Compositions and methods for silencing MYOC expression

Abstract

Provided are double-stranded ribonucleic acid (dsRNA) agents targeting MYOC, and pharmaceutical compositions comprising the dsRNA agents.SOLUTION: A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of myocilin (MYOC), wherein the dsRNA agent comprises a sense strand and an antisense strand forming a duplex region, the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides with 0, 1, 2, or 3 mismatches from one of the antisense sequences listed in any one of the plurality of specified sequences, and the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides with 0, 1, 2, or 3 mismatches from the sense sequence listed in any one of the plurality of specified sequences corresponding to the antisense sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 005,735, filed April 6, 2020, the entire contents of which are incorporated herein by reference.

[0002] Array List This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on March 31, 2021 is named A2038_7237WO_SL.txt and is 1,020,574 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 aging population. Misfolding of the MYOC protein obstructs its secretion from trabecular meshwork cells, causing 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. Novel treatments for glaucoma are needed. Summary of the Invention

[0005] The present disclosure describes the method and iRNA composition for modulating the expression of MYOC.In certain embodiments, the expression of MYOC is reduced or inhibited using MYOC-specific iRNA.Such inhibition can be useful in the treatment of disorders associated with MYOC expression, such as eye diseases (for example, glaucoma, for example, primary open-angle glaucoma (POAG)).

[0006] Therefore, the present invention provides compositions and methods for achieving RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of MYOC in cells or in subjects (e.g., mammals, e.g., human subjects). 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)).

[0007] The iRNA (e.g., dsRNA) included in the compositions featured herein comprises an RNA strand (antisense strand) having a region, e.g., 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, e.g., SEQ ID NO: 1 herein.

[0008] In some embodiments, the iRNA (e.g., 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 of a portion of the coding strand of human MYOC, with 0, 1, 2, or 3 mismatches, and the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides of a corresponding portion of the non-coding strand of human MYOC, with 0, 1, 2, or 3 mismatches, 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 present disclosure provides human cells or tissues that comprise reduced levels of MYOC mRNA or a level of MYOC protein compared to otherwise similar untreated cells or tissues; optionally, the cells or tissues may be genetically engineered (e.g., the cells or tissues comprise one or more naturally occurring mutations, e.g., MYOC mutations); and optionally, the levels may be 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 body, retinal pigment epithelium (RPE), retinal tissue, astrocytes, pericytes, Müller cells, ganglion cells, endothelial cells, photoreceptor cells, retinal vasculature (e.g., comprising endothelial cells and vascular smooth muscle cells), or choroidal tissue, e.g., choroidal vasculature.

[0012] The present disclosure also provides, in some aspects, cells containing the dsRNA agents described herein.

[0013] In another aspect, provided herein are human ocular cells, e.g., cells of the trabecular meshwork, cells of the ciliary body, RPE cells, retinal cells, astrocytes, pericytes, Müller cells, ganglion cells, endothelial cells, or photoreceptor cells, that contain reduced levels of MYOC mRNA or levels of 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%.

[0014] In some aspects, the present disclosure also provides pharmaceutical compositions that inhibit expression of a gene encoding MYOC, comprising a dsRNA agent described herein.

[0015] 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, and (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. Includes.

[0016] 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, and (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. Includes.

[0017] The disclosure also provides, in some aspects, a method of 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; and (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. Includes.

[0018] The present disclosure also provides, in some embodiments, 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.

[0019] Any of the aspects herein, eg, the compositions and methods described above, may be provided with any of the embodiments herein (eg, below).

[0020] 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.

[0021] 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.

[0022] In some embodiments, dsRNA agent comprises sense strand and antisense strand, and antisense strand comprises the nucleotide sequence of at least 17 consecutive nucleotides of a part of the nucleotide sequence of SEQ ID NO: 2, with 0, 1, 2 or 3 mismatches, so that sense strand is complementary to at least 17 consecutive nucleotides in antisense strand.In some embodiments, sense strand comprises the nucleotide sequence of at least 17 consecutive nucleotides of the corresponding part of the nucleotide sequence of SEQ ID NO: 1, with 0, 1, 2 or 3 mismatches.

[0023] In some embodiments, dsRNA agent comprises sense strand and antisense strand, and antisense strand comprises the nucleotide sequence of at least 19 consecutive nucleotides of a part of the nucleotide sequence of SEQ ID NO: 2, with 0, 1, 2 or 3 mismatches, so that sense strand is complementary to at least 19 consecutive nucleotides in antisense strand.In some embodiments, sense strand comprises the nucleotide sequence of at least 19 consecutive nucleotides of the corresponding part of the nucleotide sequence of SEQ ID NO: 1, with 0, 1, 2 or 3 mismatches.

[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 21 consecutive nucleotides with 0, 1, 2 or 3 mismatches with a portion of the nucleotide sequence of SEQ ID NO: 2, so 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 with the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

[0025] In some embodiments, the portion of the sense strand is a portion within the sense strand in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, and 5B.

[0026] In some embodiments, the portion of the antisense strand is a portion within the antisense strand in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, and 5B.

[0027] In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides with 0, 1, 2, or 3 mismatches from one of the antisense sequences listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, and 5B. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides with 0, 1, 2, or 3 mismatches from a sense sequence listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, and 5B that corresponds to the antisense sequence.

[0028] In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 17 consecutive nucleotides with 0, 1, 2, or 3 mismatches from one of the antisense sequences listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, and 5B. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 17 consecutive nucleotides with 0, 1, 2, or 3 mismatches from a sense sequence listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, and 5B that corresponds to the antisense sequence.

[0029] In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 19 consecutive nucleotides with 0, 1, 2, or 3 mismatches from one of the antisense sequences listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, and 5B. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 19 consecutive nucleotides with 0, 1, 2, or 3 mismatches from a sense sequence listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, and 5B that corresponds to the antisense sequence.

[0030] In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides with 0, 1, 2, or 3 mismatches from one of the antisense sequences listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, and 5B. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides with 0, 1, 2, or 3 mismatches from a sense sequence listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, and 5B that corresponds to the antisense sequence.

[0031] In some embodiments, the sense strand of the dsRNA agent is at least 23 nucleotides in length, eg, 23-30 nucleotides in length.

[0032] In some embodiments, at least one of sense strand and antisense strand is conjugated to one or more lipophilic moieties.In some embodiments, lipophilic moieties are conjugated to one or more positions in the double-stranded region of dsRNA agent.In some embodiments, lipophilic moieties are conjugated via linker or carrier.In some embodiments, logK ow The lipophilicity of the lipophilic moiety as measured by is greater than 0. 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.

[0033] In some embodiments, dsRNA agent comprises at least one modified nucleotide.In some embodiments, 5 or less of the nucleotides of sense strand and 5 or less of the nucleotides of antisense strand are unmodified nucleotide.In some embodiments, all of the nucleotides of sense strand and all of the nucleotides of antisense strand comprise modification.

[0034] In some embodiments, at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3'-terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-methoxyethyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide comprising a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate, a nucleotide comprising a 5'-phosphate mimic, a glycol-modified nucleotide, and a 2'-O-(N-methylacetamido)-modified nucleotide, 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).

[0035] In some embodiments, the dsRNA comprises a non-nucleotide spacer between two consecutive nucleotides in the sense strand or between two consecutive nucleotides in the antisense strand (the non-nucleotide spacer may comprise a C3-C6 alkyl).

[0036] In some embodiments, each strand is 30 or less nucleotides 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.

[0037] 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.

[0038] 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 an antisense strand. In some embodiments, the strand is a sense strand.

[0039] 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.

[0040] In some embodiments, the 5' and 3' ends of one strand each comprise a phosphorothioate or methylphosphonate internucleotide linkage. In some embodiments, the strand is the antisense strand.

[0041] In some embodiments, the base pair at one position at the 5' end of the antisense strand of the duplex is an AU base pair.

[0042] In some embodiments, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0043] In some embodiments, one or more lipophilic moieties are conjugated to one or more internal positions on at least one chain. In some embodiments, one or more lipophilic moieties are conjugated to one or more internal positions on at least one chain via a linker or carrier.

[0044] In some embodiments, internal positions include all positions except the two most terminal positions from each end of at least one strand. In some embodiments, internal positions include all positions except the three most terminal positions from each end of 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, counting from the 3' end of the sense strand and positions 12-14, counting from the 5' end of the antisense strand.

[0045] 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 in the sense strand and positions 6-10 and 15-18 in 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 in the sense strand and positions 15 and 17 in the antisense strand, counting from the 5' end of each strand.

[0046] In some embodiments, the positions in the double-stranded region exclude the cleavage site region of the sense strand.

[0047] In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, and the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, position 7, position 6, or position 2 of the sense strand or position 16 of the antisense strand. In some embodiments, the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, or position 7 of the sense strand. In some embodiments, the lipophilic moiety is conjugated to position 21, position 20, or position 15 of the sense strand. In some embodiments, the lipophilic moiety is conjugated to position 20 or position 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.

[0048] 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 lipids, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic 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.

[0049] In some embodiments, lipophilic moiety is conjugated via carrier that replaces one or more nucleotides in internal position or double-stranded region.In some embodiments, carrier is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl and decalinyl; or is based on serinol skeleton or diethanolamine skeleton acyclic moiety.

[0050] In some embodiments, the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker that contains 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.

[0051] In some embodiments, the lipophilic moiety is conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage.

[0052] 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.

[0053] 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.

[0054] In some embodiments, the dsRNA agent further comprises targeting ligand, for example, the ligand that targets 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.

[0055] In some embodiments, the ligand is conjugated to the sense strand. In some embodiments, the ligand is conjugated to the 3'-end or 5'-end of the sense strand. In some embodiments, the ligand is conjugated to the 3'-end of the sense strand.

[0056] 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 conjugates or one or more GalNAc derivatives attached by a monovalent linker or a bivalent, trivalent, or tetravalent branched linker. In some embodiments, the ligand is

[0057] [ka] In some embodiments, the dsRNA agent is conjugated to a ligand as shown in the following schematic diagram:

[0058] [ka] wherein X is O or S. In some embodiments, X is O.

[0059] In some embodiments, the dsRNA agent further includes a terminal chiral modification occurring at the first internucleotide linkage at the 3'-end of the antisense strand, the first internucleotide linkage having the linking phosphorus atom in the Sp configuration, a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand, the first internucleotide linkage having the linking phosphorus atom in the Rp configuration, and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, the first internucleotide linkage having the linking phosphorus atom in either the Rp or Sp configuration.

[0060] 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, with the linking phosphorus atom in the Sp configuration, a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration, and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration.

[0061] 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, with the linking phosphorus atom in the Sp configuration, a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration, and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration.

[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, with the linking phosphorus atom in the Sp configuration; a terminal chiral modification occurring at the third internucleotide linkage at the 3'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration; a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration; and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration.

[0063] 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, with the 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, with the linking phosphorus atom in the Rp configuration, and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration.

[0064] In some embodiments, the dsRNA agent further comprises a phosphate or a phosphate mimic at the 5'-end of the antisense strand. In some embodiments, the phosphate mimic is 5'-vinylphosphonate (VP).

[0065] In some embodiments, a cell described herein, e.g., a human cell, is produced by a process comprising contacting a human cell with a dsRNA agent described herein.

[0066] In some embodiments, a pharmaceutical composition described herein comprises a dsRNA agent and a lipid formulation.

[0067] 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 the expression gene for MYOC 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%.

[0068] 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).

[0069] In some embodiments, the ocular cell or tissue is trabecular meshwork tissue, ciliary body, RPE, retinal cells, 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, e.g., choroidal blood vessels.

[0070] 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).

[0071] In some embodiments, treatment comprises amelioration of at least one sign or symptom of the disorder, in some embodiments, the at least one sign or symptom comprises one or more measures of optic nerve damage, vision loss, tunnel vision, blurred vision, eye pain, or the presence, level, or activity of MYOC (e.g., MYOC gene, MYOC mRNA, or MYOC protein).

[0072] In some embodiments, a level of MYOC higher than the reference level indicates that the subject has glaucoma. In some embodiments, the treatment includes preventing the progression of the disorder. In some embodiments, the treatment includes one or more of: (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) improving visual acuity.

[0073] In some embodiments, treatment results in an average reduction from baseline of at least 30% of MYOC mRNA in the trabecular meshwork, ciliary body, 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, treatment results in an average reduction from baseline of at least 60% of MYOC mRNA in the trabecular meshwork, ciliary body, 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, treatment results in an average reduction from baseline of at least 90% of MYOC mRNA in the trabecular meshwork, ciliary body, retina, RPE, retinal blood vessels (e.g., comprising endothelial cells and vascular smooth muscle cells), or choroidal tissue, e.g., choroidal blood vessels.

[0074] In some embodiments, after treatment, subject experiences knockdown for at least 8 weeks after a single dose of dsRNA, as assessed by MYOC protein in retina.In some embodiments, after treatment, subject experiences knockdown for at least 12 weeks after a single dose of dsRNA, as assessed by MYOC protein in retina.In some embodiments, after treatment, subject experiences knockdown for at least 16 weeks after a single dose of dsRNA, as assessed by MYOC protein in retina.

[0075] In some embodiments, the subject is a human.

[0076] In some embodiments, the dsRNA agent is administered at a dose of about 0.01 mg / kg to about 50 mg / kg.

[0077] In some embodiments, dsRNA agent is administered to subject intraocularly.In some embodiments, intraocular administration comprises intravitreal administration, for example, intravitreal injection, transscleral administration, for example, transscleral 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.

[0078] In some embodiments, the dsRNA agent is administered to the subject intravenously. In some embodiments, the dsRNA agent is administered to the subject locally.

[0079] 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) obtained from the subject. In some embodiments, the methods described herein further comprise performing a blood test, an imaging test, or an aqueous humor biopsy (e.g., an aqueous humor tap).

[0080] In some embodiments, the method described herein further measures the level of MYOC (for example, MYOC gene, MYOC mRNA or MYOC protein) in the subject before treatment with dsRNA agent or pharmaceutical composition.In some embodiments, when it is determined that the subject has a level of MYOC higher 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 carried out after treatment with dsRNA agent or pharmaceutical composition.

[0081] In some embodiments, the methods described herein further include treating the subject with a therapy suitable for treating or preventing a MYOC-related disorder, e.g., the therapy includes laser trabeculoplasty, trabeculectomy, minimally invasive glaucoma surgery, or replacement 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-related 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 eye drops.

[0082] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0083] Details of various embodiments of the disclosure are set forth in the description that follows. Other features, objects, and advantages of the disclosure will be apparent from the description and drawings, and from the claims. DETAILED DESCRIPTION OF THE INVENTION

[0084] iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).Described herein is iRNA and the method of using them for modulating (for example, inhibiting) the expression of MYOC.Also provided are compositions and methods for treating disorders associated with MYOC expression, such as glaucoma (for example, primary open-angle glaucoma (POAG)).

[0085] Human MYOC is a secreted glycoprotein of approximately 57 kDa, which 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 normally expressed and secreted by various tissues, including the retina and structures involved in the regulation of aqueous humor, such as the trabecular meshwork and ciliary body. Abnormal MYOC is associated with glaucoma, such as primary open-angle glaucoma (POAG). Without intending to be limited by theory, abnormal MYOC may exacerbate the pathogenesis of glaucoma, for example, by impeding the drainage of aqueous humor, which then causes an increase in intraocular pressure.

[0086] The following description discloses methods for making and using 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.

[0087] Featured herein, in some aspects, 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)).

[0088] I. Definition For convenience, the meanings of certain terms and phrases used in the specification, examples, and appended claims are provided below. In the event of an apparent discrepancy between the use of a term in other parts of this specification and its definition provided in this section, the definition in this section shall control.

[0089] The term "about," when referring to a number or numerical range, means that the referenced number or numerical range is an approximation within experimental variability (or within gross experimental error), and thus the number or numerical range may vary, for example, by between 1% and 15% of the stated number or numerical range.

[0090] The term "at least" before a number or a series of numbers, if clear from the context, is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that may be logically included. 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 the term "at least" is present before a series of numbers or a range, it is understood that "at least" can modify each of the numbers and ranges in the series.

[0091] As used herein, "less than" or "less than" shall be understood as from the value adjacent to the phrase and a logically smaller value or integer than that value, up to zero, if logical from the context. For example, a duplex with a mismatch to a target site of "two or fewer nucleotides" has 2, 1, or 0 mismatches. When "less than" is present before a series of numbers or ranges, it will be understood that "less than" can modify each of the numbers or ranges in the series.

[0092] As used herein, "up to," as in "up to 10," is understood to be up to and including 10, i.e., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0093] Ranges provided herein are understood to include all individual integer values ​​and all subranges within that range.

[0094] The terms "activate," "enhance," "upregulate expression," "increase expression," and the like, 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 or detected from 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).

[0095] 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 as 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 as 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 as 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 as 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, as well as in US2007 / 0111963 and US2005 / 226848, each of which is incorporated herein by reference.

