IRNA COMPOSITIONS AND METHODS FOR SILING FILAMIN A (FLNA)

Abstract

The present disclosure relates to double-stranded ribonucleic acid (dsRNAi) agents and compositions that target the Filamin A (FLNA) gene, and methods of using the dsRNAi agents and compositions to inhibit expression of the FLNA gene and to treat subjects with a FLNA-associated disease or disorder, e.g., Alzheimer's disease.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 214,220, filed June 23, 2021, and U.S. Provisional Patent Application No. 63 / 274,248, filed November 1, 2021. The entire contents of the foregoing applications are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on June 23, 2022, is named A108868_1250WO_SL.txt and is 346,812 bytes in size. [Background technology]

[0003] The filamin A (FLNA) gene, which encodes the filamin A protein, is located in chromosome region Xq28. FLNA is an actin-binding protein of the filamin family and is involved in cross-linking actin filaments during cytoskeletal remodeling.

[0004] Alzheimer's disease is a neurodegenerative disorder characterized by abnormalities in amyloid beta and tau proteins, leading to the formation of amyloid plaques and neurofibrillary tangles. An altered conformational, or misfolded, form of FLNA has been found in Alzheimer's disease brains and is thought to be induced by amyloid beta. This modified FLNA does not aggregate but is linked to both the amyloid beta and tau signaling pathways in Alzheimer's disease.

[0005] Currently, there is no disease-modifying therapy for Alzheimer's disease, and treatments are aimed only at alleviating disease symptoms and improving patients' quality of life as the neurodegenerative disease progresses. Thus, there is a need for agents that can treat, prevent, and / or inhibit the progression of Alzheimer's disease. Summary of the Invention

[0006] The present disclosure provides an RNAi composition that performs RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of FLNA gene.The FLNA gene can be present in a cell, for example, in a subject cell such as a human.The use of these iRNAs allows the targeted degradation of the mRNA of corresponding gene (FLNA gene) in mammals.

[0007] The iRNA of the present invention is designed to target the FLNA gene, for example, the FLNA gene with missense and / or deletion mutations in the exons of the gene, and / or the wild-type gene, in subjects with Alzheimer's disease and a combination of nucleotide modifications. The iRNA of the present invention inhibits the expression of the FLNA gene by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. Without intending to be bound by theory, it is believed that the combination or subcombination of the above-mentioned characteristics and specific target sites or specific modifications in these iRNAs improves the efficacy, stability, potency, durability, and safety of the iRNA of the present invention. In one aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of FLNA, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:1 by no more than 3 nucleotides, and the antisense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:2 by no more than 3 nucleotides.

[0008] In another aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of FLNA, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a region complementary to an mRNA encoding FLNA, the complementary region comprising at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:2 by no more than 3 nucleotides.

[0009] In yet another aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of FLNA, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a region complementary to an mRNA encoding FLNA, the complementary region comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense nucleotide sequences in Tables 3-8.

[0010] In one embodiment, the sense strand comprises nucleotides 100-120, 176-196, 244-264, 375-395, 404-424, 425-445, 473-493, 500-520, 545-565, 598-618, 641-661, 665-685, 689-709, 714-734, 871-891, 906-926, 933-953, 1002-1022, 1077-1097, 1127-1147, 1151-1171, 1189-1209, 1235-1255, 1258-1278, 1305-1325, 1328-1331, 1332-1333, 1334-1335, 1336-1337, 1338-1339, 1340-1341, 1342-1343, 1344-1345, 1346-1347, 1348-1349, 1350-1351, 1352-1353, 1354-1355, 1356-1357, 1358-1360, 1359-1361, 1362-1363, 1364-1365, 1366-1367, 1368-1369, 1370-1371, 1372-1373, 1374-1375, 1376- 348, 1355-1375, 1391-1411, 1427-1447, 1448-1468, 1488-1508, 1530-1550, 1563-1583, 1660-1680, 1749-1769, 1790-1810, 1854-1874, 1888-1908, 1926-1946, 1956-1976, 2023-2043, 2068-2088, 2103-2123, 2132-2152, 2187-2207, 2219-2239, 2258-2278, 2317-2337, 2340-2360, 2376-2396, 2399 -2419, 2421-2441, 2468-2488, 2553-2573, 2615-2635, 2654-2674, 2700-2720, 2721-2741, 2742-2762, 2783-2803, 2841-2861, 2900-2920, 2930-295 0, 2954-2974, 2975-2995, 3017-3037, 3038-3058, 3080-3100, 3124-3144, 3155-3175, 3189-3209, 3214-3234, 3249-3269, 3323-3343, 3375-3395, 34 38-3458, 3503-3523, 3569-3589, 3601-3621, 3647-3667, 3714-3734, 3782-3802, 3826-3846, 3854-3874, 3876-3896, 3930-3950, 3999-4019, 4041-4 061, 4066-4086, 4122-4142, 4145-4165, 4170-4190, 4191-4211, 4217-4237, 4336-4356, 4367-4387, 4442-4462, 4491-4511, 4516-4536, 4547-4567,4569-4589、4622-4642、4652-4672、4694-4714、4746-4766、4812-4832、4869-4889、4943-4963、4977-4997、5022-5042、5060-5080、5088-5108、5180-5200、5205-5225、5255-5275、5290-5310、5314-5334、5343-5363、5364-5384、5405-5425、5521-5541、5582-5602、5612-5632、5639-5659、5703-5723、5772-5792、5811-5831、5847-5867、5876-5896、5910-5930、5967-5987、5992-6012、6038-6058、6107-6127、6128-6148、6163-6183、6243-6263、6272-6292、6300-6320、6358-6378、6391-6411、6441-6461、6479-6499、6509-6529、6545-6565、6581-6601、6711-6731、6741-6761、6798-6818、6826-6846、6849-6869、6873-6893、6987-7007、7014-7034、7088-7108、7125-7145、7152-7172、7173-7193、7206-7226、7253-7273、7288-7308、7386-7406、7408-7428、7445-7465、7466-7486、7537-7557、7560-7580、7586-7606、7657-7677、7728-7748、7832-7852、7978-7998、8002-8022、8048-8068、8081-8101、8120-8140、8359-8379、8404-8424、8447-8467、8483-8503、171-191、173-193、375-395、542-562、1442-1462、1449-1469、1751-1771、1752-1772、1753-1773、1854-1874、1855-1875、1856-1876、2722-2742、2730-2750、3080-3100、3081-3101、3082-3102、3083-3103、3084-3104、3212-3232、3217-3237, 3445-3465, 3446-3466, 3447-3467, 4068-4088, 5182-5202, 5183-5203, 5978-5998, 6046-6066, 6432-6452, 6585-6605, 6586-6606, 6587-66 07, 7079-7099, 7161-7181, 7163-7183, 7165-7185, 7166-7186, 7376-7396, 7377-7397, 7378-7398, 7390-7410, 7391-7411, 7392-7412, 7393-7413, 7394- and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NO: 7414, 7398-7418, 7435-7455, 7436-7456, 7437-7457, 7446-7466, 7654-7674, 7655-7675, 7657-7677, 7726-7746, 8404-8424, 8474-8494, 8475-8495, 8476-8496, and 8477-8497, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 2.

[0011] In one embodiment, the sense strand comprises nucleotides 923-943, 1569-1589, 1570-1590, 1572-1592, 1987-2007, 2222-2242, 2560-2580, 2561-2581, 2745-2765, 3339-3359, 3340-3360, 4023-4043, 4716-4736, 5491-5511, 5940-5960, 6088-6108, 6704-6724, 6705-6725, 6707-6727, 6708-6729, 6719-6726, 6727-6728, 6729-6730, 6731-6732, 6733-6734, 6735-6736, 6745-6746, 6747-6748, 6749-6750, 6751-6752, 6753-6754, 6755-6756, 6757-6758, 6759-6760, 6761-6762, 6763-6764, 6765-6766, 6766-6768, 6769-6770, 6771-6772, 6772-6773, 6774-6775, 6775-6776, 6776-6777, 6778-6779, 6780-6 and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NO: 864-6884, 6865-6885, 6866-6886, 6949-6969, 6965-6985, 7376-7396, 7519-7539, 7961-7981, 8100-8120, 8101-8121, 8541-8561, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 1358.

[0012] In one embodiment, the antisense strand is AD-1615378, AD-1615433, AD-1615454, AD-1615511, AD-1615540, AD-1615561, AD-1615604, AD-1615631, AD-1615676, AD-1615729, AD-1615772, AD-1615796, AD-1615820, AD-1615843, AD-1615930, AD-1615964, AD-1615991, AD-1616007, AD-1616044, AD-1616087, AD-1616111, AD-1616149, AD-1616182, AD-1616205, AD-1616222, AD-1616245, AD-1616252, AD-1616288, AD-1616313, AD-1616334, AD-1616364, AD-1616386, AD-1616399, AD-1616471, AD-1616531, AD-1616554, AD-1616579, AD-1616593, AD-1616613, AD-1616643, AD-1616682, AD-1616707, AD-1616742, AD-1616771, AD-1616821, AD-1616833, AD-1616852, AD-1616911, AD-1616934, AD-1616950, AD-1616972, AD-1616994, AD-1617041, AD-1617061, AD-1617103, AD-1617117, AD-1617127, AD-1617148, AD-1617169, AD-1617186, AD-1617219, AD-1617268, AD-1617298, AD-1617322, AD-1617343, AD-1617366, AD-1617387, AD-1617429, AD-1617455, AD-1617486, AD-1617520, AD-1617545, AD-1617580, AD-1617612, AD-1617644, AD-1617657, AD-1617679, AD-1617703, AD-1617717, AD-1617743, AD-1617790, AD-1..AD-1618049、AD-1618052、AD-1618077、AD-1618098、AD-1618124、AD-1618187、AD-1618214、AD-1618237、AD-1618286、AD-1618311、AD-1618342、AD-1618364、AD-1618404、AD-1618434、AD-1618456、AD-1618508、AD-1618572、AD-1618601、AD-1618645、AD-1618661、AD-1618706、AD-1618744、AD-1618772、AD-1618810、AD-1618835、AD-1618851、AD-1618886、AD-1618910、AD-1618939、AD-1618960、AD-1618979、AD-1619014、AD-1619034、AD-1619064、AD-1619071、AD-1619116、AD-1619178、AD-1619197、AD-1619233、AD-1619262、AD-1619296、AD-1619333、AD-1619358、AD-1619385、AD-1619434、AD-1619455、AD-1619470、AD-1619529、AD-1619540、AD-1619549、AD-1619586、AD-1619601、AD-1619651、AD-1619689、AD-1619699、AD-1619735、AD-1619751、AD-1619849、AD-1619879、AD-1619936、AD-1619946、AD-1619969、AD-1619993、AD-1620033、AD-1620060、AD-1620113、AD-1620150、AD-1620177、AD-1620198、AD-1620211、AD-1620244、AD-1620279、AD-1620330、AD-1620352、AD-1620389、AD-1620410、AD-1620426、AD-1620449、AD-1620475、AD-1620525、AD-1620574、AD-1620616、AD-1620707、AD-1620731、AD-1620737、AD-1620767、AD-1620787、AD-1620837、AD-1620879、AD-1620891、AD-1620927、AD-1615428.1、AD-1615430.1、AD-1615511.2、AD-1615673.1、AD-1616328.1、AD-1616335.1、AD-1616533.1、AD-1616534.1、AD-1616535.1、AD-1616579.2、AD-1616580.1、AD-1616581.1、AD-1617149.1、AD-1617157.1、AD-1617429.2、AD-1617430.1、AD-1617431.1、AD-1617432.1、AD-1617433.1、AD-1617543.1、AD-1617548.1、AD-1617664.1、AD-1617665.1、AD-1617666.1、AD-1618008.1、AD-1618812.1、AD-1618813.1、AD-1619344.1、AD-1619393.1、AD-1619642.1、AD-1619755.1、AD-1619756.1、AD-1619757.1、AD-1620104.1、AD-1620186.1、AD-1620188.1、AD-1620190.1、AD-1620191.1、AD-1620320.1、AD-1620321.1、AD-1620322.1、AD-1620334.1、AD-1620335.1、AD-1620336.1、AD-1620337.1、AD-1620338.1、AD-1620342.1、AD-1620379.1、AD-1620380.1、AD-1620381.1、AD-1620390.1、AD-1620522.1、AD-1620523.1、AD-1620525.2、AD-1620572.1、AD-1620879.2、AD-1620918.1、AD-1620919.1、AD-1620920.1、AD-1620921.1、AD-1687606.1、AD-1688124.1、AD-1688125.1、AD-1688127.1、AD-1688431.1、AD-1688626.1、AD-1688897.1、AD-1688898.1、AD-1689041.1、AD-1689527.1、AD-1689528.1、AD-1690032.1、AD-1690554.1、AD-1691133.1、AD-1691437.1、AD-1691559.1、AD-1692108.1、It comprises at least 15 consecutive nucleotides that differ by 3 nucleotides or less from any one of the antisense strand nucleotide sequences of the duplex selected from the group consisting of AD-1692109.1, AD-1692111.1, AD-1692242.1, AD-1692243.1, AD-1692244.1, AD-1692317.1, AD-1692333.1, AD-1692616.1, AD-1692718.1, AD-1693004.1, AD-1693103.1, AD-1693104.1, and AD-1693450.1.

[0013] In some embodiments, the nucleotide sequences of the sense and antisense strands comprise any one of the sense strand nucleotide sequences in Tables 3-8.

[0014] In one embodiment, the sense strand, the antisense strand, or both the sense and antisense strands are conjugated to one or more lipophilic moieties.

[0015] In one embodiment, the lipophilic moiety is conjugated to one or more interior positions within the double-stranded region of the dsRNA agent.

[0016] In one embodiment, the lipophilic moiety is conjugated via a linker or carrier.

[0017] In one embodiment, the lipophilicity of the lipophilic moiety is greater than 0, as measured by log Kow.

[0018] In one embodiment, 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.

[0019] In one embodiment, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

[0020] In some embodiments, the dsRNA agent includes at least one modified nucleotide.

[0021] In one embodiment, no more than 5 nucleotides in the sense strand and no more than 5 nucleotides in the antisense strand are unmodified nucleotides.

[0022] In one embodiment, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand are modified nucleotides.

[0023] In one embodiment, at least one of the modified nucleotides is a deoxy-nucleotide, a 3'-terminal deoxy-thymidine (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'-hydroxyl-modified nucleotide, a 2'-methoxyethyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified ... nucleotides containing 5'-phosphorothioate groups, nucleotides containing 5'-methylphosphonate groups, nucleotides containing 5' phosphates or 5' phosphate mimics, nucleotides containing vinyl phosphonates, nucleotides containing glycol nucleic acids (GNAs) (e.g., adenosine-glycol nucleic acids), nucleotides containing glycol nucleic acid S isomers (S-GNAs) (e.g., thymidine glycol nucleic acid S isomers), nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3' phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, 2'-5'-linked ribonucleotides (3'-RNA), and terminal nucleotides linked to cholesteryl derivatives, and dodecanoic acid bisdecylamide groups, and combinations thereof.

[0024] In one embodiment, the modified nucleotide is selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, 3'-terminal deoxy-thymine nucleotides (dT), locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and nucleotides containing unnatural bases.

[0025] In one embodiment, the modified nucleotides include a short sequence of 3'-terminal deoxy-thymine nucleotides (dT).

[0026] In one embodiment, the modifications in the nucleotide are 2'-O-methyl, GNA, and 2' fluoro modifications.

[0027] In some embodiments, the dsRNA agent further comprises at least one phosphorothioate internucleotide linkage.

[0028] In one embodiment, the dsRNA agent includes 6 to 8 phosphorothioate internucleotide linkages.

[0029] In one embodiment, each strand is 30 nucleotides or less in length.

[0030] In one embodiment, at least one strand comprises a 3' overhang of at least 1 nucleotide, hi another embodiment, at least one strand comprises a 3' overhang of at least 2 nucleotides.

[0031] The double-stranded region can be 15 to 30 nucleotide pairs in length, 17 to 23 nucleotide pairs in length, 17 to 25 nucleotide pairs in length, 23 to 27 nucleotide pairs in length, 19 to 21 nucleotide pairs in length, or 21 to 23 nucleotide pairs in length.

[0032] Each strand can have 19 to 30 nucleotides, 19 to 23 nucleotides, or 21 to 23 nucleotides.

[0033] In one embodiment, one or more lipophilic moieties are conjugated, for example via a linker or carrier, to one or more interior positions on at least one chain.

[0034] In one embodiment, the interior positions include all but the two most distal positions on each end of at least one strand.

[0035] In another embodiment, the interior positions include all but the three most distal positions on each of at least one strand.

[0036] In one embodiment, the internal position excludes the cleavage site region of the sense strand.

[0037] In one embodiment, internal positions include all positions except positions 9 to 12 counting from the 5' end of the sense strand.

[0038] In another embodiment, internal positions include all positions except positions 11-13 counting from the 3' end of the sense strand.

[0039] In one embodiment, the internal position excludes the cleavage site region of the antisense strand.

[0040] In one embodiment, internal positions include all positions except positions 12 to 14 counting from the 5' end of the antisense strand.

[0041] In one embodiment, internal positions include all positions except positions 11-13 on the sense strand counting from the 3' end and positions 12-14 on the antisense strand counting from the 5' end.

[0042] In one embodiment, the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of positions 4-8 and 13-18 on the sense strand and positions 6-10 and 15-18 on the antisense strand, counting from the 5' end of each strand.

[0043] In another embodiment, the one or more lipophilic moieties are conjugated to one or more internal positions selected from the group consisting of positions 5, 6, 7, 15, and 17 on the sense strand and positions 15 and 17 on the antisense strand, counting from the 5' end of each strand.

[0044] In one embodiment, the internal positions in the double-stranded region exclude the cleavage site region of the sense strand.

[0045] In one embodiment, the sense strand is 21 nucleotides in length, the antisense strand is 23 nucleotides in length, and the lipophilic moiety is conjugated to position 21, 20, 15, 1, 7, 6, or 2 of the sense strand or position 16 of the antisense strand.

[0046] In one embodiment, the lipophilic moiety is conjugated to position 21, 20, 15, 1, or 7 of the sense strand.

[0047] In another embodiment, the lipophilic moiety is conjugated to position 21, 20, or 15 of the sense strand.

[0048] In yet another embodiment, the lipophilic moiety is conjugated to position 20 or 15 of the sense strand.

[0049] In one embodiment, the lipophilic moiety is conjugated to position 16 of the antisense strand.

[0050] In one embodiment, the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

[0051] In some embodiments, the lipophilic moiety is selected from the group consisting of a lipid, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, a heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

[0052] In one embodiment, 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.

[0053] In one embodiment, the lipophilic moiety contains a saturated or unsaturated C6 to C18 hydrocarbon chain.

[0054] In one embodiment, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.

[0055] In one embodiment, a saturated or unsaturated C16 hydrocarbon chain is conjugated to position 6, counting from the 5' end of one chain.

[0056] In one embodiment, the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotides in an internal position or in the double-stranded region.

[0057] In one embodiment, the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl, or is an acyclic portion of a serinol or diethanolamine backbone system.

[0058] In one embodiment, 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.

[0059] In one embodiment, the lipophilic moiety is conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage.

[0060] In one embodiment, 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.

[0061] In one embodiment, 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.

[0062] In one embodiment, the targeting ligand is a GalNAc conjugate.

[0063] In one embodiment, the dsRNA agent further includes a terminal chiral modification that occurs in a first internucleotide linkage at the 3'-end of the antisense strand having a linking phosphorus atom in the Sp configuration, a terminal chiral modification that occurs in a first internucleotide linkage at the 5'-end of the antisense strand having a linking phosphorus atom in the Rp configuration, and a terminal chiral modification that occurs in a first internucleotide linkage at the 5'-end of the sense strand having a linking phosphorus atom in either the Rp or Sp configuration.

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

[0065] In yet another embodiment, the dsRNA agent further includes a terminal chiral modification occurring at the first, second, and third internucleotide linkages at the 3'-end of the antisense strand having a linking phosphorus atom in the Sp configuration, a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand having a linking phosphorus atom in the Rp configuration, and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand having a linking phosphorus atom in either the Rp or Sp configuration.

[0066] In another embodiment, the dsRNA agent further includes a terminal chiral modification occurring at the first and second internucleotide linkages at the 3'-end of the antisense strand having a linking phosphorus atom in the Sp configuration, a terminal chiral modification occurring at the third internucleotide linkage at the 3'-end of the antisense strand having a linking phosphorus atom in the Rp configuration, a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand having a linking phosphorus atom in the Rp configuration, and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand having a linking phosphorus atom in either the Rp or Sp configuration.

[0067] In another embodiment, the dsRNA agent further includes a terminal chiral modification occurring at the first and second internucleotide linkages at the 3'-end of the antisense strand having a linking phosphorus atom in the Sp configuration, a terminal chiral modification occurring at the first and second internucleotide linkages at the 5'-end of the antisense strand having a linking phosphorus atom in the Rp configuration, and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand having a linking phosphorus atom in either the Rp or Sp configuration.

[0068] In one embodiment, the dsRNA agent further comprises a phosphate or a phosphate mimic at the 5' end of the antisense strand.

[0069] In one embodiment, the phosphate mimic is a 5'-vinylphosphonate (VP).

[0070] In one embodiment, the base pair at position 1 of the 5'-end of the antisense strand of the duplex is an AU base pair.

[0071] In one embodiment, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0072] In another aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of Filamin A (FLNA) in a cell, the dsRNA agent comprising a sense strand and an antisense strand that form a duplex region. The sense strand comprises the nucleotide sequence of any one of the agents in Tables 3-8, and the antisense strand comprises the nucleotide sequence of any one of the agents in Tables 3-8. Substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, and the dsRNA agent is conjugated to a ligand.

[0073] In various embodiments of the aforementioned dsRNA agent, the dsRNA agent targets a hotspot region of the mRNA encoding FLNA. In one embodiment, the hotspot region is nucleotides 1437-1469, 1750-1773, 3078-3104, 3210-3237, 2720-2750, 3081-3104, 3444-3467, 6583-6607, 7159-7186, 7374-7418, 1440-1469, 1852-1876, 3078-3101, 7374-7398, 7389-7418, 7400-7469, 7401-7469, 7402-7469, 7403-7418, 7404-7418, 7405-7418, 7406-7418, 7407-7418, 7408-7418, 7409 ... Including 433-7466, 8472-8497, 7390-7418, 8472-8496, 7163-7186, 3852-3896, 2698-2762, 7443-7486, 402-445, 2952-2995, 4168-4211, 6105-6148, 7150-7193, 1425-1468, 3015-3058, 2698-2741, 5341-5384, or 7384-7428.dsRNA agents are AD-1616328.1, AD-1616335.1, AD-1616534.1, AD-1616535.1, AD-1617429.2, AD-1617430.1, AD-1617431.1, AD-161 7433.1, AD-1617543.1, AD-1617548.1, AD-1617149.1, AD-1617157.1, AD-1617432.1, AD-1617665.1, AD-1617666.1, AD-16197 55.1, AD-1619756.1, AD-1619757.1, AD-1620186.1, AD-1620188.1, AD-1620190.1, AD-1620320.1, AD-1620321.1, AD-162033 4.1, AD-1620335.1, AD-1620336.1, AD-1620337.1, AD-1620338.1, AD-1616579.2, AD-1616580.1, AD-1616581.1, AD-1620322. 1, AD-1620342.1, AD-1620379.1, AD-1620380.1, AD-1620381.1, AD-1620390.1, AD-1620918.1, AD-1620919.1, AD-1620920.1 , AD-1620921.1, AD-1620191.1, AD-1617853, AD-1617875, AD-1617127, AD-1617148, AD-1617169, AD-1620389, AD-1620410, AD AD-1615540, AD-1615561, AD-1617322, AD-1617343, AD-1618077, AD-1618098, AD-1619434, AD-1619455, AD-1620177, AD-1620198, AD-1616313, AD-1616334, AD-1617366, AD-1617387, AD-1618939, AD-1618960, AD-1620330, and AD-1620352.

[0074] In another aspect, the invention provides dsRNA agents that target hotspot regions of Filamin A (FLNA) mRNA.

[0075] The present invention further provides cells and pharmaceutical compositions for inhibiting expression of a gene encoding FLNA, comprising a dsRNA agent of the invention.

[0076] In one embodiment, the dsRNA agent is in an unbuffered solution, such as saline or water.

[0077] In another embodiment, the dsRNA agent is in a buffer such as a buffer containing acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof, or phosphate buffered saline (PBS).

[0078] In one aspect, the present invention provides a method for inhibiting expression of the FLNA gene in a cell, the method comprising contacting the cell with a dsRNA agent of the present invention or a pharmaceutical composition of the present invention, thereby inhibiting expression of the FLNA gene in the cell.

[0079] In one embodiment, the cell is in a subject.

[0080] In one embodiment, the subject is a human.

[0081] In one embodiment, the subject has a FLNA-associated disorder.

[0082] In one embodiment, the FLNA-associated disorder in the subject is a neurodegenerative disorder.

[0083] In one embodiment, the neurodegenerative disorder is Alzheimer's disease.

[0084] In one embodiment, the FLNA-associated disorder is selected from the group consisting of Alzheimer's disease, tauopathy, frontotemporal dementia (FTD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), chronic traumatic encephalopathy (CTE), Pick's disease (PiD), globular glial tauopathy (GGT), argyrophilic grain disease (AGD), and primary age-related tauopathy (PART).

[0085] In one embodiment, contacting a cell with a dsRNA agent inhibits expression of FLNA by at least 30%.

[0086] In one embodiment, inhibiting expression of FLNA reduces FLNA protein levels in the serum of the subject by at least 30%.

[0087] In one aspect, the invention provides a method for treating a subject having a disorder that would benefit from reduced FLNA expression, comprising administering to the subject a therapeutically effective amount of a dsRNA agent of the invention, or a pharmaceutical composition of the invention, thereby treating the subject having a disorder that would benefit from reduced FLNA expression.

[0088] In another aspect, the present invention provides a method for preventing at least one symptom in a subject with a disorder that would benefit from reduced FLNA expression, comprising administering to the subject a prophylactically effective amount of a dsRNA agent of the present invention or a pharmaceutical composition of the present invention, thereby preventing at least one symptom in the subject with a disorder that would benefit from reduced FLNA expression.

[0089] In one embodiment, the disorder is a FLNA-associated disorder.

[0090] In one embodiment, the FLNA-associated disorder is selected from the group consisting of Alzheimer's disease, tauopathy, frontotemporal dementia (FTD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), chronic traumatic encephalopathy (CTE), Pick's disease (PiD), globular glial tauopathy (GGT), argyrophilic grain disease (AGD), and primary age-related tauopathy (PART).

