Actin-binding LIM protein 3 (ABLIM3) iRNA agent composition and method of using the same

Abstract

The present disclosure relates to double-stranded ribonucleic acid (dsRNAi) agents and compositions that target the actin-binding LIM protein 3 (ABLIM3) gene, methods of inhibiting the expression of the ABLIM3 gene, and methods of treating a subject having an ABLIM3-related disease or disorder, such as PTSD or age-related memory loss, using such dsRNAi agents and compositions.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 338,645, filed May 5, 2022, and U.S. Provisional Patent Application No. 63 / 352,573, filed June 15, 2022, the entire contents of which are incorporated herein by reference.

[0002] Array List This application contains a Sequence Listing that has been submitted electronically in XML format, and is hereby incorporated by reference in its entirety. The XML copy created on May 2, 2023, is named A108868_1570WO_SL.xml and is 4,029,028 bytes in size. [Background technology]

[0003] The actin-binding LIM protein 3 (ABLIM3) gene, which encodes the actin-binding LIM protein 3 protein, is located in the chromosomal region 5q32 on chromosome 5. ABLIM3 is part of the actin-binding LIM family of proteins, which are characterized by an N-terminal LIM domain and a C-terminal dematin-like domain.

[0004] ABLIM3 is expressed in the brain and in other tissues, such as adipose tissue, heart, placenta, and liver. ABLIM3 can bind to the actin cytoskeleton, localize to adherens junctions, stabilize branched F-actin, and also function as a protein-binding boundary through its LIM domain. ABLIM3 has recently been linked to memory as a molecular brake on the connectivity between dentate gyrus granule cells and stratum lucidum interneurons. During learning, ABLIM3 levels decrease, improving the accuracy of remote memory and reducing the generalization of remote fear memory. Therefore, ABLIM3 may play a role in post-traumatic stress disorder (PTSD) and age-related memory loss.

[0005] Current treatments for PTSD and age-related memory loss are limited and ineffective. Thus, there is a need for agents that can treat, prevent, and / or inhibit the progression or formation of PTSD and age-related memory loss. Summary of the Invention

[0006] The present disclosure provides RNAi compositions, which carry out RNA-induced silencing complex (RISC) mediated cleavage of the RNA transcript of ABLIM3 gene.ABLIM3 gene can be in cells, for example, in the cells of subjects, for example, humans.The use of these iRNAs allows the targeted degradation of the mRNA of corresponding gene (ABLIM3 gene) in mammals.

[0007] The iRNAs of the present invention are designed to target the ABLIM3 gene, for example, the wild-type ABLIM3 gene and / or the mutant ABLIM3 gene. The iRNAs of the present invention inhibit ABLIM3 gene expression by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. Without intending to be limited by theory, it is believed that the combination or partial combination of the above-mentioned properties and specific target sites or specific modifications in these iRNAs confers improved efficacy, stability, potency, durability, and safety to the iRNAs of the present invention. In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of ABLIM3, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the sense strand comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2.

[0008] In another aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of ABLIM3, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a region of complementarity to an mRNA encoding ABLIM3, the region of complementarity comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2.

[0009] In yet another aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of ABLIM3, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises a region of complementarity to an mRNA encoding ABLIM3, wherein the region of complementarity comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense nucleotide sequences in Table 3 or 4.

[0010] In one embodiment, the sense strand comprises nucleotides 82 to 102, 134 to 154, 232 to 252, 276 to 296, 293 to 313, 326 to 346, 351 to 371, 366 to 386, 381 to 401, 414 to 434, 482 to 502, 500 to 520, 542 to 562, 558 to 578, 582 to 602, 622 to 642, 674 to 694, 692 to 712, 713 to 733, 728 to 748, 743 to 763, 779 to 799, 887 to 907, 942 to 962, 983 to 1003, 1001 to 1021, 1082 to 1102, 1103 to 1114, 1115 to 1116, 1117 to 1118, 1119 to 1210, 1211 to 1212, 1213 to 1214, 1215 to 1216, 1217 to 1218, 1219 to 1220, 1220 to 1230, 1221 to 1232, 1222 to 1234, 1222 to 1236, 1222 to 1238 ... 102~1122, 1117~1137, 1177~1197, 1192~1212, 1242~1262, 1269~1289, 1313~1333, 1344~1364, 1359~1379, 1374~1394, 1408~1428, 1437~1457, 1550~1560 570, 1594~1614, 1624~1644, 1645~1665, 1674~1694, 1715~1735, 1746~1766, 1767~1787, 1812~1832, 1859~1879, 1884~1904, 1912~1932, 1935~1955, 1 971~1991, 2039~2059, 2096~2116, 2127~2147, 2169~2189, 2184~2204, 2218~2238, 2245~2265, 2273~2293, 2325~2345, 2343~2363, 2385~2405, 2422~ 2442, 2459~2479, 2483~2503, 2520~2540, 2535~2555, 2561~2581, 2585~2605, 2667~2687, 2703~2723, 2729~2749, 2764~2784, 2788~2808, 2830~2850, 2861~2881, 2885~2905, 2937~2957, 2958~2978, 2974~2994, 3011~3031, 3044~3064, 3061~3081, 3079~3099, 3110~3130, 3146~3166, 3203~3223, 3220~ 3240, 3279~3299, 3303~3323, 3321~3341, 3338~3358, 3388~3408, 3407~3427, 3422~3442, 3437~3457, 3452~3472, 3532~3552, 3548~3568, 3579~3599,3594~3614, 3626~3646, 3644~3664, 3682~3702, 3706~3726, 3721~3741, 3746~3766, 3763~3783, 3796~3816, 3814~3834, 3849~3869, 3866~3886, 3897~3917, 3919~3939, 3986~4006, 4018~4038, 4037~4057, 4058~4078, 4084~4104, 4099~4119, 4131~4151, 4159~4179, The antisense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of 4225-4245, 4241-4261, 4276-4296, 4291-4311, 4306-4326, 4336-4356, 4354-4374, 4369-4389, 4387-4407, 4404-4424, or 4421-4441, and the antisense strand comprises at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 2.

[0011] In one embodiment, the antisense strand is AD-1807455, AD-1807481, AD-1807539, AD-1807561, AD-1807578, AD-1807591, AD-1807616, AD-1807631, AD-1807646, AD-1807659, AD-1807707, AD-1807725, AD-1807764, AD-1807780, AD-1807804, AD-1807844, AD-1807870, AD-1807888, AD-1807889, AD-1807904, AD-1807919, AD-1807955, AD-1808063, AD-1808098, AD-1808139, AD-1808157, AD-1808238, AD-1808258, AD-1808273, AD-1808312, AD-1808327, AD-1808356, AD-1808383, AD-1808407, AD-1808418, AD-1808433, AD-1808448, AD-1808482, AD-1808511, AD-1808561, AD-1808598, AD-1808628, AD-1808649, AD-1808678, AD-1808698, AD-1808727, AD-1808748, AD-1808773, AD-1808789, AD-1808814, AD-1808842, AD-1808865, AD-1808901, AD-1808941, AD-1808978, AD-1809009, AD-1809051, AD-1809066, AD-1809080, AD-1809107, AD-1809135, AD-1809187, AD-1809205, AD-1809246, AD-1809283, AD-1809293, AD-1809317, AD-1809354, AD-1809369, AD-1809395, AD-1809414, AD-1809456, AD-1809472, AD-1809498, AD-1809511, AD-1809535, AD-1809550, AD-1809561, AD-1809585, AD-1809596, AD-1809617, AD-1809633, AD-1809670, AD-1809702, AD-1809719, AD-1809737, AD-1809757, AD-1809773, AD-1809822,AD-1809839, AD-1809895, AD-1809919, AD-1809937, AD-1809954, AD-1810004, AD-1810 023, AD-1810038, AD-1810053, AD-1810068, AD-1810127, AD-1810143, AD-1810174, AD- 1810189, AD-1810221, AD-1810239, AD-1810255, AD-1810277, AD-1810292, AD-1810296 , AD-1810313, AD-1810345, AD-1810363, AD-1810398, AD-1810415, AD-1810446, AD-181 0468, AD-1810494, AD-1810526, AD-1810545, AD-1810566, AD-1810591, AD-1810606, AD-1810638, AD-1810666, AD-1810676, AD-1810692, AD-1810727, AD-1810742, AD-1810757, AD-1810767, AD-1810785, AD-1810800, AD-1810818, AD-1810822, and AD-1810839.

[0012] In some embodiments, the nucleotide sequences of the sense and antisense strands comprise any one of the sense strand nucleotide sequences in Tables 3 or 4.

[0013] In one embodiment, the sense strand, the antisense strand, or both the sense and antisense strands are conjugated to one or more lipophilic moieties.

[0014] In one embodiment, the lipophilic moiety is conjugated to one or more internal positions in the double-stranded region of the dsRNA agent.

[0015] In one embodiment, the lipophilic moiety is conjugated via a linker or carrier.

[0016] In one embodiment, the lipophilicity of the lipophilic moiety, as measured by log Kow, is greater than zero.

[0017] 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.

[0018] In one embodiment, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

[0019] In some embodiments, the dsRNA agent includes at least one modified nucleotide.

[0020] In one embodiment, no more than 5 nucleotides of the sense strand and no more than 5 nucleotides of the antisense strand are unmodified nucleotides.

[0021] In one embodiment, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand are modified nucleotides.

[0022] In one embodiment, at least one of the modified nucleotides is a deoxynucleotide, a 3'-terminal deoxythimidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a non-locked 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 (hydroxly) 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 ... modified nucleotides, nucleotides containing a 5'-phosphorothioate group, nucleotides containing a 5'-methylphosphonate group, nucleotides containing a 5' phosphate or a 5' phosphate mimic, nucleotides containing vinyl phosphonate, glycol nucleic acids (GNAs) (e.g., nucleotides containing adenosine-glycol nucleic acids (GNAs)), S-glycol nucleic acids (S-GNAs) (e.g., nucleotides containing thymidine-glycol nucleic acids (GNAs) S-isomers), nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3' phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, nucleotides containing 2' phosphate ("3'-RNA", e.g., G2p, C2p, A2p, U2p), and terminal nucleotides linked to cholesteryl derivatives and dodecanoic acid bisdecylamide groups; and combinations thereof.

[0023] 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 deoxythymidine nucleotides (dT), locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and nucleotides containing unnatural bases.

[0024] In one embodiment, the modified nucleotides include a short sequence of 3' terminal deoxythymidine nucleotides (dT).

[0025] In one embodiment, the modifications to the nucleotides are 2'-O-methyl, GNA, and 2'-fluoro modifications.

[0026] In some embodiments, the dsRNA agent further comprises at least one phosphorothioate internucleotide linkage.

[0027] In one embodiment, the dsRNA agent includes 6 to 8 phosphorothioate internucleotide linkages.

[0028] In one embodiment, each strand is 30 nucleotides or less in length.

[0029] 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.

[0030] The double-stranded region may 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. Each strand may have 19 to 30 nucleotides; 19 to 23 nucleotides; or 21 to 23 nucleotides.

[0031] In one embodiment, one or more lipophilic moieties are conjugated to one or more internal positions on at least one chain, eg, via a linker or carrier.

[0032] In one embodiment, internal positions include all but the two most distal positions from each end of at least one strand.

[0033] In another embodiment, the internal positions include all but the last three positions from each end of at least one strand.

[0034] In one embodiment, the internal position excludes the cleavage site region of the sense strand.

[0035] In one embodiment, internal positions include all positions except positions 9-12 counting from the 5' end of the sense strand.

[0036] In another embodiment, internal positions include all positions except positions 11-13 counting from the 3' end of the sense strand.

[0037] In one embodiment, the internal position excludes the cleavage site region of the antisense strand.

[0038] In one embodiment, internal positions include all positions except positions 12-14 counting from the 5' end of the antisense strand.

[0039] In one embodiment, internal positions include all positions except positions 11-13 counting from the 3' end of the sense strand and positions 12-14 counting from the 5' end of the antisense strand.

[0040] In one embodiment, the one or more lipophilic moieties are conjugated to one or more internal positions selected from the group consisting of positions 4-8 and 13-18 in the sense strand and positions 6-10 and 15-18 in the antisense strand, counting from the 5' end of each strand.

[0041] 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 in the sense strand and positions 15 and 17 in the antisense strand, counting from the 5' end of each strand.

[0042] In one embodiment, the internal position in the double-stranded region excludes the cleavage site region of the sense strand.

[0043] 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, position 20, position 15, position 1, position 7, position 6, or position 2 of the sense strand or position 16 of the antisense strand.

[0044] In one embodiment, the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, or position 7 of the sense strand.

[0045] In another embodiment, the lipophilic moiety is conjugated to position 21, 20, or 15 of the sense strand.

[0046] In yet another embodiment, the lipophilic moiety is conjugated to position 20 or 15 of the sense strand.

[0047] In one embodiment, the lipophilic moiety is conjugated to position 16 of the antisense strand.

[0048] In one embodiment, the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

[0049] In one embodiment, 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, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, a heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine.

[0050] 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.

[0051] In one embodiment, the lipophilic moiety contains a saturated or unsaturated C6 to C18 hydrocarbon chain.

[0052] In one embodiment, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.

[0053] In one embodiment, the ligand is conjugated to the 2' position of a nucleotide or modified nucleotide in the sense or antisense strand. For example, a C16 ligand may be conjugated as shown in the following structure:

[0054] [ka] (In the formula, * represents a bond to an adjacent nucleotide, and B is a nucleobase or nucleobase analog, where B may be adenine, guanine, cytosine, thymine, or uracil).

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

[0056] In one embodiment, the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotides at 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 an acyclic moiety based on a serinol or diethanolamine backbone.

[0058] In one embodiment, the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, the 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, disulfides, amides; 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 at the first internucleotide linkage at the 3'-end of the antisense strand, with the linking phosphorus atom in the Sp configuration, a terminal chiral modification that occurs at the first internucleotide linkage at the 5'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration, and a terminal chiral modification that occurs at the first internucleotide linkage at the 5'-end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration.

[0064] In 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, with the linking phosphorus atom in the Sp configuration, a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration, and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration.

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

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

[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 5'-vinylphosphonate (VP).

[0070] In one embodiment, the base pair at one position at 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 embodiment, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of actin-binding LIM protein 3 (ABLIM3) in a cell, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region. The sense strand comprises the nucleotide sequence of any one of the agents in Table 3 or 4, and the antisense strand comprises the nucleotide sequence of any one of the agents in Table 3 or 4. 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 dsRNA agent, the dsRNA agent targets a hotspot region of the mRNA encoding ABLIM3. In one embodiment, the hotspot region includes nucleotides 3042-3081, 3420-3457, 4223-4261, 726-763, 1080-1137, 1342-1379, 1910-1955, 2167-2204, 480-520, 1080-1122, 1622-1665, 1744-1787, 2323-2363, 540-578, 1175-1212, 274-313, 349-401, or 2518-2555 of SEQ ID NO:1. dsRNA agents are AD-1809702, AD-1809719, AD-1810038, AD-1810053, AD-1810676, AD-1810692, AD-1807904, AD-1807919, AD-180823 8, AD-1808258, AD-1808273, AD-1808418, AD-1808433, AD-1808842, AD-1808865, AD-1809051, AD-1809066, AD-1807707, AD-18 07725, AD-1808628, AD-1808649, AD-1808727, AD-1808748, AD-1809187, AD-1809205, AD-1807764, AD-1807780, AD-1808312, AD-1808327, AD-1807561, AD-1807578, AD-1807616, AD-1807631, AD-1807646, AD-1809354, and AD-1809369.