[0096] The terms "silencing," "inhibiting the expression of," "downregulating the expression of," "suppressing the expression of," 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 associated with MYOC expression. For example, inhibition of MYOC expression may be indicated by a reduction in the amount of MYOC mRNA that can be isolated or detected in a first cell or group of cells in which MYOC is transcribed and that has been treated such that MYOC expression is inhibited compared to a control. The control may be a second cell or group of cells (control cells) that is substantially identical to the first cell or group of cells, except that the second cell or group of cells has not been so treated. The degree of inhibition is usually expressed as a percentage of the control level, e.g.,

[0097]

number

[0098] Alternatively, the degree of inhibition can be expressed in terms of the parameter functionally associated with MYOC expression, for example, the amount of the protein encoded by MYOC gene.The parameter functionally associated with 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 constitutively or by genome engineering by any suitable assay.

[0099] 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 as described herein.

[0100] 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.

[0101] As used herein, the term " complementary region " refers to the region on the antisense strand that is substantially complementary to a sequence, for example, a target sequence as defined herein.When complementary region is not fully complementary to target sequence, mismatch can be in the internal or terminal region of the molecule.In some embodiments, complementary region comprises 0, 1 or 2 mismatches.

[0102] 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.

[0103] The term "blunt" or "blunt-ended" as used herein in reference to dsRNA means that there are no unpaired nucleotides or nucleotide analogs at a given end of the dsRNA, i.e., there are no nucleotide overhangs. One or both ends of the dsRNA can be blunt. When both ends of the dsRNA are blunt, the dsRNA is said to be blunt-ended. For clarity, a "blunt-ended" dsRNA is a dsRNA that is blunt at both ends, i.e., there are no nucleotide overhangs at either end of the molecule. In most cases, such a molecule will be double-stranded throughout its entire length.

[0104] As used herein, unless otherwise specified, the term "complementary," when used to describe a first nucleotide sequence in the context of a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize to form a duplex with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions, as understood by those skilled 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 may be applied, such as physiologically relevant conditions that may be encountered inside an organism. Those skilled in the art will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences according to the ultimate application of the hybridized nucleotides.

[0105] A complementary sequence in an iRNA, for example, in a dsRNA as described herein, includes base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence with an oligonucleotide or polynucleotide comprising a second nucleotide sequence across the entire length of one or both nucleotide sequences. Such sequences can be referred to herein as "fully complementary" to each other. However, when a first sequence is considered herein to be "substantially complementary" to a second sequence, the two sequences can be fully complementary, or they can form one or more, but generally no more than 5, 4, 3, or 2 mismatched base pairs during hybridization, in the case of a duplex of up to 30 base pairs, while maintaining the ability to hybridize under conditions that are most suitable for their final use, such as inhibiting gene expression through the RISC pathway. However, if two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, these overhangs are not considered mismatches when determining complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, where the longer oligonucleotide comprises a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, can still be considered "perfectly complementary" for the purposes described herein.

[0106] Complementary sequences, as used herein, may also include or be formed entirely from non-Watson-Crick base pairs and / or base pairs formed from non-natural modified nucleotides, such as, but not limited to, G:U Wobble or Hoogstein base pairing, so long as the above requirements regarding their ability to hybridize are met.

[0107] The terms "complementary," "fully complementary," and "substantially complementary" can be used herein 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 understood in connection with their use.

[0108] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a continuous portion of an mRNA of interest (e.g., an mRNA encoding a 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.

[0109] As used herein, the term "region of complementarity" refers to the region of one nucleotide sequence agent that is substantially complementary to another sequence, as defined herein, for example, the region of the sense sequence of a dsRNA and the corresponding antisense sequence, or the antisense strand of an iRNA and the target sequence, for example, the MYOC nucleotide sequence.If the region of complementarity is not completely complementary to the target sequence, the mismatch can be in the internal region or terminal region of the antisense strand of the iRNA.In general, the most tolerable mismatch is in the terminal region, for example, within 5, 4, 3, or 2 nucleotides of the 5' or 3' end of the iRNA agent.

[0110] As used herein, "contacting" includes direct contacting of cells as well as indirect contacting of cells. For example, cells within a subject can be contacted when a composition containing an iRNA is administered to the subject (e.g., intraocularly, topically, or intravenously).

[0111] "Introducing into a cell," when referring to iRNA, means promoting or achieving uptake or absorption into a cell. Absorption or uptake of iRNA can occur by spontaneous diffusive or active cellular 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" that is part of a living organism. In such instances, introducing 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 Publication No. 2005 / 0281781, which are incorporated herein by reference in their entireties. In vitro introduction into a cell includes methods known in the art, such as electroporation and lipofection. Further approaches are described herein below or known in the art. As used herein, "disorders associated with MYOC expression," "diseases associated with MYOC expression," "pathological processes associated with MYOC expression," "MYOC-related disorders," "MYOC-related diseases," and the like include any condition, disorder, or disease in which MYOC expression is altered (e.g., decreased or increased relative to a reference 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 from the subject (e.g., in an aqueous ocular fluid sample). The decrease or increase can be assessed relative to levels observed in the same individual before the onset of the disorder or relative to other individual(s) without the disorder. The decrease or increase may be limited to a particular organ, tissue, or body region (e.g., the eye). MYOC-related disorders include, but are not limited to, glaucoma (e.g., primary open-angle glaucoma (POAG)).

[0112] 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).

[0113] The terms "double-stranded RNA," "dsRNA," or "siRNA," as used herein, refer to an iRNA comprising an RNA molecule or complex of molecules having a hybridized duplex region comprising two antiparallel, substantially complementary nucleic acid strands, referred to as having "sense" and "antisense" orientations with respect to the target RNA. The duplex region allows for specific degradation of the desired target RNA, for example, by the RISC pathway, and can be of any length, typically ranging from 9 to 36 base pairs in length, e.g., 15 to 30 base pairs in length. Considering duplexes between 9 and 36 base pairs, the duplexes 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, and include, but are not limited to, 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18 The length of the dsRNA may be any subrange thereof, including up to 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. dsRNA generated in cells by processing by Dicer and similar enzymes is generally in the 19-22 base pair range. One strand of the double-stranded region of dsDNA contains a sequence that is a substantially complementary region of a target RNA. The two strands that form the duplex structure can be derived from a single RNA molecule with at least one self-complementary region, or they can 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 can have a double-stranded region (referred to herein as "hairpin loop") between the 3'-end of one strand that forms the double-stranded structure and the 5'-end of each of the other strands, separated by the nucleotides of the single-stranded strand.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 dsRNA are contained by separate RNA molecules, these molecules can, but do not necessarily, be connected by a covalent bond.In some embodiments, the two strands are connected by a covalent bond by means other than a hairpin loop, and the connecting structure is a linker.

[0114] In some embodiments, an iRNA agent can be a "single-stranded siRNA" that is 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 and may be 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., the sequences provided in Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, or 5B) can be used as single-stranded siRNAs as described herein, and can be chemically modified, e.g., as described herein, e.g., by the methods described in Lima et al., (2012) Cell 150:883-894.

[0115] In some embodiments, RNA interference agents comprise single-stranded RNA that interacts with target RNA sequences, directing the cleavage of target RNA. Without intending to be limited by theory, long double-stranded RNA introduced into cells is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer, a ribonuclease-III-like enzyme, processes dsRNA into short interfering RNAs of 19-23 base pairs with characteristic two-base 3' overhangs [Bernstein, et al., (2001) Nature 409:363]. The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing complementary antisense strands to guide target recognition [Nykanen, et al., (2001) Cell 107:309]. Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing [Elbashir, et al., (2001) Genes Dev. 15:188]. Thus, in some embodiments, the present disclosure relates to single-stranded RNAs that promote the formation of a RISC complex to silence a target gene.

[0116] " 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 ", " ribonucleotide " or " nucleotide " may also refer to modified nucleotides, as will be described in more detail below, or substitute replacement parts.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 parts.For example, but not limited to, the nucleotide that contains inosine as its base can form base pairs 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 featured in the present disclosure. In another example, adenine and cytosine in any of the oligonucleotides can be substituted with guanine and uracil, respectively, to form G-U Wobble base pairs with the target mRNA. Sequences containing such substituted moieties are suitable for the compositions and methods featured in this disclosure.

[0117] As used herein, the terms "iRNA," "RNAi," "iRNA agent," or "RNAi agent" or "RNAi molecule" refer to an agent that contains RNA, as that term is defined herein, and that mediates targeted cleavage of an RNA transcript, e.g., via the RNA-induced silencing complex (RISC) pathway. In some embodiments, an iRNA as described herein achieves inhibition of MYOC expression, e.g., in a cell or mammal. Inhibition of MYOC expression can be assessed based on a reduction in MYOC mRNA levels or a reduction in MYOC protein levels.

[0118] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound, e.g., attaches two parts of a compound by a covalent bond.

[0119] 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 in a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it can also be predicted by using 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., hydrophilic / lipophilic balance). In principle, a chemical can be expressed as a function of the log K ow is greater than 0, it is lipophilic in nature. Typically, a lipophilic moiety has a log K ow For example, the log K of 6-aminohexanol ow is expected to be approximately 0.7. Using the same method, the log K ow is expected to be 10.7.

[0120] The lipophilicity of a molecule can be modified with respect to the functional groups it carries. For example, adding a hydroxyl or amine group to the end of the lipophilic moiety can increase the partition coefficient (e.g., logK ow ) value can be increased or decreased.

[0121] Alternatively, the hydrophobicity of the double-stranded RNAi agent that is 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.

[0122] 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 in, for example, PCT / US2019 / 031170.The hydrophobicity of double-stranded RNAi agent measured by the fraction of unbound 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 for enhanced in vivo delivery of siRNA.

[0123] 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.

[0124] The term "lipid nanoparticle" or "LNP" refers to a vesicle that comprises a lipid layer that encapsulates a pharmaceutically active molecule, such as a nucleic acid molecule, for example, an RNAi agent or a plasmid that transcribes an RNAi agent.LNP is described, for example, in U.S. Patent No. 6,858,225, U.S. Patent No. 6,815,432, U.S. Patent No. 8,158,601, and U.S. Patent No. 8,058,069, the entire contents of which are incorporated herein by reference.

[0125] 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 as 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.

[0126] Those skilled in the art will recognize that the term "RNA molecule" or "ribonucleic acid molecule" encompasses not only naturally expressed or found RNA molecules, but also RNA analogs and derivatives containing one or more ribonucleotide / ribonucleoside analogs or derivatives as described herein or known in the art. Strictly speaking, a "ribonucleoside" comprises 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, the ribose structure, or the ribose-phosphate backbone structure, as described herein below. However, molecules containing ribonucleoside analogs or derivatives must retain the ability to form duplexes. As non-limiting examples, the RNA molecule can 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 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 a nucleoside containing a non-natural base, or any combination thereof. Alternatively, in combination, an RNA molecule can contain at least two modified ribonucleosides, 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 15, at least 20 or more full-length dsRNA molecules. The modifications are not necessarily identical for each of such multiple modified ribonucleosides in an 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 RNA, for example, by 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.

[0127] In some aspects, modified ribonucleosides include deoxyribonucleosides. In such examples, the iRNA agent can include, for example, one or more deoxynucleosides including deoxynucleoside overhang(s) or one or more deoxynucleosides within the double-stranded portion of the dsRNA. In certain embodiments, the RNA molecule includes, for example, in one or both strands, a percentage of deoxyribonucleosides of 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.

[0128] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of iRNA, such as dsRNA.For example, if the 3'-end of one strand of dsRNA extends beyond the 5'-end of the other strand, or vice versa, a nucleotide overhang exists.A dsRNA can comprise an overhang of at least one nucleotide; alternatively, the overhang can comprise 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.The overhang(s) can be on the sense strand, antisense strand, or any combination thereof.In addition, the nucleotide(s) of the overhang can be present at the 5'-end, 3'-end, or both of the antisense strand or sense strand of dsRNA.

[0129] In some embodiments, the antisense strand of the dsRNA has a 1-10 nucleotide overhang at the 3'-end and / or 5'-end. In one embodiment, the sense strand of the dsRNA has a 1-10 nucleotide overhang at the 3'-end and / or 5'-end. In some embodiments, one or more of the nucleotides in the overhang are replaced with nucleoside thiophosphate.

[0130] 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 misfolded MYOC protein levels, (c) reduce trabecular meshwork cell death, (d) reduce intraocular pressure, or (e) improve visual acuity). For example, if a given clinical treatment is considered effective if there is at least a 10% reduction in a measurable parameter associated with the disease or disorder, a therapeutically effective amount of a drug for treating the 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%.

[0131] The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, 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 excipients, 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, and corn starch and alginic acid are suitable disintegrants. Binders can include starch and gelatin, and lubricants, if present, are generally magnesium stearate, stearic acid, or talc. If necessary, tablets can be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the digestive tract. The agents contained in the drug formulation are further described herein below.

[0132] As used herein, the term "SNALP" refers to stable nucleic acid-lipid particles.SNALP refers to lipid vesicles that coat a reduced aqueous interior containing nucleic acid, such as iRNA or the plasmid from which iRNA is transcribed.SNALP is described, for example, in U.S. Patent Application Publication Nos. 2006 / 0240093, 2007 / 0135372, and International Application No. WO2009 / 082817.These applications are incorporated herein by reference in their entirety.In some embodiments, SNALP is SPLP.As used herein, the term "SPLP" refers to nucleic acid-lipid particles that contain plasmid DNA encapsulated in lipid vesicles.

[0133] As used herein, the term "strand comprising a sequence" means an oligonucleotide comprising a chain of nucleotides described by a sequence referenced using standard nucleotide nomenclature.

[0134] 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 can 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.

[0135] A "subject in need thereof" includes a subject having, suspected of having, or 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.

[0136] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed upon transcription of a gene, e.g., 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-directed cleavage at or near that portion. For example, target sequences will generally be 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 nucleotides, or 19-26 nucleotides. 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.

[0137] As used herein, the phrases "therapeutically effective amount," "prophylactically effective amount," and the like 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 specific amount that is therapeutically effective will 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.

[0138] In the context of the present 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 predicted progression of such a disorder. For example, when used to treat glaucoma, the methods featured herein may help reduce or prevent one or more symptoms of glaucoma as described herein, or reduce the risk or severity of an associated condition. Thus, unless the context clearly indicates otherwise, the terms "treat," "treatment," and the like are intended to encompass the prevention, e.g., prophylaxis, of a disorder associated with MYOC expression and / or symptoms of the disorder. Treatment can also mean prolonging survival compared to expected survival in the absence of treatment.

[0139] "Lower," 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.

[0140] As used herein, "MYOC" refers to "myocilin," the corresponding 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.

[0141] II. iRNA Agents Described herein are iRNA agents that modulate (eg, inhibit) the expression of MYOC.

[0142] In some embodiments, the iRNA agent activates expression of MYOC in a cell or mammal.

[0143] In some embodiments, the iRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting expression of MYOC in a cell or in a subject (e.g., in a mammal, e.g., in a human), wherein the dsRNA comprises an antisense strand having a region of complementarity that is complementary to at least a portion of an mRNA formed in the expression of MYOC, wherein the region of complementarity is 30 nucleotides or less, generally 19-24 nucleotides in length, and wherein the dsRNA inhibits expression of MYOC, e.g., by at least 10%, 20%, 30%, 40%, or 50%, upon contact with a cell that expresses MYOC.

[0144] 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, for example, by Western blot. Expression of MYOC in cell culture, for example, COS cells, ARPE-19 cells, hTERT RPE-1 cells, HeLa cells, primary hepatocytes, HepG2 cells, primary cultured cells, or in a biological sample obtained from a subject, can be assayed by measuring MYOC mRNA levels, for example, by bDNA or TaqMan assay, or by measuring protein levels, for example, by immunofluorescence using Western blotting or flow cytometry techniques.

[0145] dsRNA usually contains two RNA strands that are sufficiently complementary to hybridize under the conditions in which the dsRNA is used to form a duplex structure. One strand of the dsRNA (antisense strand) usually contains a region of complementarity that is substantially complementary, and generally completely complementary to the target sequence obtained from the sequence of the mRNA formed during the expression of MYOC. The other strand (sense strand) usually contains a region complementary to the antisense strand, so that when the two strands are combined under suitable conditions, they hybridize to form a duplex structure. Generally, the duplex structure is between 15 and 30 base pairs (inclusive), more usually between 18 and 25 base pairs (inclusive), even more usually between 19 and 24 base pairs (inclusive), and most usually between 19 and 21 base pairs (inclusive). Similarly, the region of complementarity to the target sequence is between 15 and 30 (inclusive), more commonly between 18 and 25 (inclusive), even more commonly between 19 and 24 (inclusive), and most commonly between 19 and 21 nucleotides in length (inclusive).

