose sugar can be racemic or chirally pure R or S isomer. Exemplary 4'-O-alkylated nucleosides include 4'-O-methyl nucleosides, which can be either racemic or chirally pure R or S isomers.

[0394] In some embodiments, 5'-alkylated nucleoside can be introduced at any position of the sense strand or antisense strand of dsRNA, and this modification can maintain or improve the efficacy of dsRNA.5'-alkyl can be either racemic or chiral pure R or S isomer.Exemplary 5'-alkylated nucleoside includes 5'-methyl nucleoside.5'-methyl can be either racemic or chiral pure R or S isomer.

[0395] In some embodiments, 4'-alkylated nucleoside can be introduced at any position of the sense strand or antisense strand of dsRNA, and this modification can maintain or improve the efficacy of dsRNA.4'-alkyl can be either racemic or chiral pure R or S isomer.Exemplary 4'-alkylated nucleoside includes 4'-methyl nucleoside.4'-methyl can be either racemic or chiral pure R or S isomer.

[0396] In some embodiments, 4'-O-alkylated nucleoside can be introduced at any position of the sense strand or antisense strand of dsRNA, and this modification can maintain or improve the efficacy of dsRNA.5'-Alkyl can be either racemic or chiral pure R or S isomer.Exemplary 4'-O-alkylated nucleoside includes 4'-O-methyl nucleoside.4'-O-methyl can be either racemic or chiral pure R or S isomer.

[0397] In some embodiments, the dsRNA molecules of the present disclosure can include 2'-5' linkages (having 2'-H, 2'-OH and 2'-OMe, and also having P=O or P=S). For example, 2'-5' linkage modifications can be used to promote nuclease resistance, or to inhibit binding of the sense strand to the antisense strand, or can be used at the 5' end of the sense strand to prevent activation of the sense strand by RISC.

[0398] In another embodiment, the dsRNA molecule of the present disclosure can comprise L sugar (for example, L-ribose, L-arabinose, with 2'-H, 2'-OH and 2'-OMe).For example, these L sugar modifications can be used to promote nuclease resistance, or to inhibit the binding of sense strand to antisense strand, or can be used at the 5' end of sense strand to prevent the activation of sense strand by RISC.

[0399] Multimeric siRNA has been described in various publications, and all of them can be used with the dsRNA of the present disclosure.These publications include WO2007 / 091269, US Patent No. 7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520, and each of them is incorporated herein by reference in its entirety.

[0400] In some embodiments, dsRNA molecules of the present disclosure are 5' phosphorylated or contain a phosphoryl analog at the 5' prime end. 5'-phosphate modifications include modifications compatible with RISC-mediated gene silencing. Suitable modifications include the following: 5'-monophosphate ((HO)2(O)PO-5'), 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'), 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'), 5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'). ), 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'), 5'-monothiophosphate (phosphorothioate; (HO)2(S)PO-5'), 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)PO-5') , 5'-phosphorothiolate ((HO)2(O)PS-5'), any additional combination of oxygen / sulfur substituted monophosphates, diphosphates and triphosphates (e.g., 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'), 5'-alkylphosphonates (R = alkyl = methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)-O-5'-, 5'-alkenylphosphonates (i.e., vinyl, substituted vinyl), (OH)2(O)P-5'-CH2-), 5'-alkyl ether phosphonates (R = alkyl ether = methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5'-). In one example, the modification may be placed on the antisense strand of the dsRNA molecule.

[0401] F. Linker In some embodiments, the conjugates or ligands described herein can be attached to the iRNA oligonucleotide using a variety of linkers, which may or may not be cleavable.

[0402] Typically, a linker is a direct bond or an atom such as oxygen or sulfur, a unit such as NR, C(O), C(O)NH, SO, SO, SONH, or a chain of atoms, including, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylhetero ...aryl Alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylhetero These include cyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, etc., where one or more methylenes can be interrupted or terminated by O, S, S(O), SO, N(R), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, where R is hydrogen, acyl, aliphatic, or substituted aliphatic.In some embodiments, the linker is about 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.

[0403] In some embodiments, the dsRNA of the present disclosure is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formulas (XXXI)-(XXXIV): [ka] and During the ceremony, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C each independently represent 0 to 20, and the repeat 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 is, for each occurrence, independently absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CHO; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C is independently at each occurrence absent, alkylene, or substituted alkylene, wherein one or more methylenes are selected from O, S, S(O), SO, N(R N), C(R')=C(R''), C≡C or C(O), R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C are each independently absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocyclyl, L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B , and L 5C represents a ligand, i.e., each occurrence is independently a monosaccharide (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide, and R a is H or an amino acid side chain. Trivalent conjugated GalNAc derivatives are particularly useful for use with RNAi agents that inhibit expression of target genes, such as those represented by the following formula (XXXV): [ka] , and the like, wherein L 5A , L 5B , and L 5C represents a monosaccharide, such as a GalNAc derivative.

[0404] Examples of bivalent and trivalent branched linker groups for conjugating GalNAc derivatives include, but are not limited to, the structures listed above as Formulas II, VII, XI, X, and XIII.

[0405] A cleavable linking group is one that is sufficiently stable outside a cell, but that, once inside a target cell, is cleaved to release the two moieties held together by the linker. In some embodiments, the cleavable linking group is cleaved at a rate that is at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or more, or at least about 100-fold faster in the target cell or under first reference conditions (which may, for example, be selected to mimic or represent intracellular conditions) than in the subject's blood or under second reference conditions (which may, for example, be selected to mimic or represent conditions found in blood or serum).