[0074] In another embodiment, the invention provides dsRNA agents that target hotspot regions of actin-binding LIM protein 3 (ABLIM3) mRNA.

[0075] The invention also provides cells and pharmaceutical compositions for inhibiting expression of the gene encoding ABLIM3, 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 solution, eg, a buffer solution comprising acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof; or phosphate buffered saline (PBS).

[0078] In one aspect, the invention provides a method of inhibiting expression of the ABLIM3 gene in a cell, the method comprising contacting the cell with a dsRNA agent of the invention or a pharmaceutical composition of the invention, thereby inhibiting expression of the ABLIM3 gene in the cell.

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

[0080] In one embodiment, the cell is a central nervous system (CNS) cell. In another embodiment, the cell is a brain cell. In certain embodiments, the cell is an astrocyte, neuron, or oligodendrocyte.

[0081] In one embodiment, the subject is a human.

[0082] In one embodiment, the subject has an ABLIM3-associated disorder.

[0083] In one embodiment, the subject has post-traumatic stress disorder (PTSD). In one embodiment, the subject has age-related memory loss.

[0084] In one embodiment, contacting the cell with the dsRNA agent inhibits expression of ABLIM3 by at least 30%.

[0085] In one embodiment, inhibiting expression of ABLIM3 reduces ABLIM3 protein levels in the serum of the subject by at least 30%.

[0086] In one aspect, the invention provides a method of treating a subject having a disorder that would benefit from reduced ABLIM3 expression, the method comprising administering a therapeutically effective amount of a dsRNA agent of the invention or a pharmaceutical composition of the invention to the subject, thereby treating the subject having a disorder that would benefit from reduced ABLIM3 expression.

[0087] In another aspect, the invention provides a method for preventing at least one symptom or sign in a subject having a disorder that would benefit from reduced ABLIM3 expression, the method comprising administering to the subject a prophylactically effective amount of a dsRNA agent of the invention or a pharmaceutical composition of the invention, thereby preventing at least one symptom or sign in a subject having a disorder that would benefit from reduced ABLIM3 expression.

[0088] In another aspect, the invention provides a method for preventing a subject from having a disorder for which reduced ABLIM3 expression would be beneficial, comprising administering to the subject a prophylactically effective amount of a dsRNA agent of the invention or a pharmaceutical composition of the invention, thereby preventing the subject from having the disorder for which reduced ABLIM3 expression would be beneficial.

[0089] In one embodiment, the disorder is an ABLIM3-associated disorder.

[0090] In one embodiment, the disorder is post-traumatic stress disorder (PTSD).

[0091] In one embodiment, the disorder is age-related memory loss.

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

[0093] In one embodiment, administration of the agent to a subject results in improved memory, or a decrease in anxiety, depression, fear, restlessness, hostility, and / or distress.

[0094] In one embodiment, the dsRNA agent is administered to a 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 invention further comprises determining the level of ABLIM3 in a sample from the subject.

[0098] In one embodiment, the level of ABLIM3 in a subject sample is the level of ABLIM3 protein 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 embodiment, the invention provides a vial containing a dsRNA agent of the invention or a pharmaceutical composition of the invention.

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

[0103] In another embodiment, the invention provides an intrathecal pump comprising a dsRNA agent of the invention, or a pharmaceutical composition of the invention. DETAILED DESCRIPTION OF THE INVENTION

[0104] The present disclosure provides an RNAi composition, which carries out RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of ABLIM3 gene.ABLIM3 gene can be present in cells, for example, in the cells of a subject such as human.The use of these iRNAs allows the targeted degradation of the mRNA of corresponding gene (ABLIM3 gene) in mammals.

[0105] The iRNAs of the present invention are designed to target the ABLIM3 gene, for example, the ABLIM3 gene with or without nucleotide modifications. The iRNAs of the present invention inhibit ABLIM3 gene expression by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. Without intending to be limited by theory, it is believed that the combination or partial combination of the above-mentioned properties and the specific target sites or specific modifications of these iRNAs confers improved efficiency, stability, potency, durability, and safety to the iRNAs of the present invention.

[0106] Thus, the present disclosure also provides methods of using the RNAi compositions of the present disclosure to inhibit expression of the ABLIM3 gene or to treat subjects with disorders in which inhibiting or reducing expression of the ABLIM3 gene would be beneficial, such as PTSD or age-related memory loss.

[0107] 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, 19-23, The RNAi agent of the present disclosure comprises an RNA strand (antisense strand) having a region that is 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which is substantially complementary to at least a portion of an mRNA transcript of the ABLIM3 gene, e.g., an exon of ABLIM3. 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 ABLIM3 gene.

[0108] In certain embodiments, RNAi agents of the present disclosure comprise an RNA strand (antisense strand) having a region of at least 19 contiguous nucleotides that is substantially complementary to at least a portion of an mRNA transcript of the ABLIM3 gene, and can comprise a longer length, e.g., up to 66 nucleotides, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, or 27-53 nucleotides in length. These RNAi agents with longer antisense strand lengths preferably include a second RNA strand (sense strand) of 20-60 nucleotides in length, in which case the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

[0109] The use of these RNAi agents allows for targeted degradation and / or inhibition of the mRNA of the ABLIM3 gene in mammals. Thus, methods and compositions comprising these RNAi agents are useful for treating subjects who would benefit from reduced ABLIM3 protein levels or activity, such as subjects with PTSD or age-related memory loss.

[0110] The following detailed description discloses methods for making and using compositions containing RNAi agents to inhibit expression of the ABLIM3 gene, as well as compositions and methods for treating subjects with diseases and disorders in which inhibiting or reducing expression of the gene would be beneficial.

[0111] 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.

[0112] 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 more than one element, e.g., a plurality of elements.

[0113] 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.

[0114] The term "about" is used herein to mean within a range that is typical 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 is present before a series of numbers or ranges, it will be understood that "about" can modify each number or range in the series.

[0115] The term "at least" before a number or a series of numbers, if clear from the context, is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that may be logically included. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the specified property. When the term "at least" is present before a series of numbers or a range, it is understood that "at least" can modify each of the numbers and ranges in the series.

[0116] As used herein, "less than" or "less than" shall be understood as the value adjacent to the phrase and its logically smaller value or integer, if logical from the context, to zero.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 present before a series of numbers or ranges, it shall be understood that "less than" can modify each number or range in the series.

[0117] As used herein, a method of detection can include determining that the amount of analyte present is below the detection level of the method.

[0118] In the event of a discrepancy between the nucleotide sequence for a given target site and the sense or antisense strand, the given sequence prevails.

[0119] If the chemical structure and chemical name do not agree, the chemical structure takes precedence.

[0120] As used herein, the term "actin-binding LIM protein 3", which is used interchangeably with the term "ABLIM3", refers to a well-known gene and the polypeptide encoded by the gene, and is also known in the art as "actin-binding LIM protein family member 3", "KIAA0843" and "HMFN1661".The ABLIM3 gene is expressed and active in the brain, and is also expressed in other tissues, including adipose tissue, heart, placenta and liver.ABLIM3 is also expressed in some cells of the central nervous system, including dentate gyrus granule cells.RNAi therapy can specifically target ABLIM3 in the brain, thereby avoiding peripheral side effects.

[0121] ABLIM3 has recently been implicated in memory as a molecular brake on connectivity between dentate gyrus granule cells and stratum lucidum interneurons [Guo, et al., 2018, Nat Med.24(4):438-449]. Using an animal model of learning, it was shown that ABLIM3 levels decrease during learning. This study found that downregulating ABLIM3 in dentate gyrus granule cells reduces generalization of remote memories and increases their accuracy, which may help alleviate memory impairment and PTSD.

[0122] Exemplary nucleotide and amino acid sequences for ABLIM3 can be found, for example, in GenBank Accession No. NM_001301015.3 [Homo sapiens ABLIM3, SEQ ID NO: 1, reverse complement, SEQ ID NO: 2)]; and GenBank Accession No. NM_001164491.1 [Mus musculus ABLIM3)].

[0123] The nucleotide sequence of the genomic region of a human chromosome containing the ABLIM3 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 5 containing the ABLIM3 gene can also be found, for example, in GenBank accession number NC_000005.10, which corresponds to nucleotides 149, 141, 193-14, 9260, and 542 of human chromosome 5. The nucleotide sequence of the human ABLIM3 gene can be found, for example, in GenBank accession number NG_051566.1.

[0124] Further examples of ABLIM3 sequences can be found in publicly available databases such as GenBank, OMIM, and UniProt.

[0125] Additional information regarding ABLIM3 can be found, for example, at https: / / www.ncbi.nlm.nih.gov / gene / 22885. The term ABLIM3, as used herein, also refers to variations of the ABLIM3 gene, including variants provided in clinical variant databases, for example, https: / / www.ncbi.nlm.nih.gov / clinvar / ?term=ABLIM3[gene].

[0126] 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.

[0127] As used herein, "target sequence" refers to a contiguous portion of a nucleotide sequence in an mRNA molecule formed upon transcription of the ABLIM3 gene, e.g., an mRNA that is the product of RNA processing of a primary transcript. In one embodiment, the target portion of the sequence will be at least sufficiently long to serve as a substrate for RNAi-dependent cleavage at or near a portion of the nucleotide sequence of the mRNA molecule formed upon transcription of the ABLIM3 gene.

[0128] The target sequence is about 15-30 nucleotides in length. For example, the target sequence can be about 15-30 nucleotides in length, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 1 The target sequence may be 9-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In certain embodiments, the target sequence is 19-23 nucleotides in length, and, where appropriate, may be 21-23 nucleotides in length. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of this disclosure.

[0129] 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" each generally represent a nucleotide containing guanine, cytosine, adenine, thymidine, and uracil as a base, respectively, in relation to a modified or unmodified nucleotide. However, it is understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides, as described in more detail below, or substitute replacement moieties (see, for example, Table 2). Those skilled in the art are well aware that guanine, cytosine, adenine, thymidine, and uracil can be replaced with other moieties without substantially changing the base pairing properties of an oligonucleotide containing a nucleotide with such a replacement moiety. For example, but not limited to, a nucleotide containing inosine as its base can form a base pair with a nucleotide containing adenine, cytosine, or uracil. Therefore, the nucleotide that comprises uracil, guanine or adenine can be substituted with the nucleotide that comprises inosine in the nucleotide sequence of the dsRNA that is featured in the present disclosure.In another example, the adenine and cytosine in any of oligonucleotides can be substituted with guanine and uracil, respectively, to form G-U Wobble base pairing with target mRNA.The sequence that comprises such a replacement part is suitable for the composition and method that is featured in the present disclosure.

[0130] The terms " iRNA ", " RNAi agent ", " iRNA agent ", " RNA interference agent ", " used interchangeably herein, refer to an agent that contains RNA as defined herein and mediates targeted cleavage in RNA transcription via the RNA-induced silencing complex (RISC) pathway.RNA interference (RNAi) is the process that directs the sequence-specific degradation of mRNA.RNAi modulates, for example, inhibits, the expression of ABLIM3 in cells, for example, in cells of a subject, for example, a mammalian subject.

[0131] 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 ABLIM3 target mRNA sequence, to direct cleavage of 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]. Dicer, an RNase III-like enzyme, processes this dsRNA into 19-23 base pair small interfering RNAs with a characteristic two-base 3' overhang [Bernstein, et al., (2001) Nature 409:363]. These siRNAs are then introduced into RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing complementary antisense strands to induce target recognition [Nykanen, et al., (2001) Cell 107:309]. Upon binding to the appropriate target mRNA, one or more endonucleases in RISC cleave the target to induce silencing [Elbashir, et al., (2001) Genes Dev. 15:188]. Thus, 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, promotes the formation of RISC complex, and thereby silences the target gene, i.e., the ABLIM3 gene. Therefore, the term "siRNA" is used herein to also mean the RNAi described above.

[0132] 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 and then cleaves the target mRNA. The single-stranded siRNA is generally 15-30 nucleotides and chemically modified. The design and testing of single-stranded RNA is described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein can be used as the single-stranded siRNA described herein or as the single-stranded siRNA chemically modified by the method described in Lima et al., (2012) Cell 150:883-894.

[0133] In another embodiment, the "RNAi agent" for use in the compositions and methods of the present disclosure is double-stranded RNA, and is referred to herein as a "double-stranded RNAi agent," a "double-stranded RNA (dsRNA) molecule," a "dsRNA agent," or a "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel, substantially complementary nucleic acid strands, said to have a "sense" or "antisense" orientation with respect to the target RNA, i.e., the ABLIM3 gene. In some embodiments of the present disclosure, the double-stranded RNA (dsRNA) induces the degradation of the target RNA, for example, mRNA, by a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0134] Generally, dsRNA molecules can comprise ribonucleotides, but as described in detail herein, each strand or both strands can also comprise one or more ribonucleotides, such as deoxyribonucleotides, modified nucleotides.In addition, as used herein, " RNAi agent " can comprise ribonucleotides with chemical modification; RNAi agent can comprise substantial modifications in multiple nucleotides.As used herein, the term " modified nucleotide " refers to the nucleotide that independently has modified sugar moiety, modified internucleotide linkage, or modified nucleobase.Therefore, the term modified nucleotide encompasses the substitution, addition, or removal of, for example, functional group or atom, etc., in internucleoside linkage, sugar moiety, or nucleobase.The modifications suitable for use in the agent of the present disclosure encompass 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.

[0135] In certain embodiments of the present disclosure, the inclusion of deoxynucleotides can be considered to constitute modified nucleotides when present within an RNAi agent.

[0136] The duplex region can be any length that allows for specific degradation of the desired target RNA by the RISC pathway, as well as 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 31, 18 to 32, 18 to 33, 18 to 34, 18 to 35, 18 to 36, or 36 base pairs in length. 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 in length. In certain embodiments, the duplex region is 19-21 base pairs in length, e.g., 21 base pairs in length. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of the present disclosure.

[0137] The two strands forming the duplex structure can be different portions of a larger RNA molecule, or they can be separate RNA molecules. When the two strands are part of a larger molecule and can be connected by an uninterrupted chain of nucleotides between the 3' end of one strand forming the duplex structure and the 5' end of the other strand, the connecting 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 targeted to 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 unpaired nucleotides.

[0138] The two substantially complementary strands of dsRNA are contained in separate RNA molecules, and these molecules can, but do not necessarily, be covalently linked. In certain embodiments, where the two strands are covalently linked between the 3' end of one strand and the 5' end of each other strand forming a duplex structure by means other than an uninterrupted chain of nucleotides, the connecting structure is called a "linker" (although certain other structures defined elsewhere herein may also be called "linkers"). The RNA strands can have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus all overhangs present in the duplex. In addition to the duplex structure, the RNAi can contain one or more nucleotide overhangs. In one embodiment of an RNAi agent, at least one strand contains a 3' overhang of at least one nucleotide. In another embodiment, at least one strand contains a 3' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5' overhang of at least one nucleotide. In certain embodiments, at least one strand comprises a 5' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet other embodiments, both the 3' and 5' ends of one strand of the RNAi agent comprise an overhang of at least one nucleotide.

[0139] In one embodiment, an RNAi agent of the disclosure is a dsRNA, each strand of which independently comprises 19-23 nucleotides that interact with a target RNA sequence, e.g., an ABLIM3 target mRNA sequence, to induce cleavage of the target RNA.

[0140] In some embodiments, the iRNA of the invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, eg, an ABLIM3-targeted mRNA sequence, to direct cleavage of the target RNA.