[0146] In some embodiments, the dsRNA is between 15 and 20 nucleotides in length (inclusive), and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length (inclusive). As those skilled in the art will recognize, the targeted region of an RNA targeted for cleavage will most often be a portion of a larger RNA molecule, often an mRNA molecule. In relevant cases, a "portion" of an mRNA target is a contiguous sequence of the mRNA target long enough to be a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway). dsRNAs with duplexes as short as 9 base pairs can, under some circumstances, mediate RNAi-directed RNA cleavage. Most often, the target will be at least 15 nucleotides in length, e.g., 15-30 nucleotides in length.

[0147] Those skilled in the art will recognize that the duplex region is the primary functional portion of a dsRNA, e.g., a 9-36, e.g., 15-30 base pair duplex region. Thus, in some embodiments, an RNA molecule or complex of RNA molecules having a duplex region of more than 30 base pairs is a dsRNA, to the extent that it is processed to a functional duplex of, e.g., 15-30 base pairs that targets a desired RNA for cleavage. Thus, in some embodiments, those skilled in the art will recognize that an miRNA is 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.

[0148] dsRNA as described herein can further comprise one or more single-stranded nucleotide overhangs.dsRNA can be synthesized by standard methods known in the art, as further discussed below, for example, by using automated DNA synthesizer, and is commercially available from, for example, Biosearch, Applied Biosystems, Inc.

[0149] In some embodiments, the MYOC is human MYOC.

[0150] In specific embodiments, the dsRNA comprises a sense strand that comprises or consists of a sense sequence selected from the sense sequences provided in Table 2A, 2B, 3A, 3B, 4A, 4B, 5A, or 5B and an antisense strand that comprises or consists of an antisense sequence selected from the antisense sequences provided in Table 2A, 2B, 3A, 3B, 4A, 4B, 5A, or 5B.

[0151] In some embodiments, the dsRNA comprises at least a sense and an antisense nucleotide sequence, wherein the sense strand is selected from the sequences provided in Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, or 5B, and the corresponding antisense strand is selected from the sequences provided in Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, or 5B.

[0152] In these embodiments, one of the two sequences is complementary to the other of the two sequences, and one of the sequences is substantially complementary to the sequence of the mRNA produced by expression of MYOC. As such, the dsRNA comprises two oligonucleotides, one oligonucleotide being described as the sense strand and the second oligonucleotide being described 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, such that they are opposite each other on separate oligonucleotides.

[0153] Those skilled in the art are well aware that dsRNA with a duplex structure between 20 and 23, particularly 21 base pairs, is recognized 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 duplex structures can also be effective.

[0154] In the above embodiments, due to the nature of the oligonucleotide sequences provided in Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A and 5B, the dsRNAs described herein may comprise at least one strand with a minimum length of 19 nucleotides.It can be reasonably expected that shorter duplexes having one of the sequences in Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A and 5B, minus only a few nucleotides at one or both ends, will be similarly effective compared to the above dsRNAs.

[0155] 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, 2B, 3A, 3B, 4A, 4B, 5A or 5B.

[0156] 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, 2B, 3A, 3B, 4A, 4B, 5A, or 5B and a sense sequence comprising at least 15, 16, 17, 18, or 19 contiguous nucleotides of the corresponding sense sequence provided in Table 2A, 2B, 3A, 3B, 4A, 4B, 5A, or 5B.

[0157] 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, 2B, 3A, 3B, 4A, 4B, 5A, or 5B 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, 2B, 3A, 3B, 4A, 4B, 5A, or 5B.

[0158] In some such embodiments, the dsRNA comprises only a portion of the sequences provided in Table 2A, 2B, 3A, 3B, 4A, 4B, 5A, or 5B, but is equally effective in inhibiting the level of MYOC expression as a dsRNA comprising the full-length sequences provided in Table 2A, 2B, 3A, 3B, 4A, 4B, 5A, or 5B. In some embodiments, the dsRNA differs in its inhibition of the level of expression of MYOC by no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% inhibition compared to a dsRNA comprising the full-length sequences disclosed herein.

[0159] In some embodiments, the iRNAs described herein comprise an antisense strand 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. In some embodiments, the iRNAs described herein comprise a sense strand comprising at least 15 contiguous nucleotides with 0, 1, 2, or 3 mismatches of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

[0160] Human MYOC mRNA can have the sequence of SEQ ID NO: 1 provided herein. Human (Homo sapiens) myocilin (MYOC), mRNA

[0161] The reverse complement of SEQ ID NO: 1 is provided herein as SEQ ID NO: 2:

[0162] In some embodiments, the iRNAs described herein comprise at least 15 contiguous nucleotides from one of the sequences provided in Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, and 5B, and may be coupled to an additional nucleotide sequence taken from a region contiguous to the selected sequence in MYOC.

[0163] Target sequences are generally 15-30 nucleotides in length, although there is wide variation in the suitability of specific sequences within this range to direct 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 taken in which a "window" or "mask" of a given size (for example, 21 nucleotides) is placed literally or figuratively (including in silico) on the target RNA sequence to identify sequences within a size range that can serve as target sequences. By progressively moving the sequence "window" one nucleotide upstream or downstream of the initial target sequence position, subsequent potential target sequences can be identified until a complete set of possible sequences for any given target size is identified. This process, coupled 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 can be achieved by progressively "window walking" one nucleotide upstream or downstream of a given sequence to identify sequences with equivalent or better inhibitory characteristics.

[0164] Furthermore, it is contemplated that, for example, for any sequence identified in Table 2A, 2B, 3A, 3B, 4A, 4B, 5A and 5B, further optimization can be achieved by systematically adding or removing nucleotides to generate longer or shorter sequences, and testing the sequences generated by walking up or down the target RNA from these points through longer or shorter size windows.Again, combining this approach to generate new candidate targets with testing the effectiveness of iRNA based on these target sequences in inhibition assays as known in the art or as described herein can lead to further improvement in the efficiency of inhibition. Furthermore, such optimized sequences can be adjusted, for example, by introducing modified nucleotides as described herein or known in the art, additions or changes in overhangs, or other modifications as 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 to specific locations or cell types, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.).

[0165] In some embodiments, the present disclosure provides an unmodified or unconjugated iRNA of any of Tables 2B, 3B, 4B, or 5B. In some embodiments, the RNAi agent of the present disclosure has a nucleotide sequence as provided in any of Tables 2A, 3A, 4A, and 5A, 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 in any of the positions provided herein.

[0166] iRNAs as described herein can contain one or more mismatches to the target sequence. In some embodiments, iRNAs as described herein contain three or fewer mismatches. In some embodiments, if the antisense strand of an iRNA contains mismatches to the target sequence, the region of mismatch is not located in the center of the region of complementarity. In some embodiments, if the antisense strand of an iRNA contains mismatches to the target sequence, for a 23-nucleotide iRNA agent RNA strand that is complementary to a region of MYOC, mismatches are limited to, for example, within the last five nucleotides from the 5' or 3' end of the region of complementarity; the RNA strand generally does not contain any mismatches within the central 13 nucleotides. Methods described herein or known in the art can be used to determine whether iRNAs containing mismatches to the target sequence are effective in inhibiting the expression of MYOC. Consideration of the effectiveness of iRNAs with mismatches in inhibiting the expression of MYOC is important, especially when a particular region of complementarity in the MYOC gene is known to have polymorphic sequence variation within the population.

[0167] 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 relative to their blunt-ended counterparts. In some embodiments, the RNA of an iRNA (e.g., a dsRNA) is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in this disclosure can be synthesized and / or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, SL 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'-terminal modifications (phosphorylation, conjugation, reverse linkage, etc.) and 3'-terminal modifications (conjugation, DNA nucleotides, reverse linkage, etc.); (b) base modifications, such as replacement of a stabilizing base, a destabilizing base, or a base that base-pairs with an expanded repertoire partner, removal of a base (abasic nucleotide), or a conjugated base; and (c) sugar modifications (e.g., at the 2' or 4' position, or with an acyclic sugar) or sugar replacement, as well as backbone modifications, including modification or replacement of a phosphodiester bond. Specific examples of RNA compounds useful in the present disclosure include, but are not limited to, RNAs containing modified backbones or that do not contain natural internucleoside linkages. RNAs with modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For purposes of this specification, 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, the modified RNA has a phosphorus atom in its internucleoside backbone.

[0168] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates, 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, 2'-5' linked analogs thereof, and those with reverse polarity, where 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.

[0169] 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; 286,717;5,321,131;5,399,676;5,405,939;5,453,496;5,455,233;5,466,677;5,476,925;5,519,126;5,536,821;5,541,316;5,550,111;5,563,253;5,571,799;5 ,587,361;5,625,050;6,028,188;6,124,445;6,160,109;6,169,170;6,172,209;6,239,265;6,277,603;6,326,199;6,346,614;6,444,423;6,531,590;6,534,639; 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. Reissue Patent No. RE39464, each of which is incorporated herein by reference.

[0170] Modified RNA backbones that do not contain phosphorus atoms 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. These include 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.

[0171] 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, and 5,470,967. Nos. 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437 and 5,677,439, each of which is incorporated herein by reference.

[0172] In other RNA mimetics suitable or contemplated for use in iRNA, both the sugar and internucleoside linkage, i.e., the backbone, of the nucleotide unit are replaced with novel groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimetic 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 directly or indirectly bound 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. Patent 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.

[0173] Some embodiments featured in this disclosure include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, particularly --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [known as the methylene(methylimino) or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2-- and --N(CH3)--CH2--CH2-- [the natural phosphodiester backbone is represented as --O--P--O--CH2--] of the above-referenced U.S. Pat. No. 5,489,677, and amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have the morpholino backbone structures of the above-referenced US Pat. No. 5,034,506.

[0174] Modified RNAs may also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein may contain one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl, or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl 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 contains at the 2' position: C1 to C 10The modification may include one of the following: lower 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, interfering substance, 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(CH)ON(CH), 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--CH--O--CH--N(CH).

[0175] 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., 4, 3, 2, or 1 acyclic nucleotide per strand). The one or more acyclic nucleotides can be found, for example, at the 5' end, the 3' end, or both the 5' and 3' ends of the sense or antisense strand, or both strands, in the double-stranded region of the iRNA agent. In some embodiments, the one or more acyclic nucleotides are present in positions 1-8 of the sense or 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.

[0176] The term "acyclic nucleotide" or "acyclic nucleoside" as used herein refers to any nucleotide or nucleoside having an acyclic sugar, such as an acyclic ribose. Exemplary acyclic nucleotides or nucleosides include nucleobases, such as naturally occurring or modified nucleobases (e.g., nucleobases as described herein). In certain embodiments, the bond between any of the ribose carbons (C1, C2, C3, C4, or C5) is absent, either independently or in combination, in a nucleotide. In some embodiments, the bond between the C2-C3 carbon 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 US 8,314,227, which is incorporated herein in its entirety. For example, the acyclic nucleotide can include any of monomers D-J in Figures 1-2 of US 8,314,227. In some embodiments, the acyclic nucleotide can include any of the following monomers:

[0177] [ka] wherein the base is a nucleobase, e.g., a naturally occurring or modified nucleobase (e.g., a nucleobase as described herein). Includes.

[0178] In certain embodiments, acyclic nucleotides can be modified or derivatized, for example, by coupling the acyclic nucleotide to another moiety, such as a ligand (e.g., GalNAc, cholesterol ligand), alkyl, polyamine, sugar, polypeptide, among others.

[0179] In other embodiments, the iRNA agent includes one or more acyclic nucleotides and one or more LNAs (e.g., LNAs as described herein). For example, 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 as 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).

[0180] In other embodiments, the 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.

[0181] 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 a 2'-5'-linked dsRNA and the 5' position of the 5'-terminal nucleotide. An iRNA can also have a sugar mimetic, such as a cyclobutyl moiety 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, ,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, certain of which are commonly owned with the present application and each of which is incorporated herein by reference.

[0182] iRNAs may also contain nucleobase (often simply referred to in the art as "base") modifications or substitutions. 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, 5-uracil ... These include 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and adenine, 8-azaguanine and adenine, 7-deazaguanine and adenine, and 3-deazaguanine and adenine.

[0183] Additional nucleobases include those disclosed in U.S. 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, J. L, ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Certain 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 O-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 exemplary base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

[0184] Representative United States patents that teach the preparation of certain of the above modified nucleobases as well as other modified nucleobases include, but are not limited to, U.S. Pat. No. 3,687,808 and U.S. Pat. Nos. 4,845,205, 5,130,30, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,562,563, 5,564,565, 5,566,567, 5,568,569, 5,570,571, 5,572,572, 5,576,577, 5,578,579, 5,576,579, 5,578,571, 5,578,572, 5,578,573, 5,578,574, 5,578,575, 5,578,575, 5,578,579 ... ,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, as well as U.S. Pat. No. 5,750,692, which is also incorporated herein by reference.

[0185] 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 structure. 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 with modified ribose moieties, for example, where the ribose moiety contains an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo structural conformation. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects [Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447, Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843, Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193].

[0186] 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, the 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 known 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. 8,399,845). 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' (wherein 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 U.S. patents that teach the preparation of locked nucleic acids include, but are not limited to, the following: U.S. Pat. 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 (eg, Wengel et al., International PCT Publication Nos. WO00 / 66604 and WO99 / 14226).

[0187] For example, any of the above bicyclic nucleosides can be prepared with one or more stereochemical sugar configurations including α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0188] 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 containing a bicyclic sugar moiety containing a 4'-CH(CH3)-0 to 2' bridge. In some embodiments, the constrained ethyl nucleotide is in the S conformation and is referred to herein as an "S-cEt."

[0189] The RNAi agents 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 and increases hybridization affinity to mRNA. The linker is of sufficient length to position the oxygen in an optimal position for stability and affinity, resulting in less ribose ring puckering.

[0190] Representative publications that teach the preparation of certain of the above CRNs include, but are not limited to, US2013 / 0190383 and WO2013 / 036868, the contents of each of which are incorporated herein by reference for the methods provided herein.

[0191] In some embodiments, the RNAi agent of the present disclosure comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is an unlocked acyclic nucleic acid, in which any of the sugar bonds have been removed to form an unlocked "sugar" residue. In one example, UNA also encompasses a monomer in which the bond between C1'-C4' (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon 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].

[0192] Representative U.S. publications that teach the preparation of UNAs include, but are not limited to, U.S. Pat. Nos. 8,314,227 and U.S. Patent 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.

[0193] 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 in which the modification confers the ability to hydrogen bond to both the Watson-Crick and Hoogsteen faces of complementary guanine 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 helix thermal stability and mismatch discrimination when hybridized with a complementary oligonucleotide. Inclusion of such nucleotides in an iRNA molecule can result in enhanced affinity and specificity for a nucleic acid target, complementary sequence, or template strand.

[0194] Potentially stabilizing modifications to the ends of RNA molecules include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"phosphate, inverted base dT (idT), and others. Disclosure of this modification can be found in PCT Publication No. WO2011 / 005861.

[0195] Other modifications of the RNAi agent of the present disclosure include 5' phosphate or 5' phosphate mimic, for example, the 5' terminal phosphate or phosphate mimic on the antisense strand of RNAi agent.Suitable phosphate mimic is disclosed for the method provided herein in, for example, US2012 / 0157511, the content of which is incorporated herein by reference.

[0196] 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 for the methods provided herein in WO2013 / 075035, the contents of which are incorporated herein by reference.As shown herein and in WO2013 / 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 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 disrupts the modification pattern of the sense or antisense strand, if present.The RNAi agent can also be conjugated with a lipophilic moiety or ligand, for example, a C16 moiety or ligand on the sense strand.The RNAi agent can also be modified, for example, with an (S)-glycol nucleic acid (GNA) modification at one or more residues of the antisense strand.The resulting RNAi agent exhibits excellent gene silencing activity.

[0197] In some embodiments, the sense strand sequence has formula (I): 5'n p -N a -(XXX) i -N b -YYY -N b -(ZZZ) j -N a -n q 3' (I) [In the formula, i and j each independently represent 0 or 1; p and q each independently represent 0 to 6; each N a independently represent oligonucleotide sequences comprising 0 to 25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each N b independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p and n qindependently represent an overhanging nucleotide; 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.

[0198] In some embodiments, N a and / or N b includes alternating pattern modifications.

[0199] In some embodiments, the YYY motif occurs at or near the cleavage site of the sense strand. For example, if the RNAi agent has a duplex region 17-23 nucleotides in length, the YYY motif can occur at or near the cleavage site of the sense strand (e.g., can occur at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13), and the numbers start from the first nucleotide from the 5' end, or, optionally, the numbers can start from the first paired nucleotide within the duplex region from the 5' end.

[0200] 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' n p -N a -YYY-N b -ZZZ-N a -n q 3' (Ib), 5' n p -N a -XXX-N b -YYY-N a -n q 3' (Ic), or 5' n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q3' (Id) It can be expressed as:

[0201] When the sense strand is represented by formula (Ib), N b 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 can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0202] When the sense strand is represented by formula (Ic), N b 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 may independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0203] When the sense strand is represented by formula (Id), each N b independently represent an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6. Each N a may independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0204] Each of X, Y and Z may be the same as or different from one another.