[0406] Cleavable linking groups are susceptible to cleaving agents, such as pH, redox potential, or the presence of degradable molecules. Cleavage agents are generally more widespread or found at higher levels or activity inside cells than in serum or blood. Examples of such degrading agents include redox agents that are selective for a specific substrate or have no substrate specificity, e.g., oxidases or reductases or reducing agents present in cells, such as mercaptans, that can degrade redox-cleavable linking groups by reduction; esterases; reagents that can create an endosomal or acidic environment, e.g., reagents that result in a pH of 5 or less; and enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0407] Cleavable linking groups, such as disulfide bonds, can be pH-sensitive. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers have cleavable linking groups that are cleaved at the appropriate pH, thereby releasing the cationic lipid from the ligand inside the cell or into the desired compartment of the cell.

[0408] The linker may include a cleavable linking group that is cleavable by a specific enzyme. The type of cleavable linking group incorporated into the linker may depend on the cell to be targeted.

[0409] Generally, the suitability of a candidate cleavable linking group can be evaluated by testing the ability (or conditions) of a degrading agent to cleave the candidate linking group. It may also be desirable to test the candidate cleavable linking group for its ability to resist cleavage in blood or when in contact with other non-target tissues. Thus, the relative susceptibility to cleavage between first and second conditions can be determined, where the first condition is selected to exhibit cleavage in target cells, and the second condition is selected to exhibit cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to perform initial evaluation in cell-free or culture conditions and confirm by further evaluation in whole animals. In some embodiments, useful candidate compounds are cleaved at a rate that is 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).

[0410] i. Redox-cleavable linking group In some 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 is suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, one can refer to the methods described herein. For example, candidates can be evaluated in cells by incubating with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate that would be observed in target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In some conditions, the candidate compound is cleaved at a maximum of about 10% in blood. In other embodiments, useful candidate compounds are degraded at a rate 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 the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic intracellular media and compared to conditions selected to mimic extracellular media.

[0411] ii. Phosphate-based cleavable linkers In some embodiments, the cleavable linker comprises a phosphate-based cleavable linking group. The phosphate-based cleavable linking group is cleaved by a reagent that decomposes or hydrolyzes the phosphate group. An example of a reagent that cleaves the phosphate group in a cell is an enzyme such as a phosphatase in the cell. Examples of phosphate-based linking groups include -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. In some embodiments, phosphate-based linking groups 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 some embodiments, the phosphate-based linking group is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0412] iii. Acid-cleavable linking group In some embodiments, the cleavable linker comprises an acid-cleavable linking group. An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In some embodiments, the acid-cleavable linking group is cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0 or less) or by an agent, such as an enzyme, that can act as a general acid. In cells, certain low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid-cleavable linking group. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). In some embodiments, the carbon attached 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.

[0413] iv. Ester-based cleavable linking groups In some embodiments, the cleavable linker comprises an ester-based cleavable linking group. Ester-based cleavable linking groups are cleaved in cells by enzymes such as esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0414] v. Peptide-based cleavable linking groups In some embodiments, the cleavable linker comprises a peptide-based cleavable linking group. Peptide-based cleavable linking groups are cleaved in cells by enzymes such as peptidases and proteases. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (—C(O)NH—). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. Peptide-based cleaving groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to yield peptides and proteins, but do not include all amide functional groups. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where R and R are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.Representative United States patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979, 4,948,882, 5,218,105, 5,525,465, 5,541,313, 5,545,730, 5,552,538, 5,578,717, 5,580,731, 5,591,584, 5,109,124, 5,118,802, 5,138,045, 5,414,077, No. 5,486,603, No. 5,512,439, No. 5,578,718, No. 5,608,046, No. 4,587,044, No. 4,605,735, No. 4,667,025, No. 4,762,779, No. 4,789,737, No. 4,824,941, No. 4,835,263, No. 4,876,335, No. 4,904,582, No. 4,958,013, No. 5,082,830, No. 5,112,963, No. 5,214,136, No. 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. 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 , 5,574,142, 5,585,481, 5,587,371, 5,595,726, 5,597,696, 5,599,923, 5,599,928, and 5,688,941, 6,294,664, 6,320,017, 6,576,752, 6,783,931, 6,900,297, 7,037,646, and 8,106,022, the entire contents of each of which are incorporated herein by reference.

[0415] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the foregoing modifications may be incorporated in a single compound, or even at a single nucleoside within an iRNA. The present disclosure also includes iRNA compounds that are chimeric compounds.

[0416] A "chimeric" iRNA compound or "chimera," in the context of this disclosure, is an iRNA compound, e.g., a dsRNAi agent, that contains two or more chemically distinct regions, each of which is 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 on the iRNA increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity for the target nucleic acid. Additional regions of the iRNA may serve as substrates for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNA when using chimeric dsRNA compared to the phosphorothioate deoxydsRNA hybridized to the same target region.Cleavage of RNA target can be routinely detected by gel electrophoresis, and if necessary, by related nucleic acid hybridization techniques known in the art.

[0417] In certain instances, the RNA of an iRNA can be modified with a non-ligand group. Several non-ligand molecules have been conjugated to iRNAs to enhance their activity, cellular distribution, or cellular uptake, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties include lipid moieties such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), fatty 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), polyamines or polyethylene glycol chains (Manoharan et al. al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (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 bearing an amino linker at one or more positions in the sequence. The amino group is then reacted with the molecule being conjugated using an appropriate coupling or activation reagent. The conjugation reaction can be carried out while the RNA is still attached to the solid support or after cleavage of the RNA in solution phase. Purification of the RNA conjugate by HPLC typically yields a pure conjugate.

[0418] V. iRNA Delivery Delivery of iRNA to a subject in need thereof can be achieved in many different ways. In vivo delivery can be achieved directly by administering a composition containing iRNA, such as dsRNA, to a subject. Alternatively, delivery can be achieved indirectly by administering one or more vectors that encode and induce the expression of iRNA. These options are further described below.