[0141] 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, when the 3'-end of one strand of dsRNA extends beyond the 5'-end of the other strand, and vice versa, there is a nucleotide overhang.DsRNA can comprise at least one nucleotide overhang; alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more nucleotides.The nucleotide overhang can comprise or consist of nucleotide / nucleoside analogs, such as deoxynucleotides / nucleosides.The overhang can be on the sense strand, antisense strand, or any combination thereof.In addition, the nucleotide of the overhang can be present at the 5'-end, 3'-end, or both of the antisense strand or sense strand of dsRNA.

[0142] In one embodiment, the antisense strand of the dsRNA has a 1-10 nucleotide overhang at the 3'-end or 5'-end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide overhang. In one embodiment, the sense strand of the dsRNA has a 1-10 nucleotide overhang at the 3'-end or 5'-end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide overhang. In another embodiment, one or more of the nucleotides in the overhang are replaced with nucleoside thiophosphate.

[0143] In certain embodiments, the antisense strand of dsRNA has an overhang of 1 to 10 nucleotides at the 3'-end 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 dsRNA has an overhang of 1 to 10 nucleotides at the 3'-end or 5'-end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more of the nucleotides in the overhang are replaced with nucleoside thiophosphate.

[0144] In certain embodiments, the antisense strand of the dsRNA has an overhang of 1 to 15 nucleotides at the 3' end, for example, 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, 6 to 12, or for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In another embodiment, one or more of the nucleotides in the overhang are replaced with nucleoside thiophosphates.

[0145] In certain embodiments, the overhang on the sense strand or the antisense strand can comprise an extended length of more than 10 or 15 nucleotides, e.g., 1 to 30 nucleotides, 2 to 30 nucleotides, 10 to 30 nucleotides, or 10 to 15 nucleotides. In certain embodiments, the extended overhang is on the sense strand of the duplex. In certain embodiments, the extended overhang is at the 3'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is at 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 at the 3'-end of the antisense strand of the duplex. In certain embodiments, the extended overhang is at the 5'-end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the overhang are replaced 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.

[0146] The term "blunt" or "blunt-ended" as used herein with respect to dsRNA means that there are no unpaired nucleotides or nucleotide analogs at a given end of the dsRNA, i.e., there are no nucleotide overhangs. One or both ends of the dsRNA can be blunt. When both ends of a dsRNA are blunt, the dsRNA is said to be blunt-ended. For clarity, a "blunt-ended" dsRNA is a dsRNA that is blunt at both ends, i.e., there are no nucleotide overhangs at either end of the molecule. In most cases, such a molecule will be double-stranded throughout its entire length.

[0147] The term "antisense strand" or "guide strand" refers to the strand of an RNAi agent, eg, a dsRNA, that includes a region that is substantially complementary to a target sequence, eg, ABLIM3 mRNA.

[0148] As used herein, the term "region of complementarity" refers to a region on the antisense strand that is substantially complementary to a sequence, for example, a target sequence, for example, an ABLIM3 nucleotide sequence, as defined herein. If the region of complementarity is not completely complementary to the target sequence, the mismatch may be in the internal or terminal region of the molecule. Generally, the most tolerable mismatch is in the terminal region, for example, within 5, 4, 3, or 2 nucleotides of the 5' or 3' end of the RNAi agent. In some embodiments, the double-stranded RNA agent of the present invention contains nucleotide mismatches in the antisense strand. In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention contains four or fewer mismatches with the target mRNA, for example, the antisense strand contains four, three, two, one, or zero mismatches with the target mRNA. In some embodiments, the antisense strand of a double-stranded RNA agent of the invention contains four or fewer mismatches with the sense strand, e.g., the antisense strand contains four, three, two, one, or zero mismatches with the sense strand. In some embodiments, a double-stranded RNA agent of the invention contains a nucleotide mismatch in the sense strand. In some embodiments, the sense strand of a double-stranded RNA agent of the invention contains four or fewer mismatches with the antisense strand, e.g., the sense strand contains four, three, two, one, or zero 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, at the 3' terminal nucleotide of the iRNA agent. In some embodiments, the mismatch is not in the seed region.

[0149] Thus, the RNAi agents described herein can contain one or more mismatches to the target sequence. In one embodiment, the RNAi agents described herein contain three or fewer mismatches (i.e., three, two, one, or zero mismatches). In one embodiment, the RNAi agents described herein contain two or fewer mismatches. In one embodiment, the RNAi agents described herein contain one or fewer mismatches. In one embodiment, the RNAi agents described herein contain zero mismatches. In certain embodiments, when the antisense strand of an RNAi agent contains a mismatch to the target sequence, the mismatch can be limited to within the last five nucleotides from the 5' or 3' end of the region of complementarity, as appropriate. For example, in such an embodiment, in the case of a 23-nucleotide RNAi agent, the strand complementary to a region of the ABLIM3 gene generally does not contain any mismatches within the central 13 nucleotides. 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 expression of the ABLIM3 gene. For example, Jackson et al. (Nat. Biotechnol. 2003;21:635-637) described an expression profiling study in which the expression of a small set of genes with only 12-18 nt of sequence identity to a MAPK14 siRNA on the sense strand was downregulated with kinetics similar to MAPK14. Similarly, Lin et al. (Nucleic Acids Res. 2005;33(14):4527-4535) used qPCR and reporter assays to show that 7 nt of complementarity between the siRNA and the target was sufficient to trigger target mRNA degradation. It is important to consider the efficacy of mismatched RNAi agents to inhibit ABLIM3 gene expression, especially when the specific region of complementarity in the ABLIM3 gene is known to have polymorphic sequence variation within the population.

[0150] An RNA target may have a region or section of the nucleotide sequence of the target RNA, which is relatively more susceptible or receptive than other regions of the RNA target to mediated cleavage of the RNA target through RNA interference induced by the binding of an RNAi agent to that region. High susceptibility to RNA interference within such a "hotspot region" (or simply "hotspot") means that an iRNA agent targeting that region is likely to be more effective at inducing iRNA interference than an iRNA agent targeting other regions of the target RNA. For example, without being bound by theory, the accessibility of a target region of a target RNA can affect the effectiveness of an iRNA agent targeting that region, and some hotspot regions have high accessibility. Secondary structures, such as those formed within an RNA target (e.g., within or near a hotspot region), can affect the ability of an iRNA agent to bind to a target region and induce RNA interference.

[0151] According to certain embodiments of the invention, an iRNA agent may be designed to target any hotspot region of a target RNA described herein, including any specified portion of the target RNA (e.g., a particular exon). As used herein, a hotspot region may refer to a region of approximately 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, 100-200, or 100-150 nucleotides of a target RNA sequence where targeting with an RNAi agent results in an observably higher probability of effective silencing compared to targeting other regions of the same target RNA. According to certain embodiments of the present invention, hotspot regions may comprise a limited region of target RNA, for example, less than half the length of target RNA, for example, about 5%, 10%, 15%, 20%, 25%, or 30% of the length of target RNA, or in some cases, a substantially limited region of the target. Conversely, other regions compared to hotspots may cumulatively comprise at least the majority of the length of target RNA. For example, other regions may 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.

[0152] Comparative regions of the target RNA may 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 across the target RNA may be compared for the frequency of effective iRNA agents binding to each region (e.g., the amount by which target gene expression is inhibited, such 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 sufficiently characterized as such by observing the efficacy of an iRNA agent cumulatively over at least about 60% of the target region identified as a hotspot, for example, over 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 about 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 demonstrating at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% inhibition (e.g., no more than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% mRNA remaining) over the region can be identified as effective.

[0153] The amenability of an RNA region to targeting can also be evaluated using a quantitative comparison of inhibition measurements across different regions of a predetermined size (e.g., 25, 30, 40, 50, 60, 70, 80, 90, or 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nt). For example, the average level of inhibition can be determined for each region, and the average values ​​for each region can be compared. The average level of inhibition within a hotspot region can be substantially higher than the average of all the average values ​​evaluated. According to some embodiments, the average level of inhibition within a hotspot region can be at least about 10%, 20%, 30%, 40%, or 50% higher than the average of the average values. According to some embodiments, the average level of inhibition within 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 above 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 be above a threshold amount (e.g., below a threshold amount of remaining mRNA). According to some embodiments, each measured inhibition within that region may be substantially higher than the average of all measured inhibition measurements across all measured regions. For example, each measured inhibition within 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 above 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). Standards for evaluating hotspots may include various combinations of the above standards, if applicable (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 lower than the first amount).

[0154] Therefore, it is expressly contemplated that any iRNA agent, including the specific exemplary iRNA agent described herein, that targets the hotspot region of target RNA can be preferably selected for inducing 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.The RNAi agent that targets a target sequence that substantially overlaps (for example, at least about 70%, 75%, 80%, 85%, 90%, 95% of the length of the target sequence), or preferably exists entirely within the hotspot region, can be considered to target the hotspot region.The hotspot region of the RNA target(s) of the present invention can include any region that the data disclosed herein demonstrates (for example, by any of the standards described elsewhere herein) demonstrates a higher frequency of effective targeting by the RNAi agent, regardless of whether the range(s) of such hotspot region are explicitly specified.

[0155] In various embodiments, the dsRNA agent of the invention targets a hotspot region of the mRNA encoding ABLIM3. In one embodiment, the hotspot region includes nucleotides 3042-3081, 3420-3457, 4223-4261, 726-763, 1080-1137, 1342-1379, 1910-1955, 2167-2204, 480-520, 1080-1122, 1622-1665, 1744-1787, 2323-2363, 540-578, 1175-1212, 274-313, 349-401, or 2518-2555 of SEQ ID NO:1. dsRNA agents are AD-1809702, AD-1809719, AD-1810038, AD-1810053, AD-1810676, AD-1810692, AD-1807904, AD-1807919, AD-180823 8, AD-1808258, AD-1808273, AD-1808418, AD-1808433, AD-1808842, AD-1808865, AD-1809051, AD-1809066, AD-1807707, AD-18 07725, AD-1808628, AD-1808649, AD-1808727, AD-1808748, AD-1809187, AD-1809205, AD-1807764, AD-1807780, AD-1808312, AD-1808327, AD-1807561, AD-1807578, AD-1807616, AD-1807631, AD-1807646, AD-1809354, and AD-1809369.

[0156] "Substantially all of the nucleotides are modified," as used herein, means that most, but not all, are modified, including no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0157] 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.

[0158] 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 that are directly adjacent to either end of the cleavage site.In some embodiments, the cleavage region comprises two bases that are directly adjacent to either end of the cleavage site.In some embodiments, specifically, the cleavage site occurs at the site that is bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12, and 13.

[0159] As used herein, unless otherwise specified, the term "complementary," when used to describe a first nucleotide sequence in the context of a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize to form a duplex with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions, as understood by those of skill in the art. Such conditions may be, for example, "stringent conditions," including, but not limited to, 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12-16 hours, followed by washing. (See, for example, "Molecular Cloning: A Laboratory Manual," Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press.) As used herein, "stringent conditions" or "stringent hybridization conditions" refer to conditions under which an antisense compound hybridizes to its target sequence but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances, and the "stringent conditions" that antisense compounds hybridize with target sequence are determined by the nature and composition of antisense compounds and the assay they are investigated.Other conditions, such as physiologically relevant conditions that can be encountered inside living organisms, can also be applied.Those skilled in the art will be able to determine the most suitable set of conditions for testing the complementarity of two sequences according to the final application of hybridized nucleotide.

[0160] The complementary sequence in RNAi agent, for example, in the dsRNA described herein, comprises the base pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence with the oligonucleotide or polynucleotide comprising the second nucleotide sequence throughout the entire length of one or both nucleotide sequences.Such sequences can be referred to herein as " completely complementary " with respect to each other.However, when the first sequence is considered herein as " substantially complementary " with the second sequence, the two sequences can be completely complementary, or they can form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs during hybridization, in the case of a double strand of up to 30 base pairs. In some embodiments, the "substantially complementary" sequence disclosed herein comprises a contiguous nucleotide sequence that is at least about 80%, 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 corresponding region of the target ABLIM3 sequence over its entire length. However, if two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs are not considered mismatches when determining complementarity. For example, a dsRNA comprising one 21-nucleotide long oligonucleotide and another 23-nucleotide long oligonucleotide, where the longer oligonucleotide comprises a 21-nucleotide sequence that is completely complementary to the shorter oligonucleotide, can still be considered "completely complementary" for the purposes described herein.

[0161] "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 the above requirements regarding their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsteen base pairing.

[0162] The terms "complementary," "fully complementary," and "substantially complementary" may be used herein, as understood in connection with their use, in reference to base matching between two oligonucleotides or polynucleotides, such as between the sense and antisense strands of a dsRNA, or between the antisense strand of an RNAi agent and a target sequence.

[0163] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" a messenger RNA (mRNA) means a polynucleotide that is substantially complementary to a continuous portion of an mRNA of interest (e.g., an mRNA encoding ABLIM3). For example, a polynucleotide is complementary to at least a portion of an ABLIM3 mRNA if the sequence is substantially complementary to an uninterrupted portion of the mRNA encoding ABLIM3.

[0164] Thus, in some embodiments, the antisense polynucleotides disclosed herein are fully complementary to the target ABLIM3 sequence. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target ABLIM3 sequence and comprise a contiguous nucleotide sequence that is at least 80%, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO: 1, or a fragment of any one of SEQ ID NOs: 1, 3, 5, and 7.

[0165] In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of a target ABLIM3 sequence, and include nucleotides 80-102, 132-154, 230-252, 274-296, 291-313, 324-346, 349-371, 364-386, 379-401, 412-434, 480-502, 498-520, 540-562, 556-578, 580-602, 620-642, 672-694, 690-712, 711-733, 726-748, 741-763, 777-782, 780-792, 790-802, 800-812, 810-822, 820-832, 830-842, 840-852, 850-862, 860-872, 870-882, 880-892, 890-902, 910-924, 920-932, 930-942, 940-952, 950-962, 960-972, 970-982, 980-992, 990-1002, 1000-1012, 1010-1024, 1020-1032, 1030-1042, 1040-1052, 1050-1062, 1060-1072, 1070-1082, 108 799, 885~907, 940~962, 981~1003, 999~1021, 1080~1102, 1100~1122, 1115~1137, 1175~1197, 1190~1212, 1240~1262, 1267~1289, 1311~1333, 1342~1 364, 1357-1379, 1372-1394, 1406-1428, 1435-1457, 1548-1570, 1592-1614, 1622-1644, 1643-1665, 1672-1694, 1713-1735, 1744-1766, 1765-1787, 1 810~1832, 1857~1879, 1882~1904, 1910~1932, 1933~1955, 1969~1991, 2037~2059, 2094~2116, 2125~2147, 2167~2189, 2182~2204, 2216~2238, 2243~ 2265, 2271~2293, 2323~2345, 2341~2363, 2383~2405, 2420~2442, 2457~2479, 2481~2503, 2518~2540, 2533~2555, 2559~2581, 2583~2605, 2665~2687, 2701~2723, 2727~2749, 2762~2784, 2786~2808, 2828~2850, 2859~2881, 2883~2905, 2935~2957, 2956~2978, 2972~2994, 3009~3031, 3042~3064, 3059~ 3081, 3077~3099, 3108~3130, 3144~3166, 3201~3223, 3218~3240, 3277~3299, 3301~3323, 3319~3341, 3336~3358, 3386~3408, 3405~3427, 3420~3442,3435~3457, 3450~3472, 3530~3552, 3546~3568, 3577~3599, 3592~3614, 3624~3646, 3642~3664, 3680~3702, 3704~3726, 3719~3741, 3744~3766, 3761~3773 783, 3794~3816, 3812~3834, 3847~3869, 3864~3886, 3895~3917, 3917~3939, 3984~4006, 4016~4038, 4035~4057, 4056~4078, 4082~4104, 4097~4119, 41 29-4151, 4157-4179, 4223-4245, 4239-4261, 4274-4296, 4289-4311, 4304-4326, 4334-4356, 4352-4374, 4367-4389, 4385-4407, 4402-4424, and 4419-4441. The present disclosure also contemplates ranges between the above-listed ranges.