[0091] In one embodiment, the FLNA-associated disorder is Alzheimer's disease.

[0092] In one embodiment, the subject is a human.

[0093] In one embodiment, administration of the agent to a subject causes a decrease in FLNA protein accumulation. In another embodiment, administration of the agent to a subject causes a decrease in altered FLNA protein accumulation.

[0094] In one embodiment, the dsRNA agent is administered to the subject at a dose of about 0.01 mg / kg to about 50 mg / kg.

[0095] In one embodiment, the dsRNA agent is administered to the subject subcutaneously.

[0096] In another embodiment, the dsRNA agent is administered to the subject intrathecally.

[0097] In one embodiment, the method of the present invention further comprises determining the level of FLNA in a sample from the subject.

[0098] In one embodiment, the FLNA level in the subject's sample is the FLNA protein level in a blood, serum, or cerebrospinal fluid sample.

[0099] In one embodiment, the methods of the invention further comprise administering to the subject an additional therapeutic agent.

[0100] In one aspect, the invention provides a kit comprising a dsRNA agent of the invention, or a pharmaceutical composition of the invention.

[0101] In another aspect, the invention provides a vial containing a dsRNA agent of the invention, or a pharmaceutical composition of the invention.

[0102] In yet another aspect, the invention provides a syringe comprising a dsRNA agent of the invention, or a pharmaceutical composition of the invention.

[0103] In another aspect, the invention provides an intrathecal pump comprising a dsRNA agent of the invention, or a pharmaceutical composition of the invention.

[0104] In one embodiment, the RNAi agent is a pharmaceutically acceptable salt thereof. The "pharmaceutically acceptable salt" of each RNAi agent herein includes, but is not limited to, sodium, calcium, lithium, potassium, ammonium, magnesium, and mixtures thereof. Those skilled in the art will understand that when provided as a polycationic salt, the RNAi agent will have one cation per free acid group of the optionally modified phosophodiester backbone and / or any other acidic modifications (e.g., phosphonate groups at the 5' end). For example, an oligonucleotide "n" nucleotides in length contains n-1 optionally modified phosophodiesters, such that a 21-nt oligonucleotide can be provided as a salt with up to 20 cations (e.g., 20 sodium cations). Similarly, an RNAi agent having a 21-nt sense strand and a 23-nt antisense strand can be provided as a salt with up to 42 cations (e.g., 42 sodium cations). In the foregoing examples, when the RNAi agent also includes a 5'-terminal phosphate group or a 5'-terminal vinylphosphonate group, the RNAi agent can be provided as a salt with up to 44 cations (e.g., 44 sodium cations). DETAILED DESCRIPTION OF THE INVENTION

[0105] The present disclosure provides an RNAi composition that performs RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of FLNA gene.The FLNA gene can be present in a cell, for example, in a subject cell such as a human.The use of these iRNAs allows the targeted degradation of the mRNA of corresponding gene (FLNA gene) in mammals.

[0106] The iRNA of the present invention is designed to target the FLNA gene, for example, the FLNA gene with nucleotide modification or the FLNA gene without nucleotide modification.The iRNA of the present invention inhibits the expression of the FLNA gene by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.Without intending to be bound by theory, it is believed that the combination or subcombination of the above-mentioned characteristics and the specific target site or specific modification in these iRNAs improves the efficacy, stability, potency, durability and safety of the iRNA of the present invention.

[0107] Thus, the present disclosure also provides methods of using the RNAi compositions of the present disclosure to inhibit expression of the FLNA gene or to treat subjects with disorders that would benefit from inhibiting or reducing expression of the FLNA gene, e.g., FLNA-associated diseases, e.g., neurodegenerative diseases such as Alzheimer's disease.

[0108] RNAi agents of the present disclosure can be about 30 nucleotides in length or less, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-25 ...5, 19-25, 19-26, 19-25, 19-25, 19-26, 19-25, 19-25, 19-26, 19-25, 1 In certain embodiments, the RNAi agent of the present disclosure comprises an RNA strand (antisense strand) having a region that is approximately 21-23 nucleotides in length, which is substantially complementary to at least a portion of an mRNA transcript of the FLNA gene, e.g., an FLNA exon.

[0109] In certain embodiments, RNAi agents of the present disclosure comprise an RNA strand (antisense strand) that can comprise a longer length, e.g., up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides, with a region of at least 19 contiguous nucleotides that is substantially complementary to at least a portion of an mRNA transcript of the FLNA gene. These RNAi agents with longer antisense strand lengths preferably comprise a second RNA strand (sense strand) 20-60 nucleotides in length, where the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

[0110] The use of these RNAi agents allows for the targeted degradation and / or inhibition of the mRNA of FLNA gene in mammals.Therefore, the methods and compositions comprising these RNAi agents are useful for treating subjects who will benefit from the reduction of the level or activity of FLNA protein, such as subjects with FLNA-related diseases, such as Alzheimer's disease.

[0111] The detailed description below discloses methods for making and using compositions containing RNAi agents that inhibit expression of the FLNA gene, as well as compositions and methods for treating subjects with diseases and disorders that would benefit from inhibiting or reducing expression of the gene.

[0112] I. Definition In order that this disclosure may be more readily understood, certain terms are first defined. Additionally, whenever a value or range of values ​​for a parameter is listed, it is intended that values ​​and ranges intermediate to the listed values ​​are also intended to be part of this disclosure.

[0113] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or to more than one element, e.g., a plurality of elements.

[0114] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to." The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise.

[0115] The term "about" is used herein to mean within a typical tolerance in the art. For example, "about" can be understood as about 2 standard deviations from the mean. In certain embodiments, about means ±10%. In certain embodiments, about means ±5%. When about precedes a series of numbers or ranges, it is understood that "about" can modify each of the consecutive numbers or ranges.

[0116] 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 can be logically included. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the specified property. When at least is before a series of numbers or ranges, it is understood that "at least" can modify each of the series of numbers or ranges.

[0117] As used herein, "less than" or "less than" refers to the value adjacent to the term and any logically smaller value or integer up to zero, if logical from the context. For example, a duplex with an overhang of "2 nucleotides or less" has an overhang of 2, 1, or 0 nucleotides. When "less than" is used before a series of numbers or ranges, it is understood that "less than" can modify each of the series of numbers or ranges.

[0118] As used herein, a method of detection can include determining that the amount of analyte present is below the detection level of the method.

[0119] In the event of a conflict between a given target site and the nucleotide sequence for either the sense or antisense strand, the given sequence controls.

[0120] If the chemical structure and chemical name do not match, the chemical structure takes precedence.

[0121] As used herein, the term "filamin A" is used interchangeably with the term "FLNA" and refers to a well-known gene and the polypeptide encoded by it, also known in the art as "filamin A," "FLNA," "FLN-A," "endothelial actin-binding protein," "actin-binding protein 280," "ABP-280," "alpha-filamin," "filamin-1," "FLN1," and "non-muscle filamin." The FLNA gene is active in the brain and other tissues throughout the body. FLNA is expressed in a variety of tissues, including the central nervous system.

[0122] FLNA encodes a protein known as filamin A, an actin-binding protein involved in cytoskeletal reorganization. FLNA functions as a homodimer and contains an N-terminal actin-binding domain and 24 immunoglobulin-like domains. In addition to its role in organizing filamentous actin into intracellular networks and stress fibers, FLNA also provides a scaffold for a range of signaling proteins by anchoring them to the actin cytoskeleton. FLNA interacts with a diverse repertoire of signaling molecules and transmembrane receptors, suggesting that it plays an important role in cell signaling.

[0123] FLNA protein with an altered conformation has been implicated in Alzheimer's disease. Without wishing to be bound by theory, amyloid beta 1-42 (Aβ42) induces a conformational change in FLNA, which allows it to associate with the α7-nicotinic acetylcholine receptor (α7nAChR) and toll-like receptor 4 (TLR4). This allows Aβ42 to signal through the α7nAChR, resulting in the activation of kinases that hyperphosphorylate tau protein and cause neurofibrillary tangle formation. Furthermore, the abnormal association of FLNA with TLR4 promotes Aβ42 activation of TLR4, which induces the release of inflammatory cytokines and neuroinflammation. (Burns, et al., 2017, Neuroimm. and Neuroinflam. 4:263-71)

[0124] The small molecule inhibitor PTI-125 (simufilam) binds to the altered FLNA and restores its native form, reducing the pathology associated with Alzheimer's disease. In a mouse model of Alzheimer's disease, PTI-125 administration improved synaptic plasticity, spatial and working memory, reduced tau hyperphosphorylation, neurogenic inflammation, and neurofibrillary tangles (Wang, et al., 2017, Neurobiol. Aging 55:99-114).

[0125] Examples of nucleotide and amino acid sequences for FLNA can be found, for example, in GenBank accession numbers NM_001110556.2 (Homo sapiens FLNA, SEQ ID NO: 1, reverse complement, SEQ ID NO: 2) and XM_006527911.5 (Mus musculus FLNA, SEQ ID NO: 1357, reverse complement, SEQ ID NO: 1358).

[0126] The nucleotide sequence of the genomic region of a human chromosome carrying the FLNA gene can be found, for example, in Genome Reference Consortium Human Build 38 (also referred to as Human Genome build 38 or GRCh38), available at GenBank. The nucleotide sequence of the genomic region of human chromosome X carrying the FLNA gene can also be found, for example, in GenBank accession number NC_000023.11, which corresponds to nucleotides 154348531-154374634 of human chromosome X. The nucleotide sequence of the human FLNA gene can be found, for example, in GenBank accession number NC_000023.11.

[0127] Further examples of FLNA sequences can be found in publicly available databases such as GenBank, OMIM, and UniProt.

[0128] Additional information regarding FLNA can be found, for example, at https: / / www.ncbi.nlm.nih.gov / gene / 2316. As used herein, the term FLNA also refers to variations in the FLNA gene, including variants provided in the clinical variant database, for example, at https: / / www.ncbi.nlm.nih.gov / clinvar / ?term=NM_001110556.2.

[0129] The entire contents of each of the foregoing GenBank Accession Numbers and Gene Database Numbers are incorporated herein by reference as of the filing date of this application.

[0130] In some aspects, the iRNA that is substantially complementary to the region of human FLNA mRNA cross-reacts with mouse FLNA mRNA.In some aspects, the iRNA that is substantially complementary to the region of mouse FLNA mRNA cross-reacts with human FLNA mRNA.In some embodiments, the iRNA that is substantially complementary to the region of mouse or human FLNA mRNA cross-reacts with rat, monkey and rabbit FLNA mRNA.

[0131] As used herein, " target sequence " refers to the continuous portion of the nucleotide sequence of the mRNA molecule formed during the transcription of FLNA gene, such as the mRNA that is the product of RNA processing of primary transcript.In one embodiment, the target portion of the sequence will be at least long enough to function as the substrate for RNAi-directed cleavage at or near the portion of the nucleotide sequence of the mRNA molecule formed during the transcription of FLNA gene.

[0132] The target sequence is about 15 to 30 nucleotides in length. For example, the target sequence may be about 15 to 30 nucleotides, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 The target sequence may be 19-23, 21-22, 21-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In certain embodiments, the target sequence is 19-23 nucleotides in length, and optionally 21-23 nucleotides in length. Ranges and lengths between the ranges and lengths listed above are also intended to be part of this disclosure.

[0133] As used herein, the term "strand containing a sequence" refers to an oligonucleotide containing a chain of nucleotides described by a sequence referenced using standard nucleotide nomenclature. "G", "C", "A", "T", and "U" generally refer to nucleotides containing guanine, cytosine, adenine, thymidine, and uracil as bases, respectively, in the context of modified or unmodified nucleotides. However, it will be understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides, as described in more detail below, or alternative replacement moieties (see, for example, Table 2). Those skilled in the art are well aware that guanine, cytosine, adenine, thymidine, and uracil can be substituted with other moieties without substantially changing the base pairing properties of oligonucleotides containing nucleotides with such replacement moieties. For example, but not limited to, a nucleotide containing inosine as its base can base pair with a nucleotide containing adenine, cytosine, or uracil. Therefore, the nucleotide that contains uracil, guanine or adenine can be substituted with the nucleotide that contains inosine in the nucleotide sequence of the dsRNA described in the present disclosure.In another embodiment, the adenine and cytosine at any position in oligonucleotide can be substituted with guanine and uracil, respectively, to form the GU wobble base that pairs with target mRNA.The sequence that contains such a substitution portion is suitable for the composition and method described in the present disclosure.

[0134] As used herein, the terms " iRNA ", " RNAi agent ", " iRNA agent ", " RNA interference agent " refer to the agent that contains the RNA as defined herein and mediates the targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway.RNA interference (RNAi) is the process that controls the sequence-specific degradation of mRNA.RNAi regulates (for example, inhibits) the expression of FLNA in cells, for example, in cells of a subject, such as a mammalian subject.

[0135] In one embodiment, the RNAi agent of the present disclosure comprises a single-stranded RNAi that interacts with a target RNA sequence, such as an FLNA target mRNA sequence, to cleave the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNA introduced into cells is degraded into double-stranded small interfering RNAs (siRNAs) comprising a sense strand and an antisense strand by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). The RNase III-like enzyme Dicer processes this dsRNA into 19-23 base pair small interfering RNAs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). These siRNAs are then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, thereby allowing target recognition to be induced by complementary antisense strands (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases in RISC cleave the target and induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). That is, in one aspect, the present disclosure relates to a single-stranded RNA (ssRNA) (the antisense strand of the siRNA duplex) that is produced in cells and promotes the formation of a RISC complex to silence the target gene, i.e., the FLNA gene. Therefore, the term "siRNA" is used herein to also refer to the RNAi described above.

[0136] In another embodiment, the RNAi agent can be a single-stranded RNA introduced into a cell or organism to inhibit target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides and chemically modified. The design and testing of single-stranded RNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein can be used as the single-stranded siRNA described herein or as the single-stranded siRNA that is chemically modified by the method described in Lima et al., (2012) Cell 150:883-894.

[0137] In another embodiment, the "RNAi agent" used in the compositions and methods of the present disclosure is double-stranded RNA, and is also referred to herein as "double-stranded RNAi agent", "double-stranded RNA (dsRNA) molecule", "dsRNA agent" or "dsRNA". The term "dsRNA" refers to a complex of ribonucleic acid molecules with a duplex structure, comprising two antiparallel, substantially complementary nucleic acid strands, which are referred to as having "sense" and "antisense" orientation with respect to target RNA, i.e., FLNA gene. In some embodiments of the present disclosure, double-stranded RNA (dsRNA) induces the degradation of target RNA, for example, mRNA, through a post-transcriptional gene silencing mechanism, herein referred to as RNA interference or RNAi.

[0138] Generally, dsRNA molecules can contain ribonucleotides, but as described in detail herein, each or both strands can also contain one or more non-ribonucleotides, such as deoxyribonucleotides, modified nucleotides.In addition, as used herein, " RNAi agent " can include ribonucleotides with chemical modifications, and RNAi agent can contain substantial modifications in multiple nucleotides.As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide linkage, or a modified nucleobase.Therefore, the term modified nucleotide encompasses the substitution, addition, or removal of, for example, functional groups or atoms, etc., to internucleoside linkage, sugar moiety, or nucleobase.The modifications suitable for use in the agent of the present disclosure include all types of modifications disclosed herein or known in the art.Any such modifications used in siRNA type molecules are encompassed by " RNAi agent " for the purpose of this specification and claims.

[0139] In certain embodiments of the present disclosure, the inclusion of deoxy-nucleotides, if present within an RNAi agent, can be considered to constitute modified nucleotides.

[0140] The duplex region may be of any length that allows for specific degradation of the desired target RNA via the RISC pathway, and may be about 15 to 36 base pairs in length, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, e.g., about 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 22, 18 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 18 to 29, 18 to 31, 18 to 32, 18 to 29, 18 to 33, 18 to 34, 18 to 35, 18 to 36, or 18 to 37 base pairs in length. The length may be in the range of 8, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. In certain embodiments, the duplex region is 19-21 base pairs in length, e.g., 21 base pairs in length. Ranges and lengths between the ranges and lengths listed above are also intended to be part of this disclosure.

[0141] The two strands forming the duplex structure can be different portions of a single larger RNA molecule, or they can be separate RNA molecules. When two strands are part of a single larger molecule and are therefore connected by an uninterrupted chain of nucleotides between the 3' end of one strand forming the duplex structure and the corresponding 5' end of the other strand, the connected RNA strands are called "hairpin loops." A hairpin loop can contain at least one unpaired nucleotide. In some embodiments, a hairpin loop can contain at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23, or more unpaired nucleotides or nucleotides not directed toward the target site of the dsRNA. In some embodiments, a hairpin loop can be 10 or fewer nucleotides. In some embodiments, a hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, a hairpin loop can be 4 to 10 unpaired nucleotides. In some embodiments, a hairpin loop can be 4 to 8 nucleotides.

[0142] When the two substantially complementary strands of dsRNA are composed of separate RNA molecules, these molecules can, but do not necessarily, be covalently linked. In certain embodiments, when the two strands are covalently linked by means other than an uninterrupted chain of nucleotides between the 3'-end of one strand and the corresponding 5'-end of the other strand that form a duplex structure, this connecting structure is called a "linker" (note, however, that other specific structures as defined elsewhere in this specification are also called "linkers"). The RNA strands can have the same or different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, the RNAi can include one or more nucleotide overhangs. In one embodiment of an RNAi agent, at least one strand includes a 3'-overhang of at least one nucleotide. In another embodiment, at least one strand comprises a 3' overhang of at least 2 nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5' overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5' overhang of at least 2 nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3' end and the 5' end of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide.

[0143] In one embodiment, the RNAi agent of the present disclosure is a dsRNA, each strand of which independently comprises 19-23 nucleotides that interact with a target RNA sequence, e.g., a FLNA target mRNA sequence, to induce cleavage of the target RNA.

[0144] In some embodiments, the iRNA of the invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, such as a FLNA target mRNA sequence, and mediates cleavage of the target RNA.

[0145] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of an RNAi agent, such as dsRNA.For example, a nucleotide overhang exists when the 3'-end of one strand of dsRNA extends beyond the 5'-end of the other strand, or vice versa.A dsRNA can comprise an overhang of at least one nucleotide, or the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more.A nucleotide overhang can comprise or consist of nucleotide / nucleoside analogs, such as deoxynucleotides / nucleosides.An overhang can be on the sense strand, on the antisense strand, or any combination thereof.Furthermore, a certain overhanging nucleotide can be present on the 5'-end, 3'-end, or both ends of either the antisense strand or the sense strand of dsRNA.

[0146] In one embodiment, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more of the nucleotides in the overhang are substituted with a nucleoside thiophosphate.

[0147] In certain embodiments, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, for example, 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more of the nucleotides in the overhang are substituted with nucleoside thiophosphates.

[0148] In certain embodiments, the overhang on the sense strand or the antisense strand can comprise an extended length greater than 10 nucleotides, e.g., 1 to 30 nucleotides, 2 to 30 nucleotides, 10 to 30 nucleotides, or 10 to 15 nucleotides in length. In certain embodiments, the extended overhang is on the sense strand of the duplex. In certain embodiments, the extended overhang is on the 3'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the 5'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the antisense strand of the duplex. In certain embodiments, the extended overhang is on the 3'-end of the antisense strand of the duplex. In certain embodiments, the extended overhang is on the 5'-end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the overhang are substituted with a nucleoside thiophosphate. In certain embodiments, the overhang comprises a self-complementary portion that enables the overhang to form a stable hairpin structure under physiological conditions.

[0149] The term "blunt" or "blunt-end" when used herein in relation to dsRNA means that there is no unpaired nucleotide or nucleotide analogue at the predetermined end of dsRNA, that is, there is no nucleotide overhang.One end or both ends of dsRNA can be blunt.When both ends of dsRNA are blunt, dsRNA is said to be blunt-ended.For clarity, "blunt-end" dsRNA is the dsRNA that has both ends blunt, that is, there is no nucleotide overhang at either end of the molecule.In most cases, this molecule will be double-stranded throughout its entire length.

[0150] The term "antisense strand" or "guide strand" refers to the strand of an RNAi agent, e.g., dsRNA, that includes a region that is substantially complementary to a target sequence, e.g., FLNA mRNA.

[0151] As used herein, the term "region of complementarity," as defined herein, refers to a region on the antisense strand that is substantially complementary to a sequence, for example, a target sequence, for example, an FLNA nucleotide sequence. If the complementary region is not completely complementary to the target sequence, the mismatch can be within the internal region or terminal region of the molecule. Generally, the most tolerable mismatch is within the terminal region, for example, within 5, 4, 3, or 2 nucleotides of the 5' or 3' end of the RNAi agent. In some embodiments, the double-stranded RNA agent of the present invention comprises a nucleotide mismatch within the antisense strand. In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention comprises 4 or less mismatches with the target mRNA, for example, the antisense strand comprises 4, 3, 2, 1, or 0 mismatches with the target mRNA. In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention comprises 4 or less mismatches with the sense strand, for example, the antisense strand comprises 4, 3, 2, 1, or 0 mismatches with the sense strand. In some embodiments, the double-stranded RNA agent of the present invention contains a nucleotide mismatch in the sense strand. In some embodiments, the sense strand of the double-stranded RNA agent of the present invention contains four or fewer mismatches with the antisense strand, for example, the sense strand contains 4, 3, 2, 1, or 0 mismatches with the antisense strand. In some embodiments, the nucleotide mismatch is, for example, within 5, 4, or 3 nucleotides from the 3' end of the iRNA. In another embodiment, the nucleotide mismatch is, for example, in the 3' terminal nucleotide of the iRNA agent. In some embodiments, the mismatch is not in the seed region.

[0152] Therefore, the RNAi agent described herein may contain one or more mismatches to the target sequence. In one embodiment, the RNAi agent described herein contains 3 or fewer mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, the RNAi agent described herein contains 2 or fewer mismatches. In one embodiment, the RNAi agent described herein contains 1 or fewer mismatches. In one embodiment, the RNAi agent described herein contains 0 mismatches. In certain embodiments, when the antisense strand of an RNAi agent contains a mismatch to the target sequence, the mismatch can be optionally limited to be within the last 5 nucleotides from either the 5'-end or 3'-end of the complementary region. For example, in such an embodiment, for a 23-nucleotide RNAi agent, the strand complementary to the region of the FLNA gene generally does not contain any mismatches within the central 13 nucleotides. By using the methods described herein or known in the art, it can be determined whether an RNAi agent containing a mismatch to the target sequence is effective in inhibiting the expression of the FLNA gene. It is important to consider the effectiveness of mismatched RNAi agents in inhibiting expression of the FLNA gene, especially if specific complementary regions within the FLNA gene are known to have polymorphic sequence variation within the population.

[0153] RNA targets may have regions or spans of the nucleotide sequence of target RNA that are relatively more susceptible or receptive than other regions of the RNA target to mediating RNA target cleavage through RNA interference induced by the binding of RNAi agents to those regions.The increased receptivity to RNA interference in these "hotspot regions" (or simply "hotspots") means that iRNA agents that target those regions are likely to be more effective in inducing iRNA interference than iRNA agents that target other regions of the target RNA.For example, without being bound by theory, the accessibility of the target region of target RNA can affect the effectiveness of iRNA agents that target that region, and some hotspot regions have increased accessibility.For example, secondary structures formed within an RNA target (e.g., within or near hotspot regions) can affect the ability of iRNA agents to bind to target regions and induce RNA interference.

[0154] According to certain embodiments of the invention, iRNA agents can be designed to target hotspot regions of any of the target RNAs described herein, including any specified portion of the target RNA (e.g., a particular exon). As used herein, a hotspot region can refer to a region of about 19-200, 19-150, 19-100, 19-75, 19-50, 21-200, 21-150, 21-100, 21-75, 21-50, 50-200, 50-150, 50-100, 50-75, 75-200, 75-150, 75-100, 100-20, or 100-150 nucleotides of a target RNA sequence where targeting with an RNAi agent provides a significantly higher likelihood of effective silencing compared to targeting other regions of the same target RNA. According to certain embodiments of the present invention, hotspot regions can comprise a limited region of target RNA, and in some cases, a substantially limited region of target RNA, for example, comprising less than half of the length of target RNA, for example, about 5%, 10%, 15%, 20%, 25%, or 30% of the length of target RNA. Conversely, other regions to which hotspots are compared can cumulatively comprise at least half of the length of target RNA. For example, other regions can cumulatively comprise at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95% of the length of target RNA.

[0155] Comparative regions of the target RNA can be empirically evaluated to identify hotspots using efficacy data obtained from in vitro or in vivo screening assays. For example, RNAi agents targeting various regions spanning the target RNA can be compared for the frequency of effective iRNA agents binding to each region (e.g., the amount of target gene expression inhibited, as measured by mRNA expression or protein expression). Generally, hotspots can be recognized by observing the clustering of multiple effective RNAi agents binding to a limited region of the RNA target. A hotspot can be fully characterized by observing the efficacy of iRNA agents that cumulatively cover at least about 60% of the target region identified as a hotspot, for example, about 70%, about 80%, about 90%, or about 95% or more of the length of the region, including both ends of the region (i.e., at least about 60%, 70%, 80%, 90%, or 95% or more of the nucleotides within the region, including the nucleotides at each end of the region, are targeted by the iRNA agent). According to some embodiments of the invention, an iRNA agent that exhibits at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% inhibition across the region (e.g., about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less of mRNA remaining) can be identified as effective.

[0156] The suitability of an RNA region for targeting can also be evaluated using a quantitative comparison of inhibition measurements across different regions of defined size (e.g., 25, 30, 40, 50, 60, 70, 80, 90, or 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nts). For example, the average level of inhibition can be determined for each region, and the averages of each region can be compared. The average level of inhibition within a hotspot region can be substantially higher than the average of all the evaluated regions. According to some embodiments, the average level of inhibition in a hotspot region can be at least about 10%, 20%, 30%, 40%, or 50% higher than the average of the averages. According to some embodiments, the average level of inhibition in a hotspot region may be at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 standard deviations higher than the mean of the means. The average level of inhibition may be higher by a statistically significant amount (e.g., p<0.05). According to some embodiments, each measured inhibition within a hotspot region may exceed a threshold amount (e.g., below a threshold amount of residual mRNA). According to some embodiments, each measured inhibition within a region may be substantially higher than the average of all measured inhibition measurements across all measured regions. For example, each measured inhibition in a hotspot region may be at least about 10%, 20%, 30%, 40%, or 50% higher than the average of all measured inhibition measurements. According to some embodiments, each inhibition measurement may be at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 standard deviations higher than the mean of all inhibition measurements. Each inhibition measurement may be higher than the mean of all inhibition measurements by a statistically significant amount (e.g., p<0.05). Criteria for evaluating hotspots may include meeting various combinations of the above standards (e.g., an average level of inhibition of at least about a first amount, with no inhibition measurements below a threshold level of a second amount less than the first amount).