[0419] A. Direct delivery Generally, any method for delivering nucleic acid molecules can be adapted for use with iRNA (see, e.g., Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and WO 94 / 02595, which are incorporated herein by reference in their entireties). However, three important factors to consider for successful in vivo delivery of iRNA molecules include (a) the biological stability of the delivered molecule, (2) prevention of nonspecific effects, and (3) accumulation of the delivered molecule in the target tissue. Nonspecific effects of iRNA can be minimized by local administration, such as direct injection or implantation into a tissue (e.g., the eye) or by local administration of a preparation. Local administration to the treatment site maximizes the local concentration of the agent, limits exposure to systemic tissues that may otherwise be harmed by or degrade the agent, and allows for a smaller total dosage of the iRNA molecule to be administered. Several studies have demonstrated the successful knockdown of gene products when iRNAs are administered locally. For example, intraocular delivery of VEGF dsRNA via intravitreal 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) both prevented neovascularization in experimental models of age-related macular degeneration. In addition, direct intratumoral injection of dsRNA in mice can reduce tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and prolong the survival of tumor-bearing mice (Kim, WJ., et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523).RNA interference has been shown to be successful when delivered locally to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, P.H., et al. (2005) Gene Ther. 12:59-66; Makimura, H., et al. (2002) BMC Neurosci. 3:18; Shishkina, G.T., et al. (2004) Neuroscience 129:521-528; Thakker, E.R., 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, K.A., 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). When administering iRNA systemically to treat disease, the RNA can be modified or, alternatively, delivered using a drug delivery system, either of which functions to prevent rapid degradation of dsRNA by endonucleases and exonucleases in vivo.

[0420] The modification of RNA or pharmaceutical carrier can also allow iRNA composition to target tissue and avoid undesirable off-target effects.iRNA molecule can be modified by, for example, chemical conjugation to lipid or hydrocarbon group as described herein.Using such conjugate, iRNA can be targeted to specific cells, for example, liver cells such as hepatocytes.For example, GalNAc conjugate or lipid (e.g., LNP) formulation can be used to target iRNA to specific cells, for example, liver cells such as hepatocytes.

[0421] iRNA molecules can also be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, iRNA directed to ApoB conjugated to a lipophilic cholesterol moiety was systemically injected into mice, resulting in apoB mRNA knockdown in both the liver and jejunum (Soutschek, J., et al. (2004) Nature 432:173-178). Conjugation of iRNA to aptamers has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, JO., et al. (2006) Nat. Biotechnol. 24:1005-1015). In alternative embodiments, iRNA can be delivered using drug delivery systems, such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems facilitate the binding of iRNA molecules (which are negatively charged) and also enhance their interaction with the negatively charged cell membrane, thereby enabling efficient uptake of iRNA by cells. Cationic lipids, dendrimers, or polymers can bind to iRNA or be induced to form vesicles or micelles that encapsulate iRNA (see, for example, Kim SH., et al. (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles also prevents degradation of iRNA upon systemic administration. Methods for making and administering cationic iRNA complexes are well within the capabilities of those skilled in the art (see, e.g., 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 by reference in their entireties).Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA 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), polyethyleneimine (Bonnet ME, et al., (2008) Pharm. Res. Aug 16 Epub ahead of (print; Aigner, A. (2006) J. Biomed. Biotechnol. 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, iRNAs are complexed with cyclodextrins for systemic administration. Methods of administration and pharmaceutical compositions of iRNAs and cyclodextrins are found in U.S. Patent No. 7,427,605, which is incorporated herein by reference in its entirety.

[0422] B. Vector-encoded iRNA In another embodiment, iRNAs targeting MYOC can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International Publication No. 00 / 22113; Conrad, International Publication No. 00 / 22114; and Conrad, U.S. Patent No. 6,054,299). Expression can be transient (hours to weeks) or persistent (weeks to months or longer), depending on the specific construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrative or non-integrative. Transgenes can also be constructed to allow them to be passaged as extrachromosomal plasmids (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0423] Each strand of iRNA can be transcribed from a promoter on an expression vector. When two separate strands are expressed to produce, for example, dsRNA, two separate expression vectors can be simultaneously introduced into target cells (for example, by transfection or infection). Alternatively, each separate strand of dsRNA can be transcribed by a promoter located on the same expression plasmid. In some embodiments, dsRNA is expressed as an inverted repeat linked by a linker polynucleotide sequence, so that the dsRNA has a stem-and-loop structure.

[0424] iRNA expression vectors are typically DNA plasmids or viral vectors. Recombinant constructs for expressing the iRNAs described herein can be produced using expression vectors compatible with eukaryotic cells, such as expression vectors compatible with vertebrate cells. Eukaryotic cell expression vectors are well known in the art and are available from numerous commercial sources. Typically, such vectors contain convenient restriction sites for the insertion of desired nucleic acid segments. iRNA expression vectors can be delivered systemically, for example, by intravenous or intramuscular administration, by administration to target cells removed from a patient and then reintroduced into the patient, or by any other means that allows for introduction into desired target cells.