[0166] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target ABLIM3 sequence and comprise a contiguous nucleotide sequence that is at least 80%, 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 to any one of the sense strand nucleotide sequences in Table 3 or 4, or a fragment of any one of the sense strand nucleotide sequences in Table 3 or 4, over its entire length.

[0167] In one embodiment, an RNAi agent of the present disclosure comprises a sense strand that is substantially complementary to an antisense polynucleotide, thereby ultimately being identical to the target ABLIM3 sequence, and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80%, 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 to the equivalent region of the nucleotide sequence of SEQ ID NO:1, or a fragment of any one of SEQ ID NO:1, over its entire length.

[0168] In some embodiments, an iRNA of the invention comprises a sense strand that is substantially complementary to an antisense polynucleotide that is complementary to a target ABLIM3 sequence, wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least 80%, 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 to any one of the antisense strand nucleotide sequences in Table 3 or 4, or a fragment of any one of the antisense strand nucleotide sequences in Table 3 or 4, over its entire length.

[0169] In certain embodiments, the sense and antisense strands are duplexed as follows: AD-1807455, AD-1807481, AD-1807539, AD-1807561, AD-1807578, AD-1807591, AD-1807616, AD-1807631, AD-1807646, AD-1807659, AD-1807707, AD-1807725, AD-1807764, AD-1807780, AD-1807804, AD-1807844, AD-1807870, AD-1807888, AD-1807889, AD-1807904 , AD-1807919, AD-1807955, AD-1808063, AD-1808098, AD-1808139, AD-1808157, AD-1808238, AD-1808258, AD-1808273, AD-1808312, AD-1808327, AD- 1808356, AD-1808383, AD-1808407, AD-1808418, AD-1808433, AD-1808448, AD-1808482, AD-1808511, AD-1808561, AD-1808598, AD-1808628, AD-1808 649, AD-1808678, AD-1808698, AD-1808727, AD-1808748, AD-1808773, AD-1808789, AD-1808814, AD-1808842, AD-1808865, AD-1808901, AD-1808941 , AD-1808978, AD-1809009, AD-1809051, AD-1809066, AD-1809080, AD-1809107, AD-1809135, AD-1809187, AD-1809205, AD-1809246, AD-1809283, AD- 1809293, AD-1809317, AD-1809354, AD-1809369, AD-1809395, AD-1809414, AD-1809456, AD-1809472, AD-1809498, AD-1809511, AD-1809535, AD-1809 550, AD-1809561, AD-1809585, AD-1809596, AD-1809617, AD-1809633, AD-1809670, AD-1809702, AD-1809719, AD-1809737, AD-1809757, AD-1809773,AD-1809822, AD-1809839, AD-1809895, AD-1809919, AD-1809937, AD-1809954, AD-1810004, AD-1810023, AD-1810038, AD-1810053, AD-1810068, AD-1810127, A D-1810143, AD-1810174, AD-1810189, AD-1810221, AD-1810239, AD-1810255, A D-1810277, AD-1810292, AD-1810296, AD-1810313, AD-1810345, AD-1810363, AD -1810398, AD-1810415, AD-1810446, AD-1810468, AD-1810494, AD-1810526, AD-1810545, AD-1810566, AD-1810591, AD-1810606, AD-1810638, AD-1810666, AD-1810676, AD-1810692, AD-1810727, AD-1810742, AD-1810757, AD-1810767, AD-1810785, AD-1810800, AD-1810818, AD-1810822 and AD-1810839.

[0170] In one embodiment, at least partial suppression of ABLIM3 gene expression is assessed by a reduction in the amount of ABLIM3 mRNA, e.g., sense mRNA, antisense mRNA, total ABLIM3 mRNA, which can be isolated from or detected in a first cell or group of cells in which the ABLIM3 gene is transcribed and which has been treated or has been treated to inhibit expression of the ABLIM3 gene, and compared to a second cell or group of cells (control cells) that is substantially identical to the first cell or group of cells but has been or has not been so treated. The degree of inhibition can be expressed by:

[0171]

number

[0172] 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 with cell afterwards.

[0173] Contacting cells in vitro can be achieved, for example, by incubating cells with an RNAi agent. Contacting cells in vivo can be achieved, 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), by intrathecal injection, intravitreal injection, or other injection, as appropriate, or by injecting the RNAi agent into the bloodstream (i.e., intravenous) 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 stabilizes the RNAi agent to the target site, for example, 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 may be contacted with an RNAi agent in vitro and then transferred into a subject.

[0174] 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 includes methods known in the art, such as electroporation and lipofection. Further approaches are described herein below or known in the art.

[0175] The terms "lipophilic" or "lipophilic moiety" refer broadly to any compound or chemical moiety that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is by the octanol-water partition coefficient, log K ow In this case, K ow is the ratio of the concentration of a chemical in the octanol phase to the concentration of the chemical in the aqueous phase in a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it can also be predicted by using coefficients attributable to the structural components of a chemical calculated using first principles or empirical methods [see, e.g., Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), incorporated herein by reference in its entirety]. It provides a thermodynamic measure of a substance's tendency to prefer a non-aqueous or oily environment rather than water (i.e., hydrophilic / lipophilic balance). In principle, a chemical can be expressed as a function of the log K ow is greater than 0, it is lipophilic in nature. Typically, a lipophilic moiety has a log K ow For example, the log K of 6-aminohexanol owis expected to be approximately 0.7. Using the same method, the log K ow is expected to be 10.7.

[0176] The lipophilicity of a molecule can be modified with respect to the functional groups it carries. For example, adding a hydroxyl or amine group to the end of the lipophilic moiety can increase the partition coefficient (e.g., logK ow ) value can be increased or decreased.

[0177] Alternatively, the hydrophobicity of the double-stranded RNAi agent that is conjugated with one or more lipophilic moieties can be measured by its protein binding properties.For example, in certain embodiments, the unbound fraction of the plasma protein binding assay of double-stranded RNAi agent can be determined to be positively correlated with the relative hydrophobicity of double-stranded RNAi agent, which can be positively correlated with the silencing activity of double-stranded RNAi agent.

[0178] In one embodiment, the plasma protein binding assay that is determined is electrophoretic mobility shift assay (EMSA) using human serum albumin protein.The exemplary protocol of this binding assay is described in detail in, for example, PCT / US2019 / 031170.The hydrophobicity of double-stranded RNAi agent measured by the fraction of unbound siRNA in binding assay is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45 or greater than 0.5 for enhanced in vivo delivery of siRNA.

[0179] Thus, conjugating a lipophilic moiety to an internal position of a double-stranded RNAi agent provides optimal hydrophobicity in siRNA for enhanced in vivo delivery.

[0180] 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 the 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.

[0181] As used herein, a "subject" is an animal, e.g., a mammal, e.g., a primate (e.g., a human, a non-human primate, e.g., a monkey or chimpanzee), or a non-primate (e.g., a rat or a mouse). In one embodiment, the subject is a human, e.g., a human being treated or evaluated for a disease, disorder, or condition in which reduced ABLIM3 expression would be beneficial; a human being at risk for a disease, disorder, or condition in which reduced ABLIM3 expression would be beneficial; a human being with a disease, disorder, or condition in which reduced ABLIM3 expression would be beneficial; or a human being treated for a disease, disorder, or condition as described herein in which reduced ABLIM3 expression would be beneficial. In some embodiments, the subject is a human female. In other embodiments, the subject is a human male. In one embodiment, the subject is a human adult. In one embodiment, the subject is a human child. In another embodiment, the subject is a human juvenile, i.e., a subject under the age of 20.

[0182] As used herein, the terms "treating" or "treatment" refer to a beneficial or desired result, including, but not limited to, alleviating or ameliorating one or more signs or symptoms associated with ABLIM3 gene expression or ABLIM3 protein production, such as an ABLIM3-associated disorder, e.g., PTSD or age-related memory loss. "Treatment" can also mean prolonging survival as compared to expected survival if no treatment is administered.

[0183] The term "lower" in reference to the level of ABLIM3 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%, e.g., 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more, in the disease marker. In certain embodiments, the decrease is at least about 50% in the disease marker. When referring to the level of ABLIM3 in a subject, "reducing" preferably refers to reducing to a level that is accepted as being within the normal range in an individual without such disorder. In certain embodiments, "reducing" refers to a reduction in the difference between the level of a marker or symptom in a subject suffering from a disease and a level that is accepted within the normal range in an individual, e.g., a reduction in the difference in the level of memory loss compared to an accepted normal level.

[0184] As used herein, "prevention" or "preventing," when used in reference to a disease, disorder, or condition for which reducing the expression of the ABLIM3 gene or the production of the ABLIM3 protein would be beneficial, refers to a reduction in the likelihood that a subject will develop symptoms or signs associated with such disease, disorder, or condition, such as symptoms or signs of an ABLIM3-related disease, such as PTSD or age-related memory loss. Not developing the disease, disorder, or condition, or a reduction in the onset of symptoms associated with such disease, disorder, or condition (e.g., a reduction of at least about 10% on a scale clinically acceptable for the disease or disorder), or a delay in the onset of symptoms (e.g., a delay of several days, weeks, months, or years) is considered effective prevention.

[0185] As used herein, the term "ABLIM3-associated disease," "ABLIM3-associated condition," or "ABLIM3-associated disorder" includes any disease, condition, or disorder in which reducing the expression and / or activity of ABLIM3 would be beneficial. Exemplary ABLIM3-associated conditions include PTSD or age-related memory loss.

[0186] A "therapeutically effective amount," as used herein, is intended to include an amount of an RNA agent sufficient to treat the disease (e.g., by reducing, ameliorating, or maintaining the existing disease or one or more symptoms of the disease) when administered to a subject with an ABLIM3-associated disease. A "therapeutically effective amount" may vary depending on the RNAi agent, how the agent is administered, the disease and its severity, as well as medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, if any, and other individual characteristics of the subject being treated.

[0187] As used herein, a "prophylactically effective amount" is intended to include an amount of an RNAi agent sufficient to prevent or ameliorate the disease or one or more symptoms of the disease when administered to a subject with an ABLIM3-associated disease. Amelioration of the disease includes slowing the course of the disease or reducing the severity of subsequent disease development. A "prophylactically effective amount" may vary depending on the RNAi agent, how the agent is administered, the degree of risk of the disease, and other individual characteristics of the patient being treated, such as medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, if any.

[0188] 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.

[0189] The phrase "pharmaceutically acceptable" is used herein to mean compounds, materials, compositions, or dosage forms that are suitable for use in contact with the tissues of human and animal subjects, within the scope of sound medical judgment, without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0190] The phrase "pharmaceutically acceptable carrier," as used herein, refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material, involved in the transport or transportation of a compound of interest from one organ or part of the body to another organ, e.g., part of the body. 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 function 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 stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; and (9) oils, such as peanut oil, cottonseed oil, safflower oil, and 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) buffers, 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 affinity substances used in pharmaceutical formulations.

[0191] 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, 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 obtained 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 entire brain or a specific segment of the brain, e.g., the hippocampus, striatum, or specific types of cells in the brain, e.g., dentate granule cells, 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.

[0192] The term "substituted" refers to one or more hydrogen radicals in a given structure being replaced with the radical of a specified substituent. These include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, halo, thiol, alkylthio, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, and aliphatic. It is understood that the substituent can be further substituted.

[0193] The term "alkyl" refers to saturated and unsaturated non-aromatic hydrocarbon chains (including, but not limited to, propyl, allyl, or propargyl) that may be straight or branched and contain the indicated number of carbon atoms, and which may be interrupted by N, O, or S. For example, "(C1-C6) alkyl" refers to a radical having 1 to 6 carbon atoms in a linear or branched arrangement. "(C1-C6) alkyl" includes, for example, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, pentyl, and hexyl. In certain embodiments, lipophilic moieties of the present disclosure may comprise a C6 to C18 alkyl hydrocarbon chain.

[0194] The term "alkylene" refers to an optionally substituted saturated aliphatic branched or straight-chain divalent hydrocarbon radical having the specified number of carbon atoms. For example, "(C1-C6) alkylene" refers to a divalent saturated aliphatic radical having 1 to 6 carbon atoms arranged in a linear fashion, e.g., [(CH2) n ], where n is an integer from 1 to 6. "(C1-C6) alkylene" includes methylene, ethylene, propylene, butylene, pentylene, and hexylene. Alternatively, "(C1-C6) alkylene" refers to a divalent saturated radical having from 1 to 6 carbon atoms arranged in a branched configuration, such as: [(CH2CH2CH2CH2CH(CH3)], [(CH2CH2CH2CH2C(CH3)2], [(CH2C(CH3)2CH(CH3))], and the like. The term "alkylenedioxo" refers to a divalent species of the structure -ORO-, where R represents alkylene.

[0195] The term "mercapto" refers to an -SH radical. The term "thioalkoxy" refers to an -S-alkyl radical.

[0196] The term "halo" refers to any radical of fluorine, chlorine, bromine, or iodine. "Halogen" and "halo" are used interchangeably herein.

[0197] As used herein, unless otherwise specified, the term "cycloalkyl" refers to a saturated or unsaturated non-aromatic hydrocarbon ring group having 3 to 14 carbon atoms. For example, "(C3-C10)cycloalkyl" refers to a hydrocarbon radical of a (3-10)-membered saturated aliphatic cyclic hydrocarbon ring. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, methylcyclopropyl, 2,2-dimethylcyclobutyl, 2-ethylcyclopentyl, cyclohexyl, and the like. Cycloalkyl groups may include multiple spiro or fused rings. Cycloalkyl groups may be mono-, di-, tri-, tetra-, or penta-substituted at any position, as permitted by normal valence.

[0198] As used herein, the term "alkenyl" refers to a straight-chain or branched, non-aromatic hydrocarbon radical containing at least one carbon-carbon double bond and, unless otherwise specified, having 2 to 10 carbon atoms. Up to five carbon-carbon double bonds may be present in such a group. For example, a "C2-C6" alkenyl is defined as an alkenyl radical having 2 to 6 carbon atoms. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, and cyclohexenyl. The straight-chain, branched, or cyclic portions of an alkenyl group may contain double bonds and may be mono-, di-, tri-, tetra-, or penta-substituted at any position, as allowed by the normal valence. The term "cycloalkenyl" refers to a monocyclic hydrocarbon group having the specified number of carbon atoms and at least one carbon-carbon double bond.

[0199] As used herein, the term "alkynyl," unless otherwise specified, refers to a straight-chain or branched hydrocarbon radical containing 2 to 10 carbon atoms and at least one carbon-carbon triple bond. Up to five carbon-carbon triple bonds may be present. Thus, "C2-C6 alkynyl" refers to an alkynyl radical having 2 to 6 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, 2-propynyl, and 2-butynyl. The straight-chain or branched portion of an alkynyl group may contain triple bonds as permitted by normal valence and may be mono-, di-, tri-, tetra-, or penta-substituted at any position as permitted by normal valence.