[0205] In other embodiments, i is 0, 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:

[0206] When the sense strand is represented by formula (Ia), each N a may independently comprise an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0207] In some embodiments, the antisense strand sequence of the RNAi has the formula (II): 5' n q’ -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p '3' (II) [In the formula, k and l each independently represent 0 or 1; p' and q' each independently represent 0 to 6; each N a each independently represents an oligonucleotide sequence comprising 0 to 25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each N b 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, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one of three identical modifications on three consecutive nucleotides. It can be expressed as:

[0208] In some embodiments, N a ' and / or N b ' includes alternating pattern modifications.

[0209] The Y'Y'Y' motif occurs at or near the cleavage site of the sense strand. For example, if the RNAi agent has a duplex region 17-23 nucleotides in length, the Y'Y'Y' motif can occur at positions 9, 10, 11, 10, 11, 12, 11, 12, 13, 12, 13, 14, or 13, 14, 15 of the antisense strand, with the numbers starting from the first nucleotide from the 5' end, or, where appropriate, the numbers starting from the first paired nucleotide in the duplex region from the 5' end. In some embodiments, the Y'Y'Y' motif occurs at positions 11, 12, or 13.

[0210] In some embodiments, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0211] 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.

[0212] Thus, the antisense strand has the formula: 5' n q '-N a '-Z'Z'Z'-N b '-Y'Y'Y'-N a 'n p ' 3' (IIb), 5' n q '-N a '-Y'Y'Y'-N b '-X'X'X'-n p ' 3' (IIc), or 5' n q '-N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'-N a '-n p '3' (IId) It can be expressed as:

[0213] When the antisense strand is represented by formula (IIb), N b ’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 represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0214] When the antisense strand is represented by formula (IId), each 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 In some embodiments, N' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6.

[0215] In other embodiments, k is 0, l is 0, and the antisense strand has the formula: 5' n p’ -N a’ -Y'Y'Y'- N a’ -n q’ 3' (Ia) It can be expressed as:

[0216] When the antisense strand is represented by formula (IIa), each N a ' 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 as or different from one another.

[0218] 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.

[0219] In some embodiments, the sense strand of an RNAi agent may contain a YYY motif occurring at positions 9, 10, and 11 of the strand when the duplex region is 21 nt, the numbers starting from the first nucleotide from the 5' end, or, where appropriate, the numbers may start from the 5' end with the first paired nucleotide in the duplex region, and Y represents a 2'-F modification. The sense strand may further contain a XXX motif or a ZZZ motif as a wing modification at the opposite end of the duplex region, where XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0220] In some embodiments, the antisense strand may contain a Y'Y'Y' motif occurring at positions 11, 12, 13 of the strand, where the number starts from the first nucleotide from the 5' end, or, where appropriate, the number may start from the 5' end with the first paired nucleotide in the duplex region, and Y' represents a 2'-O-methyl modification. The antisense strand may further contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the duplex region, where X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0221] 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 (IIa), (IIb), (IIc) and (IId), respectively.

[0222] Thus, certain RNAi agents for use in the methods of the disclosure can include a sense strand and an antisense strand, each strand having 14-30 nucleotides, and the RNAi duplex is represented by formula (III): Sense: 5' n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3' n p ’ -N a ’ -(X'X'X') k -N b ’ -Y'Y'Y'-N b ’ -(Z'Z'Z') l -N a ’ -n q ’ 5' (III) [In the formula, i, j, k, and l each independently represent 0 or 1; p, p', q and q' each independently represent 0 to 6; each N a and N a ’ independently represent oligonucleotide sequences comprising 0 to 25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each N b and N b ’ independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p ',n p , n q ' and n q independently represent overhanging nucleotides, each of which may or may not be present; 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. is expressed by

[0223] 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.

[0224] An exemplary combination of sense and antisense strands that form an RNAi duplex has the following formula: 5' n p - N a -YYY -N a -n q 3' 3' n p ’ -N a ’ -Y'Y'Y' -N a ’ n q ’ 5' (IIIa) 5' n p -N a -YYY -N b -ZZZ -N a -n q 3' 3' n p ’ -N a ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a ’ n q ’ 5' (IIIb) 5' n p -N a -XXX-Nb -YYY-N a -n q 3' 3' n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N a ’ -n q ’ 5' (IIIc) 5' n p -N a -XXX -N b -YYY -N b - ZZZ -N a -n q 3' 3' n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a ’ -n q ’ 5' (IIId) Includes.

[0225] When the RNAi agent is represented by formula (IIIa), each N a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0226] When the RNAi agent is represented by formula (IIIb), 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.

[0227] When the RNAi agent is represented by formula (IIIc), 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 independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0228] When the RNAi agent is represented by formula (IIId), 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 a ’ independently represent 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 comprises an alternating pattern of modifications.

[0229] Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc) and (IIId) may be the same as or different from each other.

[0230] When an RNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides can base pair with one of the Y' nucleotides, alternatively, at least two of the Y nucleotides base pair with the corresponding Y' nucleotide, or all three of the Y nucleotides base pair with the corresponding Y' nucleotide.

[0231] When the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides can base pair with one of the Z' nucleotides, alternatively, at least two of the Z nucleotides base pair with the corresponding Z' nucleotide, or all three of the Z nucleotides base pair with the corresponding Z' nucleotide.

[0232] When the RNAi agent is represented by formula (IIIc) or (IIId), at least one of the X nucleotides can base pair with one of the X' nucleotides, alternatively, at least two of the X nucleotides base pair with the corresponding X' nucleotide, or all three of the X nucleotides base pair with the corresponding X' nucleotide.

[0233] 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.

[0234] In some embodiments, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification. In some embodiments, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0, and at least one np' is linked to an adjacent nucleotide by a phosphorothioate linkage. In some embodiments, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0, and at least one np' is linked to an adjacent nucleotide by 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 bivalent or trivalent branched linker. In some embodiments, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0, and at least one np' is linked to an adjacent nucleotide by a phosphorothioate linkage, the sense strand comprises at least one 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 bivalent or trivalent branched linker.

[0235] In some embodiments, when the RNAi agent is represented by formula (IIIa), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0, and at least one np' is linked to an adjacent nucleotide by a phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand comprises at least one 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 bivalent or trivalent branched linker.

[0236] In some embodiments, the RNAi agent is a multimer that contains at least two double strands represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), and the double strands are connected by a linker.The linker can be cleavable or non-cleavable.The multimer can further comprise a ligand.Each double strand can target the same gene, or can target two different genes, or each double strand can target the same gene at two different target sites.

[0237] In some embodiments, the RNAi agent is a multimer that contains 3, 4, 5, 6 or more double strands, as represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), and the double strands are connected by a linker.The linker may be cleavable or non-cleavable.The multimer may further comprise a ligand.Each double strand may target the same gene, or may target two different genes, or each double strand may target the same gene at two different target sites.

[0238] In some embodiments, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId) are linked to each other at the 5' end, and one or both of the 3' ends may be conjugated to a ligand. Each agent may target the same gene, or may target two different genes, or each agent may target the same gene at two different target sites.

[0239] Various publications describe the multimeric RNAi agent that can be used in the method of the present disclosure.Such publications include WO2007 / 091269, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520 and US7858769, 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.

[0240] As described in more detail below, RNAi agents containing one or more carbohydrate moieties conjugated to the RNAi agent can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety is attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced 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 (RRMS). The cyclic carrier can be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring structure, i.e., one or more ring atoms can be heteroatoms, such as nitrogen, oxygen, or sulfur. The cyclic carrier can be a monocyclic ring structure or can contain two or more rings, such as fused rings. The cyclic carrier can be a fully saturated ring structure or can contain one or more double bonds.

[0241] Ligands can be attached to polynucleotides via carriers. The carriers include (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 a bond available and suitable for incorporation of the carrier into a backbone, e.g., a phosphate or modified phosphate, e.g., sulfur-containing backbone, of a ribonucleic acid. "Tethering attachment point" (TAP) refers, in some embodiments, to a constituent ring atom, e.g., a carbon atom or heteroatom (separate from the atom providing the backbone attachment point), of a cyclic carrier that connects the selected moiety. The moiety can be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. The selected moiety may be connected to the cyclic carrier by an intervening tether. Thus, cyclic carriers often include a functional group, e.g., an amino group, or generally provide a bond suitable for incorporation or tethering another chemical entity, e.g., a ligand, to the constituent ring.

[0242] The RNAi agent may be conjugated to the ligand via a carrier, which can be a cyclic or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is selected from a serinol backbone or a diethanolamine backbone.

[0243] In certain specific embodiments, the RNAi agent for use in the method of the present disclosure is an agent selected from the group of agents listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A and 4B.These agents can further comprise ligand.Ligand can be attached to sense strand, antisense strand or both strands at 3' end, 5' end or both ends.For example, ligand can be conjugated to sense strand, particularly to the 3' end of sense strand.

[0244] iRNA conjugates The iRNA agent disclosed herein can be in the form of conjugate.Conjugate can be attached to any suitable position in iRNA molecule, for example, at the 3'-end or 5'-end of sense or antisense strand.Conjugate can also be attached via linker.

[0245] In some embodiments, the iRNA agents described herein are chemically linked to one or more ligands, moieties, or conjugates, which 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. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), and the like. 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 di-hexadecyl-rac-glycerol or triethyl-ammonium 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), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0246] In some embodiments, a ligand alters the distribution, targeting, or lifespan of an iRNA agent into which it is incorporated. In some embodiments, a ligand provides enhanced affinity for a selected target, such as a molecule, a cell or cell type, a compartment, such as a cellular or organ compartment, a tissue, an organ, or a region of the body, for example, compared to a species in which such a ligand is not present. Conventional ligands do not participate in duplex pairing in double-stranded nucleic acids.

[0247] 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-glycolized) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylamide), 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 alpha-helical peptides.

[0248] The ligand can also include a targeting group, such as a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid, or protein, for example, 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 acid, polyvalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, biotin, or RGD peptide or RGD peptide mimetic.

[0249] 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-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithium, and the like. Examples of suitable cleavage inhibitors include cleavage inhibitors (e.g., 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.

[0250] The ligand can be a protein, such as a glycoprotein or peptide, a molecule with specific affinity for a co-ligand, or an antibody, such as an antibody that binds to a specific cell type, such as an ocular cell. 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, p38 MAP kinase activators, or NF-κB activators.

[0251] The ligand can be a substance, e.g., a drug, that can increase cellular uptake of an iRNA agent, for example, by disrupting the cytoskeleton of a 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.

[0252] In some embodiments, the ligand attached to the iRNA described herein acts as a pharmacokinetic modulator (PK modulator). 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 are also known to bind to serum proteins. Therefore, short oligonucleotides, e.g., oligonucleotides of about 5, 10, 15, or 20 bases containing multiple phosphorothioate linkages in the backbone, are also suitable as ligands (e.g., as PK-modulating ligands) for the present disclosure. Furthermore, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0253] Ligand-conjugated oligonucleotides of the present disclosure can be synthesized by using oligonucleotides bearing pendant reactive functionality, e.g., resulting from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide can be reacted directly with commercially available ligands, ligands that have been synthesized with any of a variety of protecting groups, or ligands that have a linking moiety attached to them.

[0254] The oligonucleotides used in the conjugates of the present disclosure can be conveniently and routinely produced by known techniques 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 also or alternatively be used.It is also known to use similar techniques to prepare other oligonucleotides, for example, phosphorothioates and alkylated derivatives.

[0255] For the ligand-conjugated oligonucleotides and ligand molecules having sequence-specific linked nucleosides of the present disclosure, the oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer using standard nucleotide or nucleoside precursors or nucleotide or nucleoside conjugate precursors that already have a linking moiety, ligand-nucleotide or nucleoside conjugate precursors that already have a ligand molecule, or non-nucleoside ligands that have building blocks.

[0256] When using a nucleotide-conjugate precursor that already has a linking moiety, the synthesis of the sequence-specific linked nucleoside is usually completed, and then a ligand molecule is reacted with the linking moiety to form a 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 standard and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

[0257] A. Lipophilic part In certain embodiments, the lipophilic moiety is an aliphatic, such as an alicyclic, cyclic, or polycyclic, such as a polyalicyclic, compound, such as a steroid (e.g., a sterol) or a straight-chain or branched aliphatic hydrocarbon. 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, but are not limited to, saturated or unsaturated C4-C6 30 Hydrocarbons (e.g., C6-C 18 hydrocarbons), saturated or unsaturated fatty acids, waxes (e.g., monohydric alcohol esters of fatty acids and fatty diamides), terpenes (e.g., C 10 Terpene, C 15 Sesquiterpene, C 20 Diterpenes, C 30 Triterpenes and C 40 tetraterpenes) and other polyalicyclic hydrocarbons. For example, the lipophilic moiety is C4-C 30 Hydrocarbon chains (e.g., C4 to C 30 In some embodiments, the lipophilic moiety may contain a saturated or unsaturated C-C 18 Hydrocarbon chains (e.g., straight chain C6-C 18 In some embodiments, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain (e.g., a straight-chain C16 alkyl or alkenyl).

[0258] Lipophilic moieties can be attached to RNAi agents by any method known in the art via functional groups already present in the lipophilic moiety or introduced into the RNAi agent, such as hydroxyl groups (e.g., -CO-CH2-OH). Functional groups already present in 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.

[0259] Conjugation of an RNAi agent and a lipophilic moiety can occur, for example, by forming an ether or carboxy 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, 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.

[0260] In some embodiments, the lipophilic moiety is conjugated to the double-stranded RNAi agent via a linker that is an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, the product of a click reaction (e.g., a triazole derived from an azide-alkyne cycloaddition), or a carbamate-containing linker.

[0261] In another embodiment, the lipophilic moiety is a steroid, for example, a sterol.Steroids are polycyclic compounds containing a perhydro-1,2-cyclopentanophenanthrene ring structure.Steroids include, but are not limited to, bile acids (e.g., cholic acid, deoxycholic acid, and dehydrocholic acid), cortisone, digoxigenin, testosterone, cholesterol, and cationic steroids, for example, cortisone."Cholesterol derivative" refers to a compound derived from cholesterol, for example, by substitution, addition or removal of a substituent.

[0262] In another embodiment, the lipophilic moiety is an aromatic moiety. In this context, the term "aromatic" broadly refers to monocyclic and polycyclic aromatic hydrocarbons. Aromatic groups include, but are not limited to, C6-C8 aromatic groups containing 1-3 aromatic rings, which may be optionally substituted. 14These include aryl moieties; "aralkyl" or "arylalkyl" groups, which comprise an aryl group covalently linked to an alkyl group, either of which may independently 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; having 6, 10, or 14 pi-electrons shared in the cyclic array; and having, in addition to carbon atoms, from 1 to about 3 heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and sulfur (S).

[0263] As used herein, a "substituted" alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclic group is one having 1 to about 4, preferably 1 to about 3, and more preferably 1 or 2 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.

[0264] In some embodiments, the lipophilic moiety is an aralkyl group, for example, 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-macroglobulin, or α-1-glycoprotein.

[0265] In certain embodiments, the ligand is naproxen or a structural derivative of naproxen. Procedures for the synthesis of naproxen can be found in U.S. Patent Nos. 3,904,682 and 4,009,197, the entire contents of which are incorporated herein by reference. Naproxen has the chemical name (S)-6-methoxy-α-methyl-2-naphthaleneacetic acid and the structure:

[0266] [ka]

[0267] In certain embodiments, the ligand is ibuprofen or a structural derivative of ibuprofen. Procedures for the synthesis of ibuprofen can be found in US 3,228,831, which is incorporated herein by reference for the methods provided herein. The structure of ibuprofen is:

[0268] [ka]

[0269] Further exemplary aralkyl groups are exemplified in US Pat. No. 7,626,014, which is incorporated herein by reference for the purposes provided herein.

[0270] 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.

[0271] In certain embodiments, more than one lipophilic moiety 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, each strand of a double-stranded RNAi agent has one or more lipophilic moieties incorporated therein.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 internucleoside linkage).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 using one or more linkers that connect the lipophilic moieties consecutively.

[0272] The lipophilic moiety can be conjugated to the RNAi agent by direct attachment 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.

[0273] In certain embodiments, the lipophilic moiety can be conjugated to the RNAi agent via one or more linkers (tethers).

[0274] In some embodiments, the lipophilic moiety is conjugated to the double-stranded RNAi agent via a linker that is an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, the product of a click reaction (e.g., a triazole derived from an azide-alkyne cycloaddition), or a carbamate-containing linker.

[0275] B. Lipid Conjugates In some embodiments, the ligand is a lipid or lipid-based molecule. Such lipid or lipid-based molecule can usually bind to serum protein, for example, human serum albumin (HSA). HSA-binding ligand allows vascular distribution of the conjugate to target tissue. For example, the target tissue may be the eye. Other molecules that can bind to 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 protein, for example, HSA.