[0425] iRNA expression plasmids are delivered in cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO TM The vector can be transfected into target cells as a complex with iRNA. Multiple lipid transfections for iRNA-mediated knockdown targeting different regions of a target RNA over a period of a week or more are also contemplated by the present disclosure. Successful introduction of the vector into host cells can be monitored using various known methods. For example, transient transfection can be signaled using a reporter, such as a fluorescent marker like green fluorescent protein (GFP). Stable transfection of cells in vitro can be ensured using a marker that provides transfected cells with resistance to certain environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

[0426] Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to, (a) adenoviral vectors, (b) retroviral vectors, such as, but not limited to, lentiviral vectors and Moloney murine leukemia virus, (c) adeno-associated viral vectors, (d) herpes simplex viral vectors, (e) SV40 vectors, (f) polyoma viral vectors, (g) papilloma viral vectors, (h) picorna viral vectors, (i) pox viral vectors, such as orthopox, e.g., vaccinia viral vectors, or avian pox, e.g., canarypox or fowlpox, and (j) helper-dependent or gutless adenoviruses. Replication-deficient viruses may also be advantageous. Different vectors may or may not become integrated into the cellular genome. The construct can include viral sequences for transfection, if desired. Alternatively, the constructs can be incorporated into vectors capable of episomal replication, such as EPV and EBV vectors. Constructs for recombinant expression of iRNAs will generally require regulatory elements, such as promoters, enhancers, etc., to ensure expression of the iRNA in target cells. Other embodiments for considering vectors and constructs are further described below.

[0427] Vectors useful for delivery of iRNA contain sufficient regulatory elements (promoters, enhancers, etc.) for expression of the iRNA in the desired target cells or tissues. Regulatory elements can be selected to provide for either constitutive or regulated / inducible expression.

[0428] iRNA expression can be precisely regulated, for example, by using inducible regulatory sequences that are sensitive to specific physiological regulators, such as circulating glucose levels or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Suitable inducible expression systems for controlling dsRNA expression in cells or mammals include, for example, regulation by ecdysone, estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-β-D1-thiogalactopyranoside (IPTG). Those skilled in the art will be able to select appropriate regulatory / promoter sequences based on the intended use of the iRNA transgene.

[0429] In certain embodiments, viral vectors containing nucleic acid sequences encoding iRNAs can be used. For example, retroviral vectors can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for correct packaging of the viral genome and integration into host cell DNA. The nucleic acid sequences encoding iRNAs are cloned into one or more vectors, which facilitates delivery of the nucleic acid to the patient. More details about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells to make them more resistant to chemotherapy. Other references demonstrating the use of retroviral vectors in gene therapy include: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, HIV-based vectors described in U.S. Patent Nos. 6,143,520, 5,665,557, and 5,981,276, which are incorporated herein by reference.

[0430] Adenoviruses are also contemplated for use in delivering iRNA. Adenoviruses are particularly attractive vehicles for delivering genes to, for example, respiratory epithelia. Adenoviruses naturally infect respiratory epithelia, causing mild disease. Other targets of adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscles. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993), provide a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994), demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other examples of the use of adenovirus in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin Invest. 91:225-234 (1993); WO 94 / 12649; and Wang, et al., Gene Therapy 2:775-783 (1995). Suitable AV vectors for expressing the iRNAs featured in this disclosure, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described in Xia H et al. (2002), Nat. Biotech. 20:1006-1010.

[0431] The use of adeno-associated virus (AAV) vectors is also contemplated (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No. 5,436,146). In some embodiments, the iRNA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either a U6 or H1 RNA promoter, or a cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in this disclosure, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described in Samulski R et al. (1987), J.Virol. 61:3096-3101, Fisher KJ et al. (1996), J.Virol., 70:520-532, Samulski R et al. (1989), J.Virol. 63:3822-3826, U.S. Patent No. 5,252,479, U.S. Patent No. 5,139,941, WO 94 / 13788, and WO 93 / 24641, the entire disclosures of which are incorporated herein by reference.

[0432] Another exemplary viral vector is a poxvirus such as vaccinia virus, eg, an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowlpox or canarypox.

[0433] The tropism of viral vectors can be modified by pseudotyping the vector with envelope proteins or surface antigens from other viruses, or by replacing different viral capsid proteins as needed.For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, etc.AAV vectors can be engineered to target different cells by engineering the vector to express different capsid protein serotypes; see, for example, Rabinowitz JE et al. (2002), J Virol 76:791-801, the entire disclosure of which is incorporated herein by reference.

[0434] The pharmaceutical preparation of the vector can include the vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, and the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0435] VI. Pharmaceutical compositions containing iRNA In some embodiments, the present disclosure provides a pharmaceutical composition containing an iRNA as described herein and a pharmaceutically acceptable carrier. The iRNA-containing pharmaceutical composition is useful for treating a disease or disorder associated with the expression or activity of MYOC (e.g., glaucoma, e.g., primary open-angle glaucoma (POAG)). Such pharmaceutical compositions are formulated based on the mode of delivery. In some embodiments, the composition can be formulated for local delivery, e.g., intraocular delivery (e.g., intravitreal administration, e.g., intravitreal injection; transscleral administration, e.g., intrascleral injection; subconjunctival administration, e.g., subconjunctival injection; retrobulbar administration, e.g., retrobulbar injection; intracameral administration, e.g., intracameral injection; or subretinal administration, e.g., subretinal injection). In other embodiments, the composition can be formulated for local delivery. In another example, the composition can be formulated for systemic administration via parenteral delivery, e.g., via intravenous (IV) delivery. In some embodiments, the compositions provided herein (eg, compositions comprising GalNAc conjugates or LNP formulations) are formulated for intravenous delivery.

[0436] The pharmaceutical compositions featured herein are administered at a dosage sufficient to inhibit MYOC expression. Generally, suitable doses of iRNA range from 0.01 to 200.0 milligrams per kilogram of recipient body weight per day. The pharmaceutical composition may be administered once daily, or the iRNA may be administered as two, three, or more subdoses at appropriate intervals throughout the day, or using continuous infusion or delivery via sustained-release formulations. In such cases, the amount of iRNA contained in each subdose must be correspondingly smaller to achieve the total daily dose. Dosage units may also be formulated for delivery over several days, for example, using conventional sustained-release formulations that provide sustained release of iRNA over several days. Sustained-release formulations are well known in the art and are particularly useful for delivering agents at specific sites, such as those that can be used in conjunction with the agents disclosed herein. In this embodiment, the dosage unit contains a multiple of the corresponding daily dose.