[0157] Therefore, it is expressly contemplated that any iRNA agent, including certain exemplary iRNA agents described herein, that targets a hotspot region of target RNA can be preferably selected to induce RNA interference of target mRNA, because targeting such hotspot region is more likely to show robust inhibitory response compared to targeting a region that is not a hotspot region. RNAi agents that target a target sequence that substantially overlaps (for example, at least about 70%, 75%, 80%, 85%, 90%, 95% of the target sequence length), or preferably, is completely within a hotspot region, can be considered to target a hotspot region. The hotspot region of the RNA target of the present invention can include any region that the data disclosed herein shows a higher frequency of targeting by effective RNAi agents, including by any of the criteria described elsewhere herein, regardless of whether the scope of such hotspot region is explicitly specified.

[0158] In various embodiments, the dsRNA agent of the present invention targets the hotspot region of the mRNA encoding FLNA.In one embodiment, the hotspot region is nucleotides 1437-1469, 1750-1773, 3078-3104, 3210-3237, 2720-2750, 3081-3104, 3444-3467, 6583-6607, 7159-7186, 7374-7418, 1440-1469, 1852-1876, 3078-3101, 7374-7398, 7389-7418, 7 ... Including 433-7466, 8472-8497, 7390-7418, 8472-8496, 7163-7186, 3852-3896, 2698-2762, 7443-7486, 402-445, 2952-2995, 4168-4211, 6105-6148, 7150-7193, 1425-1468, 3015-3058, 2698-2741, 5341-5384, or 7384-7428.dsRNA agents are AD-1616328.1, AD-1616335.1, AD-1616534.1, AD-1616535.1, AD-1617429.2, AD-1617430.1, AD-1617431.1, AD-161 7433.1, AD-1617543.1, AD-1617548.1, AD-1617149.1, AD-1617157.1, AD-1617432.1, AD-1617665.1, AD-1617666.1, AD-16197 55.1, AD-1619756.1, AD-1619757.1, AD-1620186.1, AD-1620188.1, AD-1620190.1, AD-1620320.1, AD-1620321.1, AD-162033 4.1, AD-1620335.1, AD-1620336.1, AD-1620337.1, AD-1620338.1, AD-1616579.2, AD-1616580.1, AD-1616581.1, AD-1620322. 1, AD-1620342.1, AD-1620379.1, AD-1620380.1, AD-1620381.1, AD-1620390.1, AD-1620918.1, AD-1620919.1, AD-1620920.1 , AD-1620921.1, AD-1620191.1, AD-1617853, AD-1617875, AD-1617127, AD-1617148, AD-1617169, AD-1620389, AD-1620410, AD AD-1615540, AD-1615561, AD-1617322, AD-1617343, AD-1618077, AD-1618098, AD-1619434, AD-1619455, AD-1620177, AD-1620198, AD-1616313, AD-1616334, AD-1617366, AD-1617387, AD-1618939, AD-1618960, AD-1620330, and AD-1620352.

[0159] As used herein, "substantially all of the nucleotides are modified" means extensively but not entirely modified and can include no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0160] The term "sense strand" or "passenger strand," as used herein, refers to the strand of an RNAi agent that includes a region that is substantially complementary to a region of the antisense strand, as those terms are defined herein.

[0161] As used herein, the term "cleavage region" refers to the region located directly adjacent to the cleavage site.Cleavage site is the site on the target where cleavage occurs.In some embodiments, the cleavage region comprises three bases on either end of the cleavage site and directly adjacent to the cleavage site.In some embodiments, the cleavage region comprises two bases on either end of the cleavage site and directly adjacent to the cleavage site.In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and this cleavage region comprises nucleotides 11, 12, and 13.

[0162] As used herein, unless otherwise specified, the term "complementary," when used to describe a first nucleotide sequence in the context of a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize to form a duplex structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under specified conditions, as understood by those skilled in the art. Such conditions can be, for example, stringent conditions, such as 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12-16 hours followed by a wash (see, e.g., "Molecular Cloning: A Laboratory Manual," Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions encountered in organisms, can be applied. Those skilled in the art can determine the most appropriate set of conditions for testing the complementarity of two sequences depending on the ultimate use of the hybridized nucleotides.

[0163] A complementary sequence within an RNAi agent, such as within a dsRNA described herein, involves base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence with an oligonucleotide or polynucleotide comprising a second nucleotide sequence throughout the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as being "fully complementary" to each other. However, when a first sequence is referred to herein as being "substantially complementary" to a second sequence, the two sequences may be perfectly complementary, or they may form one or more, but generally no more than five, four, three, or two, mismatched base pairs upon hybridization of a duplex of up to 30 base pairs, while maintaining the ability to hybridize under conditions most relevant to its end use, such as inhibiting gene expression via the RISC pathway. However, if two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs shall not be considered mismatches in determining complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, where the longer oligonucleotide comprises a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, may still be referred to as "fully complementary" for purposes described herein.

[0164] "Complementary" sequences, as used herein, may also include or be formed entirely of non-Watson-Crick base pairs, or base pairs formed from non-naturally occurring modified nucleotides, so long as they satisfy the above requirements regarding their ability to hybridize. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsteen base pairing.

[0165] The terms "complementary," "fully complementary," and "substantially complementary" herein may be used in reference to base matching between the sense and antisense strands of a dsRNA, or between the antisense strand of an RNAi agent and a target sequence, as will be understood from the context of their use.

[0166] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding FLNA). For example, a polynucleotide is complementary to at least a portion of an FLNA mRNA if the sequence is substantially complementary to an uninterrupted portion of the mRNA encoding FLNA.

[0167] Therefore, in some embodiments, the antisense polynucleotides disclosed herein are completely complementary to the target FLNA sequence. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target FLNA sequence, and comprise a contiguous nucleotide sequence that is at least 80% complementary, for example, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary to the equivalent region of any one of the nucleotide sequences of SEQ ID NOs: 1, 3, 5, and 7, or to a fragment of any one of SEQ ID NOs: 1, 3, 5, and 7, over its entire length.

[0168] In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of a target FLNA sequence, including nucleotides 98-120, 174-196, 242-264, 373-395, 402-424, 423-445, 471-493, 498-520, 543-565, 596-618, 639-661, 663-685, 687-709, 712-734, 869-891, 904-926, 931-953, 1000-1022, 1075-1097, 1125-1147, 1149-1171, 1180-1182, 1182-1184, 1184-1186, 1186-1188, 1188-1189, 1190-1193, 1200-1201, 1202-1203, 1204-1205, 1206-1207, 1208-1210, 1212-1213, 1214-1215, 1216-1217, 1218-1219, 1220-1221, 1222-1223, 1224-1225, 1226-1227, 1228-1230, 1228-1231, 1228-1232, 1228-1233, 1228-1234, 1228-1235, 1228-1236 7-1209, 1233-1255, 1256-1278, 1303-1325, 1326-1348, 1353-1375, 1389-1411, 1425-1447, 1446-1468, 1486-1508, 1528-1550, 1561-1583, 1658-1668 80, 1747-1769, 1788-1810, 1852-1874, 1886-1908, 1924-1946, 1954-1976, 2021-2043, 2066-2088, 2101-2123, 2130-2152, 2185-2207, 2217-2239, 22 56-2278, 2315-2337, 2338-2360, 2374-2396, 2397-2419, 2419-2441, 2466-2488, 2551-2573, 2613-2635, 2652-2674, 2698-2720, 2719-2741, 2740-2 762, 2781-2803, 2839-2861, 2898-2920, 2928-2950, ​​2952-2974, 2973-2995, 3015-3037, 3036-3058, 3078-3100, 3122-3144, 3153-3175, 3187-3209, 3 212-3234, 3247-3269, 3321-3343, 3373-3395, 3436-3458, 3501-3523, 3567-3589, 3599-3621, 3645-3667, 3712-3734, 3780-3802, 3824-3846, 3852- 3874, 3874-3896, 3928-3950, 3997-4019, 4039-4061, 4064-4086, 4120-4142, 4143-4165, 4168-4190, 4189-4211, 4215-4237, 4334-4356, 4365-4387,4440-4462、4489-4511、4514-4536、4545-4567、4567-4589、4620-4642、4650-4672、4692-4714、4744-4766、4810-4832、4867-4889、4941-4963、4975-4997、5020-5042、5058-5080、5086-5108、5178-5200、5203-5225、5253-5275、5288-5310、5312-5334、5341-5363、5362-5384、5403-5425、5519-5541、5580-5602、5610-5632、5637-5659、5701-5723、5770-5792、5809-5831、5845-5867、5874-5896、5908-5930、5965-5987、5990-6012、6036-6058、6105-6127、6126-6148、6161-6183、6241-6263、6270-6292、6298-6320、6356-6378、6389-6411、6439-6461、6477-6499、6507-6529、6543-6565、6579-6601、6709-6731、6739-6761、6796-6818、6824-6846、6847-6869、6871-6893、6985-7007、7012-7034、7086-7108、7123-7145、7150-7172、7171-7193、7204-7226、7251-7273、7286-7308、7384-7406、7406-7428、7443-7465、7464-7486、7535-7557、7558-7580、7584-7606、7655-7677、7726-7748、7830-7852、7976-7998、8000-8022、8046-8068、8079-8101、8118-8140、8357-8379、8402-8424、8445-8467、8481-8503、169-191、171-193、373-395、540-562、1440-1462、1447-1469、1749-1771、1750-1772、1751-1773、1852-1874、1853-1875、1854-1876、2720-2742、2728-2750、3078-3100、3079-3101、3080-3102, 3081-3103, 3082-3104, 3210-3232, 3215-3237, 3443-3465, 3444-3466, 3445-3467, 4066-4088, 5180-5202, 5181-5203, 5976-5998, 6044-6066, 6430-6452, 65 83-6605, 6584-6606, 6585-6607, 7077-7099, 7159-7181, 7161-7183, 7163-7185, 7164-7186, 7374-7396, 7375-7397, 7376-7398, 7388-7410, 7389-7411, 7390-7412, 7391 7413, 7392-7414, 7396-7418, 7433-7455, 7434-7456, 7435-7457, 7444-7466, 7652-7674, 7653-7675, 7655-7677, 7724-7746, 8402-8424, 8472-8494, 8473-8495, 8474-8496, and 8475-8497. Ranges intermediate to the above-listed ranges are also contemplated as part of this disclosure.

[0169] In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of a target FLNA sequence, including nucleotides 921-943, 1567-1589, 1568-1590, 1570-1592, 1985-2007, 2220-2242, 2558-2580, 2559-2581, 2743-2765, 3337-3359, 3338-3360, 4021-4043, 4714-4736, 5489-5511, 5938-5960, 6086-6108, 6702-6724, 6703-6725 of SEQ ID NO: 1357. , 6705-6727, 6862-6884, 6863-6885, 6864-6886, 6947-6969, 6963-6985, 7374-7396, 7517-7539, 7959-7981, 8098-8120, 8099-8121, and 8539-8561. Ranges intermediate to the above-listed ranges are also contemplated as part of the present disclosure.

[0170] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target FLNA sequence and comprise a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary, over its entire length to any one of the sense strand nucleotide sequences in Tables 3-8, or a fragment of any one of the sense strand nucleotide sequences in Tables 3-8.

[0171] In one embodiment, the RNAi agent of the present disclosure is substantially complementary to an antisense polynucleotide that is identical to the target FLNA sequence and comprises a sense strand, and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, for example, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary to the equivalent region of the nucleotide sequence of SEQ ID NOs: 1, 3, 5, and 7, or to a fragment of any one of SEQ ID NOs: 1, 3, 5, and 7, over its entire length.

[0172] In some embodiments, the iRNA of the present invention comprises a sense strand that is substantially complementary to an antisense polynucleotide that is in turn complementary to a target FLNA sequence, wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary over its entire length to any one of the antisense strand nucleotide sequences in Tables 3-8, or a fragment of any one of the antisense strand nucleotide sequences in Tables 3-8.

[0173] In certain embodiments, the sense and antisense strands are duplexed with AD-1615378, AD-1615433, AD-1615454, AD-1615511, AD-1615540 AD-1615561, AD-1615604, AD-1615631, AD-1615676, AD-1615729, AD-1615772, AD-1615796, AD-1615820, AD-1615843, AD-1615930, AD-1 615964, AD-1615991, AD-1616007, AD-1616044, AD-1616087, AD-1616111, AD-1616149, AD-1616182, AD-1616205, AD-1616222, AD-16162 45, AD-1616252, AD-1616288, AD-1616313, AD-1616334, AD-1616364, AD-1616386, AD-1616399, AD-1616471, AD-1616531, AD-1616554, A D-1616579, AD-1616593, AD-1616613, AD-1616643, AD-1616682, AD-1616707, AD-1616742, AD-1616771, AD-1616821, AD-1616833, AD-16 16852, AD-1616911, AD-1616934, AD-1616950, AD-1616972, AD-1616994, AD-1617041, AD-1617061, AD-1617103, AD-1617117, AD-161712 7, AD-1617148, AD-1617169, AD-1617186, AD-1617219, AD-1617268, AD-1617298, AD-1617322, AD-1617343, AD-1617366, AD-1617387, AD -1617429, AD-1617455, AD-1617486, AD-1617520, AD-1617545, AD-1617580, AD-1617612, AD-1617644, AD-1617657, AD-1617679, AD-161 7703, AD-1617717, AD-1617743, AD-1617790, AD-1617815, AD-1617843, AD-1617853, AD-1617875, AD-1617899, AD-1617939, AD-1617981,AD-1618006、AD-1618049、AD-1618052、AD-1618077、AD-1618098、AD-1618124、AD-1618187、AD-1618214、AD-1618237、AD-1618286、AD-1618311、AD-1618342、AD-1618364、AD-1618404、AD-1618434、AD-1618456、AD-1618508、AD-1618572、AD-1618601、AD-1618645、AD-1618661、AD-1618706、AD-1618744、AD-1618772、AD-1618810、AD-1618835、AD-1618851、AD-1618886、AD-1618910、AD-1618939、AD-1618960、AD-1618979、AD-1619014、AD-1619034、AD-1619064、AD-1619071、AD-1619116、AD-1619178、AD-1619197、AD-1619233、AD-1619262、AD-1619296、AD-1619333、AD-1619358、AD-1619385、AD-1619434、AD-1619455、AD-1619470、AD-1619529、AD-1619540、AD-1619549、AD-1619586、AD-1619601、AD-1619651、AD-1619689、AD-1619699、AD-1619735、AD-1619751、AD-1619849、AD-1619879、AD-1619936、AD-1619946、AD-1619969、AD-1619993、AD-1620033、AD-1620060、AD-1620113、AD-1620150、AD-1620177、AD-1620198、AD-1620211、AD-1620244、AD-1620279、AD-1620330、AD-1620352、AD-1620389、AD-1620410、AD-1620426、AD-1620449、AD-1620475、AD-1620525、AD-1620574、AD-1620616、AD-1620707、AD-1620731、AD-1620737、AD-1620767、AD-1620787、AD-1620837、AD-1620879、AD-1620891、AD-1620927、AD-1615428.1、AD-1615430.1、AD-1615511.2、AD-1615673.1、AD-1616328.1、AD-1616335.1、AD-1616533.1、AD-1616534.1、AD-1616535.1、AD-1616579.2、AD-1616580.1、AD-1616581.1、AD-1617149.1、AD-1617157.1、AD-1617429.2、AD-1617430.1、AD-1617431.1、AD-1617432.1、AD-1617433.1、AD-1617543.1、AD-1617548.1、AD-1617664.1、AD-1617665.1、AD-1617666.1、AD-1618008.1、AD-1618812.1、AD-1618813.1、AD-1619344.1、AD-1619393.1、AD-1619642.1、AD-1619755.1、AD-1619756.1、AD-1619757.1、AD-1620104.1、AD-1620186.1、AD-1620188.1、AD-1620190.1、AD-1620191.1、AD-1620320.1、AD-1620321.1、AD-1620322.1、AD-1620334.1、AD-1620335.1、AD-1620336.1、AD-1620337.1、AD-1620338.1、AD-1620342.1、AD-1620379.1、AD-1620380.1、AD-1620381.1、AD-1620390.1、AD-1620522.1、AD-1620523.1、AD-1620525.2、AD-1620572.1、AD-1620879.2、AD-1620918.1、AD-1620919.1、AD-1620920.1、AD-1620921.1、AD-1687606.1、AD-1688124.1、AD-1688125.1、AD-1688127.1、AD-1688431.1、AD-1688626.1、AD-1688897.1、AD-1688898.1、AD-1689041.1、AD-1689527.1、AD-1689528.1、AD-1690032.1、AD-1690554.1、AD-1691133.1、AD-1691437.1、AD-1691559.1、Selected from any one of AD-1692108.1, AD-1692109.1, AD-1692111.1, AD-1692242.1, AD-1692243.1, AD-1692244.1, AD-1692317.1, AD-1692333.1, AD-1692616.1, AD-1692718.1, AD-1693004.1, AD-1693103.1, AD-1693104.1, and AD-1693450.1.

[0174] In one embodiment, at least partial suppression of the expression of the FLNA gene is evaluated by a decrease in the amount of FLNA mRNA, for example, sense mRNA, antisense mRNA, total FLNA mRNA, and the amount of FLNA mRNA, for example, sense mRNA, antisense mRNA, total FLNA mRNA, which can be isolated from or detected in a first cell or group of cells in which the FLNA gene is transcribed and which has been treated so that the expression of the FLNA gene is inhibited, is compared with a second cell or group of cells (control cells) that is substantially identical to the first cell or group of cells but has not been treated in the same way as the first cell or group of cells. The degree of inhibition can be expressed by:

number

[0175] As used herein, the phrase " contacting cell with RNAi agent " such as dsRNA includes contacting cell by any possible means.Contacting cell with RNAi agent includes contacting cell with RNAi agent in vitro or contacting cell with RNAi agent in vivo.Contacting can be carried out directly or indirectly.Therefore, for example, RNAi agent can be physically contacted with cell by carrying out a method separately, or RNAi agent can be placed in a situation that can allow or cause it to contact cell afterwards.

[0176] Contacting cells in vitro can be carried out, for example, by incubating cells with an RNAi agent. Contacting cells in vivo can be carried out, for example, by injecting an RNAi agent into or near the tissue where the cells are located, or by injecting the RNAi agent into another region, for example, the central nervous system (CNS), optionally via intrathecal injection, intravitreal injection, or other injection, or by injecting the RNAi agent into the bloodstream or subcutaneous space, so that the agent then reaches the tissue where the cells to be contacted are located. For example, the RNAi agent can contain or be coupled to a ligand that directs or otherwise stabilizes the RNAi agent to the desired site, for example, in the CNS, for example, a lipophilic moiety, as described below and further detailed in, for example, PCT / US2019 / 031170, which is incorporated herein by reference. A combination of in vitro and in vivo contacting methods is also possible. For example, cells can be contacted with an RNAi agent in vitro and then transferred to a subject.

[0177] In one embodiment, contacting a cell with an RNAi agent includes "introducing" or "delivering an RNAi agent into a cell" by promoting or performing uptake or absorption into the cell. The absorption or uptake of an RNAi agent can occur by spontaneous diffusive or active cellular processes, or by auxiliary agents or devices. The introduction of an RNAi agent into a cell can be in vitro or in vivo. For example, in the case of in vivo introduction, the RNAi agent can be injected into a tissue site or administered systemically. In vitro introduction into a cell can include methods known in the art, such as electroporation and lipofection. Further approaches are described herein below or known in the art.

[0178] The terms "lipid-soluble" or "lipophilic moiety" refer broadly to any compound or chemical moiety that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is by the octanol-water partition coefficient, log K ow In this case, K ow is the ratio of the concentration of a chemical in the octanol phase to the concentration of the chemical in the aqueous phase of a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it can also be predicted by using coefficients attributable to the structural components of a chemical calculated using first principles or empirical methods (see, e.g., Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), incorporated herein by reference in its entirety). It provides a thermodynamic measure of a substance's tendency to prefer a non-aqueous or oily environment rather than water (i.e., its hydrophilic / lipophilic balance). In principle, a chemical can be expressed as its logK ow is greater than 0, the molecule is lipophilic in nature. Typically, a lipophilic moiety has a logK ow For example, the log K of 6-aminohexanol ow For example, the log K of cholesteryl N-(hexan-6-ol)carbamate is predicted to be approximately 0.7. ow is predicted to be 10.7.

[0179] The lipophilicity of a molecule can be changed depending on the functional groups it contains. For example, adding a hydroxyl or amine group to the end of the lipophilic moiety can increase the partition coefficient (e.g., logK ow ) may increase or decrease in value.

[0180] 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 property.For example, in certain embodiments, if the unbound fraction of the plasma protein binding assay of double-stranded RNAi agent is determined to be positively correlated with the relative hydrophobicity of double-stranded RNAi agent, then it will be positively correlated with the silencing activity of double-stranded RNAi agent.

[0181] In one embodiment, the plasma protein binding assay that is determined is electrophoretic mobility shift assay (EMSA) that uses 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 unbound fraction of siRNA in binding assay is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45 or greater than 0.5 in the case of enhanced siRNA in vivo delivery.

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

[0183] The term "lipid nanoparticle" or "LNP" refers to a vesicle comprising a lipid layer that encapsulates a pharmaceutically active molecule, such as a nucleic acid molecule, such as an RNAi agent or a plasmid from which an RNAi agent is transcribed. LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0184] As used herein, a "subject" refers to an animal, such as a mammal, including a primate (e.g., a human, a non-human primate, such as a monkey or chimpanzee) or a non-primate (e.g., a rat or a mouse). In a preferred embodiment, the subject is a human, such as a human being who is treated or evaluated for a disease, disorder, or condition that would benefit from reduced FLNA expression, a human being at risk for a disease, disorder, or condition that would benefit from reduced FLNA expression, a human being who has a disease, disorder, or condition that would benefit from reduced FLNA expression, or a human being who is treated for a disease, disorder, or condition that would benefit from reduced FLNA expression as described herein. In some embodiments, the subject is a female human being. In other embodiments, the subject is a male human being. In one embodiment, the subject is an adult subject. In one embodiment, the subject is a pediatric subject. In another embodiment, the subject is a juvenile subject, i.e., a subject under the age of 20.

[0185] As used herein, the term "treating" or "treatment" refers to a beneficial or desired outcome, such as, but not limited to, the alleviation or amelioration of one or more signs or symptoms associated with FLNA gene expression or FLNA protein production, for example, of a FLNA-associated disease, such as a FLNA-associated disease. "Treatment" can also mean extending survival time compared to expected survival time in the absence of treatment.

[0186] The term "lower" in the context of the level of FLNA or a disease marker or symptom in a subject refers to a statistically significant decrease in such level. The decrease can be, for example, at least 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In certain embodiments, the decrease is at least about 20%. In certain embodiments, the decrease is at least about 30% in the disease marker, for example, a 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more decrease. In certain embodiments, the decrease is at least about 50% in the disease marker. In the context of the level of FLNA in a subject, "reducing" preferably refers to reducing to a level that is within the normal range for individuals without such disorders.In certain embodiments, "reducing" refers to the reduction in the difference between the level of marker or symptom in a subject suffering from a disease and the level that an individual would be within the normal range, for example, the reduction in the level of weight between an obese individual and an individual whose weight is within the normal range.

[0187] As used herein, "prevention" or "preventing", when used in reference to a disease, disorder, or condition that would benefit from a reduction in the expression of the FLNA gene or the production of FLNA protein, refers to a reduction in the likelihood that a subject will develop symptoms associated with such a disease, disorder, or condition, such as symptoms of an FLNA-associated disease.Not developing a disease, disorder, or condition, or a reduction in the onset of symptoms associated with such a disease, disorder, or condition (for example, a reduction of at least about 10% of the clinically acceptable magnitude for the disease or disorder), or a delay in the onset of symptoms (for example, a delay of several days, weeks, months, or years) is considered effective prevention.

[0188] As used herein, the term " FLNA-related disease " or " FLNA-related disorder " comprises any disease or disorder that can benefit from the reduction of FLNA expression and / or activity.Exemplary FLNA-related diseases include those in which subject has altered or misfolded FLNA, such as Alzheimer's disease.

[0189] FLNA-associated disorders include, but are not limited to, Alzheimer's disease, tauopathies, frontotemporal dementia (FTD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), chronic traumatic encephalopathy (CTE), Pick's disease (PiD), globular glial tauopathy (GGT), argyrophilic grain disease (AGD), and primary age-related tauopathy (PART).

[0190] As used herein, a "therapeutically effective amount" is intended to include an amount of an RNAi agent that, when administered to a subject with a FLNA-associated disease, is sufficient to treat the disease (e.g., by reducing, improving, or maintaining an existing disease or one or more symptoms of the disease). A "therapeutically effective amount" may vary depending on the RNAi agent, the method of administration of the agent, the disease and its severity and medical history, age, weight, family history, genetic makeup, the type of prior or concurrent treatment, if any, and other personal characteristics of the subject to be treated.

[0191] As used herein, the term "prophylactically effective amount" is intended to include the amount of an RNAi agent that, when administered to a subject with an FLNA-associated disorder, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the progression of the disease or reducing the severity of future disease. The "prophylactically effective amount" may vary depending on the RNAi agent, the method of administration of the agent, the degree of risk of the disease, and the patient's medical history, age, weight, family history, genetic makeup, type of previous or concurrent treatment, if any, and other personal characteristics of the patient to be treated.

[0192] A "therapeutically effective amount" or a "prophylactically effective amount" also encompasses the amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The RNAi agent used in the methods of the present disclosure can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0193] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions or dosage forms that are suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0194] The phrase "pharmaceutically acceptable carrier," as used herein, means a pharmaceutically acceptable material, composition, or vehicle involved in the transport or transfer of a subject compound from one organ or part of the body to another, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject being treated. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium state, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar. (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) pH buffer solutions, (21) polyesters, polycarbonates, or polyanhydrides, (22) bulking agents, such as polypeptides and amino acids, (23) serum components, such as serum albumin, HDL, and LDL, and (22) other non-toxic, compatible substances employed in pharmaceutical formulations.

[0195] The term "sample," as used herein, encompasses similar fluids, cells, or tissues isolated from a subject, as well as collections of fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum, and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymphatic fluid, urine, saliva, and the like. Tissue samples can include samples from tissues, organs, or localized regions. For example, samples can be derived from specific organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples can be obtained from the brain (e.g., the whole brain or specific segments of the brain, such as the striatum, or specific types of cells within the brain, such as neurons and glial cells (astrocytes, oligodendrocytes, microglia)). In some embodiments, "a sample obtained from a subject" refers to blood obtained from a subject or plasma or serum obtained therefrom. In further embodiments, "a sample obtained from a subject" refers to brain tissue (or a subcomponent thereof) or retinal tissue (or a subcomponent thereof) obtained from a subject.