[0276] Lipid-based ligands can be used to modulate, e.g., manage (e.g., inhibit), the binding of the conjugate to the target tissue. For example, lipids or lipid-based ligands that bind more strongly to HSA are less likely to be targeted to the kidney and therefore less likely to be eliminated from the body. Lipids or lipid-based ligands that bind less strongly to HSA can be used to target the conjugate to the kidney.

[0277] In some embodiments, lipid-based ligand binds to HSA.For example, the ligand can bind to HSA with sufficient affinity, so that the distribution of conjugate to non-renal tissue is enhanced.However, the affinity is usually not so strong that HSA-ligand binding cannot be reversed.

[0278] In some embodiments, the lipid-based ligand binds weakly or not at all to HSA, thereby enhancing 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.

[0279] In another embodiment, the ligand is a moiety, such as 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 types, such as cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, such as 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).

[0280] Cell-penetrating agents In another aspect, the ligand is a cell-penetrating agent, for example, a helical cell-penetrating agent. In some embodiments, these cell-penetrating agents are amphipathic. Exemplary cell-penetrating agents include peptides, such as tat or antennopedia. When the agent is a peptide, it may be modified, including peptidyl mimetics, invertomers, non-peptide or pseudopeptide linkages, and the use of D-amino acids. Helical agents are typically α-helical agents and may have a lipophilic and lipophobic phase.

[0281] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules capable of folding 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 moiety 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.

[0282] 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. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 3438). A hydrophobic MTS-containing RFGF analog (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 3439)) 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, the sequence derived from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 3440)) and the sequence derived from the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 3441)) have been found to be functionalizable 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). Typically, peptides or peptidomimetics tethered to dsRNA agents via incorporated monomer units include cell-targeting peptides, such as arginine-glycine-aspartic acid (RGD) peptides or RGD mimics. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications, for example, to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

[0283] 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 tissue(s).RGD-containing peptides and peptidomimetics can include D-amino acids and synthetic RGD mimics.In addition to RGD, other moieties can be used to target integrin ligands.In some embodiments, the conjugate of this ligand targets PECAM-1 or VEGF.

[0284] 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 facilitate 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 facilitate targeting of iRNA agents to the kidney. RGD peptides can be linear or cyclic, and can be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). For example, glycosylated RGD peptides can facilitate targeting of iRNA agents to α- V It can be delivered to tumor cells that express β3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

[0285] A "cell-penetrating peptide" is capable of penetrating cells, such as microbial cells, e.g., bacterial or fungal cells, or mammalian cells, e.g., human cells. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or seropin 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 bisected 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).

[0286] Carbohydrate Conjugates and Ligands In some embodiments of the compositions and methods of the present disclosure, the iRNA oligonucleotide further comprises a carbohydrate. Carbohydrate-conjugated iRNA is advantageous for in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic use, as described herein. As used herein, "carbohydrate" refers to a compound that is either a carbohydrate itself, composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom, or a compound that has as part thereof a carbohydrate moiety composed of one or more monosaccharide units, each having at least six carbon atoms (which may be linear, branched, or cyclic), with an oxygen, nitrogen, or sulfur atom bonded 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 gums. Particular monosaccharides include sugars of C5 or greater (e.g., C5, C6, C7, or C8), and disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0287] In certain embodiments, the compositions and methods of the present disclosure comprise a C16 ligand.In an exemplary embodiment, the C16 ligand of the present disclosure has the following structure (exemplified herein below for a uracil base, but the attachment of a C16 ligand to any base (C, G, A, etc.) or any other modified nucleotide as provided herein is contemplated, as long as the 2' ribonucleotide attachment is maintained), and is attached to the 2' position of the ribonucleotide in the residue that is modified in this way:

[0288] [ka]

[0289] As shown above, the C16 ligand modified residue exhibits a linear alkyl at the 2'-ribo position of the exemplary residue being modified (herein uracil).

[0290] In some embodiments, the carbohydrate conjugate of an RNAi agent of the present disclosure further comprises one or more additional ligands as described above, for example, but not limited to, a PK modulator or a cell-penetrating peptide.

[0291] Additional carbohydrate conjugates (and linkers) suitable for use in the present disclosure include those described in WO2014 / 179620 and WO2014 / 179627, which are incorporated herein by reference.

[0292] In certain embodiments, the compositions and methods of the present disclosure include vinyl phosphonate (VP) modifications of RNAi agents as described herein. In exemplary embodiments, the vinyl phosphonates of the present disclosure have the following structure:

[0293] [ka] It has.

[0294] 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 attached to the antisense strand of the dsRNA at the 5' end of the antisense strand of the dsRNA, as appropriate.

[0295] Vinyl phosphate modifications are also contemplated for the compositions and methods of the present disclosure. Exemplary vinyl phosphate structures include:

[0296] [ka] There is.

[0297] 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 serves as a ligand that targets iRNA to specific cells. In some embodiments, the GalNAc conjugate targets iRNA to liver cells, for example, by serving as a ligand for the asialoglycoprotein receptor of liver cells (e.g., hepatocytes).

[0298] In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivative can be attached via a linker, for example, 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, for example, a linker as described herein.

[0299] In some embodiments, the GalNAc conjugate is

[0300] [ka] Formula II

[0301] In some embodiments, the RNAi agent has the following schematic representation, where X is O or S:

[0302] [ka] The carbohydrate conjugate is attached via a linker as shown in

[0303] In some embodiments, the RNAi agent is conjugated to L96, as defined in Table 1, as shown below:

[0304] [ka]

[0305] In some embodiments, the carbohydrate conjugate for use in the compositions and methods of the present disclosure is selected from the group consisting of:

[0306] [ka] Formula II, [ka] [ka] [ka] [ka] [ka] Formula XXII,

[0307] Other exemplary carbohydrate conjugates for use in the embodiments described herein include, but are not limited to, the following:

[0308] [ka] (Formula XXIII) wherein one of X or Y is an oligonucleotide and the other is hydrogen.

[0309] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, for example, but not limited to, a PK modulator or a cell-penetrating peptide.

[0310] In some embodiments, the iRNA of the present disclosure is conjugated to a carbohydrate via a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the present disclosure include, but are not limited to, the following, where one of X or Y is an oligonucleotide and the other is hydrogen:

[0311] [ka] (formula XXIV), [ka] , and [ka] (Formula XXX)

[0312] E. Thermal destabilization modification In certain embodiments, dsRNA molecules can be optimized for RNA interference by incorporating a thermally destabilizing modification into the seed region of the antisense strand (i.e., positions 2-9 at 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 duplex modification 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., 1, 2, 3, 4, 5, or more) thermally destabilizing duplex modification within the first 9 nucleotide positions of the 5' region of the antisense strand. In some embodiments, one or more thermally destabilizing duplex modifications are located in positions 2-9, or preferably positions 4-8, from the 5' end of the antisense strand. In some further embodiments, the thermally destabilizing duplex modification(s) are located at positions 6, 7, or 8 from the 5' end of the antisense strand. In still 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(s)" includes modification(s) that will result in a dsRNA having a lower overall melting temperature (Tm) (preferably 1, 2, 3, or 4 degrees lower than the Tm of a dsRNA without such modification(s). 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.

[0313] Thermally destabilizing modifications can include, but are not limited to, abasic modifications, mismatches with the opposing nucleotide 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).

[0314] Exemplary abasic modifications include, but are not limited to, the following:

[0315] [ka] wherein R=H, Me, Et, or OMe; R′=H, Me, Et, or OMe; and R″=H, Me, Et, or OMe.

[0316] [ka] wherein B is a modified or unmodified nucleobase. Examples include:

[0317] Exemplary sugar modifications include, but are not limited to, the following:

[0318] [ka] [ka] wherein B is a modified or unmodified nucleobase. Examples include:

[0319] In some embodiments, the thermally destabilizing modification of the duplex is one of the following:

[0320] [ka] where B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic. is selected from the group consisting of:

[0321] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, e.g., in which any of the bonds between the ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') are 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

[0322] [ka] or [ka] wherein B is a modified or unmodified nucleobase and R 1 and R 2 are independently H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. The term "UNA" refers to an unlocked acyclic nucleic acid in which one of the sugar bonds has been removed to form an unlocked "sugar" residue. In one example, UNA also encompasses a monomer 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 covalent carbon-carbon bond between the C2' and C3' carbons) has been removed [see Mikhailov et al., Tetrahedron Letters, 26 (17): 2059 (1985) and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), which are incorporated herein by reference in their entireties]. Acyclic derivatives offer greater backbone flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be linked via 2'-5' or 3'-5' linkages.

[0323] The term "GNA" refers to glycol nucleic acid, a polymer similar to DNA or RNA, but differing in the composition of its "backbone" in that it is made up of repeating glycerol units linked by phosphodiester bonds:

[0324] [ka]

[0325] 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 suitable for the present invention.Mismatches can occur between nucleotides that are either naturally occurring or modified nucleotides, that is, mismatch base pairing can occur between the nucleobases derived from each nucleotide independently of the modification on the ribose sugar of the nucleotide.In certain embodiments, the dsRNA molecule contains at least one nucleobase in mismatch pairing that is a 2'-deoxynucleobase, for example, the 2'-deoxynucleobase is in the sense strand.

[0326] In some embodiments, the duplex thermally destabilizing modification in the seed region of the antisense strand is a nucleotide that has impaired WCH bonding with the complementary base on the target mRNA, such as:

[0327] [ka] Includes.

[0328] Many more examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA) and mismatch modifications are described in detail in WO2011 / 133876, which is incorporated herein by reference in its entirety.

[0329] Thermally destabilizing modifications can also include universal base and phosphate modifications that have reduced or eliminated ability to form hydrogen bonds with opposing bases.

[0330] In some embodiments, the thermal destabilizing modification of duplex comprises the nucleotide with non-canonical 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 duplex, as described in WO2010 / 0011895, the entirety of which is incorporated herein by reference.Exemplary nucleobase modifications include:

[0331] [ka] There is.

[0332] In some embodiments, the duplex thermally destabilizing modifications 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 the following:

[0333] [ka] wherein R is H, OH, OCH, F, NH, NHMe, NMe, or O-alkyl. Includes:

[0334] Exemplary phosphate modifications known to reduce the thermal stability of dsRNA duplexes compared to native phosphodiester linkages include:

[0335] [ka] R=alkyl There is.

[0336] 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.

[0337] 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, for introducing destabilizing modifications into the RNAi agent of the present disclosure, but the range of modifications that can be used and generally exist on the RNAi agent of the present disclosure tends to be greater for non-nucleobase modifications, for example, modifications to the sugar group or phosphate backbone of polyribonucleotide.Such modifications are described in more detail in other sections of this disclosure, and are expressly intended for the RNAi agent of the present disclosure that has either natural nucleobase or modified nucleobase, as described above or elsewhere herein.

[0338] In addition to the antisense strand that contains thermal destabilizing modifications, dsRNA can also contain one or more stabilizing modifications.For example, dsRNA can contain at least two (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) stabilizing modifications.Without being limited, all stabilizing modifications can be present in one strand.In some embodiments, both sense and antisense strands contain at least two stabilizing modifications.Stabilizing modifications can occur at any nucleotide of sense strand or antisense strand.For example, stabilizing modifications can occur at any nucleotide on sense strand or antisense strand, and each stabilizing modification can occur in an alternating pattern on sense strand or antisense strand, or both sense strand and antisense strand contain stabilizing modifications in an alternating pattern.The alternating pattern of stabilizing modifications on sense strand can be the same or different from that of antisense strand, and the alternating pattern of stabilizing modifications on sense strand can have a shift compared to the alternating pattern of stabilizing modifications on antisense strand.

[0339] In some embodiments, the antisense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) stabilizing modifications. Without limitation, the stabilizing modifications in the antisense strand can be located at any position.

[0340] 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.

[0341] In some embodiments, antisense strand comprises at least one stabilizing modification adjacent to destabilizing modification.For example, stabilizing modification can be at the 5'-end or 3'-end of destabilizing modification, that is, at the nucleotide of position -1 or +1 from the position of destabilizing modification.In some embodiments, antisense strand comprises stabilizing modification at each of the 5'-end and 3'-end of destabilizing modification, that is, at the nucleotide of position -1 and +1 from the position of destabilizing modification.

[0342] 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.

[0343] In some embodiments, the sense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) stabilizing modifications. Without limitation, the stabilizing modifications in 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.

[0344] 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.

[0345] Exemplary thermally stabilizing modifications include, but are not limited to, 2'-fluoro modifications. Other thermally stabilizing modifications include, but are not limited to, LNA.

[0346] In some embodiments, the dsRNA of the present disclosure comprises at least four (for example, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoro nucleotides. Without being limited thereto, all 2'-fluoro nucleotides may be present in one strand. In some embodiments, both sense and antisense strands comprise at least two 2'-fluoro nucleotides. 2'-fluoro modification can occur at any nucleotide of sense strand or antisense strand. For example, 2'-fluoro modification can occur at any nucleotide on sense strand or antisense strand, and each 2'-fluoro modification can occur in an alternating pattern on sense strand or antisense strand, or both sense strand and antisense strand contain 2'-fluoro modification in an alternating pattern. The alternating pattern of 2'-fluoro modification on sense strand can be the same or different from that of antisense strand, and the alternating pattern of 2'-fluoro modification on sense strand can have a shift compared to the alternating pattern of 2'-fluoro modification on antisense strand.

[0347] In some embodiments, the antisense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 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.

[0348] In some embodiments, antisense strand comprises at least one 2'-fluoro nucleotide adjacent to destabilizing modification.For example, 2'-fluoro nucleotide can be at the 5' end or 3' end of destabilizing modification, that is, at the nucleotide of position -1 or +1 from the position of destabilizing modification.In some embodiments, antisense strand comprises 2'-fluoro nucleotide at each of the 5' end and 3' end of destabilizing modification, that is, at the position -1 and +1 from the position of destabilizing modification.

[0349] 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.

[0350] In some embodiments, the sense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modifications in 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 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 2, 3 or 4 blocks of 2'-fluoro nucleotides.

[0351] In some embodiments, the sense strand does not contain 2'-fluoro nucleotides at positions opposite or complementary to thermally destabilizing modifications of the duplex in the antisense strand.

[0352] In some embodiments, a dsRNA molecule of the present 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 in 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 and the other end comprises a 2-nt overhang, and the dsRNA further has at least one (e.g., 1, 2, 3, 4, 5, 6, or all 7) of the following features: The dsRNA may comprise: (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.

[0353] 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, which can include one or more of non-linked phosphate oxygen or one or more of linking phosphate oxygen, one or both of the modifications of ribose sugar components, for example, the 2' hydroxyl on ribose sugar, the large-scale replacement of phosphate moiety with " dephosphorylation " linker, the modification or replacement of naturally occurring base, and the replacement or modification of ribose-phosphate backbone.

[0354] 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 non-linked Os at 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' or 5' terminal position, or only in the terminal region, for example, at the terminal nucleotide position of the chain, or at the last 2, 3, 4, 5, or 10 nucleotides. 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 modifications at non-linked O positions can occur only at one or both ends, or only in the terminal region, for example, 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 or both ends can be phosphorylated.

[0355] For example, it may be possible to 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' or 3' overhang, or both.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' or 5' overhang can be modified, for example, with the modifications described herein.Modifications can 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 nucleic acid base, and modifications at the phosphate group, for example, phosphorothioate modifications.The overhang does not need to be homologous to the target sequence.

[0356] In some embodiments, each residue of sense strand and 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.Strands can contain two or more modifications.In some embodiments, each residue of sense strand and 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 double strand that exists in antisense strand.

[0357] At least two different modifications are usually present on the sense strand and the antisense strand. These two 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 of the sense strand and the antisense strand is independently modified with 2'-O-methyl nucleotides, 2'-deoxy nucleotides, 2'-deoxy-2'-fluoro nucleotides, 2'-ON-methylacetamide (2'-O-NMA) nucleotides, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotides, 2'-O-aminopropyl (2'-O-AP) nucleotides, or 2'-ara-F nucleotides. 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.

[0358] In some embodiments, the dsRNA molecules of the present disclosure comprise alternating patterns 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 with one or more modifications, where each modification occurs at alternating nucleotides in a single strand. Alternating nucleotides may refer to one every other nucleotide or one every third nucleotide, or similar patterns. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif may be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AABBBAAABBB...", or "ABCABCABCABC...", etc.

[0359] The types of modifications contained within the alternating motifs can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternating turns, 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 possibilities for modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD..." or "CDCDCD...".

[0360] In some embodiments, the dsRNA molecules of the present disclosure comprise 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 the modified groups of the nucleotides of the sense strand correspond to the differently modified groups of the nucleotides of the antisense strand, or vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, the alternating motif in the sense strand may begin with "ABABAB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand may begin with "BABABA" from the 3'-5' end of the strand within the duplex region. As another example, the alternating motif in the sense strand may begin with "AABBAABB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand may begin with "BBAABBAA" from the 3'-5' end of the strand within the duplex region, resulting in a complete or partial shift in the modification pattern between the sense strand and the antisense strand.