[0437] The effect of a single dose on MYOC levels may be sustained for an extended period of time, such that subsequent doses are administered no more than 3, 4, or 5 days apart, or no more than 1, 2, 3, 4, 12, 24, or 36 weeks apart.

[0438] Those skilled in the art will understand that certain factors, including but not limited to, the severity of the disease or disorder, previous treatments, the subject's general health and / or age, and other existing diseases, can affect the dosage and timing required to effectively treat a subject. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimation of the effective dosage and in vivo half-life of the individual iRNAs encompassed by this disclosure can be performed using conventional methodology or based on in vivo testing using suitable animal models.

[0439] Suitable animal models, such as mice or cynomolgus monkeys, e.g., animals containing a transgene expressing human MYOC, can be used to determine a therapeutically effective dose and / or an effective dosing regimen of MYOC siRNA.

[0440] The present disclosure also includes pharmaceutical compositions and formulations containing the iRNA compounds featured herein. The pharmaceutical compositions of the present disclosure can be administered in several ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration can be topical (e.g., by intraocular injection), localized (e.g., by eye drops), or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, for example, subcutaneous administration using an implant device, or intracranial administration, for example, by intraparenchymal, intrathecal, or intracerebroventricular administration.

[0441] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, solutions, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may also be necessary or desirable. Coated condoms, gloves, and the like may also be useful. Suitable topical formulations include those in which the iRNAs featured in this disclosure are mixed with topical delivery agents, such as lipids, liposomes, lipid acids, lipid acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearolyphosphatidylcholine), negative (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The iRNAs featured in this disclosure may be encapsulated within liposomes or complexed to liposomes, particularly cationic liposomes. Alternatively, the iRNAs may be complexed with lipids, particularly cationic lipids. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicapric acid, tricapric acid, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C 1-20 Examples of suitable topical formulations include alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Topical formulations are described in U.S. Patent No. 6,747,014, which is incorporated herein by reference.

[0442] A. Liposomal Formulations In addition to microemulsions, many organized surfactant structures have been studied and used in drug formulations. These include monolayers, micelles, bilayers, and vesicles. Vesicles such as liposomes have attracted significant interest due to their specificity and the duration of action they offer in terms of drug delivery. As used in this disclosure, the term "liposome" refers to a vesicle composed of a spherical bilayer or amphiphilic lipids arranged in a bilayer.

[0443] Liposomes are unilamellar or multilamellar vesicles with a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes have the advantage of being able to fuse with the cell wall. Non-cationic liposomes cannot fuse efficiently with the cell wall, but are taken up by macrophages in vivo.

[0444] To pass through intact mammalian skin, lipid vesicles must pass through a series of micropores, each with a diameter of less than 50 nm, under the influence of a suitable transdermal gradient. It is therefore desirable to use liposomes that are highly deformable and can pass through these micropores.

[0445] Additional advantages of liposomes include that liposomes derived from natural phospholipids are biocompatible and biodegradable, liposomes can incorporate a wide range of water- and lipid-soluble drugs, and liposomes can protect the encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size, and liposome water content.

[0446] Liposomes are useful for transporting and delivering active ingredients to the site of action.Because liposome membrane is structurally similar to biological membrane, when liposome is applied to tissue, liposome begins to fuse with cell membrane, and as the fusion of liposome and cell progresses, liposome contents are discharged into the cell where the active agent can act.

[0447] Liposomal formulations have been the focus of extensive research as a delivery mode for many drugs. For topical administration, there is increasing evidence that liposomes offer several advantages over other formulations. These advantages include reduced side effects associated with high systemic absorption of administered drugs, increased accumulation of administered drugs at desired targets, and the ability to deliver a wide variety of drugs to the skin in both hydrophilic and hydrophobic ways.

[0448] Several reports have detailed the ability of liposomes to deliver drugs, including high-molecular-weight DNA, to the skin. Painkillers, antibodies, hormones, and compounds containing high-molecular-weight DNA have been administered to the skin. The majority of applications have resulted in targeting of the upper epidermis. Liposomes are classified into two major types. Cationic liposomes are positively charged liposomes that interact with negatively charged DNA molecules to form stable complexes. The positively charged DNA / liposome complexes bind to the negatively charged cell surface and are internalized into endosomes. Due to the acidic pH within the endosome, the liposomes rupture, releasing their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0449] Liposomes that are pH-sensitive or negatively charged trap DNA rather than complexing with it. Because both nucleic acids and lipids have similar charges, repulsion occurs rather than complex formation. Nevertheless, some DNA is trapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. The expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0450] One major type of liposome composition contains phospholipids other than naturally occurring phosphatidylcholine. Neutral liposome compositions can be formed, for example, from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are primarily formed from dioleoylphosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0451] Several studies have evaluated topical delivery of liposomal drug formulations to the skin. Application of interferon-containing liposomes to guinea pig skin resulted in a reduction in cutaneous herpes pain, while delivery of interferon via other means (e.g., as a solution or emulsion) was ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Furthermore, additional studies have tested the effectiveness of interferon administered as part of a liposomal formulation compared with administration of interferon using an aqueous system and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

[0452] Nonionic liposome systems, especially those containing nonionic surfactants and cholesterol, have also been tested to determine their usefulness in delivering drugs to the skin.Novasome™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) nonionic liposome formulations were used to deliver cyclosporine A into the dermis of mouse skin.As a result, these nonionic liposome systems were shown to be effective in promoting the accumulation of cyclosporine A in different layers of the skin (Hu et al. STP Pharma. Sci., 1994, 4, 6, 466).