[0200] As used herein, "alkoxyl" or "alkoxy" refers to an alkyl group, as defined above, with the indicated number of carbon atoms attached through an oxygen bridge. For example, "(C-C)alkoxy" includes methoxy, ethoxy, and propoxy. For example, "(C-C)alkoxy" is intended to include C, C, C, C, C, C, C, C, and C alkoxy groups. For example, "(C-C)alkoxy" is intended to include C, C, C, C, C, C, C, C, and C alkoxy groups. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, n-pentoxy, s-pentoxy, n-heptoxy, and n-octoxy. "Alkylthio" refers to an alkyl radical attached through a sulfur linking atom. The term "alkylamino" or "aminoalkyl" refers to an alkyl radical linked through an N-H linkage. "Dialkylamino" refers to two alkyl radicals linked through a nitrogen linking atom. The amino group can be unsubstituted, monosubstituted, or disubstituted. In some embodiments, the two alkyl radicals are the same (e.g., N,N-dimethylamino). In some embodiments, the two alkyl radicals are different (e.g., N-ethyl-N-methylamino).

[0201] As used herein, "aryl" or "aromatic" refers to any stable monocyclic or polycyclic carbon ring having up to seven atoms in each ring, with at least one ring being aromatic. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, tetrahydronaphthyl, indanyl, and biphenyl. When the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that the bond is via the aromatic ring. The aryl group may be mono-, di-, tri-, tetra-, or penta-substituted at any position, as permitted by normal valences. The term "arylalkyl" or "aralkyl" refers to an alkyl substituted with an aryl. The term "arylalkoxy" refers to an alkoxy substituted with an aryl.

[0202] "Hetero" refers to at least one carbon atom in a ring structure replaced with at least one heteroatom selected from N, S, and O. "Hetero" also refers to at least one carbon atom in a non-ring structure replaced. A heterocyclic or heteroacyclic structure can have, for example, 1, 2, or 3 carbon atoms replaced by heteroatoms.

[0203] As used herein, the term "heteroaryl" refers to a stable monocyclic or polycyclic ring having up to seven atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N, and S. Examples of heteroaryl groups include, but are not limited to, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, benzimidazolonyl, benzoxazolonyl, quinolinyl, isoquinolinyl, dihydroisoindolonyl, imidazopyridinyl, isoindolonyl, indazolyl, oxazolyl, oxadiazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, and tetrahydroquinoline. It is understood that "heteroaryl" includes the N-oxide derivative of any nitrogen-containing heteroaryl. If the heteroaryl substituent is bicyclic and one ring is non-aromatic or does not contain a heteroatom, it is understood that attachment is through the aromatic ring or through the ring containing the heteroatom. Heteroaryl groups may be mono-, di-, tri-, tetra-, or penta-substituted at any position, as permitted by normal valences.

[0204] As used herein, the term "heterocycle," "heterocyclic," or "heterocyclyl" refers to a 3- to 14-membered aromatic or non-aromatic heterocycle, including polycyclic groups, containing 1 to 4 heteroatoms selected from the group consisting of O, N, and S. As used herein, the term "heterocyclic" is also considered synonymous with the terms "heterocycle" and "heterocyclyl," which are also understood to have the same definition as set forth herein. "Heterocyclyl" includes the heteroaryls described above, as well as dihydro and tetrahydro analogs thereof.Examples of heterocyclyl groups include azetidinyl, benzimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthopyridinyl, oxadiazolyl, and oxooxazolidinyl. , oxazolyl, oxazoline, oxopiperazinyl, oxopyrrolidinyl, oxomorpholinyl, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyridinonyl, pyrimidyl, pyrimidinonyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl, tetrazolyl, tetrazolopyridyl, thiadiazinyl Zolyl, thiazolyl, thienyl, triazolyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyridin-2-onyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydroo Heterocyclyl groups include, but are not limited to, oxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, dioxidothiomorpholinyl, methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, and their N-oxides. Attachment of heterocyclyl substituents can occur through a carbon atom or through a heteroatom. Heterocyclyl groups may be mono-, di-, tri-, tetra-, or penta-substituted at any position, as permitted by normal valences.

[0205] "Heterocycloalkyl" refers to a cycloalkyl residue in which one to four of the carbons are replaced by a heteroatom such as oxygen, nitrogen, or sulfur. Examples of heterocycles in which the radical is a heterocyclyl group include tetrahydropyran, morpholine, pyrrolidine, piperidine, thiazolidine, oxazole, oxazoline, isoxazole, dioxane, tetrahydrofuran, and the like.

[0206] The term "heteroaryl" refers to an aromatic 5- to 8-membered monocyclic, 8- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring system having 1 to 3 heteroatoms if monocyclic, 1 to 6 heteroatoms if bicyclic, or 1 to 9 heteroatoms if tricyclic, selected from O, N, or S (e.g., carbon atoms and 1 to 3, 1 to 6, or 1 to 9 N, O, or S heteroatoms if monocyclic, bicyclic, or tricyclic, respectively), in which 0, 1, 2, 3, or 4 atoms in each ring are optionally substituted. Examples of heteroaryl groups include pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the like. The term "heteroarylalkyl" or "heteroaralkyl" refers to an alkyl substituted with a heteroaryl. The term "heteroarylalkoxy" refers to an alkoxy substituted with a heteroaryl.

[0207] The term "cycloalkyl," as used herein, includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, e.g., 3 to 8 carbons, e.g., 3 to 6 carbons, which cycloalkyl groups may be further optionally substituted. Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0208] The term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted by substituents.

[0209] As used herein, "keto" refers to any alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, or aryl group as defined herein attached through a carbonyl bridge.

[0210] Examples of keto groups include, but are not limited to, alkanoyl (e.g., acetyl, propionyl, butanoyl, pentanoyl, hexanoyl), alkenoyl (e.g., acryloyl), alkynoyl (e.g., ethinoyl, propynoyl, butynoyl, pentinoyl, hexynoyl), aryloyl (e.g., benzoyl), heteroaryloyl (e.g., pyrrolyl, imidazoloyl, quinolinoyl, pyridinoyl).

[0211] As used herein, "alkoxycarbonyl" refers to any alkoxy group as defined above attached through a carbonyl bridge (i.e., -C(O)O-alkyl). Examples of alkoxycarbonyl groups include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, iso-propoxycarbonyl, n-propoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, or n-pentoxycarbonyl.

[0212] As used herein, "aryloxycarbonyl" refers to any aryl group, as defined herein, attached through an oxycarbonyl bridge (i.e., -C(O)O-aryl). Examples of aryloxycarbonyl groups include, but are not limited to, phenoxycarbonyl and naphthyloxycarbonyl.

[0213] As used herein, "heteroaryloxycarbonyl" refers to any heteroaryl group as defined herein attached through an oxycarbonyl bridge (i.e., -C(O)O-heteroaryl). Examples of heteroaryloxycarbonyl groups include, but are not limited to, 2-pyridyloxycarbonyl, 2-oxazolyloxycarbonyl, 4-thiazolyloxycarbonyl, or pyrimidinyloxycarbonyl.

[0214] The term "oxo" refers to an oxygen atom which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, or a sulfoxide or sulfone when attached to sulfur.

[0215] Those skilled in the art will readily understand and appreciate that the compounds and compositions disclosed herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state depending on the environment in which the compound or composition is placed. Thus, as used herein, it is assumed that the structures disclosed herein may have certain functional groups, such as OH, SH, or NH, in a protonated or deprotonated state. The disclosure of the present invention is intended to cover the disclosed compounds and compositions regardless of their protonation state based on the pH of the environment, as will be readily understood by those skilled in the art.

[0216] II. RNAi Agents of the Disclosure Described herein are RNAi agents that inhibit expression of the ABLIM3 gene. In one embodiment, the RNAi agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting expression of the ABLIM3 gene in a cell, e.g., a cell in a subject (e.g., a mammal, e.g., a human with an ABLIM3-associated disorder, e.g., PTSD or age-related memory loss). The dsRNA comprises an antisense strand having a region of complementarity that is complementary to at least a portion of the mRNA formed upon expression of the ABLIM3 gene. The region of complementarity is no more than about 15-30 nucleotides in length. Upon contact with a cell expressing the ABLIM3 gene, the RNAi agent inhibits expression of the ABLIM3 gene (e.g., a human gene, a primate gene, or a non-primate gene) by at least 30% compared to a similar cell not contacted with the RNAi agent or an RNAi agent that is not complementary to the ABLIM3 gene. Gene expression can be assayed, for example, by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, such as immunofluorescence analysis using Western blotting or flow cytometry techniques. In one embodiment, the level of knockdown is assayed in monkey Cos-7 cells using the assay method provided in Example 2 below. In another embodiment, the level of knockdown is assayed in human BE(2)-C cells. In some embodiments, the level of knockdown is assayed in DU145 cells.

[0217] dsRNA comprises two RNA strands that are complementary to each other and hybridize to form a duplex structure under the conditions in which the dsRNA is used. One strand of the dsRNA (the antisense strand) comprises a complementary region that is substantially or completely complementary to the target sequence. The target sequence can be obtained from the sequence of a hotspot in mRNA formed during expression of the ABLIM3 gene. The other strand (the sense strand) comprises a region complementary to the antisense strand, so that the two strands hybridize to form a duplex structure when combined under suitable conditions. As described elsewhere herein and known in the art, the complementary sequences of dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, such that they are opposite each other on separate oligonucleotides.

[0218] Generally, the duplex structure is 15 to 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- 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 embodiments, the duplex structure is 18 to 25 base pairs in length, e.g., 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24, 20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24, or 24-25 base pairs in length, e.g., 19-21 base pairs in length. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of this disclosure.

[0219] Similarly, the region of complementarity to the target sequence may be 15 to 30 nucleotides 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, 19-29, 19-28, 19- 27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, e.g., 19-23 nucleotides in length or 21-23 nucleotides in length. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of this disclosure.

[0220] 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.

[0221] In some embodiments, the dsRNA is 15 to 23 nucleotides long, 19 to 23 nucleotides long, or 25 to 30 nucleotides long. Generally, the dsRNA is long enough to function as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNA longer than about 21 to 23 nucleotides can function as a substrate for Dicer. As those skilled in the art will recognize, the region of RNA targeted for cleavage is almost always a portion of a longer RNA molecule, often an mRNA molecule. In relevant cases, a "portion" of the mRNA target is a continuous sequence of the mRNA target that is long enough to be a substrate for RNAi-dependent cleavage (i.e., cleavage by the RISC pathway).

[0222] Those skilled in the art will appreciate that the duplex region may be a primary functional portion of a dsRNA, e.g., 15 to 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, 18-22, 18-23, 18-24, 18-25, 18-24, 18-23, 18-22, 18-21, 18-25, 18-26, 18-27, 18-28, 18-29, 18-30, 18-31, 18-32, 18-33, 18-34, 18-35, 18-36, 18-37, 18-38, 18-39, 18-40, 18-41, 18-42, 18-43, 18-44, 18-45, 18-46, 18-47, 18-48, 18-49, 18-50, 18-51, 18-52, 18- It will also be appreciated that the duplex region may be 8-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs, e.g., 19-21 base pairs. Therefore, in one embodiment, an RNA molecule or a complex of RNA molecules having a duplex region of more than 30 base pairs is a 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 a dsRNA.In another embodiment, the dsRNA is not a naturally occurring miRNA.In another embodiment, the RNAi agent useful for targeting ABLIM3 expression is not generated in target cells by cleavage of a larger dsRNA.

[0223] 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 overhang can be on the 5' end, 3' end, or both of the antisense strand or sense strand of dsRNA.

[0224] 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 the components are annealed.The individual strands of dsRNA compounds can be prepared using solution phase or solid phase organic synthesis or both.Organic synthesis has the advantage that it can easily prepare oligonucleotide chains 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.

[0225] In one embodiment, the dsRNA of the present disclosure comprises at least two nucleotide sequences, i.e., a sense strand and an antisense strand. The sense strand sequence for ABLIM3 can be selected from the group of sequences provided in Table 3 or 4, and the corresponding nucleotides of the sense strand and the antisense strand can be selected from the group of sequences in Table 3 or 4. 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 the mRNA produced upon expression of the ABLIM3 gene. Thus, in this embodiment, the dsRNA will comprise two oligonucleotides, one oligonucleotide described as the sense strand (passenger strand) in Table 3 or 4, and the second oligonucleotide described as the corresponding antisense strand (guide strand) to the sense strand in Table 3 or 4.

[0226] In one embodiment, the sequences substantially complementary to the dsRNA are contained in separate oligonucleotides, hi another embodiment, the sequences substantially complementary to the dsRNA are contained in a single oligonucleotide.

[0227] Although the sequences in Tables 3 and 4 are described as modified or conjugated sequences, it is understood that the RNA of the RNAi agent of the present disclosure, for example, the dsRNA of the present disclosure, can comprise any one of the sequences described in Tables 3 and 4, unmodified, unconjugated, or modified or conjugated differently from those described. For example, the sense strand of the agent of the present invention can be conjugated to a GalNAc ligand, but these agents can also be conjugated to a moiety that directs delivery to the CNS, such as a C16 ligand as described herein. The lipophilic ligand can be included in any of the positions provided herein.

[0228] Those skilled in the art are well aware that dsRNAs with duplex structures of about 20 to 23 base pairs, for example, 21 base pairs, are hailed as being particularly effective in inducing RNA interference [Elbashir et al., (2001) EMBO J., 20:6877-6888]. However, others have found 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 embodiment, due to the nature of the oligonucleotide sequences provided herein, the dsRNAs described herein can comprise at least one strand with a minimum length of 21 nucleotides. It can be reasonably expected that shorter duplexes, minus a few nucleotides at one or both ends, can be similarly effective compared to the dsRNAs described above. Thus, dsRNAs that have a sequence of at least 15, 16, 17, 18, 19, 20, or more consecutive nucleotides derived from one of the sequences provided herein and that differ in their ability to inhibit ABLIM3 gene expression from dsRNAs containing the full-length sequence by 10, 15, 20, 25, 30, 35, 40, 45, or 50% or less when using in vitro assays using, for example, BE(2)-C cells or DU145 cells and an RNA agent at a concentration of 10 nM, as well as PCR assays, as provided in the Examples herein, are contemplated to be within the scope of the present disclosure. In some embodiments, inhibition from dsRNAs containing the full-length sequence is measured using an in vitro assay using primary mouse hepatocytes.

[0229] In addition, the RNA agent described herein identifies the site of ABLIM3 mRNA transcript that is susceptible to RISC-mediated cleavage.Therefore, the present disclosure further features the RNAi agent that targets within this site.As used herein, if an RNAi agent promotes the cleavage of the mRNA transcript at any of the specific sites, it is said to target within the specific site of mRNA transcript.Such RNAi agent will generally comprise at least about 15 nucleotides, preferably at least 19 nucleotides, from one of the sequences provided herein, coupled with additional nucleotide sequences taken from the region adjacent to the selected sequence in ABLIM3 gene.

[0230] III. Modified RNAi Agents of the Present Disclosure In one embodiment, the RNA of the RNAi agent of the present disclosure, for example, dsRNA, is unmodified and does not contain modified nucleotides, for example, chemical modifications or conjugations known in the art and described herein.In one embodiment, the RNA of the RNAi agent of the present disclosure, for example, 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.The RNAi agent of the present disclosure in which "substantially all of the nucleotides are modified" is mostly, but not entirely, modified and may contain 5, 4, 3, 2, or unmodified nucleotides.In yet other embodiments of the present disclosure, the RNAi agent of the present disclosure may contain 5, 4, 3, 2, or 1 modified nucleotide.