[0196] II. RNAi Agents of the Disclosure Described herein are RNAi agents that inhibit expression of the FLNA gene. In one embodiment, the RNAi agent comprises a double-stranded ribonucleic acid (dsRNA) molecule that inhibits expression of the FLNA gene in a cell, e.g., a cell in a mammalian subject, e.g., a human, having an FLNA-associated disorder, e.g., an FLNA-associated disorder. The dsRNA comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed upon expression of the FLNA gene. The complementary region is no longer than about 15-30 nucleotides in length. When contacted with cells expressing the FLNA gene, the RNAi agent inhibits expression of the FLNA gene (e.g., human, primate, or non-primate gene) by at least 30%, as analyzed, e.g., by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, e.g., immunofluorescence, e.g., using Western blotting or flow cytometry techniques. In one embodiment, the level of knockdown is analyzed in monkey Cos-7 cells using the assay method provided in Example 2, below. In another embodiment, the level of knockdown is analyzed in human BE(2)-C cells. In some embodiments, the level of knockdown is analyzed in mouse Neuro-2a cells.

[0197] dsRNA comprises two RNA strands, which are complementary and hybridize to form a duplex structure under the conditions in which dsRNA is used.One strand of dsRNA (antisense strand) comprises a complementary region that is substantially complementary to the target sequence, and is generally completely complementary.The target sequence can be obtained from the sequence of mRNA formed during the expression of FLNA gene.The other strand (sense strand) comprises a region that is complementary to the antisense strand, so that when the two strands are combined under suitable conditions, they hybridize to form a duplex structure.As described elsewhere herein and known in the art, the complementary sequence of dsRNA can also be contained as a self-complementary region of a single nucleic acid molecule, so that it is opposite on separate oligonucleotides.

[0198] Generally, the duplex structure is 15-30 base pairs in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-22, 19-23, 19-24, 19-25, 19-26, 19-27, 19-28, 19-29, 19-30, 19-31, 19-32, 19-33, 19-34, 19-35, 19-36, 19-37, 19-38, 19-39, 19-40, 19-41, 19-42, 19-43, 19-44, 19-45, 19-46, 19-47, 19-48, 19-49, 19-50, 19-51, 19-52, 19-53, 19-54, 19-55, 19-56, 19-57, 19-58, 19-59, 19-60, 19-61, 19-62, 19-63, 19-64, 19-65, 19-6 9, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. In certain preferred embodiments, the duplex structure is 18 to 25 base pairs in length, e.g., 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 25, 21 to 24, 21 to 23, 21 to 22, 22 to 25, 22 to 24, 22 to 23, 23 to 25, 23 to 24, or 24 to 25 base pairs in length, e.g., 19 to 21 base pairs in length. Ranges and lengths between the ranges and lengths listed above are also intended to be part of this disclosure.

[0199] Similarly, the region of complementarity to the target sequence may be 15 to 30 nucleotides in length, e.g., 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 21-23, or 21-22 nucleotides in length, e.g., 19-23 nucleotides in length or 21-23 nucleotides in length. Ranges and lengths that lie between the ranges and lengths listed above are also intended to be part of this disclosure.

[0200] In some embodiments, the duplex structure is 19 to 30 base pairs in length. Similarly, the region of complementarity to the target sequence is 19 to 30 nucleotides in length.

[0201] In some embodiments, the dsRNA is 15-23 nucleotides long, 19-23 nucleotides long, or 25-30 nucleotides long. Generally, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNA longer than about 21-23 nucleotides can serve as a substrate for Dicer. As those skilled in the art will recognize, the region of RNA targeted for cleavage will most often be a portion of a longer RNA molecule, often an mRNA molecule. In relevant cases, a "portion" of an mRNA target is a contiguous sequence of the mRNA target long enough to allow it to serve as a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

[0202] Those skilled in the art will appreciate that the duplex region is a primary functional portion of the dsRNA, e.g., about 15-36 base pairs, e.g., 15-36, 15-35, 15-34, 15-33, 15-32, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21 , 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs, for example, 19-21 base pairs. That is, in one embodiment, an RNA molecule or a complex of RNA molecules having a duplex region of more than 30 base pairs is dsRNA, as long as it is processed into a functional duplex of, for example, 15-30 base pairs that targets the desired RNA for cleavage.Therefore, those skilled in the art will recognize that in one embodiment, miRNA is dsRNA.In another embodiment, the dsRNA is not a naturally occurring miRNA.In another embodiment, the RNAi agent useful for targeting FLNA expression is not generated in target cells by cleavage of a larger dsRNA.

[0203] dsRNA described herein can further comprise one or more single-stranded nucleotide overhangs, for example, 1, 2, 3 or 4 nucleotides.Nucleotide overhangs can comprise or consist of nucleotide / nucleoside analogs, such as deoxynucleotide / nucleoside.Overhangs can be on sense strand, antisense strand, or any combination thereof.In addition, the nucleotide of a certain overhang can be present on the 5'-end, 3'-end, or both ends of either antisense strand or sense strand of dsRNA.

[0204] dsRNA can be synthesized by standard methods known in the art.Double-stranded RNAi compounds of the present invention can be prepared using a two-step method.First, each strand of double-stranded RNA molecules is prepared separately.Then, the strands of these components are annealed.The individual strands of siRNA compounds can be prepared using solution phase or solid phase organic synthesis or both.Organic synthesis has the advantage that it can easily prepare the oligonucleotide strands that contain unnatural nucleotides or modified nucleotides.Similarly, single-stranded oligonucleotides of the present invention can be prepared using solution phase or solid phase organic synthesis or both.

[0205] In one embodiment, the dsRNA of the present disclosure comprises at least two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand sequence for FLNA can be selected from the sequences provided in Tables 3-8, and the corresponding nucleotide sequence of the antisense strand of the sense strand can be selected from the sequences provided in Tables 3-8. In this embodiment, one of the two sequences is complementary to the other of the two sequences, where one of the sequences is substantially complementary to the sequence of mRNA produced upon expression of the FLNA gene. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, one of which is designated in Tables 3-8 as the sense strand (passenger strand) and the second oligonucleotide is designated in Tables 3-8 as the corresponding antisense strand (guide strand) of the sense strand.

[0206] In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides, hi another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

[0207] Although the sequences provided herein are described as modified or conjugated sequences of the sequences in Tables 3-8, it will be understood that the RNA of the RNAi agents of the present disclosure, e.g., the dsRNA of the present disclosure, can comprise any one of the sequences specified in Tables 3-8, unmodified, unconjugated, or modified or conjugated differently from those described therein. For example, the sense strand of the agents of the present invention can be conjugated to a GalNAc ligand, but these agents can also be conjugated to a moiety responsible for delivery to the CNS, e.g., a C16 ligand, as described herein. A lipophilic ligand can be included at any of the positions provided in the present application.

[0208] Those skilled in the art are well aware that dsRNAs having duplex structures of approximately 20-23 base pairs, e.g., 21 base pairs, have been hailed as particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888). However, others have discovered that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the above-described embodiments, due to the nature of the oligonucleotide sequences provided herein, the dsRNAs described herein can comprise at least one strand that is a minimum of 21 nucleotides in length. It can be reasonably predicted that shorter duplexes, minus only a few nucleotides at one or both ends, can be similarly effective compared to the dsRNAs described above. Therefore, the dsRNA that has at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotide sequences obtained from one of the sequences provided herein, and that differs in their ability to inhibit the expression of FLNA gene by 10, 15, 20, 25, 30, 35, 40, 45 or 50% or less from the dsRNA that comprises complete sequence, for example, using A549 cells and 10nM concentration of RNA agent in vitro assay and PCR assay provided in Examples herein.In some embodiments, the inhibition of the dsRNA that comprises complete sequence is measured using the in vitro assay that uses primary mouse hepatocytes.

[0209] In addition, the RNA described herein identifies the site in FLNA transcript that is susceptible to RISC-mediated cleavage.Therefore, the present disclosure also features the RNAi agent that targets within this site.As used herein, if the RNAi agent promotes the cleavage of the transcript at any of the specific sites, it is said that the RNAi agent targets within the specific site of RNA transcript.This RNAi agent generally comprises at least about 15 consecutive nucleotides, preferably at least 19 nucleotides, from one of the sequences provided herein, which is combined with additional nucleotide sequences taken from the region adjacent to the selected sequence in FLNA gene.

[0210] III. Modified RNAi Agents of the Present Disclosure In one embodiment, the RNA of the RNAi agent of the present disclosure, e.g., dsRNA, is unmodified and does not contain, for example, chemical modifications or conjugations known in the art and described herein. In a preferred embodiment, the RNA of the RNAi agent of the present disclosure, e.g., dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the present disclosure, substantially all of the nucleotides of the RNAi agent of the present disclosure are modified. In other embodiments of the present disclosure, all of the nucleotides of the RNAi agent of the present disclosure are modified. An RNAi agent of the present disclosure in which "substantially all of the nucleotides are modified" may be widely modified but not entirely modified, and may contain 5, 4, 3, 2 or less, or unmodified nucleotides. In yet other embodiments of the present disclosure, an RNAi agent of the present disclosure may contain 5, 4, 3, 2, or less than 1 modified nucleotide.

[0211] Nucleic acids featured in this disclosure can be synthesized or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, SLet et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, inverted linkage) or 3'-end modifications (conjugation, DNA nucleotides, inverted linkage, etc.), base modifications, such as substitution with stabilizing bases, destabilizing bases, or bases that base-pair with partners in an extended repertoire, base removal (abasic nucleotides) or conjugated bases, sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, or backbone modifications, including phosphodiester bond modifications or substitutions. Specific examples of RNAi agents useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or lacking natural internucleoside linkages. Among the RNAs with modified backbone, those that do not have phosphorus atom in backbone are included.For the purpose of this specification, and as sometimes referred to in the art, the modified RNAs that do not have phosphorus atom in their internucleoside backbone can also be considered as oligonucleoside.In some embodiments, modified RNAi agent will have phosphorus atom in its internucleoside backbone.

[0212] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and those with reversed polarity, where adjacent pairs of nucleoside units are linked 3'-5' and 5'-3', or 2'-5' and 5'-2'. Also included are various salts, mixed salts, and free acid forms. In some embodiments of the present invention, the dsRNA agent of the present invention is in free acid form. In other embodiments of the present invention, the dsRNA agent of the present invention is in salt form. In one embodiment, the dsRNA agent of the present invention is in sodium salt form.In certain embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ion exists in the agent as counterion to substantially all of the phosphodiester and / or phosphorothioate groups present in the agent.The agent in which substantially all of the phosphodiester and / or phosphorothioate bond has sodium counterion comprises 5, 4, 3, 2 or 1 or less phosphodiester and / or phosphorothioate bond that does not have sodium counterion.In some embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ion exists in the agent as counterion to all of the phosphodiester and / or phosphorothioate groups present in the agent.

[0213] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Patent Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,711, and the like. No. 7, No. 5,321,131, No. 5,399,676, No. 5,405,939, No. 5,453,496, No. 5,455,233, No. 5,466,677, No. 5,47 No. 6,925, No. 5,519,126, No. 5,536,821, No. 5,541,316, No. 5,550,111, No. 5,563,253, No. 5,571,799, No. 5 , 587,361, 5,625,050, 6,028,188, 6,124,445, 6,160,109, 6,169,170, 6,172,209 No. 6,239,265, No. 6,277,603, No. 6,326,199, No. 6,346,614, No. 6,444,423, No. 6,531,590, No. 6,534, Nos. 6,639, 6,608,035, 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029, and U.S. Patent No. RE39464, the contents of each of which are incorporated herein by reference in their entirety.

[0214] Modified RNA backbones that do not contain a phosphorus atom in the backbone have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages, including those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 constituent moieties.

[0215] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,64,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, Nos. 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439, the entire contents of each of which are incorporated herein by reference.

[0216] In another embodiment, RNA mimics suitable for use in RNAi agents are considered, in which both the sugar and internucleoside linkages, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimic known to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly linked 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, the entire contents of each of which are incorporated herein by reference. Additional PNA compounds suitable for use in the RNAi agents of the present disclosure are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0217] Some embodiments featured in this disclosure include RNA 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 the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNA featured herein has the morpholino backbone structure of the above-referenced US Pat. No. 5,034,506.

[0218] Modified RNAs can also contain one or more substituted sugar moieties. The RNAi agents featured herein, e.g., dsRNAs, can include one of the following at the 2' position: OH, F, O-, S-, or N-alkyl, O-, S-, or N-alkenyl, O-, S-, or N-alkynyl, or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 It can be alkenyl or alkynyl. Exemplary suitable modifications include O[(CH) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10. In other embodiments, the dsRNA includes one of the following at the 2' position: C1 to C 10lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH, OCN, Cl, Br, CN, CF, OCF, SOCH, SOCH, ONO, NO, N, NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, interfering substances, groups for improving the pharmacokinetic properties of RNAi agents or groups for improving the pharmacodynamic properties of RNAi agents, 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 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described herein below in the Examples, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2)2. Further exemplary modifications include 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both R and S isomers within these three families), 2'-alkoxyalkyl, and 2'-NMA (N-methylacetamide).

[0219] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHNH), 2'-O-hexadecyl, and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an RNAi agent, 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. RNAi agents can also have sugar mimetics, 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, and 5,56 Nos. 7,811, 5,576,427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, and 5,700,920, some of which are commonly owned with this application, the entire contents of each of the foregoing being incorporated herein by reference.

[0220] The RNAi agent of the present disclosure can also comprise modification or substitution of nucleobase (often simply referred to in the art as "base").As used herein, "unmodified" or "natural" nucleobase includes purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C) and uracil (U).Modified nucleobases include other synthetic nucleobases and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, These include 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine.Additional nucleobases include those disclosed in United States Patent No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P.ed.Wiley-VCH, 2008, those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, JL, ed.John Wiley & Sons, 1990, those disclosed in Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and those disclosed in Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993.Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, such as 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, Y.S., Crooke, S.T., and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are exemplary base substitutions, especially when combined with 2'-O-methoxyethyl sugar modifications.

[0221] 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, the above-referenced U.S. Patent Nos. 3,687,808, 4,845,205, 5,130,30, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,587,469, Nos. 5,594,121, 5,596,091, 5,614,617, 5,681,941, 5,750,692, 6,015,886, 6,147,200, 6,166,197, 6,222,025, 6,235,887, 6,380,368, 6,528,640, 6,639,062, 6,617,438, 7,045,610, 7,427,672, and 7,495,088, the entire contents of each of which are incorporated herein by reference.

[0222] The RNAi agent of the present disclosure can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide with a modified ribose moiety, in which the ribose moiety contains an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo 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).

[0223] The RNAi agents of the present disclosure can also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanose ring modified by bridging two atoms. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety containing a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Thus, in some embodiments, the agents of the present disclosure can include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety contains an additional bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety containing a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in a 3'-endo structural conformation. The addition of a locked nucleic acid to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of 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 agent of the present disclosure includes one or more bicyclic nucleosides comprising a 4' to 2' bridge.Examples of such 4' to 2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH2)-O-2' (LNA), 4'-(CH2)-S-2', 4'-(CH2)2-O-2' (ENA), 4'-CH(CH3)-O-2' (also referred to as "constrained ethyl" or "cEt") and 4'-CH(CHOCH3)-O-2' (and analogs, see, e.g., U.S. Pat. No. 7,399,845), 4'-C(CH3)(CH3)-O-2' (and analogs, e.g., No. 8,278,283), 4'-CH2-N(OCH3)-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,425), 4'-CH2-ON(CH3)-2' (see, e.g., U.S. Patent Application Publication No. 2004 / 0171570), 4'-CH2-N(R)-O-2', where R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S. Pat. No. 7,427,672), 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134), and 4'-CH2-C(=CH2)-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing are incorporated herein by reference.

[0224] Additional representative U.S. patents and publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490, 6,525,191, 6,670,461, 6,770,748, 6,794,499, 6,998,484, 7,053,207, 7,034,133, 7,084,125, 7,399,845, 7,427,672, 7,569,686, 7,741,457, 8,022,193, 8,030,467, 8,278,425, 8,278,426, 8,278,283, No. 2008 / 0039618, and US2009 / 0012281, the entire contents of each of which are incorporated herein by reference.

[0225] Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0226] The RNAi agents of the present disclosure can also be modified to include one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid that includes a bicyclic sugar moiety that includes a 4'-CH(CH3)-0-2' bridge. In one embodiment, the constrained ethyl nucleotide is in the S conformation and is referred to herein as an "S-cEt."

[0227] The RNAi agent of the present disclosure may also contain one or more "conformation-restricting nucleotides" ("CRNs"). A CRN is a nucleotide analogue with a linker connecting the C2' and C4' carbons of ribose or the C3 and C5' carbons of ribose. The CRN locks the ribose ring into a stable conformation, increasing hybridization affinity to mRNA. The linker is long enough to position the oxygen at an optimal position for stability and affinity, thereby reducing puckering of the ribose ring.

[0228] Representative publications that teach the preparation of certain of the above CRNs include, but are not limited to, US2013 / 0190383 and WO2013 / 036868, the entire contents of each of which are incorporated herein by reference.

[0229] In some embodiments, the RNAi agent of the present disclosure includes one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNAs are acyclic unlocked nucleic acids in which any of their sugar linkages have been removed to form an unlocked "sugar" residue. In one example, UNAs also encompass monomers in which the C1'-C4' bond has been removed (i.e., a covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another example, the C2'-C3' bond of the sugar has been removed (i.e., a covalent carbon-carbon bond between the C2' and C3' carbons) (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference).

[0230] Representative U.S. publications that teach the preparation of UNAs include, but are not limited to, U.S. Pat. No. 8,314,227, and U.S. Patent Application Publication Nos. 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020, the entire contents of each of which are incorporated herein by reference.

[0231] Potential stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"-phosphate, inverted base dT (idT), and others. Disclosure of this modification can be found in WO 2011 / 005861.

[0232] 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 example, in US2012 / 0157511, the entire content of which is incorporated herein by reference.

[0233] In one embodiment, the double-stranded RNAi agent of the present invention further comprises a 5'-phosphate or 5'-phosphate mimic of the 5' nucleotide of the antisense strand. In another embodiment, the double-stranded RNAi agent further comprises a 5'-phosphate mimic of the 5' nucleotide of the antisense strand. In a specific embodiment, the 5' phosphate mimic is 5'-vinylphosphonate (5'-VP). In one embodiment, the phosphate mimic is 5'-cyclopropylphosphonate (VP). In some embodiments, the 5'-end of the antisense strand of the double-stranded iRNA agent does not contain a 5'-vinylphosphonate (VP).

[0234] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of deoxy-nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, glycol-modified nucleotides (GNAs) such as Ggn, Cgn, Tgn, or Agn, nucleotides having a 2' phosphate such as G2p, C2p, A2p, U2p, and vinyl-phosphonate nucleotides, and combinations thereof. In other embodiments, each of the duplexes of Tables 4, 6, or 8 may be specifically modified to provide an alternative double-stranded iRNA agent of the disclosure. In one example, the 3' end of each sense duplex may be modified by removing the 3'-terminal L96 ligand and replacing the two phosphodiester internucleotide linkages with phosphorothioate internucleotide linkages between the three 3'-terminal nucleotides, i.e., the three 3'-terminal nucleotides (N) of the sense sequence of the following formula: 5'-N1-...-N n-2 N n-1 N n L963' can be replaced with: 5'-N1-...-N n-2 s N n-1 s N n 3'

[0235] A. Modified RNAi Agents Comprising Motifs of the Present Disclosure In certain aspects of the present disclosure, the double-stranded RNAi agent of the present disclosure includes an agent having chemical modifications, such as those disclosed in International Publication No. 2013 / 075035, the entire contents of which are incorporated herein by reference. As shown herein and in International Publication No. 2013 / 075035, excellent results can be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense or antisense strand of the RNAi agent, particularly at or near the cleavage site. In some embodiments, the sense and antisense strands of the RNAi agent can be otherwise completely modified. The introduction of these motifs interrupts the modification pattern of the sense or antisense strand, if present. The RNAi agent can optionally be conjugated with a lipophilic ligand, for example, a C16 ligand, for example, on the sense strand. The RNAi agent can optionally be modified with an (S)-glycol nucleic acid (GNA) modification, for example, at one or more residues of the antisense strand. The resulting RNAi agent exhibits excellent gene silencing activity.

[0236] Thus, the present disclosure provides a double-stranded RNAi agent capable of inhibiting expression of a target gene (i.e., the FLNA gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent can be 15 to 30 nucleotides in length. Each strand can be, for example, 16 to 30 nucleotides in length, 17 to 30 nucleotides in length, 25 to 30 nucleotides in length, 27 to 30 nucleotides in length, 17 to 23 nucleotides in length, 17 to 21 nucleotides in length, 17 to 19 nucleotides in length, 19 to 25 nucleotides in length, 19 to 23 nucleotides in length, 19 to 21 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 23 nucleotides in length. In certain embodiments, each strand is 19 to 23 nucleotides in length.

[0237] The sense and antisense strands typically form a duplex, double-stranded RNA ("dsRNA"), also referred to herein as an "RNAi agent." The duplex region of an RNAi agent can be 15-30 nucleotide pairs in length. For example, the duplex region can be 16-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length. In a preferred embodiment, the duplex region is 19 to 21 nucleotide pairs in length.

[0238] In one embodiment, the RNAi agent may contain one or more overhang regions or capping groups at the 3'-end, 5'-end, or both ends of one or both strands. The overhangs may be 1 to 6 nucleotides in length, e.g., 2 to 6 nucleotides in length, 1 to 5 nucleotides in length, 2 to 5 nucleotides in length, 1 to 4 nucleotides in length, 2 to 4 nucleotides in length, 1 to 3 nucleotides in length, 2 to 3 nucleotides in length, or 1 to 2 nucleotides in length. In a preferred embodiment, the nucleotide overhang region is 2 nucleotides in length. The overhang may be the result of one strand being longer than the other, or the result of two strands of the same length being twisted. The overhang may form a mismatch with the target mRNA, be complementary to the targeted gene sequence, or be a different sequence. The first and second strands may also be linked by additional bases, for example, to form a hairpin, or by other non-basic linkers.

[0239] In one embodiment, the nucleotides in the overhang region of an RNAi agent can each independently be modified or unmodified nucleotides, including, but not limited to, 2'-sugar modifications, such as 2-F, 2'-O-methyl, thymidine (T), and any combination thereof.

[0240] For example, TT can be an overhang sequence for either end on either strand, which can form a mismatch with the target mRNA, or can be complementary to the targeted gene sequence, or can be another sequence.

[0241] The 5'- or 3'-overhang of the sense strand, antisense strand, or both strands of the RNAi agent can be phosphorylated. In some embodiments, the overhang region contains two nucleotides with a phosphorothioate between them, and the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3'-end of the sense strand, antisense strand, or both strands. In one embodiment, the 3'-overhang is present in the antisense strand. In one embodiment, the 3'-overhang is present in the sense strand.

[0242] RNAi agent can contain only a single overhang, which can enhance the interference activity of RNAi without affecting its overall stability.For example, the single-stranded overhang can be located at the 3'-end of the sense strand or at the 3'-end of the antisense strand.RNAi can also have a blunt end, which is located at the 5'-end of the antisense strand (or the 3'-end of the sense strand), or vice versa.Generally, the antisense strand of RNAi has a nucleotide overhang at the 3'-end, and its 5'-end is blunt.Without wishing to be bound by theory, the asymmetry between the blunt end at the 5'-end of the antisense strand and the 3'-end overhang of the antisense strand is favorable for the insertion of guide strand into RISC process.

[0243] In one embodiment, the RNAi agent is a double-ended bluntmer 19 nucleotides in length, the sense strand containing at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 7, 8, and 9 from the 5' end, and the antisense strand containing at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0244] In another embodiment, the RNAi agent is a double-ended bluntmer 20 nucleotides in length, the sense strand containing at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 from the 5' end, and the antisense strand containing at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0245] In yet another embodiment, the RNAi agent is a double-ended bluntmer 21 nucleotides in length, the sense strand containing at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and the antisense strand containing at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0246] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and the antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, and one end of the RNAi agent is blunt, and the other end comprises a two-nucleotide overhang.Preferably, the two-nucleotide overhang is at the 3' end of the antisense strand.When the two-nucleotide overhang is at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the three terminal nucleotides, two of which are overhanging nucleotides, and the third nucleotide is a paired nucleotide adjacent to the overhanging nucleotide. In one embodiment, the RNAi agent further comprises two phosphorothioate internucleotide linkages between the three terminal nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand.In one embodiment, every nucleotide in the sense strand and antisense strand of the RNAi agent, including the nucleotide that is part of a motif, is a modified nucleotide.In one embodiment, each residue is independently modified with 2'-O-methyl or 3'-fluoro, for example, within an alternating motif.Optionally, the RNAi agent further comprises a ligand (for example, a lipophilic ligand, optionally a C16 ligand).

[0247] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length and, starting from the 5'-most nucleotide (position 1), comprises at least 8 ribonucleotides at positions 1-23 of the first strand; and the antisense strand is 36-66 nucleotide residues in length and, starting from the 3'-most nucleotide, comprises at least 8 ribonucleotides at positions 1-23 of the sense strand, forming a duplex; wherein at least the 3'-most nucleotide of the antisense strand is unpaired with the sense strand, and up to 6 consecutive 3'-most nucleotides are unpaired with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides; and wherein the 5'-end of the antisense strand comprises 10-30 consecutive ribonucleotides that are not paired with the sense strand. the sense strand comprises nucleotides corresponding to the 2'-F modification, thereby forming a 10-30 nucleotide single-stranded 5' overhang, at least the 5'- and 3'-terminal nucleotides of the sense strand base-pair with nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplex region between the sense and antisense strands, and the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length, such that the double-stranded nucleic acid reduces target gene expression when introduced into a mammalian cell, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, at least one of the motifs occurring at or near the cleavage site. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0248] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, the RNAi agent comprising a first strand having a length of at least 25 nucleotides and at most 29 nucleotides, and a second strand having a length of at most 30 nucleotides with at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, the 3' end of the first strand and the 5' end of the second strand forming a blunt end, the second strand being 1 to 4 nucleotides longer at its 3' end than the first strand, the duplex region being at least 25 nucleotides in length, and the second strand being sufficiently complementary to a target mRNA along at least 19 nucleotides of the length of the second strand, the RNAi agent reducing target gene expression when introduced into a mammalian cell, and Dicer cleavage of the RNAi agent preferentially resulting in an siRNA comprising the 3' end of the second strand, thereby reducing target gene expression in a mammal. Optionally, the RNAi agent further comprises a ligand.