[0453] Liposomes also include "sterically stabilized" liposomes, which term, as used herein, refers to liposomes that contain one or more specialized lipids that, when incorporated into the liposome, result in improved circulation longevity compared to liposomes lacking the specialized lipid. Examples of sterically stabilized liposomes include liposomes in which the vesicle-forming lipid portion of the liposome is (A) e.g., monosialoganglioside G M1or (B) liposomes that are derivatized with one or more hydrophilic polymers, such as, for example, polyethylene glycol (PEG) moieties. Without wishing to be bound by any particular theory, it is believed in the art that, at least in the case of sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes results from reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0454] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., 1987, 507, 64) describe monosialoganglioside G M1 reported the ability of galactocerebroside sulfate, galactocerebroside sulfate, and phosphatidylinositol to enhance the blood half-life of liposomes. These findings were commented upon by Gabizon et al. (Proc. Natl. Acad. Sci. USA, (1988), 85, :6949). Allen et al., in both U.S. Pat. No. 4,837,028 and WO 88 / 04924, reported the ability of (1) sphingomyelin and (2) ganglioside G to enhance the blood half-life of liposomes. M1 Liposomes containing galactocerebroside sulfate or galactocerebroside sulfate have been disclosed. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimyristoylphosphatidylcholine have been disclosed in WO 97 / 13499 (Lim et al.).

[0455] Many liposomes containing lipids derivatized with one or more hydrophilic polymers, and methods for their preparation, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) describe the use of 2C, a non-ionic detergent containing a PEG moiety. 1215Ghave described liposomes containing PEG- or PEG-stearate-derivatized phosphatidylethanolamine (PE). Illum et al. (FEBS Lett., 1984, 167, 79) found that hydrophilic coating of polystyrene particles with polymeric glycols significantly extended their blood half-life. Synthetic phospholipids modified by the attachment of carboxylic acid groups of polyalkylene glycols (e.g., PEG) were described by Sears (see U.S. Patents Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes containing phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate had significantly increased blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended these observations to other PEG-derivatized phospholipids, such as DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes with covalently attached PEG moieties on their exterior surfaces are described in Fisher's European Patent No. EP 0 445 131(B1) and International Publication No. WO 90 / 04384. Liposome compositions containing 1 to 20 mole percent PE derivatized with PEG, and methods for their use, are described in Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. 0 496 813(B1)). Liposomes containing several other lipid-polymer conjugates are described in WO 91 / 05545 and U.S. Pat. No. 5,225,212 (both Martin et al.) and WO 94 / 20073 (Zalipsky et al.). Liposomes containing PEG-modified ceramide lipids are described in WO 96 / 10391 (Choi et al.).US Pat. No. 5,540,935 (Makiaki et al.) and US Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

[0456] Numerous liposomes containing nucleic acids are known in the art. Thierry et al., WO 96 / 40062, discloses a method for encapsulating high molecular weight nucleic acids in liposomes. Tagawa et al., U.S. Patent No. 5,264,221, discloses protein-bound liposomes, and claims that the contents of such liposomes can contain dsRNA. Rahman et al., U.S. Patent No. 5,665,710, describes a specific method for encapsulating oligodeoxynucleotides in liposomes. Love et al., WO 97 / 04787, discloses liposomes containing dsRNA that target the raf gene.

[0457] Transfersomes are yet another type of liposome, highly deformable lipid aggregates that are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets that are so highly deformable that they can easily pass through pores smaller than liquid droplets. Transfersomes are adaptable to the environment in which they are used; for example, they self-optimize (adapt to the shape of pores in the skin), self-repair, frequently reach their targets without fragmentation, and are often self-loading. To create transfersomes, surface edge activators, usually surfactants, can be added to standard liposome compositions. Transfersomes have been used to deliver serum albumin to the skin. Transfersome-mediated delivery of serum albumin has been found to be as effective as subcutaneous injection of a solution containing serum albumin.

[0458] Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common method for classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by using the hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for classifying the various surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0459] If the surfactant molecules are not ionized, they are classified as nonionic surfactants. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and can be used over a wide range of pH values. Generally, their HLB values ​​range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers, are also included in this class. Polyoxyethylene surfactants are the most common members of the nonionic surfactant class.

[0460] When the surfactant molecule holds a negative charge when dissolved or dispersed in water, the surfactant is classified as anionic.Anionic surfactants include carboxylic acid esters, such as soaps, acyl lactates, acyl amides of amino acids, sulfuric acid esters, such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates, such as alkyl benzene sulfonates, acyl isethionates, acyltaurates, sulfosuccinates, and phosphates.The most important members of the anionic surfactant class are alkyl sulfates and soaps.

[0461] If the surfactant molecule carries a positive charge when dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used members of this class.

[0462] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phosphatides.

[0463] The use of surfactants in drugs, formulations, and emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0464] B. Nucleic acid lipid particles In some embodiments, the MYOC dsRNA featured in this disclosure is fully encapsulated in a lipid formulation, forming, for example, SPLP, pSPLP, SNALP, or other nucleic acid-lipid particles. SNALP and SPLP typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). SNALP and SPLP exhibit extended circulatory life after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically distant from the administration site), making them highly useful for systemic administration. SPLPs include "pSPLP," which contain encapsulated condensing agent-nucleic acid complexes such as those described in WO 00 / 03683. Particles of this disclosure typically have an average particle size of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, when nucleic acid is present in the nucleic acid-lipid particles of the present disclosure, it is resistant to degradation by nucleases in aqueous solution.Nucleic acid-lipid particles and their preparation methods are disclosed in, for example, U.S. Patent Nos. 5,976,567, 5,981,501, 6,534,484, 6,586,410, 6,815,432 and WO 96 / 40964.