[0361] 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 at any nucleotide of sense strand or antisense strand or both at any position of chain.For example, internucleotide linkage modification can occur at any nucleotide on sense strand or antisense strand, and 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 have a shift with respect to the alternating pattern of internucleotide linkage modification on antisense strand.

[0362] In some embodiments, dsRNA molecule comprises phosphorothioate or methylphosphonate internucleotide linkage modification in overhang region.For example, overhang region comprises two nucleotides with phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides.Internucleotide linkage modification can also be performed to connect overhang nucleotide with the terminal pairing nucleotide in duplex region.For example, at least 2, 3, 4 or all overhang nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkage, and there can be additional phosphorothioate or methylphosphonate internucleotide linkage that connects overhang nucleotide with the nucleotide that is adjacent to the overhang nucleotide.For example, there can be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of which are overhang nucleotides, and the third is the nucleotide that is adjacent to the overhang nucleotide that is adjacent to the overhang nucleotide.Preferably, these terminal three nucleotides can be the 3' end of antisense strand.

[0363] In some embodiments, the sense strand of the dsRNA molecule comprises 1 to 10 blocks of 2 to 10 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, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is positioned at any position in the oligonucleotide sequence, and the sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0364] In some embodiments, the antisense strand of the 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 located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages and an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0365] 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 the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0366] 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 the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0367] In some embodiments, the antisense strand of the dsRNA molecule comprises 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 the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate linkages.

[0368] In some embodiments, the antisense strand of the dsRNA molecule comprises 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 the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0369] In some embodiments, the antisense strand of the dsRNA molecule comprises 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 the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0370] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, or 6 phosphate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0371] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by one, two, three, or four phosphate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate linkages.

[0372] In some embodiments, dsRNA molecules of the present disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within the terminal positions of positions 1 through 10 of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkages at one or both ends of the sense or antisense strand.

[0373] 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 internal region of the duplex of each of the sense or antisense strands. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides can be linked by phosphorothioate methylphosphonate internucleotide linkages at positions 8-16 of the duplex region, counting from the 5' end of the sense strand. The dsRNA molecule can further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within the terminal positions of positions 1-10.

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

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

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

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

[0378] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 (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 within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 (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 within positions 18-23 (counting from the 5' end) of the antisense strand.

[0380] In some embodiments, dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 and one within positions 18-23 (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 positions 18-23 (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 within positions 1-5 of the sense strand (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5' end).

[0382] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications (counting from the 5' end) within positions 1-5 of the sense strand and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications (counting from the 5' end) within positions 18-23 of the antisense strand.

[0383] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and one within positions 18-23 (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 positions 18-23 (counting from the 5' end) of the antisense strand.

[0384] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 (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 within positions 18-23 (counting from the 5' end) of the antisense strand.

[0385] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 (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 18-23 (counting from the 5' end) of the antisense strand.

[0386] In some embodiments, dsRNA molecules of the present disclosure further comprise 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 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.

[0387] In some embodiments, dsRNA molecules of the present 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.

[0388] In some embodiments, dsRNA molecules of the present disclosure further comprise 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 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.

[0389] In some embodiments, dsRNA molecules of the present 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.

[0390] In some embodiments, dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 22 and 23 (counting from the 5' end) 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.

[0391] In some embodiments, dsRNA molecules of the present 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.

[0392] In some embodiments, the compounds of the present disclosure comprise a pattern of backbone chiral centers. In some embodiments, the general pattern of backbone chiral centers comprises at least 5 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 6 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 7 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 8 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 9 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 16 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 17 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 18 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 19 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises no more than 8 internucleotide linkages in the Rp configuration.In some embodiments, the general pattern of backbone chiral centers comprises seven or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises six or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises five or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises four or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises three or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises two or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises one or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises eight or fewer non-chiral internucleotide linkages (phosphodiesters as a non-limiting example). In some embodiments, the general pattern of backbone chiral centers comprises seven or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises six or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises five or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises four or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises three or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises two or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises one or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration and eight or fewer non-chiral internucleotide linkages.In some embodiments, the general pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration and no more than 7 nonchiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration and no more than 6 nonchiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration and no more than 6 nonchiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration and no more than 5 nonchiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration and no more than 4 nonchiral internucleotide linkages. In some embodiments, the internucleotide linkages in the Sp configuration may be contiguous or noncontiguous. In some embodiments, the internucleotide linkages in the Rp configuration may be contiguous or noncontiguous. In some embodiments, the nonchiral internucleotide linkages may be contiguous or noncontiguous.

[0393] 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 do not include Sp blocks. In some embodiments, provided oligonucleotides include one or more Sp blocks but do not include Rp blocks. In some embodiments, provided oligonucleotides include one or more PO blocks, in which each internucleotide linkage is a natural phosphate linkage.

[0394] 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 includes a 2'-F modification. In some embodiments, the 3'-block includes 4 or more nucleoside units. In some embodiments, the 3'-block includes 5 or more nucleoside units. In some embodiments, the 3'-block includes 6 or more nucleoside units. In some embodiments, the 3'-block includes 7 or more nucleoside units.

[0395] In some embodiments, compounds of the disclosure include a certain type of nucleoside in a region, or an oligonucleotide is followed by a particular type of internucleotide linkage, e.g., 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.

[0396] In some embodiments, the dsRNA molecule of the present disclosure comprises a mismatch (or mismatches) or combinations thereof within the double strand with the target. Mismatches can occur in the overhang region or the double-stranded region. Base pairs can be ranked based on their tendency to promote dissociation or melting (for example, the free energy of association or dissociation of a particular pairing; the simplest approach is to examine pairs on an individual basis, but next-neighbor analysis or similar analysis can also be used). In terms of promoting dissociation, A:U is preferred over G:C, G:U is preferred over G:C, and I:C is preferred over G:C (I=inosine). Mismatches, such as non-canonical pairings or those other than canonical pairings (as described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings, and pairings involving universal bases are preferred over canonical pairings.

[0397] In some embodiments, the dsRNA molecules of the present disclosure comprise at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex region from the 5' end of the antisense strand, which can be independently selected from the group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical or other than canonical pairings or pairings including universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0398] 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 1, 2 or 3 base pairs in the double-stranded region from the 5' end of the antisense strand is an AU base pair.For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0399] 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, thus protecting it from nucleases and stabilizing it.

[0400] 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 is 5'-methyl nucleoside.5'-methyl can be racemic or chirally pure R or S isomer.

[0401] 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 5' 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 group at 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.

[0402] In some embodiments, 5'-alkylated nucleoside is introduced at any position of the sense strand or antisense strand of dsRNA, and this modification maintains or improves the efficacy of dsRNA.5'-alkyl can be either racemic or chirally pure R or S isomer.Exemplary 5'-alkylated nucleoside is 5'-methyl nucleoside.5'-methyl can be either racemic or chirally pure R or S isomer.

[0403] In some embodiments, 4'-alkylated nucleoside is introduced at any position of the sense strand or antisense strand of dsRNA, and this modification maintains or improves the efficacy of dsRNA.4'-alkyl can be either racemic or chirally pure R or S isomer.Exemplary 4'-alkylated nucleoside is 4'-methyl nucleoside.4'-methyl can be either racemic or chirally pure R or S isomer.

[0404] In some embodiments, 4'-O-alkylated nucleosides are introduced at any position of the sense strand or antisense strand of dsRNA, and such modifications maintain or improve the efficacy of dsRNA.5'-Alkyl can be either racemic or chirally pure R or S isomer.Exemplary 4'-O-alkylated nucleosides include 4'-O-methyl nucleosides.4'-O-methyl can be either racemic or chirally pure R or S isomer.

[0405] In some embodiments, the dsRNA molecules of the present disclosure may contain 2'-5' linkages (having 2'-H, 2'-OH, and 2'-OMe, with P=O or P=S). For example, 2'-5' linkage modifications can be used to promote nuclease resistance or inhibit binding of the sense strand to the antisense strand, or can be used at the 5' end of the sense strand to prevent sense strand activation by RISC.

[0406] In another embodiment, the dsRNA molecules of the present disclosure can contain L sugars (e.g., L-ribose, L-arabinose, with 2'-H, 2'-OH and 2'-OMe).For example, these L sugar modifications can be used to promote nuclease resistance or inhibit binding of the sense strand to the antisense strand, or can be used at the 5' end of the sense strand to prevent sense strand activation by RISC.

[0407] Various publications have described multimeric siRNA, and all of them can be used with the dsRNA of the present disclosure.Such publications include WO2007 / 091269, US7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520, all of which are incorporated herein in their entirety.

[0408] In some embodiments, the dsRNA molecules of the present disclosure are 5' phosphorylated or contain a phosphoryl analog at the 5' prime end. 5' phosphate modifications include those compatible with RISC-mediated gene silencing. Suitable modifications include 5'-monophosphate ((HO)2(O)PO-5'), 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'), 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'), 5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)PO -(HO)(O)POP(HO)(O)-O-5'), 5'-adenosine cap (Appp) and any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'), 5'-monothiophosphate (phosphorothioate, (HO)2(S)PO-5'), 5'-monodithiophosphate (phosphorodithioate, (HO)(HS) (S)PO-5'), 5'-phosphorothiolates ((HO)2(O)PS-5'), any further combination of oxygen / sulfur substituted monophosphates, diphosphates and triphosphates (e.g., 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidites ((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 modifications can be placed in the antisense strand of the dsRNA molecule.

[0409] 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.

[0410] A linker is typically 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 group including, but not limited to, a 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, alkylheteroarylalkynyl, alkylheteroarylalkynyl

[0044] In some embodiments, R8 is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc.In some embodiments, the linker is about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.

[0411] 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):

[0412] [ka] [In the formula, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C independently for each occurrence 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, independently for each occurrence, absent, CO, NH, O, S, OC(O), NHC(O), CH, CHNH, or CHO; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C is, independently for each occurrence, absent, alkylene, substituted alkylene, and one or more methylenes are 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 is independently for each occurrence 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,

[0413] [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 independently for each occurrence, a monosaccharide (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; R a is H or an amino acid side chain].

[0414] Of formula (XXXV):

[0415] [ka] Trivalent conjugating GalNAc derivatives such as are particularly useful for use with RNAi agents to inhibit expression of target genes. [In the formula, L 5A , L 5B and L 5C represents a monosaccharide, e.g., a GalNAc derivative]

[0416] Examples of suitable bivalent and trivalent branched linker groups for conjugating GalNAc derivatives include, but are not limited to, the structures listed above, such as Formulas II, VII, XI, X, and XIII.

[0417] A cleavable linking group is one that is sufficiently stable outside a cell, but that, upon entry into 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 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 a first reference condition (which may, for example, be selected to mimic or represent intracellular conditions) than in the subject's blood or under a second reference condition (which may be selected to mimic or represent conditions found in blood or serum).

[0418] Cleavable linking groups are susceptible to the influence of cleaving agents, such as pH, redox potential, or the presence of degradable molecules.Generally, cleaving agents are more common or found at higher levels or activity inside cells than in serum or blood.Examples of such degrading agents include redox agents that are selected for specific substrates or do not have substrate specificity, such as oxidizing enzymes or reductases or reducing agents present in cells, such as mercaptans, which can degrade redox-cleavable linking groups by reduction, esterases, endosomes, or agents that can create an acidic environment, such as those that produce a pH of 5 or less, general acids, peptidases (can be substrate-specific), and enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as phosphatases.

[0419] 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 approximately 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 will have a cleavable linking group that is cleaved at an appropriate pH, thereby releasing the cationic lipid from the ligand inside the cell into the desired compartment of the cell.

[0420] 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 vary depending on the cell to be targeted.

[0421] Generally, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degrading agent (or condition) to cleave the candidate linking group. It may also be desirable to test the candidate cleavable linking group for its ability to resist cleavage when in blood or in contact with other non-target tissues. Thus, the relative susceptibility to cleavage between a first and a second condition can be determined, with the first being selected to exhibit cleavage in target cells, and the second being selected to exhibit cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be carried out in a cell-free system, in cells, in cell culture, in organs 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 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).

[0422] 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 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 cells, e.g., target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In some cases, candidate compounds are cleaved at a maximum of about 10% in blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic intracellular media compared to conditions selected to mimic extracellular media.

[0423] 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 an agent that degrades or hydrolyzes the phosphate group. An example of an agent that cleaves phosphate groups in cells is an enzyme such as a phosphatase in the cell. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, and -OP(S)(Rk)-S-. In some embodiments, the phosphate-based linking group is -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-, or -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.

[0424] Acid-cleavable linking groups 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 having a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower) or by 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 bonded to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group, such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0425] 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 by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester-cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0426] Peptide-Based Cleavable Linkers In some embodiments, the cleavable linker comprises a peptide-based cleavable linking group. Peptide-based cleavable linking groups are cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids, resulting in 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, resulting in peptides and proteins. Peptide-based cleaving groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids, resulting in peptides and proteins, and do not include the entire amide functionality. 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.

[0427] 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, 5,414,07 ... 5,486,603, 5,512,439, 5,578,718, 5,608,046, 4,587,044, 4,605,735, 4,667,025, 4,762,779, 4,789,737, 4,824,941, 4,835,263, 4,876,335, 4,904,582, 4,958,013, 5,082,830, 5,112,963, 5,214,136, 5,082,830, No. 5,112,963, No. 5,214,136, No. 5,245,022, No. 5,254,469, No. 5,258,506, No. 5,262,536, No. 5,272,250, No. 5,292,873, No. 5,317,098, No. No. 5,371,241, No. 5,391,723, No. 5,416,203, No. 5,451,463, No. 5,510,475, No. 5,512,667, No. 5,514,785, No. 5,565,552, No. 5,567,810, No. Nos. 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.

[0428] Not all positions in a given compound need be uniformly modified, and in fact more than one of the above modifications can 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.

[0429] In the context of this disclosure, a "chimeric" iRNA compound or "chimera" refers to an iRNA compound, e.g., dsRNA, that contains two or more chemically distinct regions, each composed of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region in which the RNA has been modified to confer 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 iRNAs when chimeric dsRNAs are used compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis, optionally followed by associated nucleic acid hybridization techniques known in the art.

[0430] In certain instances, the RNA of an iRNA can be modified with a non-ligand group. To enhance the activity, cellular distribution, or cellular uptake of an iRNA, several non-ligand molecules have been conjugated to the iRNA, and procedures for performing such conjugation 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, e.g., 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 such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids such as 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., Nucleosides & Nucleotides, 1995, 14:969) or adamantaneacetic 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 activating 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 usually yields a pure conjugate.

[0431] iRNA delivery Delivery of iRNA to a subject in need thereof can be achieved in several different ways. In vivo delivery can be performed directly by administering a composition containing iRNA, e.g., dsRNA, to a subject. Alternatively, delivery can be performed indirectly by administering one or more vectors that encode and induce the expression of iRNA. These alternatives are further described below.

[0432] Direct Delivery Generally, any method for delivering nucleic acid molecules can be adapted for use with iRNAs [see, e.g., Akhtar S. and Julian RL., (1992) Trends Cell. Biol. 1999, 1999, incorporated herein by reference in its entirety]. 2(5):139-144 and WO94 / 02595]. However, there are three important factors to consider for successful in vivo delivery of iRNA molecules: (a) 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, e.g., direct injection or implantation into tissue (such as, but not limited to, the eye) or local administration of the preparation. Local administration at the treatment site maximizes the local concentration of the drug, limits exposure to systemic tissues that may be harmed by or degrade the drug, and allows for a smaller total dose of the administered iRNA molecule. Several studies have shown successful knockdown of gene products when iRNA is administered locally. For example, intraocular delivery of VEGF dsRNA by 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] have both been shown to prevent neovascularization in experimental models of age-related macular degeneration. In addition, direct intratumoral injection of dsRNA into mice can reduce tumor volume [Pille, J. et al. (2005) Mol. Ther. 11:267-274] and prolong the survival of 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 can be delivered to the CNS by direct injection [Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, PH. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al. (2002) BMC Neurosci. 3:18; Shishkina, GT., et al. (2004) Neuroscience 129:521-528; Thakker, ER., et al. (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya,Y., et al. (2005) J. Neurophysiol. 93 :594-602] and to the lungs by intranasal administration [Howard, KA. et al., (2006) Mol. Ther. 14:476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55], and local delivery has also shown success. When administering iRNA systemically to treat disease, the RNA can be modified or, alternatively, delivered using a drug delivery system; both methods function to prevent the rapid degradation of dsRNA by endonucleases and exonucleases in vivo.

[0433] Modification of RNA or pharmaceutical carrier can also enable iRNA composition to target tissue and avoid undesired off-target effects.iRNA molecules can be modified by chemical conjugation to other groups, for example, lipid or carbohydrate groups as described herein.Such conjugates can be used to target iRNA to specific cells, for example, liver cells, for example, hepatocytes.For example, GalNAc conjugates or lipid (e.g., LNP) formulations can be used to target iRNA to specific cells, for example, liver cells, for example, hepatocytes.