[0249] In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, one of which motifs occurs at the cleavage site within the sense strand.

[0250] In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, one of which occurs at or near the cleavage site in the antisense strand.

[0251] For RNAi agents with a duplex region 17-23 nucleotides in length, the cleavage sites of the antisense strand are typically approximately 10, 11, and 12 positions from the 5' end. Thus, three identical modification motifs can occur at positions 9, 10, 11, 10, 11, 12, 11, 12, 13, 12, 13, 14, or 13, 14, 15 of the antisense strand, starting from the first nucleotide from the 5' end of the antisense strand, or starting from the first paired nucleotide within the duplex region from the 5' end of the antisense strand. The cleavage site within the antisense strand can also vary depending on the length of the duplex region of the RNAi from the 5' end.

[0252] The sense strand of RNAi agent can contain at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of its strand, and antisense strand can have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of strand.When sense strand and antisense strand form dsRNA duplex, sense strand and antisense strand can be aligned so that one motif of three nucleotides on sense strand and one motif of three nucleotides on antisense strand have at least one nucleotide overlap, that is, at least one of the three nucleotides of the motif in sense strand and at least one of the three nucleotides of the motif in antisense strand form base pairing.Alternatively, at least two nucleotides can overlap, or all three nucleotides can overlap.

[0253] In one embodiment, the sense strand of an RNAi agent may contain multiple motifs of three identical modifications on three consecutive nucleotides. The first motif may occur at or near the site of strand cleavage, and the other motifs may be wing modifications. As used herein, the term "wing modification" refers to a motif that occurs in another portion of the strand separated from a motif at or near the site of cleavage of the same strand. The wing modifications may be adjacent to the first motif or separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other, the chemical nature of the motifs is distinct from each other, and when the motifs are separated by one or more nucleotides, the chemical nature may be the same or different. Two or more wing modifications may be present. For example, when two wing modifications are present, each wing modification may occur at one end of the first motif at or near the cleavage site, or on either side of the lead motif.

[0254] Like the sense strand, the antisense strand of an RNAi agent may contain multiple motifs of three identical modifications on three consecutive nucleotides, at least one of which occurs at or near the cleavage site of that strand. The antisense strand may also contain one or more wing modifications in the same alignment as the wing modifications that may be present on the sense strand.

[0255] In one embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0256] In another embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two paired nucleotides in the duplex region at the 3' end, 5' end, or both ends of the strand.

[0257] When the sense and antisense strands of an RNAi agent each contain at least one wing modification, the wing modifications can be at the same end of the duplex region and have an overlap of 1, 2, or 3 nucleotides.

[0258] When the sense and antisense strands of an RNAi agent each contain at least two wing modifications, the sense and antisense strands can be aligned such that the two modifications from each single strand are at one end of a duplex region with 1, 2, or 3 nucleotide overlap, the two modifications from each single strand are at the other end of the duplex region with 1, 2, or 3 nucleotide overlap, and the two modifications from the single strand are on either side of a lead motif within the duplex region with 1, 2, or 3 nucleotide overlap.

[0259] In one embodiment, the RNAi agent contains mismatches or combinations thereof in the duplex with the target. Mismatches can occur in the overhang region or in the duplex region. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., based on the free energy of association or dissociation of a particular pairing; the simplest approach is to examine each pair individually, but adjacent or similar analysis can also be used). In terms of promoting dissociation, A:U is preferred to G:C, G:U is preferred to G:C, and I:C is preferred to G:C (I=inosine). Mismatches, such as non-standard pairings or non-standard pairings (described elsewhere herein), are preferred to standard pairings (A:T, A:U, G:C), and pairings containing universal bases are preferred to standard pairings.

[0260] In one embodiment, the RNAi agent comprises the first 1, 2, 3, 4, or 5 base pairs within the duplex region from the 5' end of the antisense strand independently selected from the group of A:U, G:U, I:C, and at least one mismatch pair, e.g., a non-canonical pairing or a non-canonical pairing or a pairing containing a universal base, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0261] In one embodiment, the nucleotide at position 1 from the 5' end of the antisense strand to the duplex region is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first one, two, or three base pairs from the 5' end of the antisense strand to the duplex region is an AU base pair. For example, the first base pair from the 5' end of the antisense strand to the duplex region is an AU base pair.

[0262] In another embodiment, the nucleotide at the 3' end of the sense strand is deoxythymine (dT). In another embodiment, the nucleotide at the 3' end of the antisense strand is deoxythymine (dT). In one embodiment, there is a short sequence of deoxythymine nucleotides, for example, two dT nucleotides on the 3' end of either the sense strand or the antisense strand.

[0263] In one embodiment, the sense strand sequence has formula (Ia): 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3'(Ia) It can be expressed as During the ceremony, i and j each independently represent 0 or 1; p and q each independently represent 0 to 6; each N aindependently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing 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 q independently represent overhanging nucleotides, wherein Nb and Y do not have the same modification, and XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. Preferably, YYY are all 2'-F modified nucleotides.

[0264] In one embodiment, N a or N b includes alternating pattern modifications.

[0265] In one embodiment, YYY motif occurs at or near the cleavage site of sense strand.For example, when RNAi agent has a double-stranded region of 17-23 nucleotides in length, YYY motif can occur at or near the cleavage site of sense strand (for example, can occur at position 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13), and this number starts from the first nucleotide from the 5' end, or optionally this number starts from the first paired nucleotide in the double-stranded region from the 5' end.

[0266] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. 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 q 3'(Id) It can be expressed as:

[0267] 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.

[0268] each N a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0269] When the sense strand is represented by formula (Ic), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 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.

[0270] When the sense strand is represented by formula (Id), each N b independently represent an oligonucleotide sequence containing 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 can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0271] Each of X, Y and Z may be the same or different from one another.

[0272] In other embodiments, i is 0 and j is 0, and the sense strand has the formula 5'n p -N a -YYY-N a -n q 3'(Ie) It can be expressed as:

[0273] When the sense strand is represented by formula (Ie), each N a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0274] In one embodiment, the antisense strand sequence of the RNAi has the formula (If): 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' (If) It can be expressed as During the ceremony, k and l each independently represent 0 or 1; p' and q' each independently represent 0 to 6; each N a each independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b ' 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 motif of three identical modifications on three consecutive nucleotides.

[0275] In one embodiment, N a ' or N b ' includes alternating pattern modifications.

[0276] Y'Y'Y' motif occurs at or near the cleavage site of antisense strand.For example, when RNAi agent has a double-stranded region of 17-23 nucleotides in length, Y'Y'Y' motif can occur at position 9, 10, 11, 10, 11, 12, 11, 12, 13, 12, 13, 14, or 13, 14, 15 of antisense strand, this number starting from the first nucleotide from the 5' end, or optionally this number starting from the first paired nucleotide in the double-stranded region from the 5' end.Preferably, Y'Y'Y' motif occurs at position 11, 12, 13.

[0277] In one embodiment, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0278] 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.

[0279] 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'(IIk), 5'n q’ -N a '-Y'Y'Y'-N b '-X'X'X'-n p’ 3'(IIl), or 5'n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'-N a '-n p’ 3'(IIm) It can be expressed as:

[0280] When the antisense strand is represented by formula (IIk), N b ’ represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 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.

[0281] When the antisense strand is represented by formula (IIl), N b ' represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 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.

[0282] When the antisense strand is represented by formula (IIm), each N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 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. b is 0, 1, 2, 3, 4, 5 or 6.

[0283] In other embodiments, k is 0 and l is 0, and the antisense strand has the formula 5'n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 3'(Ie) It can be expressed as:

[0284] When the antisense strand is represented by formula (Ij), each N a ' independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0285] Each of X', Y' and Z' may be the same as or different from one another.

[0286] Each nucleotide of sense strand and antisense strand can be independently modified with LNA, HNA, CeNA, 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.

[0287] In one embodiment, the sense strand of the RNAi agent can 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 optionally, the numbers starting from the 5' end with the first paired nucleotide in the duplex region, and Y represents a 2'-F modification. The sense strand can further contain a XXX motif or a ZZZ motif as a wing modification at the opposite end of the duplex region, and XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0288] In one embodiment, the antisense strand can contain a Y'Y'Y' motif occurring at positions 11, 12, or 13 of the strand, where the numbering starts from the first nucleotide from the 5' end, or optionally, the numbering starts from the 5' end with the first paired nucleotide in the duplex region, and Y' represents a 2'-O-methyl modification. The antisense strand can further contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the duplex region, and X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0289] The sense strand represented by any one of the above formulas (Ie), (Ib), (Ic), and (Id) forms a duplex with the antisense strand represented by any one of formulas (Ij), (Ik), (Il), and (Im), respectively.

[0290] Thus, the RNAi agent used in the methods of the present disclosure can include a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the RNAi duplex can have the formula (In): 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'(In) During the ceremony, i, j, k, and l each independently represent 0 or 1; p, p', q and q' each independently represent 0 to 6; each N a and N a ’ independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b and N b ’ independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; During the ceremony, each n p ',n p , nq ' and n q each of which may or may not be present independently represents an overhanging nucleotide; 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.

[0291] In one embodiment, i is 0 and j is 0, or i is 1 and j is 0, or i is 0 and j is 1, or i and j are both 0, or i and j are both 1. In another embodiment, 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.

[0292] Exemplary combinations of sense and antisense strands that form RNAi duplexes include the following formulas: 5'n p -N a -YYY-N a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y'-N a ’ n q ’ 5'(Io) 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'(Ip) 5'np -N a -XXX-N b -YYY-N a -n q 3' 3'n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N a ’ -n q ’ 5'(IIIq) 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'(IIIr)

[0293] When the RNAi agent is represented by formula (Io), each N a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0294] When the RNAi agent is represented by formula (Ip), 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.

[0295] When the RNAi agent is represented by formula (Iq), each N b , N bEach N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 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.

[0296] When the RNAi agent is represented by formula (Ir), each N b , N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 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.

[0297] In one embodiment, when the RNAi agent has formula (Ir), N a The modification is a 2'-O-methyl modification or a 2'-fluoro modification. In another embodiment, when the RNAi agent has formula (Ir), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, and p '>0 and at least one n p In yet another embodiment, when the RNAi agent has formula (Ir), N' is linked to the adjacent nucleotide via a phosphorothioate linkage. a The modification is 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p In another embodiment, when an RNAi agent has formula (Ir), N' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand is conjugated to one or more C16 (or related thereto) moieties attached via a bivalent or trivalent branched linker (described below).a The modification is 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p ' are linked to adjacent nucleotides via phosphorothioate linkages, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic moieties, e.g., C16 (or related) moieties, which may optionally be attached via a bivalent or trivalent branched linker.

[0298] In one embodiment, when the RNAi agent has formula (Io), N a The modification is 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p ' are linked to adjacent nucleotides via phosphorothioate linkages, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic moieties, e.g., C16 (or related) moieties, which may be attached via a bivalent or trivalent branched linker.

[0299] In one embodiment, the RNAi agent is a multimer containing at least two duplexes represented by formula (In), (Io), (Ip), (Iq) and (Ir), and these duplexes are connected by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each duplex may target the same gene or two different genes, or each duplex may target the same gene at two different target sites.

[0300] In one embodiment, the RNAi agent is a multimer that contains three, four, five, six or more double strands represented by formula (In), (Io), (Ip), (Iq) and (Ir), and the double strands are connected by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each double strand can target the same gene or two different genes, or each double strand can target the same gene at two different target sites.

[0301] In one embodiment, two RNAi agents represented by formula (In), (Io), (Ip), (Iq), and (Ir) are linked to each other at their 5' ends and optionally conjugated to a ligand at one or both of their 3' ends. The agents can each target the same gene or two different genes, or the agents can each target the same gene at two different target sites.

[0302] Various publications describe the multimeric RNAi agent that can be used in the method of the present disclosure.These publications include International Publication No. 2007 / 091269, International Publication No. 2010 / 141511, International Publication No. 2007 / 117686, International Publication No. 2009 / 014887 and International Publication No. 2011 / 031520 and United States Patent No. 7858769, each of whose entire contents is incorporated herein by reference.

[0303] 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: [ka]

[0304] In an exemplary embodiment, a 5' vinylphosphonate modified nucleotide of the disclosure has the structure: [ka] wherein X is O or S; R is hydrogen, hydroxy, fluoro, or C 1-20 alkoxy (e.g., methoxy or n-hexadecyloxy); R 5’ is =C(H)-P(O)(OH)2, and the C5' carbon and R 5’ and the double bond between is in the E or Z conformation (e.g., E conformation), and B is a nucleobase or modified nucleobase, optionally B is adenine, guanine, cytosine, thymine, or uracil.

[0305] In one embodiment, R 5’ is ═C(H)—P(O)(OH) and the double bond between the C5′ carbon and R5′ is in the E configuration. In another embodiment, R is methoxy and R 5’ is ═C(H)—P(O)(OH) and the double bond between the C5′ carbon and R5′ is in the E configuration. In another embodiment, X is S, R is methoxy and R 5’ is =C(H)-P(O)(OH)2, and the double bond between the C5' carbon and R5' is in the E configuration.

[0306] 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. The dsRNAi agent can include a phosphorus-containing group at the 5'-end of the sense or antisense strand. The 5'-terminal phosphorus-containing group can be 5'-terminal phosphate (5'-P), 5'-terminal phosphorothioate (5'-PS), 5'-terminal phosphorodithioate (5'-PS2), 5'-terminal vinyl phosphonate (5'-VP), 5'-terminal methyl phosphonate (MMePhos), or 5'-deoxy-5'-C-malonyl. When the 5'-terminal phosphorus-containing group is 5'-terminal vinyl phosphonate (5'-VP), the 5'-VP can be a 5'-E-VP isomer (i.e., trans-vinyl phosphonate, [ka] ), 5'-Z-VP isomer (i.e., cis-vinyl phosphonate, [ka] ), or a mixture thereof.

[0307] Vinyl phosphate modifications are also contemplated for the compositions and methods of the present disclosure. Exemplary vinyl phosphate structures are as follows: [ka]

[0308] Another exemplary vinyl phosphate structure includes the structure previously described, where R 5’ is =C(H)-OP(O)(OH)2, and the C5' carbon and R 5’ The double bond between them can be in the E or Z conformation (e.g., E conformation). For example, if the phosphate mimetic is 5'-vinyl phosphate, the 5'-terminal nucleotide can have an immediately adjacent structure in which the phosphonate group is replaced by a phosphate.

[0309] i. Thermal destabilization modification In certain embodiments, dsRNA molecules can be optimized for RNA interference by incorporating thermodestabilizing modifications into the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand) to reduce or inhibit off-target gene silencing. It has been discovered that dsRNAs having an antisense strand containing at least one thermodestabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end of the antisense strand have reduced off-target gene silencing activity. Thus, in some embodiments, the antisense strand contains at least one (e.g., one, two, three, four, five, or more) thermodestabilizing modification of the duplex within the first 9 nucleotide positions of the 5' region of the antisense strand. In some embodiments, the thermodestabilizing modification of one or more duplexes is located between positions 2-9, or preferably between positions 4-8, from the 5' end of the antisense strand. In some further embodiments, the thermodestabilizing modification of the duplex is located at positions 6, 7, or 8 from the 5' end of the antisense strand. In some further embodiments, the thermally destabilizing modification of the duplex is located at position 7 from the 5' end of the antisense strand. The term "thermally destabilizing modification" includes modifications that result in a dsRNA having a lower overall melting temperature (Tm), preferably one, two, three or four degrees lower than the melting temperature (Tm) of a dsRNA that does not have such a modification. In some embodiments, the thermally destabilizing modification of the duplex is located at position 2, 3, 4, 5 or 9 from the 5' end of the antisense strand.

[0310] Thermally destabilizing modifications can include, but are not limited to, abasic modifications, mismatches with opposing nucleotides in the opposing strand, and sugar modifications, such as 2'-deoxy modifications or acyclic nucleotides, such as unlocked nucleic acids (UNAs) or glycol nucleic acids (GNAs).

[0311] Exemplary abasic modifications include, but are not limited to, the following: [ka] wherein R=H, Me, Et or OMe, R'=H, Me, Et or OMe, R"=H, Me, Et or OMe [ka] wherein B is a modified or unmodified nucleobase.

[0312] Exemplary sugar modifications include, but are not limited to, the following: [ka] wherein B is a modified or unmodified nucleobase.

[0313] In some embodiments, the thermally destabilizing modification of the duplex is selected from the group consisting of: [ka] where B is a modified or unmodified nucleobase, and the asterisk on each structure represents either R, S, or racemic.

[0314] In some embodiments, the thermally destabilizing modification of the duplex is selected from the group consisting of: [ka] wherein B is a modified or unmodified nucleobase, and the asterisk represents either R, S, or racemic (e.g., S).

[0315] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, for example, one in which any of the bonds of the ribose carbon ring (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 [ka] B is a modified or unmodified nucleobase; 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 acyclic unlocked nucleic acid in which any of the sugar bonds has been removed to form an unlocked "sugar" residue. In one example, UNA also encompasses monomers in which the C1'-C4' bond has been removed (i.e., a carbon-oxygen-carbon covalent bond between the C1' and C4' carbons). In another example, the C2'-C3' bond of the sugar has been removed (i.e., a carbon-carbon covalent bond between the C2' and C3' carbons) (see Mikhailov et al., Tetrahedron Letters, 26(17):2059 (1985), and Fluiter et al., Mol. Biosyst., 10:1039 (2009), which are incorporated 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.

[0316] The term "GNA" refers to glycol nucleic acid, which is a polymer similar to DNA or RNA, but differs in the composition of its "backbone" in that it is made up of repeating glycerol units linked by phosphodiester bonds. [ka]

[0317] The thermally destabilizing modification of the duplex can be a mismatch (i.e., non-complementary base pair) between the thermally destabilizing nucleotide and the opposite nucleotide in the opposite strand of the dsRNA duplex. Exemplary mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or combinations thereof. Other mismatch base pairings known in the art are also contemplated by the present invention. Mismatches can occur between nucleotides that are either naturally occurring or modified nucleotides, i.e., mismatch base pairing can occur between the nucleobases derived from each nucleotide, regardless of the modification on the ribose sugar of the nucleotide. In certain embodiments, the dsRNA molecule contains at least one nucleobase in the mismatch pairing that is a 2'-deoxynucleobase, for example, the 2'-deoxynucleobase is in the sense strand.

[0318] In some embodiments, the thermodestabilizing modifications of the duplex in the seed region of the antisense strand include nucleotides that impair WCH bonding with complementary bases on the target mRNA, such as: [ka] Many examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA) and mismatch modifications are described in detail in WO 2011 / 133876, which is incorporated herein by reference in its entirety.

[0319] Thermodestabilizing modifications can also include universal base and phosphate modifications that have reduced or eliminated ability to form hydrogen bonds with opposing bases.

[0320] In some embodiments, the thermal destabilizing modification of duplex comprises the nucleotide with non-standard base, for example but not limited to, the nucleobase modification that the ability to form hydrogen bond with the base in the opposite strand is impaired or completely lost.These nucleobase modifications have been evaluated for destabilizing the central region of dsRNA duplex, as described in International Publication No. 2010 / 0011895, the entire contents of which are incorporated herein by reference.Exemplary nucleobase modifications include: [ka] In some embodiments, the thermodestabilizing modifications of the duplex in the seed region of the antisense strand include one or more α-nucleotides that are complementary to bases on the target mRNA, such as: [ka] wherein R is H, OH, OCH3, F, NH2, NHMe, NMe2 or O-alkyl.

[0321] Exemplary phosphate modifications that have been shown to reduce the thermal stability of dsRNA duplexes compared to natural phosphodiester linkages include the following: [ka] 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.

[0322] As those skilled in the art will recognize, considering that the functional role of nucleobase defines the specificity of the RNAi agent of the present disclosure, nucleobase modification can be carried out in various ways as described herein, for example, for the purpose of enhancing on-target effect against off-target effect, for example, introducing destabilizing modification into the RNAi agent of the present disclosure, and generally the range of modifications present on the RNAi agent of the present disclosure tends to be much greater than the non-nucleobase modifications, for example, the modification of the sugar group or phosphate backbone of polyribonucleotide.Such modifications will be described in more detail in other sections of the present 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.

[0323] In addition to the antisense strand containing a thermally destabilizing modification, the dsRNA may also contain one or more stabilizing modifications. For example, the dsRNA may contain at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilizing modifications. Without limitation, all of the stabilizing modifications may be present in one strand. In some embodiments, both the sense and antisense strands contain at least two stabilizing modifications. The stabilizing modifications may occur at any nucleotide in either the sense strand or the antisense strand. For example, the stabilizing modifications may occur at any nucleotide in the sense strand or the antisense strand, and each stabilizing modification may occur in an alternating pattern on the sense strand or the antisense strand, or the sense strand or the antisense strand may contain both stabilizing modifications in an alternating pattern. The alternating pattern of stabilizing modifications on the sense strand may be the same as or different from that of the antisense strand, and the alternating pattern of stabilizing modifications on the sense strand may be shifted relative to the alternating pattern of stabilizing modifications on the antisense strand.

[0324] In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilizing modifications. Without limitation, the stabilizing modifications within the antisense strand can be located at any position. 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.

[0325] In some embodiments, the antisense strand comprises at least one stabilizing modification adjacent to the destabilizing modification.For example, the stabilizing modification can be at the 5'-end or 3'-end of the destabilizing modification, i.e., at the -1 or +1 position from the position of the destabilizing modification, nucleotide.In some embodiments, the antisense strand comprises a stabilizing modification at each of the 5'-end and 3'-end of the destabilizing modification, i.e., at the -1 and +1 positions from the position of the destabilizing modification.

[0326] 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.

[0327] In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilizing modifications. Without limitation, the stabilizing modifications within the sense strand can be located at any position. In some embodiments, the sense strand comprises stabilizing modifications at positions 7, 10, and 11 from the 5' end. In some other embodiments, the sense strand comprises stabilizing modifications at positions 7, 9, 10, and 11 from the 5' end. In some embodiments, the sense strand comprises stabilizing modifications at positions opposite or complementary to positions 11, 12, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some other embodiments, the sense strand comprises stabilizing modifications at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some embodiments, the sense strand comprises blocks of two, three, or four stabilizing modifications.

[0328] 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.

[0329] Exemplary thermally stabilizing modifications include, but are not limited to, 2'-fluoro modifications. Other thermally stabilizing modifications include, but are not limited to, LNA.

[0330] In some embodiments, the dsRNA of the present disclosure comprises at least four (e.g., four, five, six, seven, eight, nine, ten or more) 2'-fluoro nucleotides. Without limitation, all of the 2'-fluoro nucleotides can be present in one strand. In some embodiments, both the sense and antisense strands comprise at least two 2'-fluoro nucleotides. The 2'-fluoro modification can occur on either the sense strand or the antisense strand's nucleotide. For example, the 2'-fluoro modification can occur on any nucleotide on the sense strand or the antisense strand, and each 2'-fluoro modification can occur in an alternating pattern on the sense strand or the antisense strand, or both the sense strand and the antisense strand contain 2'-fluoro modifications in an alternating pattern. The alternating pattern of 2'-fluoro modifications on the sense strand can be the same as or different from that of the antisense strand, and the alternating pattern of 2'-fluoro modifications on the sense strand can be shifted relative to the alternating pattern of 2'-fluoro modifications on the antisense strand.

[0331] In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modifications in the antisense strand can be present at any position. In some embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 8, 9, 14, and 16 from the 5' end. In some other embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end. In yet some other embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 14, and 16 from the 5' end.

[0332] 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 -1 position or +1 position from the position of destabilizing modification, nucleotide.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 -1 and +1 positions from the position of destabilizing modification.

[0333] 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.

[0334] In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modifications within the sense strand can be present at any position. In some embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 7, 10, and 11 from the 5'-end. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 7, 9, 10, and 11 from the 5'-end. In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to positions 11, 12, and 15 of the antisense strand, counting from the 5'-end of the antisense strand. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5'-end of the antisense strand. In some embodiments, the sense strand comprises two, three, or four blocks of 2'-fluoro nucleotides.

[0335] In some embodiments, the sense strand does not contain a 2'-fluoro nucleotide at a position opposite or complementary to a thermally destabilizing modification of the duplex in the antisense strand.

[0336] In some embodiments, a dsRNA molecule of the disclosure comprises a 21-nucleotide (nt) sense strand and a 23-nucleotide (nt) antisense strand, wherein the antisense strand contains at least one thermolabile nucleotide, the at least one thermolabile nucleotide occurring within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), one end of the dsRNA is blunt while the other end comprises a 2-nt overhang, and the dsRNA optionally further comprises at least one (e.g., one, two, three, four, five, six, or all seven) of the following features: (i) the antisense strand comprises two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; or (iv) the sense strand comprises two, three, four, or five 2'-fluoro modifications. (v) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, (vi) the dsRNA contains at least four 2'-fluoro modifications, and (vii) the dsRNA contains 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.

[0337] In some embodiments, a dsRNA molecule of the present disclosure comprises a sense strand and an antisense strand, wherein the sense strand is 25 to 30 nucleotide residues in length, and starting from the 5'-terminal nucleotide (position 1), positions 1 to 23 of the sense strand comprise at least 8 ribonucleotides; the antisense strand is 36 to 66 nucleotide residues in length, and starting from the 3'-terminal nucleotide, at least 8 ribonucleotides at these positions are paired with positions 1 to 23 of the sense strand to form a duplex; at least the 3'-terminal nucleotide of the antisense strand is not paired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are not paired with the sense strand, thereby forming a 1-6 nucleotide 3' single-stranded overhang; the 5'-end of the sense strand is composed of 10 to 30 contiguous nucleotides that are not paired with the sense strand, thereby forming a 10 to 30 nucleotide single-stranded 5' overhang; at least the 5'- and 3'-terminal nucleotides of the sense strand base-pair with nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplex region between the sense and antisense strands; the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the length of the antisense strand to reduce target gene expression when the double-stranded nucleic acid is introduced into a mammalian cell; and the antisense strand contains at least one thermally destabilizing nucleotide, the at least one thermally destabilizing nucleotide being within the seed region of the antisense strand (i.e., at positions 2 to 9 of the 5'-end of the antisense strand).For example, the thermally destabilizing nucleotide occurs between positions 14-17 opposite or complementary to positions 14-17 of the 5' end of the sense strand, and the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, or all seven) of the following features: (i) the antisense strand contains two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains two, three, four, or five 2'-fluoro modifications; (v) the sense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; and (vi) the dsRNA contains at least four 2'-fluoro modifications; and (vii) the dsRNA contains a duplex region 12-30 nucleotide pairs in length.