[0465] In some embodiments, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to dsRNA ratio) will range from about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1.

[0466] Cationic lipids include, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLinDMA), and the like. -C-DAP), 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-Dilinoleic acid-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanediol (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K) -DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or mixtures thereof. The cationic lipid may comprise from about 20 mol % to about 50 mol %, or about 40 mol % of the total lipid present in the particle.

[0467] In some embodiments, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. The synthesis of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. Provisional Patent Application No. 61 / 107,998, filed October 23, 2008, which is incorporated herein by reference.

[0468] In some embodiments, the lipid-siRNA particles comprise 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane:10% DSPC:40% cholesterol:10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA / lipid ratio.

[0469] Non-cationic lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4-(N- The lipid may be an anionic or neutral lipid, including, but not limited to, maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid may represent about 5 mol% to about 90 mol%, about 10 mol%, or, if cholesterol is included, about 58 mol% of the total lipid present in the particle.

[0470] The conjugated lipid that inhibits particle aggregation may be, for example, a polyethylene glycol (PEG) lipid, including, but not limited to, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (C]8). The conjugated lipid that prevents particle aggregation may be 0 mol% to about 20 mol%, or about 2 mol% of the total lipid present in the particle.

[0471] In some embodiments, the nucleic acid-lipid particle further comprises cholesterol, for example, from about 10 mol % to about 60 mol %, or about 48 mol % of the total lipid present in the particle. In some embodiments, the iRNA is formulated in a lipid nanoparticle (LNP).

[0472] LNP01 In some embodiments, lipid-dsRNA nanoparticles (e.g., LNP01 particles) can be prepared using lipidoid ND98-4HCl (MW 1487) (see U.S. Patent Application No. 12 / 056,230, filed March 26, 2008, incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar Lipids). Stock solutions of each can be prepared in ethanol as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml; PEG-ceramide C16, 100 mg / ml. The ND98, cholesterol, and PEG-ceramide C16 stock solutions can then be combined in a molar ratio of, for example, 42:48:10. The combined lipid solution can be mixed with an aqueous dsRNA solution (e.g., sodium acetate, pH 5) so that the final ethanol concentration is approximately 35-45% and the final sodium acetate concentration is approximately 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously during mixing. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cutoff) using, for example, a thermobarrel extruder such as Lip Extruder (Northern Lipids, Inc.). In some cases, the extrusion step can be omitted. Buffer exchange simultaneously with ethanol removal can be achieved, for example, by dialysis or tangential flow filtration. The buffer can be exchanged with phosphate buffered saline (PBS), for example, at about pH 7, for example, about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

[0473] [ka]

[0474] LNP01 formulations are described, for example, in International Application Publication No. WO2008 / 042973, which is incorporated herein by reference.

[0475] Further exemplary lipid-dsRNA formulations are provided in the table below. Table 4: Exemplary lipid formulations [Table 1] [Table 2] DSPC: Distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholine PEG-DMG: PEG-dimyristoylglycerol (C14-PEG, or PEG-C14) (PEG with an average molecular weight of 2000) PEG-DSG: PEG-distyrylglycerol (C18-PE...

Claims

**Claim 1** A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of myosin (MYOC), wherein the dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, and the antisense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides having one of the antisense sequences listed in SEQ ID NO: 1889, 2093, 1794, 1885, 557, 761, 462, 553, or any one of Tables 2A and 2B with 0, 1, 2, or 3 mismatches, and the sense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides having 0, 1, 2, or 3 mismatches with the sense sequence listed in SEQ ID NO: 1445, 1649, 1350, 1441, 113, 317, 18, 109, or any one of Tables 2A and 2B corresponding to the antisense sequence, dsRNA agent. **Claim 2** (a) The antisense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides having 0, 1, 2, or 3 mismatches with the antisense strand nucleotide sequence of double-stranded AD-1565804, and the sense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides having 0, 1, 2, or 3 mismatches with the sense strand nucleotide sequence of double-stranded AD-1565804, (b) The antisense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides having 0, 1, 2, or 3 mismatches with the antisense strand nucleotide sequence of double-stranded AD-1565837, and the sense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides having 0, 1, 2, or 3 mismatches with the sense strand nucleotide sequence of double-stranded AD-1565837; (c) The sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1445 and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1889; (d) The sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1649 and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 2093; (e) The sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 113 and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 557; or (f) The sense strand contains the nucleotide sequence set forth in SEQ ID NO: 317, and the antisense strand contains the nucleotide sequence set forth in SEQ ID NO: 761, The dsRNA agent according to claim 1. **Claim 3** The dsRNA agent according to claim 1, wherein at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties. **Claim 4** The dsRNA agent according to claim 2, wherein at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties. **Claim 5** (a) One or more lipophilic moieties are conjugated via a linker or a carrier; (b) One or more lipophilic moieties are conjugated to one or more inner positions on at least one strand; (c) One or more lipophilic moieties are an aliphatic compound, an alicyclic compound, or a polycyclic compound; (d) One or more lipophilic moieties contain a saturated or unsaturated C16 hydrocarbon chain; (e) One or more lipophilic moieties are conjugated via a carrier that replaces one or more nucleotides at the inner position or in the double-stranded region; (f) One or more lipophilic moieties are conjugated to the double-stranded iRNA agent via a linker containing an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide linkage, a product of a click reaction, or a carbamate; and / or (g) One or more lipophilic moieties are conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage, The dsRNA agent according to claim 3. **Claim 6** The dsRNA agent according to claim 1, wherein the dsRNA agent contains at least one modified nucleotide. **Claim 7** The dsRNA agent according to claim 2, wherein the dsRNA agent contains at least one modified nucleotide. **Claim 8** The dsRNA agent according to claim 3, wherein the dsRNA agent contains at least one modified nucleotide. **Claim 9** (a) Five or fewer nucleotides of the sense strand and five or fewer nucleotides of the antisense strand are unmodified nucleotides; or (b) All nucleotides of the sense strand and all nucleotides of the antisense strand contain modifications; Here, at least one of the modified nucleotides is selected from the group consisting of deoxy-nucleotide, 3'-terminal deoxy-thymidine (dT) nucleotide, 2'-O-methyl modified nucleotide, 2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide, locked nucleotide, unlocked nucleotide, conformationally restricted nucleotide, constrained ethyl nucleotide, abasic nucleotide, 2'-amino modified nucleotide, 2'-O-allyl modified nucleotide, 2'-C-alkyl modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, morpholino nucleotide, phosphoramidate, unnatural base containing nucleotide, tetrahydropyran modified nucleotide, 1,5-anhydrohexitol modified nucleotide, cyclohexenyl modified nucleotide, nucleotide containing phosphorothioate group, nucleotide containing methylphosphonate group, nucleotide containing 5'-phosphate, nucleotide containing 5'-phosphate mimetic, glycol modified nucleotide, and 2-O-(N-methylacetamide) modified nucleotide, and combinations thereof. The dsRNA agent according to claim 6.