[0231] Nucleic acids featured in this disclosure can be synthesized or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, reverse ligation) or 3'-end modifications (conjugation, DNA nucleotides, reverse ligation, etc.), base modifications, such as replacement with a stabilizing base, a destabilizing base, or a base that base-pairs with an expanded repertoire partner, removal of a base (abasic nucleotide) or a conjugated base, sugar modifications (e.g., at the 2' or 4' position) or sugar replacement, or backbone modifications, including modification or replacement of a phosphodiester bond. Specific examples of RNAi agents useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or that do not contain natural internucleoside linkages. Among the RNAs with modified backbone, those that do not have phosphorus atom in backbone include.For the purpose of this specification, as sometimes referred to in the art, the modified RNA that does not have phosphorus atom in its internucleoside backbone can also be considered as oligonucleoside.In some embodiments, modified RNAi agent has phosphorus atom in its internucleoside backbone.

[0232] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates, and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates; phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates; thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their analogs linked in 2'-5', and those with reverse polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. 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 the counterion for 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 linkages have sodium counterion comprises 5, 4, 3, 2 or 1 or less phosphodiester and / or phosphorothioate linkages that do not have sodium counterion.In some embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ion exists as the counterion for all of the phosphodiester and / or phosphorothioate groups present in the agent.

[0233] 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 5,286,712. No. 7, No. 5,321,131, No. 5,399,676, No. 5,405,939, No. 5,453,496, No. 5,455,233, No. 5,466,677, No. 5,476 , No. 925, No. 5,519,126, No. 5,536,821, No. 5,541,316, No. 5,550,111, No. 5,563,253, No. 5,571,799, No. 5,5 87,361, 5,625,050, 6,028,188, 6,124,445, 6,160,109, 6,169,170, 6,172,209, No. 6,239,265, No. 6,277,603, No. 6,326,199, No. 6,346,614, No. 6,444,423, No. 6,531,590, No. 6,534,639 , 6,608,035, 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029 and U.S. Reissue Patent No. RE39464, the entire contents of each of which are incorporated herein by reference.

[0234] Modified RNA backbones that do not contain phosphorus atoms have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 constituent moieties.

[0235] 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.

[0236] In another embodiment, RNA mimetics suitable for use in RNAi agents are contemplated, in which both the sugar and internucleoside linkages, i.e., backbone, of nucleotide units are replaced with alternative groups. Nucleobase units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimetic known to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. Nucleobases are retained and are directly or indirectly linked to the aza nitrogen atom 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.

[0237] Some embodiments featured in the present disclosure include RNAs with phosphorothioate backbones and heteroatom backbones, particularly --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- (known as methylene (methylimino) or MMI backbones), --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- of the above-referenced U.S. Pat. No. 5,489,677, and oligonucleosides with amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have the morpholino backbone structure of the above-referenced U.S. Pat. No. 5,034,506. The natural phosphodiester backbone can be represented as --OP(O)(OH)--OCH2--.

[0238] Modified RNAs may also contain one or more substituted sugar moieties. The RNAi agents, e.g., dsRNAs, featured herein may contain one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl, or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 It can be alkenyl and 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 has at the 2' position one of the following: C1 to C 10The modification may include one of alkyl, substituted alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH, OCN, Cl, Br, CN, CF, OCF, SOCH, SOCH, ONO, NO, N, NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, interfering substance, group for improving the pharmacokinetic properties of RNAi agents or group 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(CH3)2. Further exemplary modifications include 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both R and S isomers in these three families), 2'-alkoxyalkyl, and 2'-NMA (N-methylacetamide).

[0239] 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 at the 3'-position of the sugar on the 3'-terminal nucleotide or in a 2'-5'-linked dsRNA and at the 5'-position of the 5'-terminal nucleotide. RNAi agents can also have sugar mimetics, such as cyclobutyl moieties instead of pentofuranosyl sugars. 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, certain of which are commonly owned with the present application, the entire contents of each of which are incorporated herein by reference.

[0240] The RNAi agent of the present disclosure may also include nucleobase (often simply referred to in the art as "base") modifications or substitutions.As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), the pyrimidine bases thymine (T), cytosine (C) and uracil (U).Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil ... These include 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and adenine, 8-azaguanine and adenine, 7-deazaguanine and adenine, and 3-deazaguanine and adenine.Further modified nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008, those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Certain of these modified nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present disclosure.These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-methylcytosine substitution has been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST, and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and is an exemplary base substitution, particularly when combined with 2'-O-methoxyethyl sugar modification.

[0241] Representative U.S. patents that teach the preparation of certain of the above-mentioned modified nucleobases, as well as other modified nucleobases, include, but are not limited to, the above-mentioned U.S. Patent Nos. 3,687,808, 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, and 5,587,469. , 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.

[0242] The RNAi agents of the present disclosure can also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by bridging two atoms. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety containing a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring structure. In certain embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Thus, in some embodiments, 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 into a 3'-endo 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 known 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, see e.g., U.S. Pat. No. 8,399,845). 278,283), 4'-CH2-N(OCH3)-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,425), 4'-CH2-ON(CH3)-2' (see, e.g., U.S. Patent Publication No. 2004 / 0171570), 4'-CH2-N(R)-O-2' (wherein R is H, C1-C12 alkyl, or a protecting group) (see, e.g., U.S. Pat. No. 7,427,672), 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134) and 4'-CH2-C(-CH2)-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,426), the entire contents of each of which are incorporated herein by reference.

[0243] 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, Nos. 7,399,845, 7,427,672, 7,569,686, 7,741,457, 8,022,193, 8,030,467, 8,278,425, 8,278,426, 8,278,283, US2008 / 0039618 and US2009 / 0012281, the entire contents of each of which are incorporated herein by reference.

[0244] For example, any of the above bicyclic nucleosides can be prepared with one or more stereochemical sugar configurations including α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0245] RNAi agents of the present disclosure can also be modified to include one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid containing a bicyclic sugar moiety containing a 4'-CH(CH3)-0 to 2' bridge. In one embodiment, the constrained ethyl nucleotide is in the S conformation and is referred to herein as an "S-cEt."

[0246] The RNAi agents of the present disclosure may also contain one or more "conformation-restricting nucleotides" ("CRNs"). A CRN is a nucleotide analogue with a linker connecting the C2' and C4' carbons of ribose or the C3 and C5' carbons of ribose. The CRN locks the ribose ring into a stable conformation and increases hybridization affinity to mRNA. The linker is of sufficient length to position the oxygen in an optimal position for stability and affinity, resulting in less ribose ring puckering.

[0247] 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.

[0248] In some embodiments, the RNAi agent of the present disclosure comprises one or more monomers that are UNA (non-locked nucleic acid) nucleotides. UNA is a non-locked acyclic nucleic acid, and any of the sugar bonds have been removed to form a non-locked "sugar" residue. In one example, UNA also encompasses a monomer in which the bond between C1'-C4' (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) has been removed [see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference].

[0249] Representative U.S. publications that teach the preparation of UNAs include, but are not limited to, U.S. Pat. Nos. 8,314,227 and U.S. Patent Publication Nos. 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020, the entire contents of each of which are incorporated herein by reference.

[0250] The RNAi agent of the present disclosure may also contain one or more "cyclohexene nucleic acids" or ("CeNA"). CeNA is a nucleotide analog in which the furanose moiety of DNA is replaced with a cyclohexene ring. Incorporating cyclohexenyl nucleosides into a DNA strand increases the stability of DNA / RNA hybrids. CeNA is stable against degradation in serum, and CeNA / RNA hybrids can activate Escherichia coli (E. coli) RNase H, thereby cleaving the RNA strand. (See Wang et al., Am. Chem. Soc. 2000, 122, 36, 8595-8602, incorporated herein by reference.)

[0251] Potentially stabilizing modifications to the ends of RNA molecules include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"-phosphate, inverted base dT (idT), and others. Disclosure of this modification can be found in WO2011 / 005861.

[0252] Other modifications of the RNAi agent of the present disclosure include 5' phosphate or 5' phosphate mimic, for example, 5' terminal phosphate or phosphate mimic on the antisense strand of the RNAi agent. Suitable phosphate mimics are disclosed, for example, in US2012 / 0157511, the entire contents of which are incorporated herein by reference. In one embodiment, the double-stranded RNAi agent of the present invention further comprises a 5' phosphate or 5' phosphate mimic at the 5' nucleotide of the antisense strand. In another embodiment, the double-stranded RNAi agent further comprises a 5' phosphate mimic at the 5' nucleotide of the antisense strand. In certain embodiments, the 5' phosphate mimic is 5'-vinyl phosphonate (5'-VP). In one embodiment, the phosphate mimic is 5'-cyclopropyl phosphonate (VP). In some embodiments, the 5' end of the antisense strand of the double-stranded iRNA agent does not contain 5'-vinyl phosphonate (VP).

[0253] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of deoxynucleotides, 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 with a 2' phosphate, such as G2p, C2p, A2p, or U2p, and vinylphosphonate nucleotides; and combinations thereof. In other embodiments, each of the duplexes in Tables 3 and 4 may be individually modified to provide another double-stranded iRNA agent of the present 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 between the three 3'-terminal nucleotides with phosphorothioate internucleotide linkages. That is, the following formula: 5'-N1-...-N n-2 N n-1 N n L96 3' The three 3' terminal nucleotides (N) of the sense sequence of 5'-N1-...-N n-2 sN n-1 sN n 3' while leaving the antisense sequence unchanged, resulting in another double-stranded iRNA agent of the present disclosure.

[0254] A. Modified RNAi Agents Containing Motifs of the Disclosure In certain aspects of the present disclosure, the double-stranded RNAi agent of the present disclosure includes an agent having chemical modifications, for example, as disclosed in WO2013 / 075035, the entire contents of which are incorporated herein by reference.As shown herein and in WO2013 / 075035, excellent results can be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense or antisense strand of the RNAi agent at or near the cleavage site.In some embodiments, the sense and antisense strands of the RNAi agent can be otherwise completely modified.The introduction of these motifs disrupts the modification pattern of the sense or antisense strand, if present.The RNAi agent can also be conjugated with a lipophilic ligand, for example, a C16 ligand on the sense strand.The RNAi agent can also be modified, for example, with (S)-glycol nucleic acid (GNA) modification at one or more residues of the antisense strand.The resulting RNAi agent exhibits excellent gene silencing activity.

[0255] Thus, the present disclosure provides double-stranded RNAi agents capable of inhibiting expression of a target gene (i.e., the ABLIM3 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. For example, each strand can be 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.

[0256] 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 another embodiment, the duplex region is 19-21 nucleotide pairs in length.

[0257] In one embodiment, the RNAi agent may contain one or more overhang regions or capping groups at the 3', 5', or both ends of one or both strands. The overhangs may be 1 to 6 nucleotides in length, e.g., 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides in length. In another 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 staggered. 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 joined by additional bases, e.g., to form a hairpin, or by other non-basic linkers.

[0258] In one embodiment, the nucleotides in the overhang region of an RNAi agent can each independently be a modified or unmodified nucleotide, including, but not limited to, a 2'-sugar modified, e.g., 2-F, 2'-O-methyl, thymidine (T), and any combination thereof.

[0259] For example, TT can be an overhang sequence at either end on either strand. The overhang can form a mismatch with the target mRNA, or can be complementary to the targeted gene sequence, or can be another sequence.

[0260] 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(s) contain two nucleotides with 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.

[0261] dsRNAi 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 located at the 5' end of the antisense strand (i.e., the 3' end of the sense strand), or vice versa.Generally, the antisense strand of RNAi has a nucleotide overhang at the 3' end, and its 5' end is blunt.Without wishing to be bound by theory, the blunt end at the 5' end of the asymmetric antisense strand and the 3' end overhang of the antisense strand are favorable for the guide strand loading into RISC process.

[0262] In one embodiment, the RNAi agent is blunt-ended at both ends and is 19 nucleotides in length, and the sense strand contains at least one motif with three 2'-F modifications on three consecutive nucleotides at positions 7, 8, and 9 from the 5' end. The antisense strand contains at least one motif with three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0263] In another embodiment, the RNAi agent is blunt-ended at both ends and is 20 nucleotides in length, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0264] In yet another embodiment, the RNAi agent is blunt-ended at both ends and is 21 nucleotides in length, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0265] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, 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.

[0266] When a two-nucleotide overhang is at the 3'-end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the three 3'-nucleotides at the end of the antisense strand, where two of the three nucleotides are overhanging nucleotides and the third nucleotide is the next paired nucleotide after the overhanging nucleotide. In one embodiment, the RNAi agent further has 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 the motif, is a modified nucleotide. In one embodiment, each residue is independently modified with 2'-O-methyl or 2'-fluoro, for example, in the alternating motif. The RNAi agent may further comprise a ligand (e.g., a lipophilic ligand, optionally a C16 ligand).

[0267] In one embodiment, the RNAi agent comprises a sense and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, and starting from the 5'-terminal nucleotide (position 1), positions 1-23 of the first strand comprise at least 8 ribonucleotides; and the antisense strand is 36-66 nucleotide residues in length, and starting from the 3'-terminal nucleotide, comprises at least 8 ribonucleotides at positions that pair with positions 1-23 of the sense strand to form a duplex, wherein at least the 3'-terminal nucleotide of the antisense strand is not paired with the sense strand, and up to six consecutive 3'-terminal nucleotides are not paired with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides. The double-stranded nucleic acid comprises at least one nucleotide sequence consisting of three 2'-F modifications on three consecutive nucleotides, thereby forming a 10-30 nucleotide single-stranded 5' overhang, at least the 5'- and 3'-terminal nucleotides of the sense strand being base-paired with nucleotides in the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands, the antisense strand being sufficiently complementary to the target RNA along at least 19 ribonucleotides of the length of the antisense strand, such that when the double-stranded nucleic acid is introduced into a mammalian cell, target gene expression is reduced, 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, and the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0268] In one embodiment, the dsRNAi agent comprises a sense and an antisense strand, the dsRNAi agent comprising a first strand having a length of at least 25 and at most 29 nucleotides, and a second strand having a length of at most 30 nucleotides and having at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, the 3' end of the first strand and the 5' end of the second strand 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 long, the second strand being sufficiently complementary to a target mRNA along the length of at least 19 nucleotides 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 producing siRNAs comprising the 3' end of the second strand, thereby reducing target gene expression in a mammal. Optionally, the RNAi agent may further comprise a ligand.

[0269] 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 the motifs occurring at the cleavage site in the sense strand.

[0270] 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 the motifs occurring at or near the cleavage site in the antisense strand.

[0271] For RNAi agents with a duplex region 17-23 nucleotides long, the cleavage sites in the antisense strand are typically approximately positions 10, 11, and 12 from the 5' end. Thus, three identical modification motifs may occur in the antisense strand at positions 9, 10, and 11; positions 10, 11, and 12; positions 11, 12, and 13; positions 12, 13, and 14; or positions 13, 14, and 15, with the numbers starting from the first nucleotide from the 5' end of the antisense strand, or the numbers starting from the first paired nucleotide in the duplex region from the 5' end of the antisense strand. The cleavage site in the antisense strand may also vary depending on the length of the duplex region of the RNAi agent from the 5' end.

[0272] The sense strand of RNAi agent can contain at least one motif of three identical modifications on three consecutive nucleotides at the site of strand breakage, and antisense strand can have at least one motif of three identical modifications on three consecutive nucleotides at or near the site of strand breakage.When sense strand and antisense strand form dsRNA duplex, sense strand and antisense strand can be arranged 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 pairs.Alternatively, at least two nucleotides can overlap, or all three nucleotides can overlap.

[0273] In one embodiment, the sense strand of an RNAi agent may contain two or more motifs of three identical modifications on three consecutive nucleotides. The first motif may occur at or near the cleavage site of the strand, and the other motifs may be wing modifications. As used herein, the term "wing modification" refers to a motif occurring in another part of the strand, away from a motif at or near the cleavage site 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 chemistry of the motifs is distinct from each other, and when the motifs are separated by one or more nucleotides, the chemistry 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.