[0338] In some embodiments, a dsRNA molecule of the present disclosure comprises a sense strand and an antisense strand, the dsRNA molecule comprising a sense strand having a length of at least 25 nucleotides and at most 29 nucleotides, and an antisense strand having a length of at most 30 nucleotides, the sense strand comprising a modified nucleotide at position 11 from the 5' end that is susceptible to enzymatic degradation, the 3' end of the sense strand and the 5' end of the antisense strand forming a blunt end, and the antisense strand is 1 to 4 nucleotides longer at its 3' end than the sense strand, the duplex region being at least 25 nucleotides in length, and the antisense strand being sufficiently complementary to a target mRNA along at least 19 nt of the antisense strand, such that the dsRNA molecule reduces target gene expression when introduced into a mammalian cell, and Dicer cleavage of the dsRNA preferentially results in an siRNA comprising the 3' end of the antisense strand, thereby inhibiting target gene expression in a mammal. and (ii) the antisense strand contains at least one thermally destabilized nucleotide, the at least one thermally destabilized nucleotide being within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, or all seven) of the following features: (i) the antisense strand contains two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense strand contains two, three, four, five, or six 2'-fluoro modifications; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains two, three, four, or five 2'-fluoro modifications; (v) the sense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; and (vi) the dsRNA contains at least four 2'-fluoro modifications; and (vii) the dsRNA has a duplex region 12 to 29 nucleotide pairs in length.

[0339] In some embodiments, any nucleotide in the sense strand and antisense strand of dsRNA molecule can be modified.Each nucleotide can be modified with the same or different modifications, and these modifications can include one or both of non-linked phosphate oxygen or one or more of linked phosphate oxygen, modifying the ribose sugar component, for example, modifying the 2' hydroxyl on the ribose sugar, replacing phosphate moiety extensively with " dephosphorylation " linker, modifying or replacing naturally occurring base, and replacing or replacing ribose phosphate backbone.

[0340] Because nucleic acids are polymers of subunits, many modifications occur at positions that are repeated within nucleic acids, such as modifications of bases or phosphate moieties, or unlinked Os in phosphate moieties.In some cases, modifications occur at all target positions in nucleic acids, but in many cases, they do not occur.For example, modifications can occur only at the 3'-end or 5'-end positions, or only in terminal regions, such as at the terminal nucleotide position of the chain or at the last 2, 3, 4, 5, or 10 nucleotides of the chain.Modifications can occur in double-stranded regions, single-stranded regions, or both.Modifications can occur only in the double-stranded region of RNA, or only in the single-stranded region of RNA.For example, phosphorothioate modifications at unlinked O positions can occur only at one or both ends, or only in terminal regions, such as at the terminal nucleotide position of the chain or within the last 2, 3, 4, 5, or 10 nucleotides, or in double-stranded and single-stranded regions, especially at the ends. The 5' end can be phosphorylated.

[0341] This may, for example, enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in the single-stranded overhang, for example, in the 5' overhang or 3' overhang, or in both overhangs.For example, it may be desirable to include purine nucleotides in the overhang.In some embodiments, all or part of the bases in the 3' overhang or 5' overhang can be modified, for example, with the modifications described herein.Modifications may include, for example, the use of modifications at the 2' position of the ribose sugar with modifications known in the art, for example, the use of 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.

[0342] In some embodiments, each residue of the sense strand and the antisense strand is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, or 2'-fluoro. These strands may contain two or more modifications. In some embodiments, each residue of the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro. It should be understood that these modifications are in addition to at least one thermostabilizing modification of the duplex present in the antisense strand.

[0343] At least two different modifications are typically present on the sense strand and the antisense strand. These modifications can be 2'-deoxy, 2'-O-methyl, or 2'-fluoro modifications, acyclic nucleotides, etc. In some embodiments, the sense strand and the antisense strand each contain two differently modified nucleotides selected from 2'-O-methyl or 2'-deoxy. In some embodiments, each residue in the sense strand and the antisense strand is independently modified with a 2'-O-methyl nucleotide, a 2'-deoxy nucleotide, a 2'-deoxy-2'-fluoro nucleotide, a 2'-ON-methylacetamide (2'-O-NMA) nucleotide, a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotide, a 2'-O-aminopropyl (2'-O-AP) nucleotide, or a 2'-ara-F nucleotide. Again, it should be understood that these modifications are in addition to at least one thermostabilizing modification of the duplex present in the antisense strand.

[0344] In some embodiments, the dsRNA molecules of the present disclosure contain alternating patterns of modifications, particularly in the B1, B2, B3, B1', B2', B3', and B4' regions. The terms "alternating motif" or "alternating pattern," as used herein, refer to a motif having one or more modifications, with each modification occurring at alternating nucleotides in a single strand. The alternating nucleotides may refer to one every other nucleotide, one every three nucleotides, or similar patterns. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif may be "ABABABABABAB...," "AABBAABBAABB...," "AABAABAABAAB...," "AAABAAABAAAB...," "AAABBBAAABBB...," or "ABCABCABCABC...," etc.

[0345] The types of modifications contained within an alternating motif can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternation pattern, i.e., the modifications on every other nucleotide, can be the same, but each of the sense or antisense strands can be selected from several possible modifications within the alternating motif, such as "ABABAB...," "ACACAC...," "BDBDBD...," or "CDCDCD...," etc.

[0346] In some embodiments, dsRNA molecules of the present disclosure include an alternating motif modification pattern on the sense strand that is shifted relative to the alternating motif modification pattern on the antisense strand. The shift can be such that modified groups on nucleotides in the sense strand correspond to differently modified groups on nucleotides in the antisense strand, or vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, the alternating motif in the sense strand can begin with "ABABAB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand can begin with "BABABA" from the 3'-5' end of the strand in the duplex region. As another example, the alternating motif in the sense strand can begin with "AABBAABB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand can begin with "BBAABBAA" from the 3'-5' end of the strand in the duplex region, resulting in a complete or partial shift in the modification pattern between the sense and antisense strands.

[0347] The dsRNA molecule of the present disclosure can further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage.Phosphorothioate or methylphosphonate internucleotide linkage modification can occur on the nucleotide of either sense strand or antisense strand or both strands at any position of the strand.For example, internucleotide linkage modification can occur on any nucleotide on sense strand or antisense strand, each internucleotide linkage modification can occur in an alternating pattern on sense strand or antisense strand, or sense strand or antisense strand contains both internucleotide linkage modifications in an alternating pattern.The alternating pattern of internucleotide linkage modification on sense strand can be the same or different from that of antisense strand, and the alternating pattern of internucleotide linkage modification on sense strand can be shifted relative to the alternating pattern of internucleotide linkage modification on antisense strand.

[0348] In some embodiments, the dsRNA molecule comprises phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region.For example, the overhang region comprises two nucleotides, and the two nucleotides comprise phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides.Internucleotide linkage modification can also be made to link the overhang nucleotide to the terminal paired nucleotide in the double-stranded region.For example, at least 2, 3, 4 or all of the overhang nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there can be an additional phosphorothioate or methylphosphonate internucleotide linkage connecting the overhang nucleotide to the paired nucleotide adjacent to the overhang nucleotide.For example, there can be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, such that two of the three nucleotides are overhang nucleotides and the third is the paired nucleotide adjacent to the overhang nucleotide.Preferably, these terminal three nucleotides can be at the 3'-end of the antisense strand.

[0349] 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, one of which is positioned at any position within 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.

[0350] 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 positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0351] 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 positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0352] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0353] 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 which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0354] In some embodiments, the antisense strand of the dsRNA molecule contains two blocks of six phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand containing either phosphorothioate or methylphosphonate or phosphate linkages.

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

[0356] In some embodiments, the antisense strand of a 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 which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0357] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by one, two, three, or four phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate linkages.

[0358] In some embodiments, dsRNA molecules of this disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1 to 10 of the termini of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides can be linked via phosphorothioate or methylphosphonate internucleotide linkages at one or both termini of the sense or antisense strand.

[0359] In some embodiments, the dsRNA molecules of the present disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within 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 via phosphorothioate methylphosphonate internucleotide linkage modifications at positions 8-16 of the duplex region, counting from the 5' end of the sense strand, and the dsRNA molecule can optionally further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-10 of that end.

[0360] In some embodiments, dsRNA molecules of the disclosure comprise one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the sense strand, and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand and one to five within positions 18-23 of the antisense strand.

[0361] In some embodiments, dsRNA molecules of the disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 5' end) and one phosphorothioate or methylphosphonate internucleotide linkage modification within positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the antisense strand.

[0362] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18-23 of the antisense strand.

[0363] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1 to 5 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18 to 23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18 to 23 of the antisense strand.

[0364] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1-5 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the antisense strand (counting from the 5' end).

[0365] In some embodiments, dsRNA molecules of the disclosure further comprise one phosphorothioate internucleotide linkage modification within the range of positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within the range of positions 18-23 of the antisense strand (counting from the 5' end).

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

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

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

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

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

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

[0372] In some embodiments, the dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one at position 21 (counting from the 5' end) of the antisense strand.

[0373] In some embodiments, dsRNA molecules of the 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.

[0374] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0375] In some embodiments, dsRNA molecules of the 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.

[0376] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 22 and 23 of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0377] In some embodiments, the dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 (counting from the 5' end) of the antisense strand.

[0378] In some embodiments, the compounds of the present disclosure comprise a pattern of backbone chiral centers. In some embodiments, the regular pattern of backbone chiral centers comprises at least 5 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 6 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 7 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 8 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 9 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 16 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 17 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 18 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 19 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 8 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 7 or fewer internucleotide linkages in the Rp configuration.In some embodiments, the regular pattern of backbone chiral centers comprises 6 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 5 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 4 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 3 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 2 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 1 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 8 or fewer non-chiral internucleotide linkages (phosphodiesters as a non-limiting example). In some embodiments, the regular pattern of backbone chiral centers comprises 7 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 6 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 5 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 4 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 3 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 2 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 1 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration and 8 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration and 7 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration and 6 or fewer non-chiral internucleotide linkages.In some embodiments, the regular pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration and no more than 6 non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration and no more than 5 non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration and no more than 4 non-chiral internucleotide linkages. In some embodiments, the internucleotide linkages in the Sp configuration may be contiguous or non-contiguous. In some embodiments, the internucleotide linkages in the Rp configuration may be contiguous or non-contiguous. In some embodiments, the non-chiral internucleotide linkages may be contiguous or non-contiguous.

[0379] In some embodiments, compounds of the present disclosure include blocks that are stereochemical blocks. In some embodiments, the blocks are Rp blocks, in that each internucleotide linkage of the block is Rp. In some embodiments, the 5'-block is an Rp block. In some embodiments, the 3'-block is an Rp block. In some embodiments, the blocks are Sp blocks, in that each internucleotide linkage of the block is Sp. In some embodiments, the 5'-block is an Sp block. In some embodiments, the 3'-block is an Sp block. In some embodiments, provided oligonucleotides include both Rp and Sp blocks. In some embodiments, provided oligonucleotides include one or more Rp blocks but no Sp blocks. In some embodiments, provided oligonucleotides include one or more Sp blocks but no Rp blocks. In some embodiments, provided oligonucleotides include one or more PO blocks, where each internucleotide linkage is a natural phosphate linkage.

[0380] In some embodiments, compounds of the present disclosure include a 5'-block that is an Sp block in which each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each internucleotide linkage is a modified internucleotide linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each internucleotide linkage is a phosphorothioate linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block comprises four or more nucleoside units. In some embodiments, the 5'-block comprises five or more nucleoside units. In some embodiments, the 5'-block comprises six or more nucleoside units. In some embodiments, the 5'-block comprises seven or more nucleoside units. In some embodiments, the 3'-block is an Sp block in which each sugar moiety comprises a 2'-F modification. In some embodiments, the 3'-block is an Sp block in which each internucleotide linkage is a modified internucleotide linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 3'-block is an Sp block, in which each of the internucleotide linkages is a phosphorothioate linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 3'-block comprises 4 or more nucleoside units. In some embodiments, the 3'-block comprises 5 or more nucleoside units. In some embodiments, the 3'-block comprises 6 or more nucleoside units. In some embodiments, the 3'-block comprises 7 or more nucleoside units.

[0381] In some embodiments, compounds of the disclosure include a region of nucleosides or oligonucleotides followed by a particular type of internucleotide linkage, such as a natural phosphate linkage, a modified internucleotide linkage, an Rp chiral internucleotide linkage, an Sp chiral internucleotide linkage, etc. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by a natural phosphate linkage (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by a natural phosphate linkage (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by a natural phosphate linkage (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by a natural phosphate linkage (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by a natural phosphate linkage (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.

[0382] In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23, the antisense strand contains at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven, or all eight) of the following features: (i) the antisense strand contains two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense strand comprises 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to 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; (vii) the dsRNA comprises a duplex region 12 to 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0383] In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, the antisense strand contains at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally further has at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or all 8) of the following features: (i) the antisense strand comprises 2, 3, (ii) the sense strand is conjugated to a ligand; (iii) the sense strand contains two, three, four, or five 2'-fluoro modifications; (iv) the sense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; (v) the dsRNA contains at least four 2'-fluoro modifications; (vi) the dsRNA contains a duplex region 12 to 40 nucleotide pairs in length; (vii) the dsRNA contains a duplex region 12 to 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0384] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, and the antisense strand contains at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally further comprises at least one (e.g., one, two, three, four, five, six, seven, or all eight) of the following features: (i) the antisense strand comprises two, three, four, five, or six 2'-fluoro modifications; (i) the antisense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (v) the sense strand comprises three, four, or five phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; (vii) the dsRNA comprises a duplex region 12 to 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0385] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, and the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, the antisense strand contains at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally has at least one of the following characteristics: (ii) the sense strand is conjugated to a ligand; (iii) the sense strand contains two, three, four, or five 2'-fluoro modifications; (iv) the sense strand contains three, four, or five phosphorothioate internucleotide linkages; (v) the dsRNA contains at least four 2'-fluoro modifications; (vi) the dsRNA contains a duplex region 12 to 40 nucleotide pairs in length; and (vii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0386] In some embodiments, the dsRNA molecule of the present disclosure comprises mismatches in the duplex with the target, or a combination thereof. Mismatches can occur in the overhang region or the duplex region. Base pairs can be ranked based on their tendency to promote dissociation or melting (for example, based on the free energy of association or dissociation of a particular pairing; the simplest approach is to examine each pair individually, but then adjacent or similar analysis can also be used). In terms of promoting dissociation, A:U is more preferable than G:C, G:U is more preferable than G:C, and I:C is more preferable than G:C (I=inosine). Mismatches, such as non-standard pairings or non-standard pairings (described elsewhere herein), are more preferable than standard pairings (A:T, A:U, G:C), and pairings that include universal bases are more preferable than standard pairings.

[0387] In some embodiments, the dsRNA molecules of the present disclosure comprise at least one mismatch pair within the duplex region from the 5' end of the antisense strand, which may be independently selected from the group of A:U, G:U, I:C, and a non-canonical or non-standard or universal base pair, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0388] In some embodiments, the nucleotide at position 1 from the 5' end of the antisense strand to the duplex region is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first one, two, or three base pairs from the 5' end of the antisense strand to the duplex region is an AU base pair. For example, the first base pair from the 5' end of the antisense strand to the duplex region is an AU base pair.

[0389] It has been found that the introduction of a 4'- or 5'-modified nucleotide at the 3' end of a dinucleotide phosphodiester (PO), phosphorothioate (PS), or phosphorodithioate (PS2) linkage at any position in a single- or double-stranded oligonucleotide can exert a steric effect on the internucleotide linkage, thereby protecting or stabilizing it against nucleases.

[0390] In some embodiments, 5'-modified nucleoside is introduced at the 3' end of dinucleotide at any position in single-stranded or double-stranded siRNA.For example, 5'-alkylated nucleoside can be introduced at the 3' end of dinucleotide at any position in single-stranded or double-stranded siRNA.The alkyl group at the 5' position of ribose sugar can be racemic or chirally pure R or S isomer.Exemplary 5'-alkylated nucleoside includes 5'-methyl nucleoside.5'-methyl can be racemic or chirally pure R or S isomer.

[0391] In some embodiments, 4'-modified nucleosides are introduced at the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA.For example, 4'-alkylated nucleosides can be introduced at the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA.The alkyl group at the 4' position of ribose sugar can be racemic or chirally pure R or S isomer.Exemplary 4'-alkylated nucleosides include 4'-methyl nucleosides.4'-methyl can be racemic or chirally pure R or S isomer.Alternatively, 4'-O-alkylated nucleosides can be introduced at the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA.The 4'-O-alkyl of 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.

[0392] 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 chiral pure R or S isomer.Exemplary 5'-alkylated nucleoside includes 5'-methyl nucleoside.5'-methyl can be either racemic or chiral pure R or S isomer.

[0393] 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 chiral pure R or S isomer.Exemplary 4'-alkylated nucleoside includes 4'-methyl nucleoside.4'-methyl can be either racemic or chiral pure R or S isomer.

[0394] In some embodiments, 4'-O-alkylated nucleoside can be introduced at any position of the sense strand or antisense strand of dsRNA, and this modification maintains or improves the efficacy of dsRNA.5'-Alkyl can be either racemic or chiral pure R or S isomer.Exemplary 4'-O-alkylated nucleoside includes 4'-O-methyl nucleoside.4'-O-methyl can be either racemic or chiral pure R or S isomer.

[0395] In some embodiments, the dsRNA molecules of the present disclosure may contain 2'-5' linkages (having 2'-H, 2'-OH, and 2'-OMe, and also having P=O or P=S). For example, 2'-5' linkage modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5' end of the sense strand to prevent activation of the sense strand by RISC.

[0396] In another embodiment, the dsRNA molecule of the present disclosure can comprise L sugar (for example, L-ribose, L-arabinose, with 2'-H, 2'-OH and 2'-OMe).For example, these L sugar modifications can be used to promote nuclease resistance, or to inhibit the binding of sense strand to antisense strand, or can be used at the 5' end of sense strand to prevent the activation of sense strand by RISC.

[0397] Multimeric siRNA has been described in various publications, and all of them can be used with the dsRNA of the present disclosure.These publications include WO2007 / 091269, US Patent No. 7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520, and each of them is incorporated herein by reference in its entirety.

[0398] As described in more detail below, RNAi agents containing one or more carbohydrate moieties conjugated to them can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, such as a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is attached. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose-replacement modified subunit (PRMS). The cyclic carrier can be a carbon-cyclic system, i.e., all ring atoms are carbon atoms, or a heterocyclic system, i.e., one or more ring atoms can be heteroatoms, such as nitrogen, oxygen, or sulfur. The cyclic carrier can be a monocyclic ring system or can contain two or more rings, e.g., fused rings. The cyclic carrier can be a fully saturated ring system or can contain one or more double bonds.

[0399] The ligand can be attached to the polynucleotide via a carrier. The carrier comprises (i) at least one "backbone attachment point," preferably two "backbone attachment points," and (ii) at least one "tethering attachment point." As used herein, "backbone attachment point" refers to a functional group, e.g., a hydroxyl group, or generally a bond, available and suitable for incorporating the carrier into the backbone of a ribonucleic acid, e.g., a phosphate or a modified phosphate backbone, e.g., sulfur-containing. In some embodiments, a "tethering attachment point" (TAP) refers to a ring atom, e.g., a carbon atom or heteroatom (other than the atom providing the backbone attachment point), of the cyclic carrier to which the selected moiety is attached. This moiety can be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is attached to the cyclic carrier by an intervening tether. Thus, cyclic carriers often contain functional groups, e.g., amino groups, or generally bonds, that provide a linkage suitable for the incorporation or tethering of another chemical entity, e.g., a ligand, to the constituent ring.

[0400] The RNAi agent may be conjugated to the ligand via a carrier, which may be a cyclic or acyclic group, preferably the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalin, preferably the acyclic group is selected from a serinol backbone or a diethanolamine backbone.

[0401] In certain embodiments, the RNAi agent used in the methods of the present disclosure is an agent selected from the group of agents listed in Tables 3 to 8. These agents may further comprise a ligand.

[0402] IV. iRNA conjugated to a ligand Another modification of the iRNA of the invention involves chemically linking the iRNA to one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, or cellular uptake, for example, of the iRNA. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acids Sci. USA, 1989, 86:6553-6556), cholic acid (Manoharan et al., Bior. Med. Chem. Let., 1994, 4:1053-1060), thioethers such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Bior. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), fatty chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids such as dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmityl moieties (Mishra et al., Biochim.Biophys. Acta, 1995, 1264:229-237), or octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0403] In certain embodiments, ligands change the distribution, targeting or life span of the iRNA agent that they are incorporated into.In some embodiments, ligands cause enhanced affinity to selected targets, for example, molecules, cells or cell types, compartments such as cell or organ compartments, tissues, organs or regions of the body, for example, when compared with species that do not have such ligands.Normal ligands do not participate in double-stranded pairing in double-stranded nucleic acids.

[0404] Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL) or globulins), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid), or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryl acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.

[0405] The ligand can also include a targeting group, such as a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid, or protein, e.g., an antibody that binds to a specific cell type, such as a CNS 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 an RGD peptide or RGD peptide mimetic. In certain embodiments, the ligand is a polyvalent galactose, e.g., N-acetyl-galactosamine.

[0406] 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 nucleotides include hydroxybenzoates (e.g., hydroxybenzoates ...

[0407] Ligands can be proteins, e.g., glycoproteins or peptides, e.g., molecules with specific affinity for a co-ligand, or antibodies, e.g., antibodies that bind to specific cell types, such as cancer cells, endothelial cells, or bone cells. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. Ligands can be, for example, lipopolysaccharides, activators of p38 MAP kinase, or activators of NF-κB.

[0408] The ligand can be a substance, e.g., a drug, that can increase the uptake of an iRNA agent into a cell, for example, by disrupting the cytoskeleton of the cell, e.g., by disrupting the cell's microtubules, microfilaments, or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0409] In some embodiments, the ligands binding to the iRNAs described herein act as pharmacokinetic modulators (PK modulators). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing several phosphorothioate linkages have also been shown to bind to serum proteins, and therefore short oligonucleotides containing multiple phosphorothioate linkages in the backbone, e.g., oligonucleotides of about 5, 10, 15, or 20 bases, are also suitable as ligands (e.g., as PK-modulating ligands) for the present invention. Furthermore, aptamers that bind serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0410] The ligand-conjugated iRNAs of the invention can be synthesized by using oligonucleotides bearing reactive functional pendant side chains, such as those derived from the attachment of a linking molecule onto the oligonucleotide (described below). The reactive oligonucleotides can be reacted directly with commercially available ligands, synthesized ligands bearing any of a variety of protecting groups, or ligands bearing a linking moiety attached thereto.

[0411] The oligonucleotides used in the conjugates of the present invention can be conveniently and routinely prepared by the well-known technique of solid-phase synthesis. 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 may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

[0412] In the ligand-conjugated oligonucleotides and sequence-specific linked nucleosides bearing ligand molecules of the present invention, the oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors already bearing a linking moiety, ligand-nucleotide or ligand-nucleoside conjugate precursors already bearing a ligand molecule, or non-nucleoside ligand-bearing building blocks.

[0413] When using a nucleotide-conjugate precursor that already possesses a linking moiety, typically, synthesis of the sequence-specifically linked nucleoside is completed, and then a ligand molecule is reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the invention are synthesized by automated synthesizers using phosphoramidites derived from ligand-nucleoside conjugates, in addition to commercially available standard and non-standard phosphoramidites routinely used in oligonucleotide synthesis.

[0414] A. Lipid Conjugates In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule.This lipid or lipid-based molecule can usually bind serum protein, for example, human serum albumin (HSA).HSA-binding ligand allows the distribution of conjugate to target tissue in the body, for example, to target tissue other than the kidney.For example, the target tissue can be the liver, including the liver parenchymal cells.Other molecules that can bind HSA can also be used as ligand.For example, naproxen or aspirin can be used.Lipid or lipid-based ligand can (a) increase the resistance of conjugate to degradation, (b) increase targeting or transport into target cell or cell membrane, or (c) be used to adjust the binding to serum protein, for example, HSA.

[0415] Lipid-based ligands can be used to regulate, for example, control (for example, inhibit) the binding of conjugate to target tissue.For example, lipid or lipid-based ligands that bind more strongly to HSA are less likely to target the kidney, and therefore less likely to be eliminated from the body.A lipid or lipid-based ligand that binds less strongly to HSA can be used so that the conjugate targets the kidney.

[0416] In certain embodiments, the lipid-based ligand binds HSA. For example, the ligand can bind to HSA with sufficient affinity, thereby enhancing the distribution of the conjugate to non-renal tissues. However, this affinity is usually not so strong that the HSA-ligand binding is irreversible.

[0417] In certain 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.

[0418] In another aspect, the ligand is a moiety, for example, a vitamin, that is taken up by target cells, for example, proliferating cells.These are particularly useful for treating disorders characterized by unwanted cell proliferation, for example, malignant or non-malignant, for example, cancer cells.Exemplary vitamins include vitamins A, E and K.Other exemplary vitamins include vitamin B, for example, folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by cancer cells.Also included are HSA and low-density lipoprotein (LDL).

[0419] B. Cell-penetrating agents In another aspect, the ligand is a cell-penetrating agent, such as a helical cell-penetrating agent. In certain embodiments, the cell-penetrating agent is amphipathic. Exemplary cell-penetrating agents include peptides such as tat or antennapedia. When the cell-penetrating agent is a peptide, it can be modified, including peptidyl mimics, invertomers, non-peptide or pseudopeptide linkages, and the use of D-amino acids. Helical agents are usually α-helical agents, and can have lipophilic and lipophobic phases.

[0420] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules that can fold into defined three-dimensional structures similar to natural peptides. Attachment of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic distribution of iRNAs, such as by enhancing cellular recognition and uptake. The peptide or peptidomimetic portion can be about 5 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0421] The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. Alternatively, the peptide moiety can contain a hydrophobic membrane transport sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 3). An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 4)) can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, capable of transporting large polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 5)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 6)) have been shown to function as delivery peptides. Peptides or peptidomimetics can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). Peptides or peptidomimetics that are typically tethered to dsRNA agents via incorporated monomeric units include cell-targeting peptides, such as arginine-glycine-aspartic acid (RGD) peptides or RGD mimetics. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications, such as those that increase stability or affect conformational properties. Any of the structural modifications described below can be utilized.