[0434] iRNA molecules can also be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, systemic injection of iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety into mice resulted in knockdown of apoB mRNA in both the liver and jejunum [Soutschek, J. et al., (2004) Nature 432:173-178]. Conjugation of iRNA to an aptamer 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 promote 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 can 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 when administered systemically. Methods for making and administering cationic iRNA complexes are well within the capabilities of those skilled in the art (see, e.g., Sorensen, D.R., et al. (2003) J. Mol. Biol 327:761-766; Verma, U.N. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A.S. 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], and polyethyleneamine [Bonnet ME. et al., (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659], Arg-Gly-Asp (RGD) peptides [Liu, S. (2006) Mol. Pharm. 3:472-487], and polyamidoamines [Tomalia, D. A. 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 can be found in U.S. Patent No. 7,427,605, which is incorporated herein by reference in its entirety.

[0435] iRNA encoded by the vector 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 PCT Publication No. WO 00 / 22113; Conrad, International PCT Publication No. WO 00 / 22114; and Conrad, U.S. Patent No. 6,054,299). Expression can be transient (hours to weeks) or sustained (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. The transgene can also be constructed to allow it to be inherited as an extrachromosomal plasmid [Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292].

[0436] The individual strand(s) of iRNA can be transcribed from a promoter on an expression vector.When two separate strands are expressed, for example, to produce dsRNA, two separate expression vectors can be co-introduced into target cells (for example, by transfection or infection).Alternatively, each individual 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 polynucleotide linked by a linker polynucleotide sequence, so that dsRNA has a stem and loop structure.

[0437] iRNA expression vectors are usually DNA plasmids or viral vectors. Recombinant constructs for expressing the iRNAs described herein can be generated using expression vectors compatible with eukaryotic cells, for example, vertebrate cells. Eukaryotic cell expression vectors are known in the art and are available from several commercial sources. Typically, such vectors contain convenient restriction sites for the insertion of desired nucleic acid segments. Delivery of iRNA expression vectors can be systemic, for example, by intravenous or intramuscular administration, by administration to target cells explanted from the patient and then reintroduced into the patient, or by any other means that allows introduction into desired target cells.

[0438] iRNA expression plasmids can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections targeting different regions of the target RNA for iRNA-mediated knockdown over a period of one week or longer are also contemplated by the present disclosure. Successful introduction of the vector into the host cells can be monitored using various known methods. For example, transient transfection can be signaled using a reporter such as a fluorescent marker, e.g., green fluorescent protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cells with resistance to certain environmental factors (e.g., antibiotics and drugs), e.g., hygromycin B resistance.

[0439] Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, such as, but not limited to, lentivirus vectors, Moloney murine leukemia virus, and the like; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) vesicular virus vectors, such as orthopox, e.g., vaccinia virus 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 integrate into the cellular genome. The constructs can contain 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 iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure expression of the iRNA in the target cell. Other aspects to consider for vectors and constructs are further described below.

[0440] Vectors useful for delivery of iRNA will 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 either constitutive or regulated / inducible expression.

[0441] The expression of iRNA can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, such as circulating glucose levels or hormones (Docherty et al., 1994, FASEB J. 8:20-24).Such inducible expression systems suitable 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 iRNA transgene.

[0442] In a specific embodiment, a viral vector containing a nucleic acid sequence encoding an iRNA can be used. For example, a retroviral vector 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 sequence encoding the iRNA is cloned into one or more vectors, which facilitates delivery of the nucleic acid to a patient. Further details regarding retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells to make them more resistant to chemotherapy. Other references illustrating 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.

[0443] Adenoviruses are also contemplated for use in the delivery of iRNA. Adenoviruses are particularly attractive vehicles for delivering genes to, for example, respiratory epithelia. Adenoviruses naturally infect respiratory epithelia, causing mild disease there. Other targets for adenovirus-based delivery systems include 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 an overview of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994), demonstrated the use of adenovirus vectors to introduce genes into 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); PCT Publication WO94 / 12649; and Wang, et al., Gene Therapy 2:775-783 (1995). AV vectors suitable for expressing iRNAs featured in this disclosure, methods for constructing recombinant AV vectors, and methods for delivering the vectors into target cells are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.

[0444] 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, e.g., having either a U6 or H1 RNA promoter or a cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing dsRNAs featured in this disclosure, methods for constructing recombinant AAV vectors, and methods for delivering vectors into 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; International Patent Application No. WO94 / 13788; and International Patent Application No. WO93 / 24641, the entire disclosures of which are incorporated herein by reference.

[0445] Other common viral vectors are poxviruses, such as vaccinia viruses, eg, attenuated vaccinia, eg, Modified Virus Ankara (MVA) or NYVAC, and avipox, eg, fowlpox or canarypox.

[0446] The tropism of viral vectors can be modified by pseudotyping the vector with envelope proteins or other 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 express different capsid protein serotypes, thereby making them target different cells; for example, see Rabinowitz JE et al. (2002), J Virol 76:791-801, the entire disclosure of which is incorporated herein by reference.

[0447] The vector pharmaceutical preparation can include the vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0448] III. 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. Pharmaceutical compositions containing iRNA are useful for treating diseases or disorders associated with MYOC expression or activity (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 localized delivery, e.g., by intraocular delivery (e.g., intravitreal administration, e.g., intravitreal injection; transscleral administration, e.g., transscleral 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 topical delivery. In another example, the composition can be formulated for systemic administration by parenteral delivery, e.g., intravenous (IV) delivery. In some embodiments, the compositions provided herein (eg, compositions comprising a GalNAc conjugate or LNP formulation) are formulated for intravenous delivery.

[0449] 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 can be administered once daily, or the iRNA can be administered as two, three, or more subdoses at appropriate intervals throughout the day, or even using continuous infusion or sustained-release delivery. In such cases, the amount of iRNA contained in each subdose must be appropriately small to achieve the total daily dose. Dosage units can also be formulated for delivery over several days, for example, using conventional sustained-release formulations that provide sustained release of the iRNA over a period of several days. Sustained-release formulations are known in the art and are particularly useful for delivering agents at specific sites, such as those used with the agents disclosed herein. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

[0450] The effect of a single dose on MYOC levels may be long-lasting, 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.

[0451] 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, may affect the dosage and timing required to effectively treat a subject. Moreover, treatment of a subject with a therapeutically effective amount of a composition may include a single treatment or a series of treatments. Estimation of the effective dosage and in vivo half-life of individual iRNAs encompassed by the present disclosure can be performed using conventional methodologies based on in vivo studies using appropriate animal models.

[0452] Suitable animal models, such as mice or cynomolgus monkeys, eg, animals containing a transgene expressing human MYOC, can be used to determine a therapeutically effective dose and / or effective dosing regimen of MYOC siRNA.

[0453] 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 local (e.g., by intraocular injection), topical (e.g., by eye drop solution), or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, subcutaneous, e.g., by implanted device, or intracranial, e.g., by intraparenchymal, intrathecal, or intraventricular administration.

[0454] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may be necessary or desirable. Coated condoms, gloves, and the like may also be useful. Suitable topical formulations include those in which the iRNA featured in this disclosure is mixed with a topical delivery agent, such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidylethanolamine DOPE, dimyristoylphosphatidylcholine DMPC, distearoylphosphatidylcholine), cationic (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The iRNAs featured in this disclosure can be encapsulated within liposomes or complexed to liposomes, particularly cationic liposomes. Alternatively, the iRNAs can be complexed to 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, dicaproate, tricaproate, monoolein, dilaurin, glyceryl 1-monocaproate, 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 detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference.

[0455] Liposomal formulation Besides microemulsions, there are many other organized surfactant structures that have been studied and used for drug formulation. These include monolayers, micelles, bilayers, and vesicles. Vesicles such as liposomes have attracted great interest in terms of drug delivery due to the specificity and duration of action they offer. As used in this disclosure, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in a spherical bilayer(s).

[0456] 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.

[0457] 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 capable of passing through such micropores.

[0458] Additional advantages of liposomes include: liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a variety 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 the amount of water in the liposomes.

[0459] Liposomes are useful for transporting and delivering active ingredients to the site of action. Because liposome membranes are structurally similar to biological membranes, when liposomes are applied to tissues, they begin to merge with the cell membrane, and as the merging of liposomes with cells progresses, the liposomal contents flow into the cells, where the active agent can act.

[0460] Liposomal formulations have been the focus of extensive research as the delivery mode of many drugs.For topical administration, there is increasing evidence that liposomes show advantages over other formulations.These advantages include the high systemic absorption of administered drugs and the associated reduction of side effects, the increased accumulation of administered drugs at desired targets, and the ability to administer a wide range of drugs, both hydrophilic and hydrophobic, into the skin.

[0461] Several reports have detailed the ability of liposomes to deliver drugs, including high-molecular-weight DNA, into the skin. Compounds including analgesics, antibodies, hormones, and high-molecular-weight DNA have been administered to the skin. The majority of applications have resulted in targeting of the upper epidermis.

[0462] Liposomes are divided into two broad classes. 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 in endosomes. The acidic pH within the endosome causes the liposomes to rupture, releasing their contents into the cytoplasm [Wang et al. (1987) Biochem. Biophys. Res. Commun., 1987, 147:980-985].

[0463] Liposomes are pH-sensitive or negatively charged, trapping DNA rather than complexing with it. Because both DNA and lipids are similarly charged, repulsion occurs rather than complexation. 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. Expression of exogenous genes was detected in the target gene [Zhou et al. Journal of Controlled Release, 1992, 19:269-274].

[0464] 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 mainly 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.

[0465] Several studies have evaluated the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction in cutaneous herpes scores, while delivery of interferon by 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 versus administration using an aqueous system, concluding that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

[0466] Nonionic liposomal systems, particularly those containing nonionic surfactants and cholesterol, have also been investigated to determine their usefulness in delivering drugs to the skin. Nonionic liposomal formulations containing Novasome™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver cyclosporine A into the dermis of mouse skin. The results showed that such nonionic liposomal systems were effective in promoting the accumulation of cyclosporine A in different layers of the skin (Hu et al., STP Pharma. Sci., 1994, 4, 6, 466).

[0467] Liposomes also include "sterically stabilized" liposomes, which term, as used herein, refers to liposomes containing one or more specialized lipids that, when incorporated into the liposome, result in enhanced circulation life compared to liposomes lacking such specialized lipids. An example of a sterically stabilized liposome is one in which a portion of the vesicle-forming lipid portion of the liposome is (A) one or more glycolipids, e.g., monosialoganglioside G M1 or (B): derivatized with one or more hydrophilic polymers, such as 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].

[0468] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. [Ann. NY Acad. Sci., 1987, 507, 64] reported the ability of monosialoganglioside GM1, galactocerebroside sulfate, and phosphatidylinositol to improve the blood half-life of liposomes. These findings are discussed by Gabizon et al. [Proc. Natl. Acad. Sci. USA, 1988, 85, 6949]. U.S. Patent No. 4,837,028 and WO88 / 04924 (both by Allen et al.) disclose liposomes containing (1) sphingomyelin and (2) ganglioside GM1 or galactocerebroside sulfate. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97 / 13499 (Lim et al.).

[0469] Numerous 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 a method for preparing liposomes containing lipids derivatized with one or more hydrophilic polymers. 1215GLiposomes containing PEG moieties have been described. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899) described synthetic phospholipids modified by the attachment of carboxyl groups of polyalkylene glycols (e.g., PEG). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes containing phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have 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 a combination of disteroylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their outer surface are described by Fisher in European Patent No. EP 0 445 131 B1 and WO 90 / 04384. Liposomal compositions containing 1 to 20 mole percent PEG-derivatized PE and methods for their use are described by 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. EP 0 496 813 B1). Liposomes containing several other lipid-polymer conjugates are disclosed in WO 91 / 05545 and U.S. Patent No. 5,225,212 (both to Martin et al.), and in 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 (Miyazaki 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 surface.

[0470] Some liposomes containing nucleic acids are known in the art. Thierry et al., WO96 / 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 asserts that the contents of such liposomes may contain dsRNA. Rahman et al., U.S. Patent No. 5,665,710, describes a specific method for encapsulating oligodeoxynucleotides in liposomes. Love et al., WO97 / 04787, discloses liposomes containing dsRNA targeted to the raf gene.

[0471] Transfersomes are another type of liposome, highly deformable lipid aggregates that are attractive candidates for drug delivery systems. Transfersomes can be described as lipid droplets that are so deformable that they can easily penetrate through pores smaller than lipid droplets. Transfersomes adapt to the environment in which they are used; for example, they are self-optimizing (adaptable to the shape of pores in the skin), self-repairing, frequently reach their target 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.

[0472] Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way to classify and rank the properties of the many different types of surfactants, both natural and synthetic, is through the use of the hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for categorizing the various surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0473] 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.

[0474] When dissolved or dispersed in water, if surfactant molecule holds negative charge, surfactant is classified as anionic.Anionic surfactants include carboxylate, such as soap, acyl lactylate, acyl amide of amino acid, sulfuric acid ester, such as alkyl sulfate and ethoxylated alkyl sulfate, sulfonate, such as alkylbenzene sulfonate, acyl isethionate, acyl taurate, and sulfosuccinate and phosphate.The most common members of anionic surfactant class are alkyl sulfate and soap.

[0475] 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.

[0476] 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.

[0477] 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).

[0478] nucleic acid lipid particles In some embodiments, the MYOC dsRNA featured in the present disclosure is fully encapsulated in a lipid formulation to form 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 injection (iv) and accumulate at distal sites (e.g., sites physically separated from the administration site), making them highly useful for systemic administration. SPLPs include "pSPLP," which contain encapsulated condensing agent-nucleic acid complexes as described in detail in PCT Publication No. WO 00 / 03683. The particles of the present disclosure typically have an average diameter 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 non-toxic. In addition, when nucleic acid is present in the nucleic acid-lipid particle of the present disclosure, it is resistant to nuclease degradation in aqueous solution.Nucleic acid-lipid particle and its preparation method are disclosed in, for example, United States Patent No. 5,976,567; United States Patent No. 5,981,501; United States Patent No. 6,534,484; United States Patent No. 6,586,410; United States Patent No. 6,815,432; PCT Publication No. WO96 / 40964.

[0479] 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.

[0480] Examples of cationic lipids include 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-dilenoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilenole ... Dicarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleoyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleoyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-dilenoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilenoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP).Cl), 1,2-dilenoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ) or 3-(N,N-dilenoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanedio (DOAP), 1,2-dilenoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilenoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or similar. The cationic lipid may be (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 a mixture thereof. The cationic lipid may comprise about 20 mol% to about 50 mol% or about 40 mol% of the total lipid present in the particle. .

[0481] In some embodiments, the compound 2,2-dilenoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. The synthesis of 2,2-dilenoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. Provisional Patent Application No. 61 / 107,998, filed October 23, 2008, and is incorporated herein by reference.

[0482] In some embodiments, the lipid-siRNA particles comprise 40% 2,2-dilenoleyl-4-dimethylaminoethyl-[1,3]-dioxolane, 10% DSPC, 40% cholesterol, 10% PEG-C-DOMG (mol percent), and have a particle size of 63.0±20 nm and an siRNA / lipid ratio of 0.027.

[0483] Non-cationic lipids may be anionic or neutral lipids, including, but not limited to, disteroylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (P Non-cationic lipids include 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl phosphatidiethanolamine (SOPE), cholesterol, or mixtures thereof. The non-cationic lipid may represent about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% if cholesterol is included, of the total lipid present in the particle.

[0484] The conjugated lipid that inhibits particle aggregation can 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 can be, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (C]8). The conjugated lipid that prevents particle aggregation can be 0 mol% to about 20 mol%, or about 2 mol%, of the total lipid present in the particle.

[0485] In some embodiments, the nucleic acid-lipid particles further comprise cholesterol, for example, from about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

[0486] In some embodiments, the iRNA is formulated in a lipid nanoparticle (LNP).

[0487] 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, for example, in a molar ratio of 42:48:10. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate, pH 5) so that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles usually form spontaneously upon 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 the Lipex Extruder (Northern Lipids, Inc.). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration. The buffer can be exchanged, for example, with phosphate-buffered saline (PBS) at about pH 7, e.g., 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.

[0488] [ka]

[0489] LNP01 formulations are described, for example, in International Application Publication No. WO2008 / 042973, which is incorporated herein by reference.

[0490] Further exemplary lipid-dsRNA formulations are provided in the table below.

[0491] [Table 1-1]

[0492] [Table 1-2]

[0493] [Table 1-3]

[0494] Formulations containing SNALP (1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) are described in International Publication No. WO2009 / 127060, filed April 15, 2009, which is incorporated herein by reference.