[0274] Similar to the sense strand, the antisense strand of an RNAi agent may contain two or more motifs of three identical modifications on three consecutive nucleotides, with at least one of the motifs occurring at or near the site of strand cleavage.The antisense strand may also contain one or more wing modifications in the same sequence as the wing modifications that may be present on the sense strand.

[0275] 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.

[0276] 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.

[0277] When the sense and antisense strands of an RNAi agent each contain at least one wing modification, the wing modifications can fall at the same end of the duplex region and have an overlap of 1, 2, or 3 nucleotides.

[0278] When the sense or antisense strand of an RNAi agent each contains at least two wing modifications, the sense and antisense strands can be arranged such that two modifications from one strand each occupy one end of the duplex region, with an overlap of 1, 2, or 3 nucleotides; two modifications from one strand each occupy the other end of the duplex region, with an overlap of 1, 2, or 3 nucleotides; and two modifications from one strand occupy either side of the lead motif, with an overlap of 1, 2, or 3 nucleotides in the duplex region.

[0279] In one embodiment, the RNAi agent contains mismatch(es) or combinations thereof within the double strand with the target. Mismatches can occur in overhang regions or double-stranded regions. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., the free energy of association or dissociation of a particular pairing; the simplest approach is to examine pairs on an individual basis, but next-neighbor analysis or similar analysis can also be used). In terms of promoting dissociation, A:U is preferred over G:C, G:U is preferred over G:C, and I:C is preferred over G:C (I=inosine). Mismatches, such as non-canonical pairings or those other than canonical pairings (as described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings, and pairings involving universal bases are preferred over canonical pairings.

[0280] In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex region from the 5' end of the antisense strand independently selected from the group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical pairings or other than canonical pairings or pairings including universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0281] In one embodiment, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U and dT.Alternatively, at least one of the first 1, 2 or 3 base pairs in the double-stranded region from the 5' end of the antisense strand is an AU base pair.For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0282] In another embodiment, the nucleotide at the 3' end of the sense strand is deoxythymidine (dT). In another embodiment, the nucleotide at the 3' end of the antisense strand is deoxythymidine (dT). In one embodiment, there is a short sequence of deoxythymidine nucleotides, for example, two dT nucleotides at the 3' end of the sense or antisense strand.

[0283] In one embodiment, the sense strand sequence has formula (I): 5'n p -N a -(XXX) i -N b -YYY -N b -(ZZZ) j -N a -n q 3' (I) [In the formula, i and j each independently represent 0 or 1; p and q each independently represent 0 to 6; each N a independently represent oligonucleotide sequences comprising 0 to 25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each Nb independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p and n q independently represent an overhanging nucleotide; Nb and Y do not have the same modification, and XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. Preferably, all of YYY are 2'-F modified nucleotides.

[0284] In one embodiment, N a or N b includes alternating pattern modifications.

[0285] In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. For example, if the RNAi agent has a duplex region 17-23 nucleotides in length, the YYY motif can occur at or near the cleavage site of the sense strand (e.g., can occur at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13), with the number starting from the first nucleotide from the 5' end, or, optionally, the number starting from the first paired nucleotide within the duplex region from the 5' end.

[0286] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or i and j are both 1. Thus, the sense strand has the following formula: 5' n p -N a -YYY-N b -ZZZ-N a -n q 3' (Ib), 5' n p -N a -XXX-N b -YYY-N a -n q 3' (Ic), or 5' n p-N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' (Id) It can be expressed as:

[0287] 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.

[0288] each N a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0289] 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 may independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0290] 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 may independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0291] Each of X, Y and Z may be the same as or different from one another.

[0292] In other embodiments, i is 0, j is 0, and the sense strand has the formula: 5' n p -Na -YYY-N a -n q 3' (Ia) It can be expressed as:

[0293] When the sense strand is represented by formula (Ia), each N a may independently comprise an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0294] In one embodiment, the antisense strand sequence of the RNAi has formula (Ie): 5' n q’ -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p ' 3' (IE) [In the formula, k and l each independently represent 0 or 1; p' and q' each independently represent 0 to 6; each N a each independently represents an oligonucleotide sequence comprising 0 to 25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each N b independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides, each n p ' and n q ' independently represent an overhanging nucleotide; N b ' and Y' do not have the same modification, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides. It can be expressed as:

[0295] In one embodiment, N a ' or N b' includes alternating pattern modifications.

[0296] The Y'Y'Y' motif occurs at or near the cleavage site of the sense strand. For example, if the RNAi agent has a duplex region 17-23 nucleotides long, the Y'Y'Y' motif can occur at positions 9, 10, 11, 10, 11, 12, 11, 12, 13, 12, 13, 14, or 13, 14, 15 of the antisense strand, the numbers starting from the first nucleotide from the 5' end, or, where appropriate, the numbers starting from the first paired nucleotide in the duplex region from the 5' end. Preferably, the Y'Y'Y' motif occurs at positions 11, 12, 13.

[0297] In one embodiment, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0298] 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.

[0299] 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' (Ig), 5' n q’ -N a '-Y'Y'Y'-N b '-X'X'X'-n p’ 3' (Ih), or 5' n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'-N a '-n p’ 3' (II) It can be expressed as:

[0300] When the antisense strand is represented by formula (Ig), Nb ' represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0301] When the antisense strand is represented by formula (Ih), 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 represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0302] When the antisense strand is represented by formula (Ii), 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.

[0303] In other embodiments, k is 0, l is 0, and the antisense strand has the formula: 5' n p’ -N a’ -Y'Y'Y'- N a’ -n q’ 3' (If) It can be expressed as:

[0304] When the antisense strand is represented by the formula (If), each N a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0305] Each of X', Y' and Z' may be the same as or different from one another.

[0306] 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.

[0307] In one embodiment, the sense strand of the RNAi agent may contain a YYY motif occurring at positions 9, 10, and 11 of the strand when the duplex region is 21 nt, the numbers starting from the first nucleotide from the 5' end, or, where appropriate, the numbers may start from the 5' end with the first paired nucleotide in the duplex region, and Y represents a 2'-F modification. The sense strand may further contain a XXX motif or a ZZZ motif as a wing modification at the opposite end of the duplex region, where XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0308] In one embodiment, the antisense strand may contain a Y'Y'Y' motif occurring at positions 11, 12, 13 of the strand, where the number starts from the first nucleotide from the 5' end, or, where appropriate, the number may start from the 5' end with the first paired nucleotide in the duplex region, and Y' represents a 2'-O-methyl modification. The antisense strand may further contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the duplex region, where X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0309] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic) and (Id) forms a duplex with the antisense strand represented by any one of the formulas (If), (Ig), (Ih) and (Ii), respectively.

[0310] Thus, an RNAi agent for use in the methods of the 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 (Ij): 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' (Ij) [In the formula, i, j, k, and l each independently represent 0 or 1; p, p', q and q' each independently represent 0 to 6; each N a and N a ’ independently represent oligonucleotide sequences comprising 0 to 25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each N b and N b ’ independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p ',n p , n q ' and n q independently represent overhanging nucleotides, each of which may or may not be present; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides. is expressed by

[0311] 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 and k is 0 and l is 1, or k and l are both 0, or k and l are both 1.

[0312] An exemplary combination of sense and antisense strands that form an RNAi duplex has the following formula: 5' n p - N a -YYY -N a -n q 3' 3' n p ’ -N a ’ -Y'Y'Y' -N a ’ n q ’ 5' (Ik) 5' n p -N a -YYY -N b -ZZZ -N a -n q 3' 3' n p ’ -N a ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a ’ n q ’ 5' (Il) 5' n p -N a -XXX-N b-YYY-N a -n q 3' 3' n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N a ’ -n q ’ 5' (Im) 5' n p -N a -XXX -N b -YYY -N b - ZZZ -N a -n q 3' 3' n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a -n q ’ 5' (In) Includes.

[0313] When the RNAi agent is represented by formula (Ik), each N a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0314] When the RNAi agent is represented by formula (II), each N b independently represent an oligonucleotide sequence containing 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0315] When the RNAi agent is represented by formula (Im), 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.

[0316] When an RNAi agent is represented by the formula (In), each N b , N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 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.

[0317] In one embodiment, when the RNAi agent is represented by formula (In), N a The modification is a 2'-O-methyl or a 2'-fluoro modification. In another embodiment, when the RNAi agent is represented by formula (In), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p In yet another embodiment, when the RNAi agent is represented by formula (In), N' is linked to the adjacent nucleotide via a phosphorothioate linkage. a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n pIn another embodiment, when the RNAi agent is represented by the formula (In), N' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand is conjugated to one or more C16 (or related) moieties attached by a divalent or trivalent branched linker (described below). a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p ' are linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic, e.g., C16 (or related) moieties, which may be attached by a divalent or trivalent branched linker.

[0318] In one embodiment, when the RNAi agent is represented by formula (Ik), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p ' are linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic, e.g., C16 (or related) moieties attached by a divalent or trivalent branched linker.

[0319] In one embodiment, the RNAi agent is a multimer containing at least two duplexes represented by formula (Ij), (Ik), (Il), (Im) and (In), and the duplexes are connected by a linker. The linker may be cleavable or non-cleavable. The multimer may further comprise a ligand. Each duplex may target the same gene, or may target two different genes, or each duplex may target the same gene at two different target sites.

[0320] In one embodiment, the RNAi agent is a multimer that contains 3, 4, 5, 6 or more double strands represented by formula (Ij), (Ik), (Il), (Im) and (In), and the double strands are connected by a linker. The linker may be cleavable or non-cleavable. The multimer may further comprise a ligand. Each double strand may target the same gene, or may target two different genes, or each double strand may target the same gene at two different target sites.

[0321] In one embodiment, two RNAi agents represented by formula (Ij), (Ik), (Il), (Im) and (In) are linked to each other at the 5' end, and one or both of the 3' ends may be conjugated to a ligand. Each of the agents may target the same gene, or may target two different genes, or each of the agents may target the same gene at two different target sites.

[0322] Various publications describe the multimeric RNAi agent that can be used in the method of the present disclosure.Such publications include WO2007 / 091269, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520 and US7858769, each of whose entire contents is incorporated herein by reference.

[0323] 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 5'-vinyl phosphonate modified nucleotides of the present disclosure have the following structure:

[0324] [ka] wherein X is O or S; R is hydrogen, hydroxy, fluoro, or C 1~20alkoxy (e.g., methoxy or n-hexadecyloxy); R 5’ is =C(H)-P(O)(OH)2, and the double bond between the C5' carbon and R5' is in the E or Z configuration (e.g., the E configuration); B is a nucleobase or a modified nucleobase, where B may be adenine, guanine, cytosine, thymine, or uracil. It has.

[0325] 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) 2 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.

[0326] The vinyl phosphonate of the present disclosure can be attached to either the antisense or sense strand of a dsRNA of the present disclosure. In certain embodiments, the vinyl phosphonate of the present disclosure is attached to the antisense strand of a dsRNA at the 5'-end of the antisense strand of the dsRNA, as appropriate. The dsRNA agent can include a phosphorus-containing group at the 5'-end of the sense or antisense strand. The 5'-end phosphorus-containing group can be 5'-end phosphate (5'-P), 5'-end phosphorothioate (5'-PS), 5'-end phosphorodithioate (5'-PS2), 5'-end vinyl phosphonate (5'-VP), 5'-end methyl phosphonate (MePhos), or 5'-deoxy-5'-C-malonyl. When the 5'-end phosphorus-containing group is 5'-end vinyl phosphonate (5'-VP), the 5'-VP can be a 5'-E-VP isomer (i.e., trans-vinyl phosphonate,

[0327] [ka] ), 5'-Z-VP isomer (i.e., cis-vinyl phosphonate,

[0328] [ka] ), or a mixture thereof.

[0329] Vinyl phosphate modifications are also contemplated for the compositions and methods of the present disclosure. An exemplary vinyl phosphate structure is as follows:

[0330] [ka]

[0331] For example, if the phosphate mimic is 5'-vinyl phosphate, the 5' terminal nucleotide can have an immediately adjacent structure in which the phosphonate group is replaced by a phosphate.

[0332] i.Thermal destabilization modification In certain embodiments, dsRNA molecules can be optimized for RNA interference by incorporating thermally destabilizing modifications into the seed region of antisense strand.As used herein, " seed region " refers to the 2-9 position of the 5' end of the strand being referred to.For example, thermally destabilizing modifications can be incorporated into the seed region of antisense strand to reduce or inhibit off-target gene silencing.

[0333] The term "thermally destabilizing modification(s)" refers to a modification that is lower than the melting temperature (T m ) than the overall melting temperature (T m For example, thermally destabilizing modification(s) may result in a dsRNA having a T mcan be decreased by 1-4° C., for example, 1, 2, 3, or 4 degrees Celsius. Also, the term "thermally destabilized nucleotide" refers to a nucleotide that contains one or more thermally destabilizing modifications.

[0334] It has been discovered that dsRNAs having an antisense strand containing at least one duplex thermally destabilizing modification within the first 9 nucleotide positions counting from the 5' end of the antisense strand have reduced off-target gene silencing activity. Thus, in some embodiments, the antisense strand contains at least one (e.g., 1, 2, 3, 4, 5, or more) duplex thermally destabilizing modification within the first 9 nucleotide positions of the 5' region of the antisense strand. In some embodiments, one or more duplex thermally destabilizing modifications are located in positions 2-9, or preferably positions 4-8, from the 5' end of the antisense strand. In some further embodiments, the duplex thermally destabilizing modification(s) are located in positions 6, 7, or 8 from the 5' end of the antisense strand. In yet some further embodiments, the duplex thermally destabilizing modification is located in position 7 from the 5' end of the antisense strand. In some embodiments, the duplex thermally destabilizing modification is located in positions 2, 3, 4, 5, or 9 from the 5' end of the antisense strand.

[0335] 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 non-locked nucleic acids (UNAs) or glycol nucleic acids (GNAs).

[0336] Exemplary abasic modifications include, but are not limited to, the following:

[0337] [ka] wherein R=H, Me, Et, or OMe; R′=H, Me, Et, or OMe; and R″=H, Me, Et, or OMe.

[0338] [ka] wherein B is a modified or unmodified nucleobase. Examples include:

[0339] Exemplary sugar modifications include, but are not limited to, the following:

[0340] [ka] wherein B is a modified or unmodified nucleobase. Examples include:

[0341] In some embodiments, the thermally destabilizing modification of the duplex is one of the following:

[0342] [ka] where B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic. is selected from the group consisting of:

[0343] In some embodiments, the thermally destabilizing modification of the duplex is one of the following: [ka] wherein B is a modified or unmodified nucleobase and the asterisk represents either R, S or racemic (e.g., S). is selected from the group consisting of:

[0344] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, e.g., in which any of the bonds between the ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') are absent, or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4'), independently or in combination, is absent from the nucleotide. In some embodiments, an acyclic nucleotide is

[0345] [ka] wherein B is a modified or unmodified nucleobase and R 1 and R 2 are independently H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. The term "UNA" refers to a non-locked acyclic nucleic acid in which any of the sugar linkages have been removed, forming a non-locked "sugar" residue. In one example, UNA also encompasses a monomer in which the C1'-C4' bond (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar has been removed [see Mikhailov et al., Tetrahedron Letters, 26 (17): 2059 (1985); and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), which are incorporated herein by reference in their entireties]. Acyclic derivatives offer greater backbone flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be linked via 2'-5' or 3'-5' linkages.