[0422] The RGD peptides used in the compositions and methods of the present invention can be linear or cyclic, and can be modified, for example, glycosylated or methylated, to facilitate targeting of specific tissues.RGD-containing peptides and peptidiomimetics can contain D-amino acids and synthetic RGD mimics.In addition to RGD, other moieties that target integrin ligands can be used.Preferred conjugates of this ligand target PECAM-1 or VEGF.

[0423] 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 tumor targeting of dsRNA agents to 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 to facilitate targeting to specific tissues, e.g., glycosylated or methylated. For example, glycosylated RGD peptides can be used to target α V iRNA agents can be delivered to tumor cells that express β3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

[0424] 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 Ceropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain a nuclear localization signal (NLS). For example, a cell-penetrating peptide can be a bipartite amphipathic peptide, such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0425] C. Carbohydrate conjugates In some embodiments of the compositions and methods of the present invention, the iRNA further comprises a carbohydrate. Carbohydrate-conjugated iRNAs are advantageous for in vivo delivery of nucleic acids, as described herein, and are suitable for in vivo therapeutic use. As used herein, "carbohydrate" refers to a compound that is either a carbohydrate itself (which may be linear, branched, or cyclic) composed of one or more monosaccharide units having at least six carbon atoms with an oxygen, nitrogen, or sulfur atom attached to each carbon atom, or a compound having as its part a carbohydrate moiety (which may be linear, branched, or cyclic) composed of one or more monosaccharide units, each having at least six carbon atoms with an oxygen, nitrogen, or sulfur atom attached to each carbon atom. Exemplary carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide resins. Particular monosaccharides include C5 and above (e.g., C5, C6, C7 or C8) sugars, and disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7 or C8).

[0426] In certain embodiments, the carbohydrate conjugate comprises a monosaccharide.

[0427] In certain embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). GalNAc conjugates containing one or more N-acetylgalactosamine (GalNAc) derivatives are described, for example, in U.S. Patent No. 8,106,022, the entire contents of which are incorporated herein by reference. In some embodiments, the GalNAc conjugate functions as a ligand that targets iRNA to specific cells. In some embodiments, the GalNAc conjugate targets iRNA to liver cells, for example, by functioning as a ligand for the asialoglycoprotein receptor of liver cells (e.g., hepatocytes).

[0428] In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives can be attached via a linker, e.g., via a bivalent or trivalent branched linker. In some embodiments, the GalNAc conjugate is conjugated to the 3' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 3' end of the sense strand) via a linker, e.g., as described herein. In some embodiments, the GalNAc conjugate is conjugated to the 5' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 5' end of the sense strand) via a linker, e.g., as described herein.

[0429] In certain embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a monovalent linker. In some embodiments, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a bivalent linker. In yet other embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a trivalent linker. In other embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a tetravalent linker.

[0430] In certain embodiments, a double-stranded RNAi agent of the invention comprises one GalNAc or GalNAc derivative attached to an iRNA agent, hi certain embodiments, a double-stranded RNAi agent of the invention comprises multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each of which is independently attached to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers.

[0431] In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of a larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the corresponding 5' end of the other strand, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop can independently comprise a GalNAc or GalNAc derivative linked via a monovalent linker. This hairpin loop can also be formed by an extended overhang in one strand of the duplex.

[0432] In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of a larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the corresponding 5' end of the other strand, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop can independently comprise a GalNAc or GalNAc derivative linked via a monovalent linker. This hairpin loop can also be formed by an extended overhang in one strand of the duplex.

[0433] In some embodiments, the GalNAc conjugate is [ka] Formula I is.

[0434] In some embodiments, the RNAi agent is attached to the carbohydrate conjugate via a linker as shown in the following schematic diagram, where X is O or S: [ka]

[0435] In some embodiments, the RNAi agent is conjugated to L96, as defined in Table 2 and shown below. [ka]

[0436] In certain embodiments, the carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of: [ka] Formula I, [ka] Formula II, [ka] Formula III, [ka] Formula IV, [ka] Formula V, [ka] Formula VI, [ka] Formula VII, [ka] Formula VIII, [ka] Formula IX, [ka] Formula X, [ka] Formula XI, [ka] Formula XII, [ka] Formula XIII, [ka] Formula XIV, [ka] Formula XV, [ka] Formula XVI, [ka] Formula XVII, [ka] Formula XVIII, [ka] Formula XIX, [ka] Formula XX, [ka] Formula XXI, [ka] Formula XXII, [ka] , wherein Y is O or S and n is 3 to 6 (Formula XXIII), [ka] , wherein Y is O or S and n is 3 to 6 (Formula XXIV), [ka] Formula XXV, [ka] , wherein X is O or S (Formula XXVI), [ka] Formula XXVII, [ka] Formula XXVIII, [ka] Formula XXIX, [ka] Formula XXX, [ka] Formula XXXI, [ka] , Formula XXXII, and [ka] , formula XXXIII is.

[0437] In certain embodiments, the carbohydrate conjugate used in the compositions and methods of the present invention is a monosaccharide. In certain embodiments, the monosaccharide is N-acetylgalactosamine, e.g., [ka] Formula I is.

[0438] Other exemplary carbohydrate conjugates for use in the embodiments described herein include, but are not limited to: [ka] (formula XXXIV), When one of X or Y is an oligonucleotide, the other is hydrogen.

[0439] In some embodiments, suitable ligands are those disclosed in WO 2019 / 055633, the entire contents of which are incorporated herein by reference. In one embodiment, the ligand comprises the following structure: [ka] Formula XXXV

[0440] In certain embodiments, RNAi agents of the present disclosure may include GalNAc ligands, even though such GalNAc ligands are currently expected to be of limited value for the preferred intrathecal / CNS delivery route of the present disclosure.

[0441] In certain embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a monovalent linker. In some embodiments, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a bivalent linker. In yet other embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a trivalent linker.

[0442] In one embodiment, the double-stranded RNAi agent of the present invention comprises one or more GalNAc or GalNAc derivatives linked to the iRNA agent. GalNAc can be linked to any nucleotide via a linker on the sense strand or antisense strand. GalNAc can be linked to the 5' end of the sense strand, the 3' end of the sense strand, the 5' end of the antisense strand, or the 3' end of the antisense strand. In one embodiment, GalNAc is linked to the 3' end of the sense strand, for example, via a trivalent linker.

[0443] In other embodiments, a double-stranded RNAi agent of the invention comprises multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently linked to multiple nucleotides of the double-stranded RNAi agent via multiple linkers, e.g., monovalent linkers.

[0444] In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of a single larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the corresponding 5' end of the other strand, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop can independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker.

[0445] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator or a cell-penetrating peptide.

[0446] Further carbohydrate conjugates and linkers suitable for use in the present invention include those described in WO 2014 / 179620 and WO 2014 / 179627, the entire contents of each of which are incorporated herein by reference.

[0447] D. 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.

[0448] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound, e.g., covalently bonds the two parts of a compound.Typically, a linker is a direct bond or an atom such as oxygen or sulfur, a unit such as NR, C(O), C(O)NH, SO, SO, SONH, or a chain of atoms, including, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylhetero ...alkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkynylarylalkynyl, alkylheteroarylalkynyl, alkyl alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclyl and the like, wherein one or more methylenes can be interrupted or terminated by O, S, S(O), SO, N(R), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, where R is hydrogen, acyl, aliphatic, or substituted aliphatic.In certain embodiments, the linker is about 1 to 24 atoms, 2 to 24, 3 to 24, 4 to 24, 5 to 24, 6 to 24, 6 to 18, 7 to 18, 8 to 18 atoms, 7 to 17, 8 to 17, 6 to 16, 7 to 16, or 8 to 16 atoms.

[0449] A cleavable linking group is one that is sufficiently stable outside a cell, but that, once inside a target cell, is cleaved to release the two moieties held together by the linker. In preferred embodiments, the cleavable linking group is cleaved at a rate that is at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or more, or at least about 100-fold faster in the target cell or under first reference conditions (which may, for example, be selected to mimic or represent intracellular conditions) than in the subject's blood or under second reference conditions (which may, for example, be selected to mimic or represent conditions found in blood or serum).

[0450] Cleavable linking groups are susceptible to cleaving agents, such as pH, redox potential, or the presence of degradable molecules. Cleavage agents are generally more widespread or found at higher levels or activity inside cells than in serum or blood. Examples of such degrading agents include redox agents that are selective for a specific substrate or have no substrate specificity, e.g., oxidases or reductases or reducing agents present in cells, such as mercaptans, that can degrade redox-cleavable linking groups by reduction; esterases; reagents that can create an endosomal or acidic environment, e.g., reagents that result in a pH of 5 or less; and enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0451] 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 have cleavable linking groups that are cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand inside the cell or into a desired compartment of the cell.

[0452] Linker can contain a cleavable linking group that can be cleaved by specific enzyme.The type of cleavable linking group incorporated into linker can depend on the target cell.For example, liver targeting ligand can be linked to cationic lipid through a linker that contains ester group.Hepatocytes are rich in esterase, so linker will be cleaved more efficiently in hepatocytes than in cell types that are not rich in esterase.Other cell types that are rich in esterase include lung, renal cortex and testicular cells.

[0453] When targeting cell types rich in peptidases, such as hepatocytes and synoviocytes, linkers containing peptide bonds can be used.

[0454] Generally, the suitability of a candidate cleavable linking group can be evaluated by testing the ability (or conditions) of a degrading agent to cleave the candidate linking group. It may also be desirable to test the candidate cleavable linking group for its ability to resist cleavage in blood or when in contact with other non-target tissues. Thus, the relative susceptibility to cleavage between first and second conditions can be determined, where the first condition is selected to exhibit cleavage in target cells, and the second condition is selected to exhibit cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to perform initial evaluation in cell-free or culture conditions and confirm by further evaluation in whole animals. In preferred embodiments, useful candidate compounds are cleaved at a rate that is at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0455] i. Redox-cleavable linking group In certain embodiments, the cleavable linking group is a redox-cleavable linking group that is cleaved upon reduction or oxidation. An example of a reductively cleavable linking group is a disulfide linking group (-SS-). To determine whether a candidate cleavable linking group is a suitable "reductively cleavable linking group" or is suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, one can refer to the methods described herein. For example, candidates can be evaluated in cells by incubating with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate that would be observed in target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In some conditions, the candidate compound is cleaved at a maximum of about 10% in blood. In other embodiments, useful candidate compounds are degraded at a rate at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic intracellular media and compared to conditions selected to mimic extracellular media.

[0456] ii. Phosphate-based cleavable linkers In certain 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 the phosphate group within a cell is an enzyme such as a phosphatase within the cell. Examples of phosphate-based linking groups include -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. Preferred embodiments include OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. Preferred embodiments include -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0457] iii. Acid-cleavable linking group In certain 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 preferred embodiments, the acid-cleavable linking group is cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0 or less) or by an agent, such as an enzyme, that can act as a general acid. Within a cell, 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 preferred 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.

[0458] iv. Ester-based cleavable linking groups In certain embodiments, the cleavable linker comprises an ester-based cleavable linking group. Ester-based cleavable linking groups are cleaved in cells by enzymes such as esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0459] v. Peptide-based cleavable linking groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linking group. Peptide-based cleavable linking groups are cleaved intracellularly by enzymes such as peptidases and proteases. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. Peptide-based cleaving groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to yield peptides and proteins, and do not include all amide functional groups. Peptide-based cleavable linking groups have the general formula -NHCHR A C(O)NHCHR B C(O)—, wherein R A and R B are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0460] In some embodiments, the iRNA of the present invention 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 invention include, but are not limited to: [ka] (formula XXXVI), [ka] (formula XXXVII), [ka] (formula XXXVIII), [ka] (formula XXXIX), [ka] (formula XL), [ka] (formula XLI), [ka] (Formula XLII), and [ka] (Formula XLIII), and when one of X or Y is an oligonucleotide, the other is hydrogen.

[0461] In certain embodiments of the compositions and methods of the present invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a bivalent or trivalent branched linker.

[0462] In certain embodiments, the dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formulas (XLIV) to (XLVII): [ka] Formula XLIV, [ka] Formula XLV, [ka] Equation XLVI, or [ka] Formula XLVII, During the ceremony, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C each independently represent 0 to 20, and the repeat units may be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C is, for each occurrence, independently absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CHO; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C is independently at each occurrence absent, alkylene, or substituted alkylene, wherein one or more methylenes are selected from O, S, S(O), SO, N(R N ), C(R')=C(R''), C≡C, or C(O); R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C are each independently absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocyclyl, L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B , and L 5C represents a ligand, i.e., each occurrence is independently a monosaccharide (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide, and R a is H or an amino acid side chain. Trivalent conjugated GalNAc derivatives are particularly useful for use with RNAi agents that inhibit expression of target genes, such as those of formula (XLVIII): [ka] Formula XLVIII, In the formula, L 5A , L 5B , and L 5C represents a monosaccharide, such as a GalNAc derivative.

[0463] 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 I, VI, IX, X, and XII.

[0464] Representative United States patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979, 4,948,882, 5,218,105, 5,525,465, 5,541,313, 5,545,730, 5,552,538, 5,578,717, 5,580,731, 5,591,584, 5,109,124, 5,118,802, 5,138,045, 5,414,077, and 5,414,078. No. 7, No. 5,486,603, No. 5,512,439, No. 5,578,718, No. 5,608,046, No. 4,587,044, No. 4,605,735, No. 4,667,025, No. 4,762,779, No. 4,789,73 No. 7, No. 4,824,941, No. 4,835,263, No. 4,876,335, No. 4,904,582, No. 4,958,013, No. 5,082,830, No. 5,112,963, No. 5,214,136, No. 5,082,830 No. 5,112,963, No. 5,214,136, No. 5,245,022, No. 5,254,469, No. 5,258,506, No. 5,262,536, No. 5,272,250, No. 5,292,873, No. 5,317,098 No. 5,371,241, No. 5,391,723, No. 5,416,203, No. 5,451,463, No. 5,510,475, No. 5,512,667, No. 5,514,785, No. 5,565,552, No. 5,567,810 Nos. 5,574,142, 5,585,481, 5,587,371, 5,595,726, 5,597,696, 5,599,923, 5,599,928, 5,688,941, 6,294,664, 6,320,017, 6,576,752, 6,783,931, 6,900,297, 7,037,646, and 8,106,022, the entire contents of each of which are incorporated herein by reference.

[0465] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the foregoing modifications can be incorporated within a single compound, or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0466] In the context of this invention, a "chimeric" iRNA compound or "chimera" refers to an iRNA compound, preferably a dsRNA agent, that contains two or more chemically distinct regions, each of which is composed of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region in which the RNA is modified to confer on the iRNA increased resistance to nuclease degradation, increased cellular uptake, or increased binding affinity for the target nucleic acid. Additional regions of the iRNA may serve as substrates for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNA when using chimeric dsRNA compared to the phosphorothioate deoxydsRNA hybridized to the same target region.Cleavage of RNA target can be routinely detected by gel electrophoresis, and if necessary, by related nucleic acid hybridization techniques known in the art.

[0467] In certain instances, the RNA of an iRNA can be modified with a non-ligand group. Several non-ligand molecules have been conjugated to iRNAs to enhance their activity, cellular distribution, or cellular uptake, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties include lipid moieties such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), fatty chains, e.g., dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative U.S. patents teaching the preparation of such RNA conjugates are listed above. A typical conjugation protocol involves the synthesis of RNA bearing an amino linker at one or more positions in the sequence. The amino group is then reacted with the molecule being conjugated by using an appropriate coupling or activating reagent. The conjugation reaction can be performed while the RNA is still attached to the solid support or after cleavage of the RNA in solution phase. Purification of the RNA conjugate by HPLC typically yields a pure conjugate.

[0468] V. Delivery of RNAi Agents of the Present Disclosure Delivery of the RNAi agent of the present disclosure to a cell, e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, e.g., a subject with an FLNA-associated disorder, e.g., Alzheimer's disease), can be achieved in several different ways. For example, delivery can be performed by contacting a cell with the RNAi agent of the present disclosure either in vitro or in vivo. In vivo delivery can be performed directly by administering a composition comprising the RNAi agent, e.g., dsRNA, to the subject. Alternatively, in vivo delivery can be performed indirectly by administering one or more vectors that encode and induce the expression of the RNAi agent. These options are further described below.

[0469] Generally, any method of delivering nucleic acid molecules (in vitro or in vivo) can be adapted to use the RNAi agent of the present disclosure (see, for example, Akhtar S. and Julian RL., (1992) Trends Cell.Biol.2(5):139-144 and International Publication No. 94 / 02595, which are incorporated herein by reference in their entirety). For in vivo delivery, factors to consider for delivering RNAi agents include, for example, the biological stability of the delivered agent, prevention of non-specific effects, and the accumulation of the delivered agent in target tissue. The non-specific effects of RNAi agents can be minimized by local administration, for example, by direct injection or implantation into tissue or local administration of preparations. Local administration to the treatment site maximizes the local concentration of the agent, limiting the exposure of the agent to systemic tissues that may otherwise be harmed by the agent or decompose the agent, and also allows for a smaller total dosage of the RNAi agent to be administered. Several studies have demonstrated successful knockdown of gene products when RNAi agents are administered locally. For example, intraocular delivery of VEGF dsRNA via intravitreal injection in cynomolgus monkeys (Tolentino, MJ. et al., (2004) Retina 24:132-138) and subretinal injection in mice (Reich, SJ. et al., (2003) Mol. Vis. 9:210-216) both prevented neovascularization in experimental models of age-related macular degeneration. In addition, direct intratumoral injection of dsRNA in mice can reduce tumor volume (Pille, J. et al. (2005) Mol. Ther. 11:267-274) and prolong the survival of tumor-bearing mice (Kim, WJ. et al., (2006) Mol. Ther. 14:343-350, Li, S. et al., (2007) Mol. Ther. 15:515-523).RNA interference has been shown to be successful when delivered locally to the CNS by direct injection (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, P. H. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al. (2002) BMC Neurosci. 3:18; Shishkina, G. T. et al. (2004) Neuroscience 129:521-528; Thakker, E. R. et al. (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y. et al. (2005) J. Neurophysiol. 93:594-602), and to the lungs by intranasal administration (Howard, K. A. et al. (e.g., Zhang, X. et al., (2006) Mol. Ther. 14: 476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279: 10677-10684; Bitko, V. et al., (2005) Nat. Med. 11: 50-55). When RNAi agents are administered systemically to treat disease, the RNA can be modified or alternatively delivered using a drug delivery system, either of which functions to prevent rapid degradation of dsRNA by endonucleases and exonucleases in vivo. Modification of RNA or pharmaceutical carriers can also enable targeting of RNAi agents to target tissues and avoid undesirable off-target effects (e.g., without wishing to be bound by theory, the use of GNAs described herein has been determined to destabilize the seed region of dsRNA, thereby increasing the preference for on-target effects over off-target effects of the dsRNA, thereby significantly reducing off-target effects due to the destabilization of the seed region). RNAi agents can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation.For example, systemic injection of ApoB-directed RNAi agents conjugated to lipophilic cholesterol moieties 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 RNAi agents to aptamers has been shown to inhibit tumor growth and mediate tumor regression in mouse models of prostate cancer (McNamara, JO. et al., (2006) Nat. Biotechnol. 24:1005-1015). In alternative embodiments, RNAi agents can be delivered using drug delivery systems such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems promote the binding of molecular RNAi agents (negatively charged) and also improve interaction with negatively charged cell membranes, thereby enabling efficient uptake of RNAi agents by cells. Cationic lipids, dendrimers, or polymers can be bound to RNAi agents, or can be induced to form vesicles or micelles that encapsulate RNAi agents (see, for example, Kim SH. et al., (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents the degradation of RNAi when administered systemically. The method of making and administering cationic RNAi agent complexes is well within the capabilities of those skilled in the art (see, for example, Sorensen, DR., et al. (2003) J. Mol. Biol 327:761-766; Verma, UN. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al. (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety).Some non-limiting examples of drug delivery systems useful for systemic delivery of RNAi agents include DOTAP (Sorensen, D.R., et al. (2003), supra; Verma, U.N. et al. (2003), supra), oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, T.S. et al. (2006) Nature 441:111-114), cardiolipin (Chien, P.Y. et al. (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al. (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M.E. et al. (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamine (Tomalia, D. A. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In some embodiments, the RNAi agent is complexed with cyclodextrin for systemic administration. Methods for administering RNAi agents and cyclodextrins and pharmaceutical compositions comprising RNAi agents and cyclodextrins can be found in U.S. Patent No. 7,427,605, which is incorporated herein by reference in its entirety.

[0470] Particular aspects of the present disclosure relate to a method for reducing the expression of FLNA target gene in cell, comprising contacting the cell with the double-stranded RNAi agent of the present disclosure.In one embodiment, the cell is extrahepatic cell, and optionally CNS cell.Another aspect of the present disclosure relates to a method for reducing the expression of FLNA target gene in subject, comprising administering the double-stranded RNAi agent of the present disclosure to subject.

[0471] Another aspect of the present disclosure relates to a method for treating a subject with a CNS disorder (neurodegenerative disorder), comprising administering to the subject a therapeutically effective amount of a double-stranded FLNA-targeting RNAi agent of the present disclosure, thereby treating the subject. Exemplary CNS disorders that can be treated by the method of the present disclosure include FLNA-associated CNS disorders, such as Alzheimer's disease.

[0472] In one embodiment, double-stranded RNAi agent is administered intrathecally.This method can reduce the expression of FLNA target gene in brain (for example, striatum) or spinal tissue, for example, cortex, cerebellum, cervical vertebrae, lumbar vertebrae and thoracic vertebrae, immune cells such as monocytes and T cells, by administering double-stranded RNAi agent intrathecally.

[0473] For ease of explanation, the formulations, compositions, and methods in this section will be primarily described with respect to modified siRNA compounds. However, it will be understood that these formulations, compositions, and methods can be implemented with other siRNA compounds, such as unmodified siRNA compounds, and such implementations are within the scope of the present disclosure. Compositions containing RNAi agents can be delivered to a subject by various routes. Exemplary routes include intrathecal, intravenous, topical, rectal, anal, vaginal, nasal, pulmonary, and intraocular.

[0474] The RNAi agent of the present disclosure can be incorporated into pharmaceutical compositions suitable for administration.Such compositions usually comprise one or more kinds of RNAi agents and pharmaceutically acceptable carriers.As used herein, the phrase " pharmaceutically acceptable carriers " is intended to include any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration.The use of such media and agents for pharmaceutically active substances is well known in the art.Except where any conventional media or agent is incompatible with active ingredient, its use in composition is intended.A supplementary active ingredient can also be incorporated into composition.

[0475] The pharmaceutical composition of the present disclosure can be administered in several ways, depending on whether local or systemic treatment is desired and the area to be treated.Administration can be topical (e.g., intraocular, vaginal, rectal, intranasal, transdermal, etc.), intrathecal, oral, or parenteral administration.Parenteral administration includes intravenous drip infusion, subcutaneous, intraperitoneal, or intramuscular injection, or intrathecal or intracerebroventricular administration.

[0476] The route and site of administration can be selected to enhance targeting.For example, to target nerve or spinal cord tissue, intrathecal injection is a logical choice.Lung cells can be targeted by administering RNAi agents in aerosol form.Vascular endothelial cells can be targeted by coating a balloon catheter with RNAi agents and mechanically introducing RNA.

[0477] Preparations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, solutions, and powders.Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may also be necessary or desirable.Coated condoms, gloves, and the like may also be useful.

[0478] Oral administration compositions include powders or granules, suspensions or solutions in water, syrups, elixirs or non-aqueous media, tablets, capsules, drops, or lozenges. For tablets, carriers that can be used include lactose, sodium citrate, and salts of phosphoric acid. In tablets, various disintegrants, such as starch, and lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc, are commonly used. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycol. When aqueous suspensions are required for oral administration, the nucleic acid composition can be combined with emulsifiers and suspending agents. If desired, certain sweeteners or flavorings can be added.