10. (a) At least one of the sense strand and the antisense strand is conjugated to one or more of arginine-glycine-aspartic acid (RGD)-peptide or RGD peptide mimetic; (b) At least one strand contains a 3' overhang of at least 2 nucleotides; (c) The double-stranded region has a length of 15 to 30 nucleotide pairs, or a length of 17 to 23 nucleotide pairs; (d) Each strand has 19 to 30 nucleotides; and / or (e) The dsRNA agent contains at least one phosphorothioate or methylphosphonate internucleotide linkage. The dsRNA agent according to claim 1.

11. The dsRNA agent according to claim 3, further comprising a targeting ligand.

12. The dsRNA agent according to claim 8, further comprising a targeting ligand.

13. The dsRNA agent according to claim 11, wherein the targeting ligand targets ocular tissue, trabecular meshwork tissue, ciliary body-like, retinal tissue, retinal pigment epithelium (RPE) or choroidal tissue or choroidal blood vessels.

14. The dsRNA agent according to claim 1, further comprising a phosphate, a phosphate mimetic, or 5'-vinylphosphonate (VP) at the 5' end of the antisense strand.

15. The dsRNA according to claim 1, wherein the dsRNA agent targets a hotspot region of mRNA encoding MYOC.

16. A dsRNA agent targeting a hotspot region of myocilin (MYOC) mRNA.

17. A cell containing the dsRNA agent according to claim 1.

18. A pharmaceutical composition for inhibiting the expression of MYOC, comprising the dsRNA agent according to any one of claims 1 to 16.

19. An in vitro method for inhibiting the expression of MYOC in a cell, the method comprising: (a) introducing into the cell the dsRNA agent according to any one of claims 1 to 16, or a pharmaceutical composition comprising the dsRNA agent according to any one of claims 1 to 16; (b) maintaining the cell produced in step (a) for a time sufficient to reduce the levels of MYOC mRNA, MYOC protein, or both MYOC mRNA and protein, thereby inhibiting the expression of MYOC in the cell.

20. The dsRNA agent according to any one of claims 1 to 16, or a pharmaceutical composition comprising the dsRNA agent according to any one of claims 1 to 16, for use in inhibiting MYOC expression in a subject.

21. the subject is a human; and / or the subject is diagnosed with a MYOC-related disorder, glaucoma, primary open-angle glaucoma (POAG), angle-closure glaucoma, congenital glaucoma, or secondary glaucoma, The dsRNA agent or pharmaceutical composition according to claim 20.

22. The dsRNA agent according to any one of claims 1 to 16, or a pharmaceutical composition comprising the dsRNA agent according to any one of claims 1 to 16, for use in treating a subject diagnosed with a MYOC-related disorder, wherein the treatment comprises administering to the subject a therapeutically effective amount of the dsRNA agent or the pharmaceutical composition, thereby treating the disorder.

23. (i) the MYOC-related disorder is glaucoma or primary open-angle glaucoma (POAG); (ii) the treatment comprises improving at least one sign or symptom of the disorder. (iii) said treating comprising: (a) inhibiting or reducing the expression or activity of MYOC; (b) reducing the level of misfolded MYOC protein; (c) reducing trabecular meshwork cell death; (d) reducing intraocular pressure; or (e) increasing visual acuity; (iv) the medicament being formulated for intravitreal, intravenous, or topical administration to said subject; and / or (v) said use further comprising the use of an additional agent or therapy suitable for the treatment or prevention of MYOC-related disorders, The dsRNA agent, or pharmaceutical composition, according to claim 22. [

24. ] The dsRNA agent, or pharmaceutical composition, according to claim 23, wherein the medicament is formulated for intravitreal administration, intravitreal injection, transscleral administration, transscleral injection, subconjunctival administration, subconjunctival injection, retrobulbar administration, retrobulbar injection, intracameral administration, intracameral injection, subretinal administration, or subretinal injection. [

25. ] The dsRNA agent, or pharmaceutical composition, according to claim 23, wherein said additional agent or therapy suitable for treatment comprises laser trabeculoplasty, trabeculectomy, minimally invasive glaucoma surgery, placement of a drainage tube in the eye, an oral medication, or an eye drop.

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