[0495] Formulations containing XTC are described, for example, in U.S. Provisional Patent Application No. 61 / 148,366, filed January 29, 2009, U.S. Provisional Patent Application No. 61 / 156,851, filed March 2, 2009, U.S. Provisional Patent Application No. 61 / 185,712, filed June 10, 2009, U.S. Provisional Patent Application No. 61 / 228,373, filed July 24, 2009, U.S. Provisional Patent Application No. 61 / 239,686, filed September 3, 2009, and International Application No. PCT / US2010 / 022614, filed January 29, 2010, which are incorporated herein by reference.

[0496] Formulations containing MC3 are described, for example, in U.S. Provisional Patent Application No. 61 / 244,834, filed September 22, 2009, U.S. Provisional Patent Application No. 61 / 185,800, filed June 10, 2009, and International Application No. PCT / US10 / 28224, filed June 10, 2010, which are incorporated herein by reference.

[0497] Formulations containing ALNY-100 are described, for example, in International Patent Application No. PCT / US09 / 63933, filed November 10, 2009, which is incorporated herein by reference.

[0498] Formulations containing C12-200 are described in U.S. Provisional Patent Application No. 61 / 175,770, filed May 5, 2009, and International Application No. PCT / US10 / 33777, filed May 5, 2010, which are incorporated herein by reference.

[0499] Synthesis of cationic lipids Any of the compounds used in the nucleic acid-lipid particles featured in this disclosure, such as cationic lipids, can be prepared by known organic synthesis techniques, and all substituents are as defined below unless otherwise indicated.

[0500] "Alkyl" means a straight-chain or branched, acyclic or cyclic, saturated aliphatic hydrocarbon containing 1 to 24 carbon atoms. Representative saturated straight-chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, etc., while saturated branched alkyls include isopropyl, s-butyl, isobutyl, t-butyl, isopentyl, etc. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc., while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, etc.

[0501] "Alkenyl" refers to an alkyl as defined above containing at least one double bond between adjacent carbon atoms. Alkenyl includes both cis and trans isomers. Representative straight-chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, etc.

[0502] "Alkynyl" refers to any alkyl or alkenyl as defined above, further containing at least one triple bond between adjacent carbons. Representative straight-chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.

[0503] "Acyl" means any alkyl, alkenyl, or alkynyl, as defined below, in which the carbon at the point of attachment is substituted with an oxo group. For example, -C(=O)alkyl, -C(=O)alkenyl, and -C(=O)alkynyl are acyl groups.

[0504] "Heterocycle" refers to a 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic ring, either saturated, unsaturated, or aromatic, containing one or two heteroatoms independently selected from nitrogen, oxygen, and sulfur, where the nitrogen and sulfur heteroatoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heteroatoms are fused to a benzene ring. The heterocycle may be bonded through any heteroatom or carbon atom. Heterocycles include heteroaryls, as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

[0505] The terms "optionally substituted alkyl," "optionally substituted alkenyl," "optionally substituted alkynyl," "optionally substituted acyl," and "optionally substituted heterocycle" mean that, when substituted, at least one hydrogen atom is replaced by a substituent. In the case of an oxo substituent (=O), two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocycle, -CN, -OR x , -NR x R y , -NR x C(=O)R y 、 -NR x SO2R y , -C(=O)R x , -C(=O)OR x , -C(=O)NR x R y , -SO n R x and -SO n NR x R y where n is 0, 1 or 2; R x and R y are the same or different and independently hydrogen, alkyl, or heterocycle, and each of said alkyl and heterocycle substituents is selected from oxo, halogen, —OH, —CN, alkyl, —OR x , heterocycle, -NR x R y , -NR x C(=O)R y 、 -NR x SO2R y , -C(=O)R x , -C(=O)OR x , -C(=O)NR x R y , -SO n R x and -SO n NR x R y may be further substituted with one or more of:

[0506] "Halogen" means fluoro, chloro, bromo and iodo.

[0507] In some embodiments, the methods featured in the present disclosure may require the use of protecting groups. Protecting group methodology is known to those skilled in the art (see, for example, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, Green, TW et al., Wiley-Interscience, New York City, 1999). Briefly, within the context of this disclosure, a protecting group is any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during a certain reaction and then removed to reveal the original functional group. In some embodiments, an "alcohol protecting group" is used. An "alcohol protecting group" is any group that reduces or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques known in the art.

[0508] Synthesis of Formula A In some embodiments, the nucleic acid-lipid particles featured in this disclosure are formulated using a cationic lipid of Formula A:

[0509] [ka] [Wherein R1 and R2 are independently alkyl, alkenyl or alkynyl, each of which may be optionally substituted, and R3 and R4 are independently lower alkyl, or R3 and R4 may join together to form an optionally substituted heterocyclic ring.] In some embodiments, the cationic lipid is XTC (2,2-dilenoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). Generally, the lipid of the above formula A can be prepared by the following reaction scheme 1 or 2, and all substituents are as defined above unless otherwise specified.

[0510] [ka]

[0511] Lipid A, in which R1 and R2 are independently alkyl, alkenyl, or alkynyl, each optionally substituted, and R3 and R4 are independently lower alkyl, or R3 and R4 together may form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those skilled in the art. Reaction of 1 and 2 provides ketal 3. Treatment of ketal 3 with amine 4 provides lipids of formula A. Lipids of formula A can be converted to the corresponding ammonium salts using an organic base of formula 5, in which X is an anionic counterion selected from halogen, hydroxide, phosphate, sulfate, and the like.

[0512] [ka]

[0513] Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those skilled in the art. Reaction of reactions 6 and 7 gives ketone 1. Conversion of ketone 1 to the corresponding lipid of formula A is as described in Scheme 1.

[0514] Synthesis of MC3 DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) was prepared as follows: A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred overnight at room temperature. The solution was washed with dilute hydrochloric acid and then with dilute aqueous sodium bicarbonate. The organic fraction was dried over anhydrous magnesium sulfate, filtered, and the solvent removed on a rotary evaporator. The residue was passed through a 20 g silica gel column using a 1-5% methanol / dichloromethane elution gradient. The fractions containing the purified product were combined, and the solvent was removed to give a colorless oil (0.54 g).

[0515] Synthesis of ALNY-100 The synthesis of ketal 519 [ALNY-100] was carried out using Scheme 3 below:

[0516] [ka]

[0517] Synthesis of 515: To a stirred suspension of LiAlH (3.74 g, 0.09852 mol) in 200 mL of anhydrous THF in a two-necked RBF (1 L) was slowly added a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF at 0 °C under a nitrogen atmosphere. After complete addition, the reaction mixture was warmed to room temperature and then heated to reflux for 4 h. The reaction progress was monitored by TLC. After completion of the reaction (by TLC), the mixture was cooled to 0 °C and quenched by careful addition of saturated NaSO solution. The reaction mixture was stirred at room temperature for 4 h and filtered. The residue was washed thoroughly with THF. The filtrate and washings were combined, diluted with 400 mL of dioxane and 26 mL of concentrated HCl, and stirred at room temperature for 20 min. Volatiles were stripped under vacuum to afford the hydrochloride salt of 515 as a white solid. Yield: 7.12 g. 1H-NMR (DMSO, 400MHz): δ= 9.34 (wide, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).

[0518] Synthesis of 516: To a stirred solution of compound 515 in 100 mL of anhydrous DCM in a 250 mL two-necked RBF, NEt (37.2 mL, 0.2669 mol) was added and cooled to 0 °C under a nitrogen atmosphere. N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL of anhydrous DCM was slowly added, and the reaction mixture was allowed to warm to room temperature. After the reaction was complete (by TLC, 2-3 h), the mixture was washed successively with 1 N HCl solution (1 × 100 mL) and saturated NaHCO solution (1 × 50 mL). The organic layer was then dried over anhydrous NaSO, and the solvent was evaporated to give crude material, which was purified by silica gel column chromatography to give 516 as a sticky mass. Yield: 11 g (89%). 1H-NMR (CDCl3, 400MHz): δ = 7.36-7.27(m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60(m, 2H), 2.30-2.25(m, 2H). LC-MS [M+H] -232.3 (96.94%).

[0519] Synthesis of 517A and 517B: Cyclopentene 516 (5 g, 0.02164 mol) was dissolved in 220 mL of a 10:1 solution of acetone and water in a one-necked 500 mL RBF. N-methylmorpholine-N-oxide (7.6 g, 0.06492 mol) was added at room temperature, followed by 4.2 mL of a 7.6% solution of OsO (0.275 g, 0.00108 mol) in t-butanol. After the reaction was complete (approximately 3 h), solid NaSO was added to quench the mixture, and the resulting mixture was stirred at room temperature for 1.5 h. The reaction mixture was diluted with DCM (300 mL) and washed with water (2 × 100 mL), followed by saturated NaHCO (1 × 50 mL) solution, water (1 × 30 mL), and finally brine (1 × 50 mL). The organic phase was dried over anhydrous NaSO, and the solvent was removed in vacuo. Silica gel column chromatography purification of the crude material gave a mixture of diastereomers which were separated by preparative HPLC. Yield: -6 g crude material. 517A-Peak-1 (white solid), 5.13g (96%). 1H-NMR (DMSO, 400MHz): δ= 7.39-7.31(m, 5H), 5.04(s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47(d, 2H), 3.94-3.93(m, 2H), 2.71(s, 3H), 1.72- 1.67(m, 4H). LC-MS - [M+H]-266.3, [M+NH4 +]-283.5 present, HPLC-97.86%. The stereochemistry was confirmed by X-ray.

[0520] Synthesis of 518: Using a procedure similar to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. 1H-NMR (CDCl3, 400MHz): δ= 7.35-7.33(m, 4H), 7.30-7.27(m, 1H), 5.37-5.27(m, 8H), 5.12(s, 2H), 4.75(m,1H), 4.58-4.57(m,2H), 2.78-2.74(m,7H), 2.06-2.00(m,8H), 1.96-1.91(m, 2H), 1.62(m, 4H), 1.48(m, 2H), 1.37-1.25(br m, 36H), 0.87(m, 6H). HPLC-98.65%.

[0521] General procedure for the synthesis of compound 519: To a solution of compound 518 (1 equivalent) in hexane (15 mL) was added dropwise an ice-cold solution of LAH in THF (1 M, 2 equivalents). After complete addition, the mixture was heated to 40° C. for 0.5 h and then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous NaSO, filtered through Celite, and reduced to an oil. Column chromatography provided pure 519 (1.3 g, 68%), which was obtained as a colorless oil. 13C NMR = 130.2, 130.1 (x2), 127.9 (x3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (x2), 29.7, 29.6 (x2), 29.5 (x3), 29.3 (x2), 27.2 (x3), 25.6, 24.5, 23.3, 226, 14.1; electrospray MS (+ve): MW calculated for C44H80NO2 (M + H)+ 654.6, found 654.6.

[0522] Formulations prepared by either standard or non-extrusion methods can be characterized in a similar manner. For example, formulations are typically characterized by visual inspection. They should be a whitish, translucent solution without aggregates or precipitates. The particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering, for example, using a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be approximately 20-300 nm in size, e.g., 40-100 nm. The particle size distribution should be monomodal. The total dsRNA concentration in the formulation and the entrapped fraction can be estimated using a dye exclusion assay. Samples of formulated dsRNA can be incubated with an RNA-binding dye, e.g., Ribogreen (Molecular Probes), in the presence or absence of a formulation-disrupting detergent, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by comparing the signal from the detergent-containing sample against a standard curve. The captured fraction is determined by subtracting the "free" dsRNA content (as measured by the signal in the absence of surfactant) from the total dsRNA content. The percent of captured dsRNA is typically >85%. For SNALP formulations, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, or at least 120 nm. Suitable ranges are typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.

[0523] Oral administration compositions and preparations include powder or granules, microparticles, nanoparticles, suspension or solution in water or non-aqueous medium, capsules, gel capsules, sachets, tablets or mini-tablets.Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders can be desirable.In some embodiments, oral preparations are the preparations that the dsRNA characterized in the present disclosure is administered in conjunction with one or more penetration enhancers, surfactants and chelating agents.Suitable surfactants include fatty acids and / or esters or their salts, bile acids and / or their salts. Suitable bile acids / salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, l...

Claims

1. 1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of myocilin (MYOC), the dsRNA agent comprising a sense strand and an antisense strand forming 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 from one of the antisense sequences listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A and 5B, and the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides with 0, 1, 2 or 3 mismatches from the sense sequence listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A and 5B that corresponds to the antisense sequence.

2. 10. The dsRNA agent of claim 1, wherein at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties.

3. 3. The dsRNA agent of claim 2, wherein the lipophilic moiety is conjugated via a linker or carrier.

4. 4. The dsRNA agent of claim 2 or 3, wherein the one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand.

5. 5. The dsRNA agent of claim 4, wherein the one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand via a linker or carrier.

6. 6. The dsRNA agent of any one of claims 2 to 5, wherein the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

7. 7. The dsRNA agent of claim 6, wherein the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.

8. 8. The dsRNA agent of any one of claims 2 to 7, which is conjugated via a lipophilic moiety, a carrier that replaces one or more nucleotides in an internal position or double-stranded region.

9. The dsRNA agent of any one of claims 2-7, wherein the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker that contains 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.

10. 9. The double-stranded iRNA agent of any one of claims 2-8, wherein the lipophilic moiety is conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage.

11. 10. The dsRNA agent of any of the preceding claims, comprising at least one modified nucleotide.

12. 12. The dsRNA agent of claim 11, wherein no more than five of the nucleotides in the sense strand and no more than five of the nucleotides in the antisense strand are unmodified nucleotides.

13. 12. The dsRNA agent of claim 11, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise the modification.

14. 14. The dsRNA agent of any one of claims 11 to 13, wherein at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3'-terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-methoxyethyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide comprising a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate, a nucleotide comprising a 5'-phosphate mimic, a glycol-modified nucleotide, and a 2-O-(N-methylacetamido)-modified nucleotide, and combinations thereof.

15. 4. The dsRNA agent of any preceding claim, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides.

16. 8. The dsRNA agent of any of the preceding claims, wherein the double-stranded region is 15 to 30 nucleotide pairs in length.

17. 17. The dsRNA agent of claim 16, wherein the double-stranded region is 17 to 23 nucleotide pairs in length.

18. 8. The dsRNA agent of any preceding claim, wherein each strand has 19-30 nucleotides.

19. 10. The dsRNA agent of any preceding claim, comprising at least one phosphorothioate or methylphosphonate internucleotide linkage.

20. 20. The dsRNA agent of any one of claims 2 to 19, further comprising a targeting ligand, e.g., a ligand that targets eye tissue or liver tissue.

21. 21. The dsRNA agent of claim 20, wherein the ocular tissue is trabecular meshwork tissue, ciliary body, retinal tissue, retinal pigment epithelium (RPE), or choroidal tissue, e.g., choroidal blood vessels.

22. 4. The dsRNA agent of any one of the preceding claims, further comprising a phosphate or phosphate mimic at the 5' end of the antisense strand.

23. 23. The dsRNA agent of claim 22, wherein the phosphate mimic is 5'-vinylphosphonate (VP).

24. 24. A cell containing the dsRNA agent of any one of claims 1 to 23.

25. 24. A pharmaceutical composition for inhibiting expression of MYOC, comprising the dsRNA agent of any one of claims 1 to 23.

26. 1. A method for inhibiting expression of MYOC in a cell, comprising: (a) contacting a cell with the dsRNA agent of any one of claims 1 to 23 or the pharmaceutical composition of claim 25; and (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. A method comprising:

27. 27. The method of claim 26, wherein the cell is in a subject.

28. 28. The method of claim 27, wherein the subject is a human.

29. 29. The method of claim 28, wherein the subject has been diagnosed with a MYOC-related disorder, e.g., glaucoma (e.g., primary open-angle glaucoma (POAG), angle-closure glaucoma, congenital glaucoma, and secondary glaucoma).

30. 26. A method of treating a subject diagnosed with a MYOC-associated disorder, comprising administering to the subject a therapeutically effective amount of the dsRNA agent of any one of claims 1-23 or the pharmaceutical composition of claim 25, thereby treating the disorder.

31. 31. The method of claim 30, wherein the MYOC-related disorder is glaucoma.

32. 32. The method of claim 31, wherein the glaucoma is primary open-angle glaucoma (POAG).

33. 33. The method of any one of claims 30 to 32, wherein treatment comprises amelioration of at least one sign or symptom of the disorder.

34. 34. The method of any one of claims 30 to 33, wherein the treatment comprises (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) improving visual acuity.

35. 35. The method of any one of claims 27-34, wherein the dsRNA agent is administered to the subject intraocularly, intravenously, or topically.

36. 36. The method of claim 35, wherein the intraocular administration comprises intravitreal administration (e.g., intravitreal injection), transscleral administration (e.g., transscleral 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).

37. 37. The method of any one of claims 27 to 36, further comprising administering to the subject an additional agent or therapy suitable for the treatment or prevention of a MYOC-related disorder (e.g., laser trabeculoplasty, trabeculectomy, minimally invasive glaucoma surgery, replacement of drainage tubes in the eye, oral medications, eye drops).

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