[0479] Compositions for intrathecal or intracerebroventricular administration may include sterile aqueous solutions which may also contain buffers, diluents, and other ...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of FLNA, comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand contains a complementary region to the mRNA encoding FLNA, and the complementary region is any one of the antisense nucleotide sequences shown in SEQ ID NOs: 1013, 1009, 1010, 1011, 1012 or any one of the antisense nucleotide sequences in the following table 【Table 1-1】 【Table 1-2】 【Table 1-3】 【Table 1-4】 【Table 1-5】 【Table 1-6】 【Table 1-7】 【Table 1-8】 【Table 2-1】 【Table 2-2】 【Table 2-3】 【Table 2-4】 【Table 2-5】 【Table 2-6】 【Table 2-7】 【Table 2-8】 【Table 3-1】 【Table 3-2】 【Table 3-3】 【Table 4-1】 【Table 4-2】 【Table 4-3】 and contains at least 15 consecutive nucleotides that differ from any one of the antisense nucleotide sequences in the following table by 3 nucleotides or less, a double-stranded ribonucleic acid (dsRNA) agent. **Claim 2** (a) The sense strand is nucleotides 7398 - 7418, 7391 - 7411, 7392 - 7412, 7393 - 7413, 7394 - 7414, 100 - 120, 176 - 196, 244 - 264, 375 - 395, 404 - 424, 425 - 445, 473 - 493, 500 - 520, 545 - 565, 598 - 618, 641 - 661, 665 - 685, 689 - 709, 714 - 734, 871 - 891, 906 - 926, 933 - 953, 1002 - 1022, 1077 - 1097, 1127 - 1147, 1151 - 1171, 1189 - 1209, 1235 - 1255, 1258 - 1278, 1305 - 1325, 1328 - 1348, 1355 - 1375, 1391 - 1411, 1427 - 1447, 1448 - 1468, 1488 - 1508, 1530 - 1550, 1563 - 1583, 1660 - 1680, 1749 - 1769, 1790 - 1810, 1854 - 1874, 1888 - 1908, 1926 - 1946, 1956 - 1976, 2023 - 2043, 2068 - 2088, 2103 - 2123, 2132 - 2152, 2187 - 2207, 2219 - 2239, 2258 - 2278, 2317 - 2337, 2340 - 2360, 2376 - 2396, 2399 - 2419, 2421 - 2441, 2468 - 2488, 2553 - 2573, 2615 - 2635, 2654 - 2674, 2700 - 2720, 2721 - 2741, 2742 - 2762, 2783 - 2803, 2841 - 2861, 2900 - 2920, 2930 - 2950, 2954 - 2974, 2975 - 2995, 3017 - 3037, 3038 - 3058, 3080 - 3100, 3124 - 3144, 3155 - 3175, 3189 - 3209, 3214 - 3234, 3249 - 3269, 3323 - 3343, 3375 - 3395, 3438 - 3458, 3503 - 3523, 3569 - 3589, 3601 - 3621, 3647 - 3667, 3714 - 3734, 3782 - 3802, 3826 - 3846, 3854 - 3874, 3876 - 3896, 3930 - 3950, 3999 - 4019, 4041 - 4061, 4066 - 4086, 4122 - 4142, 4145 - 4165, 4170 - 4190, 4191 - 4211, 4217 - 4237, 4336 - 4356 of SEQ ID NO: 1;４３６７－４３８７、４４４２－４４６２、４４９１－４５１１、４５１６－４５３６、４５４７－４５６７、４５６９－４５８９、４６２２－４６４２、４６５２－４６７２、４６９４－４７１４、４７４６－４７６６、４８１２－４８３２、４８６９－４８８９、４９４３－４９６３、４９７７－４９９７、５０２２－５０４２、５０６０－５０８０、５０８８－５１０８、５１８０－５２００、５２０５－５２２５、５２５５－５２７５、５２９０－５３１０、５３１４－５３３４、５３４３－５３６３、５３６４－５３８４、５４０５－５４２５、５５２１－５５４１、５５８２－５６０２、５６１２－５６３２、５６３９－５６５９、５７０３－５７２３、５７７２－５７９２、５８１１－５８３１、５８４７－５８６７、５８７６－５８９６、５９１０－５９３０、５９６７－５９８７、５９９２－６０１２、６０３８－６０５８、６１０７－６１２７、６１２８－６１４８、６１６３－６１８３、６２４３－６２６３、６２７２－６２９２、６３００－６３２０、６３５８－６３７８、６３９１－６４１１、６４４１－６４６１、６４７９－６４９９、６５０９－６５２９、６５４５－６５６５、６５８１－６６０１、６７１１－６７３１、６７４１－６７６１、６７９８－６８１８、６８２６－６８４６、６８４９－６８６９、６８７３－６８９３、６９８７－７００７、７０１４－７０３４、７０８８－７１０８、７１２５－７１４５、７１５２－７１７２、７１７３－７１９３、７２０６－７２２６、７２５３－７２７３、７２８８－７３０８、７３８６－７４０６、７４０８－７４２８、７４４５－７４６５、７４６６－７４８６、７５３７－７５５７、７５６０－７５８０、７５８６－７６０６、７６５７－７６７７、７７２８－７７４８、７８３２－７８５２、７９７８－７９９８、８００２－８０２２、８０４８－８０６８、８０８１－８１０１、８１２０－８１４０、８３５９－８３７９、８４０４－８４２４、８４４７－８４６７、８４８３－８５０３、１７１－１９１、１７３－１９３、３７５－３９５、５４２－５６２、１４４２－１４６２、１４４９－１４６９、１７５１－１７７１、１７５２－１７７２、１７５３－１７７３、１８５４－１８７４、１８５５－１８７５、１８５６－１８７６、２７２２－２７４２、２７３０－２７５０、３０８０－３１００、Any one of the nucleotide sequences of 3081-3101, 3082-3102, 3083-3103, 3084-3104, 3212-3232, 3217-3237, 3445-3465, 3446-3466, 3447-3467, 4068-4088, 5182-5202, 5183-5203, 5978-5998, 6046-6066, 6432-6452, 6585-6605, 6586-6606, 6587-6607, 7079-7099, 7161-7181, 7163-7183, 7165-7185, 7166-7186, 7376-7396, 7377-7397, 7378-7398, 7390-7410, 7435-7455, 7436-7456, 7437-7457, 7446-7466, 7654-7674, 7655-7675, 7657-7677, 7726-7746, 8404-8424, 8474-8494, 8475-8495, 8476-8496, or 8477-8497, and containing at least 15 consecutive nucleotides that differ from any one of them by 3 nucleotides or less, and wherein said antisense strand contains at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 2 (b) The antisense strand is AD-1620342.1, AD-1620335.1, AD-1620336.1, AD-1620337.1, AD-1620338.1, AD-1615378, AD-1615433, AD-1615454, AD-1615511, AD-1615540, AD-1615561, AD-1615604, AD-1615631, AD-1615676, AD-1615729, AD-1615772, AD-1615796, AD-1615820, AD-1615843, AD-1615930, AD-1615964, AD-1615991, AD-1616007, AD-1616044, AD-1616087, AD-1616111, AD-1616149, AD-1616182, AD-1616205, AD-1616222, AD-1616245, AD-1616252, AD-1616288, AD-1616313, AD-1616334, AD-1616364, AD-1616386, AD-1616399, AD-1616471, AD-1616531, AD-1616554, AD-1616579, AD-1616593, AD-1616613, AD-1616643, AD-1616682, AD-1616707, AD-1616742, AD-1616771, AD-1616821, AD-1616833, AD-1616852, AD-1616911, AD-1616934, AD-1616950, AD-1616972, AD-1616994, AD-1617041, AD-1617061, AD-1617103, AD-1617117, AD-1617127, AD-1617148, AD-1617169, AD-1617186, AD-1617219, AD-1617268, AD-1617298, AD-1617322, AD-1617343, AD-1617366, AD-1617387, AD-1617429, AD-1617455, AD-1617486, AD-1617520, AD-1617545, AD-1617580, AD-1617612, AD-1617644, AD-1617657, AD-1617679, AD-1617703, AD-1617717, AD-1617743, AD-1617790, AD-1617815, AD-1617843,AD-1617853, AD-1617875, AD-1617899, AD-1617939, AD-1617981, AD-1618006, AD-1618049, AD-1618052, AD-1618077, AD-1618098, AD-1618124, AD-1618187, AD-1618214, AD-1618237, AD-1618286, AD-1618311, AD-1618342, AD-1618364, AD-1618404, AD-1618434, AD-1618456, AD-1618508, AD-1618572, AD-1618601, AD-1618645, AD-1618661, AD-1618706, AD-1618744, AD-1618772, AD-1618810, AD-1618835, AD-1618851, AD-1618886, AD-1618910, AD-1618939, AD-1618960, AD-1618979, AD-1619014, AD-1619034, AD-1619064, AD-1619071, AD-1619116, AD-1619178, AD-1619197, AD-1619233, AD-1619262, AD-1619296, AD-1619333, AD-1619358, AD-1619385, AD-1619434, AD-1619455, AD-1619470, AD-1619529, AD-1619540, AD-1619549, AD-1619586, AD-1619601, AD-1619651, AD-1619689, AD-1619699, AD-1619735, AD-1619751, AD-1619849, AD-1619879, AD-1619936, AD-1619946, AD-1619969, AD-1619993, AD-1620033, AD-1620060, AD-1620113, AD-1620150, AD-1620177, AD-1620198, AD-1620211, AD-1620244, AD-1620279, AD-1620330, AD-1620352, AD-1620389, AD-1620410, AD-1620426, AD-1620449, AD-1620475, AD-1620525, AD-1620574, AD-1620616, AD-1620707, AD-1620731, AD-1620737Any one of the antisense strand nucleotide sequences of the double-stranded selected from the group consisting of AD-1620767, AD-1620787, AD-1620837, AD-1620879, AD-1620891, and AD-1620927, AD-1615428.1, AD-1615430.1, AD-1615511.2, AD-1615673.1, AD-1616328.1, AD-1616335.1, AD-1616533.1, AD-1616534.1, AD-1616535.1, AD-1616579.2, AD-1616580.1, AD-1616581.1, AD-1617149.1, AD-1617157.1, AD-1617429.2, AD-1617430.1, AD-1617431.1, AD-1617432.1, AD-1617433.1, AD-1617543.1, AD-1617548.1, AD-1617664.1, AD-1617665.1, AD-1617666.1, AD-1618008.1, AD-1618812.1, AD-1618813.1, AD-1619344.1, AD-1619393.1, AD-1619642.1, AD-1619755.1, AD-1619756.1, AD-1619757.1, AD-1620104.1, AD-1620186.1, AD-1620188.1, AD-1620190.1, AD-1620191.1, AD-1620320.1, AD-1620321.1, AD-1620322.1, AD-1620334.1, AD-1620379.1, AD-1620380.1, AD-1620381.1, AD-1620390.1, AD-1620522.1, AD-1620523.1, AD-1620525.2, AD-1620572.1, AD-1620879.2, AD-1620918.1, AD-1620919.1, AD-1620920.1, and AD-1620921.1, and containing at least 15 consecutive nucleotides that differ by 3 nucleotides or less. (c) The nucleotide sequences of the sense strand and the antisense strand contain any one of the sense strand and antisense strand nucleotide sequences in the following table 【Table 5-1】 【Table 5-2】 【Table 5-3】 【Table 5-4】 【Table 5-5】 【Table 5-6】 【Table 5-7】 【Table 5-8】 【Table 6-1】 【Table 6-2】 【Table 6-3】 【Table 6-4】 【Table 6-5】 【Table 6-6】 【Table 6-7】 【Table 6-8】 【Table 7-1】 【Table 7-2】 【Table 7-3】 【Table 8-1】 【Table 8-2】 【Table 8-3】 (d) The base pair at one position at the 5'-end of the antisense strand of the double strand is an AU base pair, (e) The sense strand has a total of 21 nucleotides, and the antisense strand has a total of 23 nucleotides, (f) The dsRNA agent further contains at least one phosphorothioate nucleotide internucleotide linkage, (g) The dsRNA agent further contains at least one phosphorothioate nucleotide internucleotide linkage, and the dsRNA agent further contains 6 to 8 phosphorothioate nucleotide internucleotide linkages, (h) Each strand of the sense strand and the antisense strand has a length of 30 nucleotides or less, (i) At least one strand of the sense strand and the antisense strand contains at least 1 nucleotide 3'-overhang or at least 2 nucleotides 3'-overhang, (j) The double-stranded region has a length of 15 to 30, 17 to 23, 17 to 25, 23 to 27, 19 to 21, or 21 to 23 nucleotide pairs, and / or (k) Each strand of the sense strand and the antisense strand has 19 to 30 nucleotides, 19 to 23 nucleotides, or 21 to 23 nucleotides, The dsRNA agent according to claim 1.

3. The dsRNA agent according to claim 1, wherein the sense strand, the antisense strand, or both the sense strand and the antisense strand are conjugated to one or more lipophilic moieties.

4. (a) The one or more lipophilic moieties are conjugated to one or more inner positions in the double-stranded region of the dsRNA agent, (b) the one or more lipophilic moieties are conjugated via a linker or a carrier; (c) the lipophilicity of the one or more lipophilic moieties measured by logKow is greater than 0; (d) the hydrophobicity of the double-stranded RNA agent, as measured by the unbound fraction in the plasma protein binding assay of the double-stranded RNA agent, is greater than 0.2; (e) the hydrophobicity of the double-stranded RNA agent, as measured by the unbound fraction in the plasma protein binding assay of the double-stranded RNA agent, is greater than 0.2, and the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein; (f) the one or more lipophilic moieties are conjugated at one or more inner positions in at least one strand, and / or (g) the one or more lipophilic moieties are conjugated via a linker or a carrier at one or more inner positions in at least one strand; The dsRNA agent according to claim 3.

5. The dsRNA agent according to claim 3, wherein the dsRNA agent comprises at least one modified nucleotide.

6. (a) 5 or less of the nucleotides of the sense strand and 5 or less of the nucleotides of the antisense strand are unmodified nucleotides; (b) all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides; (c) the at least one modified nucleotide is selected from the group consisting of deoxy-nucleotide, 3'-terminal deoxy-thymine (dT) nucleotide, 2'-O-methyl modified nucleotide, 2'-fluoro modified nucleotide, 2'-deoxy-modified nucleotide, locked nucleotide, unlocked nucleotide, conformationally restricted nucleotide, constrained ethyl nucleotide, abasic nucleotide, 2'-amino-modified nucleotide, 2'-O-allyl-modified nucleotide, 2'-C-alkyl-modified nucleotide, 2'-hydroxyl modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-O-alkyl-modified nucleotide, morpholino nucleotide, phosphoramidate, nucleotide containing unnatural base, tetrahydropyran modified nucleotide, 1,5-anhydrohexitol modified nucleotide, cyclohexenyl modified nucleotide, nucleotide containing 5'-phosphorothioate group, nucleotide containing 5'-methylphosphonate group, nucleotide containing 5' phosphate or 5' phosphate mimetic, nucleotide containing vinylphosphonate, nucleotide containing glycol nucleic acid (GNA), nucleotide containing glycol nucleic acid S isomer (S-GNA), nucleotide containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotide containing 2'-deoxythymidine-3' phosphate, nucleotide containing 2'-deoxyguanosine-3'-phosphate, 2'-5'-linked ribonucleotide (3'-RNA) and terminal nucleotide linked to a cholesteryl derivative, and dodecanoic acid bisdecylamide group, and combinations thereof, (d) the at least one modified nucleotide is selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotide, 2'-deoxy-modified nucleotide, 3'-terminal deoxy-thymine nucleotide (dT), locked nucleotide, abasic nucleotide, 2'-amino-modified nucleotide, 2'-alkyl modified nucleotide, morpholino nucleotide, phosphoramidate, and nucleotide containing unnatural base, (e) the at least one modified nucleotide contains a short sequence of 3'-terminal deoxy-thymine nucleotide (dT), and / or (f) the at least one modified nucleotide comprises a modification selected from the group consisting of 2'-O-methyl, GNA, and 2'-fluoro modification; The dsRNA agent according to claim 5.

7. (a) the inner positions include all positions of the at least one strand except for the two terminal positions from each end of the at least one strand; (b) the inner positions include all positions of the at least one strand except for the three terminal positions from each end of the at least one strand; (c) the inner positions exclude the cleavage site region of the sense strand; (d) the inner positions include all positions of the sense strand except for positions 9 to 12 counted from the 5' end of the sense strand; (e) the inner positions include all positions of the sense strand except for positions 11 to 13 counted from the 3' end of the sense strand; (f) the inner positions exclude the cleavage site region of the antisense strand; (g) the inner positions include all positions of the antisense strand except for positions 12 to 14 counted from the 5' end of the antisense strand; (h) the inner positions include all positions of the sense strand except for positions 11 to 13 counted from the 3' end and positions 12 to 14 counted from the 5' end of the antisense strand; (i) the one or more lipophilic moieties are conjugated to one or more of the inner positions selected from the group consisting of positions 4 to 8 and 13 to 18 on the sense strand and positions 6 to 10 and 15 to 18 on the antisense strand counted from the 5' end of each strand; (j) the one or more lipophilic moieties are conjugated to one or more of the inner positions selected from the group consisting of positions 5, 6, 7, 15, and 17 on the sense strand and positions 15 and 17 on the antisense strand counted from the 5' end of each strand; (k) the inner positions in the double-stranded region exclude the cleavage site region of the sense strand; (l) the sense strand is 21 nucleotides in length, the antisense strand is 23 nucleotides in length, and the one or more lipophilic moieties are conjugated to position 21, 20, 15, 1, 7, 6, or 2 of the sense strand, or position 16 of the antisense strand; (m) the one or more lipophilic moieties are conjugated to position 21, 20, 15, 1, or 7 of the sense strand; (n) the one or more lipophilic moieties are conjugated to position 21, 20, or 15 of the sense strand; (o) The one or more lipophilic moieties are conjugated to the 20th or 15th position of the sense strand, and / or (p) The one or more lipophilic moieties are conjugated to the 16th position of the antisense strand, The dsRNA agent according to claim 4.

8. (a) The one or more lipophilic moieties are an aliphatic compound, an alicyclic compound, or a polyalicyclic compound, (b) The one or more lipophilic moieties are 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) chenodeoxycholic acid, dimethoxytrityl, or phenoxazine, (c) The one or more lipophilic moieties contain a saturated or unsaturated C4-C30 hydrocarbon chain and any functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne, (d) The one or more lipophilic moieties contain a saturated or unsaturated C6-C18 hydrocarbon chain, (e) The one or more lipophilic moieties contain a saturated or unsaturated C16 hydrocarbon chain, (f) The one or more lipophilic moieties contain a saturated or unsaturated C16 hydrocarbon chain, and the saturated or unsaturated C16 hydrocarbon chain is conjugated to the 6th position counted from the 5'-end of the chain, (g) The one or more lipophilic moieties are conjugated via a carrier that replaces one or more nucleotides in the inner position or the double-stranded region, (h) the one or more lipophilic moieties are conjugated via a carrier that replaces one or more nucleotides in the inner position or the double-stranded region, and the 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 an acyclic moiety of a serinol backbone or a diethanolamine backbone system, (i) the one or more lipophilic moieties are conjugated to the double-stranded iRNA agent via a linker containing ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction, or a carbamate, (j) the one or more lipophilic moieties are conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage, (k) the one or more lipophilic moieties are conjugated via a bio-cleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, and functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof, and / or (l) the 3' end of the sense strand is protected via an end cap that is a cyclic group having an amine, and the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl, The dsRNA agent according to claim 3.

9. (a) A targeting ligand that targets nerve cells, (b) The targeting ligand is a GalNAc conjugate, a targeting ligand, (c) A terminal chiral modification occurring in the first internucleotide linkage at the 3' end of the antisense strand having a linking phosphorus atom in the Sp configuration, The chiral modification of the terminus occurring in the first internucleotide linkage at the 5'-end of the antisense strand having a linking phosphorus atom in the Rp configuration, and The chiral modification of the terminus occurring in the first internucleotide linkage at the 5'-end of the sense strand having a linking phosphorus atom in either the Rp or Sp configuration, (d) The chiral modification of the terminus occurring in the first and second internucleotide linkages at the 3'-end of the antisense strand having a linking phosphorus atom in the Sp configuration, The chiral modification of the terminus occurring in the first internucleotide linkage at the 5'-end of the antisense strand having a linking phosphorus atom in the Rp configuration, and The chiral modification of the terminus occurring in the first internucleotide linkage at the 5'-end of the sense strand having a linking phosphorus atom in either the Rp or Sp configuration; (e) The chiral modification of the terminus occurring in the first, second, and third internucleotide linkages at the 3'-end of the antisense strand having a linking phosphorus atom in the Sp configuration, The chiral modification of the terminus occurring in the first internucleotide linkage at the 5'-end of the antisense strand having a linking phosphorus atom in the Rp configuration, and The chiral modification of the terminus occurring in the first internucleotide linkage at the 5'-end of the sense strand having a linking phosphorus atom in either the Rp or Sp configuration; (f) The chiral modification of the terminus occurring in the first and second internucleotide linkages at the 3'-end of the antisense strand having a linking phosphorus atom in the Sp configuration, The chiral modification of the terminus occurring in the third internucleotide linkage at the 3'-end of the antisense strand having a linking phosphorus atom in the Rp configuration, The chiral modification of the terminus occurring in the first internucleotide linkage at the 5'-end of the antisense strand having a linking phosphorus atom in the Rp configuration, and The chiral modification of the terminus occurring in the first internucleotide linkage at the 5'-end of the sense strand having a linking phosphorus atom in either the Rp or Sp configuration; (g) The chiral modification of the terminus occurring in the first and second internucleotide linkages at the 3'-end of the antisense strand having a linking phosphorus atom in the Sp configuration, The chiral modification of the terminus occurring at the internucleotide linkage between the first and second nucleotides at the 5'-end of the antisense strand, having a linking phosphorus atom in Rp configuration, and the chiral modification of the terminus occurring at the first internucleotide linkage at the 5'-end of the sense strand, having a linking phosphorus atom in either Rp or Sp configuration; (h) a phosphate or phosphate mimetic at the 5'-end of the antisense strand, and / or (i) a phosphate or phosphate mimetic at the 5'-end of the antisense strand, wherein the phosphate mimetic is 5'-vinylphosphonate (VP), the phosphate or phosphate mimetic The dsRNA agent according to claim 3, further comprising.

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

11. (a) The hotspot region comprises nucleotides 7389 - 7418, 1437 - 1469, 1750 - 1773, 3078 - 3104, 3210 - 3237, 2720 - 2750, 3081 - 3104, 3444 - 3467, 6583 - 6607, 7159 - 7186, 7374 - 7418, 1440 - 1469, 1852 - 1876, 3078 - 3101, 7374 - 7398, 7433 - 7466, 8472 - 8497, 7390 - 7418, 8472 - 8496, 7163 - 7186, 3852 - 3896, 2698 - 2762, 7443 - 7486, 402 - 445, 2952 - 2995, 4168 - 4211, 6105 - 6148, 7150 - 7193, 1425 - 1468, 3015 - 3058, 2698 - 2741, 5341 - 5384, or 7384 - 7428 of SEQ ID NO: 1, and / or (b) the dsRNA agent is selected from the group consisting of AD-1620342.1, AD-1620335.1, AD-1620336.1, AD-1620337.1, AD-1620338.1, AD-1616328.1, AD-1616335.1, AD-1616534.1, AD-1616535.1, AD-1617429.2, AD-1617430.1, AD-1617431.1, AD-1617433.1, AD-1617543.1, AD-1617548.1, AD-1617149.1, AD-1617157.1, AD-1617432.1, AD-1617665.1, AD-1617666.1, AD-1619755.1, AD-1619756.1, AD-1619757.1, AD-1620186.1, AD-1620188.1, AD-1620190.1, AD-1620320.1, AD-1620321.1, AD-1620334.1, AD-1616579.2, AD-1616580.1, AD-1616581.1, AD-1620322.1, AD-1620379.1, AD-1620380.1, AD-1620381.1, AD-1620390.1, AD-1620918.1, AD-1620919.1, AD-1620920.1, AD-1620921.1, AD-1620191.1, AD-1617853, AD-1617875, AD-1617127, AD-1617148, AD-1617169, AD-1620389, AD-1620410, AD-1615540, AD-1615561, AD-1617322, AD-1617343, AD-1618077, AD-1618098, AD-1619434, AD-1619455, AD-1620177, AD-1620198, AD-1616313, AD-1616334, AD-1617366, AD-1617387, AD-1618939, AD-1618960, AD-1620330, and AD-1620352; The dsRNA agent according to claim 10.

12. (a) the sense strand contains the nucleotide sequence shown in SEQ ID NO: 953, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 1013; (b) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 949, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1009; (c) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 950, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1010; (d) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 951, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1011; or (e) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 952, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1012, The dsRNA agent according to claim 1.

13. (a) the sense strand comprises all of the nucleotide sequence set forth in SEQ ID NO: 1073 and modifications, and the antisense strand comprises all of the nucleotide sequence set forth in SEQ ID NO: 1133 and modifications; (b) the sense strand comprises all of the nucleotide sequence set forth in SEQ ID NO: 1069 and modifications, and the antisense strand comprises all of the nucleotide sequence set forth in SEQ ID NO: 1129 and modifications; (c) the sense strand comprises all of the nucleotide sequence set forth in SEQ ID NO: 1070 and modifications, and the antisense strand comprises all of the nucleotide sequence set forth in SEQ ID NO: 1130 and modifications; (d) the sense strand comprises all of the nucleotide sequence set forth in SEQ ID NO: 1071 and modifications, and the antisense strand comprises all of the nucleotide sequence set forth in SEQ ID NO: 1131 and modifications; or (e) the sense strand comprises all of the nucleotide sequence set forth in SEQ ID NO: 1072 and modifications, and the antisense strand comprises all of the nucleotide sequence set forth in SEQ ID NO: 1132 and modifications, The dsRNA agent according to claim 1.

14. A cell containing the dsRNA agent according to any one of claims 1 to 13.

15. (a) the dsRNA agent according to any one of claims 1 to 13, or (b) the dsRNA agent according to any one of claims 1 to 13 and a lipid formulation A pharmaceutical composition for inhibiting the expression of a gene encoding FLNA, comprising.

16. (a) the dsRNA agent is in a non-buffered solution, (b) The dsRNA agent is in a non-buffered solution, and the non-buffered solution is physiological saline or water. (c) The dsRNA agent is in a buffered solution. (d) The dsRNA agent is in a buffered solution, and the buffered solution contains acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof, or (e) The dsRNA agent is in a buffered solution, and the buffered solution is phosphate-buffered saline (PBS). The pharmaceutical composition according to claim 15.

17. A pharmaceutical composition comprising the dsRNA agent according to any one of claims 1 to 13 for use in inhibiting the expression of the FLNA gene in a cell, wherein the inhibiting comprises introducing the dsRNA agent into the cell, thereby inhibiting the expression of the FLNA gene in the cell.

18. (a) The cell is in a subject. (b) The cell is in a human subject. (c) The cell is in a subject having an FLNA-related disorder. (d) The cell is in a subject having an FLNA-related disorder, and the FLNA-related disorder is a neurodegenerative disorder. (e) The cell is in a subject having an FLNA-related disorder, and the FLNA-related disorder is Alzheimer's disease. (f) By contacting the cell with the dsRNA agent, at least 30% of the expression of FLNA is inhibited, and / or (g) By inhibiting the expression of FLNA, the FLNA protein level in the serum of the subject is reduced by at least 30%. The pharmaceutical composition according to claim 17.

19. (a) Administering a therapeutically effective amount of the pharmaceutical composition to a subject having a disorder that would benefit from a decrease in FLNA expression, thereby treating the subject, or (b) Administering a prophylactically effective amount of the pharmaceutical composition to a subject having a disorder that would benefit from a decrease in FLNA expression, thereby preventing at least one symptom in the subject, A pharmaceutical composition comprising the dsRNA agent according to any one of claims 1 to 13 for use in.

20. (a) The disorder is an FLNA-related disorder. (b) the disorder is an FLNA-related disorder, and the FLNA-related disorder is Alzheimer's disease, tauopathy, frontotemporal dementia (FTD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP). selected from the group consisting of corticobasal degeneration (CBD), chronic traumatic encephalopathy (CTE), Pick's disease (PiD), globular glial tauopathy (GGT), argyrophilic grain disease (AGD), and primary age-related tauopathy (PART), (c) the disorder is an FLNA-related disorder and the disorder is Alzheimer's disease, (d) the subject is human, (e) administration of the agent to the subject causes a decrease in the accumulation of FLNA protein, (f) the dsRNA agent is administered to the subject at a dose of about 0.01 mg / kg to about 50 mg / kg, (g) the dsRNA agent is administered intrathecally to the subject, (h) determining the level of FLNA in a sample from the subject, (i) determining the level of FLNA in a sample from the subject, wherein the level of FLNA in the sample of the subject is the level of FLNA protein in a blood, serum, or cerebrospinal fluid sample, and / or (j) administering an additional therapeutic agent to the subject, The pharmaceutical composition according to claim 19.

21. A kit, vial, syringe, or intrathecal pump comprising the dsRNA agent according to any one of claims 1 to 13.

22. An in vitro method of inhibiting the expression of the FLNA gene in a cell, the method comprising introducing into the cell the dsRNA agent according to any one of claims 1 to 13, thereby inhibiting the expression of the FLNA gene in the cell. An in vitro method.

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