[0346] The term "GNA" refers to glycol nucleic acid, a polymer similar to DNA or RNA, but differing in the composition of its "backbone" in that it is made up of repeating glycerol units linked by phosphodiester bonds:

[0347] [ka]

[0348] The thermally destabilizing modification of the double strand can be a mismatch (i.e., non-complementary base pair) between the thermally destabilizing nucleotide and the opposite nucleotide in the opposite strand of the dsRNA duplex.Exemplary mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or combinations thereof.Other mismatch base pairings known in the art are also suitable for the present invention.Mismatches can occur between nucleotides that are either naturally occurring or modified nucleotides, that is, mismatch base pairing can occur between the nucleobases derived from each nucleotide independently of the modification on the ribose sugar of the nucleotide.In certain embodiments, the dsRNA molecule contains at least one nucleobase in mismatch pairing that is a 2'-deoxynucleobase, for example, the 2'-deoxynucleobase is in the sense strand.

[0349] In some embodiments, the duplex thermally destabilizing modification in the seed region of the antisense strand is a nucleotide that has impaired WCH bonding with a complementary base on the target mRNA, such as:

[0350] [ka] Includes.

[0351] Many more examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA) and mismatch modifications are described in detail in WO2011 / 133876, which is incorporated herein by reference in its entirety.

[0352] Thermally destabilizing modifications can also include universal base and phosphate modifications that have reduced or eliminated ability to form hydrogen bonds with opposing bases.

[0353] In some embodiments, the thermal destabilizing modification of duplex comprises the nucleotide with non-canonical base, for example but not limited to, the nucleobase modification that the ability to form hydrogen bond with the base in opposite strand is impaired or completely lost.These nucleobase modifications have been evaluated for the destabilization of the central region of dsRNA duplex, as described in WO2010 / 0011895, the entirety of which is incorporated herein by reference.Exemplary nucleobase modifications include:

[0354] [ka] There is.

[0355] In some embodiments, the duplex thermally destabilizing modifications in the seed region of the antisense strand include one or more α-nucleotides that are complementary to bases on the target mRNA, such as the following:

[0356] [ka] wherein R is H, OH, OCH, F, NH, NHMe, NMe, or O-alkyl. Includes:

[0357] Exemplary phosphate modifications known to reduce the thermal stability of dsRNA duplexes compared to native phosphodiester linkages include:

[0358] [ka] There is.

[0359] 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.

[0360] As those skilled in the art will recognize, considering that the functional role of nucleobase defines the specificity of the RNAi agent of the present disclosure, nucleobase modification can be carried out in various ways as described herein, for example, for the purpose of enhancing on-target effect against off-target effect, for example, for introducing destabilizing modifications into the RNAi agent of the present disclosure, but the range of modifications available and generally present on the RNAi agent of the present disclosure tends to be greater for non-nucleobase modifications, for example, modifications to the sugar group or phosphate backbone of polyribonucleotide.Such modifications are described in more detail in other sections of this disclosure, and are expressly intended for the RNAi agent of the present disclosure that has either natural nucleobase or modified nucleobase, as described above or elsewhere herein.

[0361] In addition to the antisense strand that contains thermal destabilizing modifications, dsRNA can also contain one or more stabilizing modifications.For example, dsRNA can contain at least two (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) stabilizing modifications.Without being limited, all stabilizing modifications can be present in one strand.In some embodiments, both sense and antisense strands contain at least two stabilizing modifications.Stabilizing modifications can occur at any nucleotide of sense strand or antisense strand.For example, stabilizing modifications can occur at any nucleotide on sense strand or antisense strand, and each stabilizing modification can occur in an alternating pattern on sense strand or antisense strand, or both sense strand and antisense strand contain stabilizing modifications in an alternating pattern.The alternating pattern of stabilizing modifications on sense strand can be the same or different from that of antisense strand, and the alternating pattern of stabilizing modifications on sense strand can have a shift compared to the alternating pattern of stabilizing modifications on antisense strand.

[0362] In some embodiments, the antisense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) stabilizing modifications. Without limitation, the stabilizing modifications in the antisense strand can be located at any position. 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.

[0363] In some embodiments, antisense strand comprises at least one stabilizing modification adjacent to destabilizing modification.For example, stabilizing modification can be at the 5'-end or 3'-end of destabilizing modification, that is, at the nucleotide of position -1 or +1 from the position of destabilizing modification.In some embodiments, antisense strand comprises stabilizing modification at each of the 5'-end and 3'-end of destabilizing modification, that is, at the nucleotide of position -1 and +1 from the position of destabilizing modification.

[0364] 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.

[0365] In some embodiments, the sense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) stabilizing modifications. Without limitation, the stabilizing modifications in the sense strand can be located at any position. In some embodiments, the sense strand comprises stabilizing modifications at positions 7, 10, and 11 from the 5' end. In some other embodiments, the sense strand comprises stabilizing modifications at positions 7, 9, 10, and 11 from the 5' end. In some embodiments, the sense strand comprises stabilizing modifications at positions opposite or complementary to positions 11, 12, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some other embodiments, the sense strand comprises stabilizing modifications at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some embodiments, the sense strand comprises blocks of two, three, or four stabilizing modifications.

[0366] 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.

[0367] Exemplary thermally stabilizing modifications include, but are not limited to, 2'-fluoro modifications. Other thermally stabilizing modifications include, but are not limited to, LNA.

[0368] In some embodiments, the dsRNA of the present disclosure comprises at least four (for example, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoro nucleotides. Without being limited thereto, all 2'-fluoro nucleotides may be present in one strand. In some embodiments, both sense and antisense strands comprise at least two 2'-fluoro nucleotides. 2'-fluoro modification can occur at any nucleotide of sense strand or antisense strand. For example, 2'-fluoro modification can occur at any nucleotide on sense strand or antisense strand, and each 2'-fluoro modification can occur in an alternating pattern on sense strand or antisense strand, or both sense strand and antisense strand contain 2'-fluoro modification in an alternating pattern. The alternating pattern of 2'-fluoro modification on sense strand can be the same or different from that of antisense strand, and the alternating pattern of 2'-fluoro modification on sense strand can have a shift compared to the alternating pattern of 2'-fluoro modification on antisense strand.

[0369] In some embodiments, the antisense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modifications in the antisense strand can be present at any position. In some embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 8, 9, 14 and 16 from the 5' end. In some other embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 14 and 16 from the 5' end. In yet some other embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 14 and 16 from the 5' end.

[0370] In some embodiments, antisense strand comprises at least one 2'-fluoro nucleotide adjacent to destabilizing modification.For example, 2'-fluoro nucleotide can be at the 5' end or 3' end of destabilizing modification, that is, at the nucleotide of position -1 or +1 from the position of destabilizing modification.In some embodiments, antisense strand comprises 2'-fluoro nucleotide at each of the 5' end and 3' end of destabilizing modification, that is, at position -1 and +1 from the position of destabilizing modification.

[0371] 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.

[0372] In some embodiments, the sense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modifications in the sense strand can be present at any position. In some embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 7, 10 and 11 from the 5' end. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 7, 9, 10 and 11 from the 5' end. In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to positions 11, 12 and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to positions 11, 12, 13 and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to positions 11, 12, 13 and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some embodiments, the sense strand comprises 2, 3 or 4 blocks of 2'-fluoro nucleotides.

[0373] In some embodiments, the sense strand does not contain 2'-fluoro nucleotides at positions opposite or complementary to thermally destabilizing modifications of the duplex in the antisense strand.

[0374] In some embodiments, a dsRNA molecule of the present disclosure comprises a 21-nucleotide (nt) sense strand and a 23-nucleotide (nt) antisense strand, wherein the antisense strand contains at least one thermally destabilized nucleotide, wherein the at least one thermally destabilized nucleotide occurs in the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), one end of the dsRNA is blunt and the other end comprises a 2-nt overhang, and the dsRNA further has at least one (e.g., 1, 2, 3, 4, 5, 6, or all 7) of the following features: The dsRNA may have: (i) two, three, four, five, or six 2'-fluoro modifications in the antisense strand; (ii) one, two, three, four, or five phosphorothioate internucleotide linkages in the antisense strand; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand has two, three, four, or five 2'-fluoro modifications; (v) the sense strand has one, two, three, four, or five phosphorothioate internucleotide linkages; (vi) the dsRNA has at least four 2'-fluoro modifications; and (vii) the dsRNA has 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.

[0375] In some embodiments, a dsRNA molecule of the present disclosure comprises a sense 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 in positions paired with positions 1 to 23 of the sense strand 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 3' single-stranded overhang of 1 to 6 nucleotides; and the 5' end of the antisense strand is 10 to 30 consecutive nucleotides that are not paired with the sense strand. wherein at least the 5'- and 3'-terminal nucleotides of the sense strand are base-paired with nucleotides in the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands; the antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the length of the antisense strand, such that when the double-stranded nucleic acid is introduced into a mammalian cell, target gene expression is reduced; and the antisense strand contains at least one thermally destabilizing nucleotide, the at least one thermally destabilizing nucleotide being in the seed region of the antisense strand (i.e., at positions 2-9 of the 5'-end of the antisense strand).For example, the thermally destabilizing nucleotide occurs between positions 14-17 at the 5' end of the sense strand and the positions opposite or complementary to them, and the dsRNA may further have 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; (vi) the dsRNA contains at least four 2'-fluoro modifications; and (vii) the dsRNA contains a duplex region 12-30 nucleotide pairs in length.

[0376] In some embodiments, a dsRNA molecule of the present disclosure comprises a sense and an antisense strand, wherein the dsRNA molecule comprises a sense strand having a length of at least 25 and at most 29 nucleotides and an antisense strand having a length of at most 30 nucleotides, wherein the sense strand comprises a modified nucleotide at position 11 from its 5' end that is susceptible to enzymatic degradation, wherein the 3' end of the sense strand and the 5' end of the antisense strand form a blunt end, wherein the antisense strand is 1-4 nucleotides longer at its 3' end than the sense strand, wherein the duplex region is at least 25 nucleotides in length, and wherein the antisense strand is sufficiently complementary to a target mRNA along at least 19 nucleotides of the length of the antisense strand, wherein the dsRNA molecule reduces target gene expression when introduced into a mammalian cell, wherein Dicer cleavage of the dsRNA preferentially yields siRNA comprising the 3' end of the antisense strand, thereby reducing target gene expression in the mammal, and wherein the antisense strand wherein at least one thermally destabilized nucleotide is in the seed region of the antisense strand (i.e., positions 2-9 at the 5' end of the antisense strand), and the dsRNA may further have at least one (e.g., one, two, three, four, five, six, or all seven) of the following features: (i) the antisense contains two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense contains two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (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.

[0377] In some embodiments, any nucleotide in the sense strand and antisense strand of dsRNA molecule can be modified.Each nucleotide can be modified with the same or different modifications, which can include one or more of non-linked phosphate oxygen or one or more of linking phosphate oxygen, one or both of the modifications of ribose sugar components, for example, the 2' hydroxyl on ribose sugar, the large-scale replacement of phosphate moiety with " dephosphorylation " linker, the modification or replacement of naturally occurring base, and the replacement or modification of ribose-phosphate backbone.

[0378] Because nucleic acids are polymers of subunits, many modifications occur at positions that are repeated within nucleic acids, such as modifications of bases or phosphate moieties or non-linked Os at phosphate moieties. In some cases, modifications occur at all target positions in nucleic acids, but in many cases, they do not occur. For example, modifications can occur only at the 3' or 5' terminal position, or only in the terminal region, for example, at the terminal nucleotide position of the chain, or at the last 2, 3, 4, 5, or 10 nucleotides. Modifications can occur in double-stranded regions, single-stranded regions, or both. Modifications can occur only in the double-stranded region of RNA, or only in the single-stranded region of RNA. For example, phosphorothioate modifications at non-linked O positions can occur only at one or both ends, or only in the terminal region, for example, at the terminal nucleotide position of the chain, or at the last 2, 3, 4, 5, or 10 nucleotides, or in double-stranded and single-stranded regions, especially at the ends. The 5' end or both ends can be phosphorylated.

[0379] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in the single-stranded overhang, for example, in the 5' or 3' overhang, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' or 5' overhang can be modified, for example, with the modifications described herein. Modifications can include, for example, the use of modifications at the 2' position of the ribose sugar with modifications known in the art, for example, the use of 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl modified deoxyribonucleotides instead of the ribosugar of the nucleobase, and modifications at the phosphate group, for example, phosphorothioate modifications. The overhang does not need to be homologous to the target sequence.

[0380] In some embodiments, each residue of sense strand and antisense strand is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy or 2'-fluoro.Strands can contain two or more modifications.In some embodiments, each residue of sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.It should be understood that these modifications are in addition to at least one thermally destabilizing modification of the double strand that exists in the antisense strand.

[0381] At least two different modifications are usually present on the sense strand and the antisense strand. These two modifications can be 2'-deoxy, 2'-O-methyl or 2'-fluoro modifications, acyclic nucleotides, etc. In some embodiments, the sense strand and the antisense strand each contain two differently modified nucleotides selected from 2'-O-methyl or 2'-deoxy. In some embodiments, each residue of the sense strand and the antisense strand is independently modified with 2'-O-methyl nucleotides, 2'-deoxy nucleotides, 2'-deoxy-2'-fluoro nucleotides, 2'-ON-methylacetamide (2'-O-NMA) nucleotides, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotides, 2'-O-aminopropyl (2'-O-AP) nucleotides, or 2'-ara-F nucleotides. It should be understood that these modifications are in addition to at least one thermally destabilizing modification of the duplex present in the antisense strand.

[0382] In some embodiments, the dsRNA molecule of the present disclosure comprises alternating pattern modifications, particularly in the B1, B2, B3, B1', B2', B3', and B4' regions.The term "alternating motif" or "alternating pattern" as used herein refers to a motif with one or more modifications, where each modification occurs at alternating nucleotides in a single strand.Alternating nucleotides can refer to one every other nucleotide or one every three nucleotides, or similar patterns.For example, if A, B, and C each represent one type of modification to nucleotide, the alternating motif can be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AABBBAAABBB..." or "ABCABCABCABC...", etc.

[0383] The types of modifications contained within the alternating motifs can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternating turns, i.e., the modifications on every other nucleotide, can be the same, but each of the sense or antisense strands can be selected from several possibilities for modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD..." or "CDCDCD...".

[0384] In some embodiments, the dsRNA molecules of the present disclosure comprise an alternating motif modification pattern on the sense strand that is shifted relative to the alternating motif modification pattern on the antisense strand. The shift can be such that the modified groups of the nucleotides of the sense strand correspond to the differently modified groups of the nucleotides of the antisense strand, or vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, the alternating motif in the sense strand may start with "ABABAB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand may start with "BABABA" from the 3'-5' end of the strand within the duplex region. As another example, the alternating motif in the sense strand may start with "AABBAABB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand may start with "BBAABBAA" from the 3'-5' end of the strand within the duplex region, resulting in a complete or partial shift in the modification pattern between the sense strand and the antisense strand.

[0385] The dsRNA molecule of the present disclosure can further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage.Phosphorothioate or methylphosphonate internucleotide linkage modification can occur at any nucleotide of sense strand or antisense strand or both at any position of chain.For example, internucleotide linkage modification can occur at any nucleotide on sense strand or antisense strand, and each internucleotide linkage modification can occur in an alternating pattern on sense strand or antisense strand, or sense strand or antisense strand contains both internucleotide linkage modifications in an alternating pattern.The alternating pattern of internucleotide linkage modification on sense strand can be the same or different from that of antisense strand, and the alternating pattern of internucleotide linkage modification on sense strand can have a shift with respect to the alternating pattern of internucleotide linkage modification on antisense strand.

[0386] In some embodiments, dsRNA molecule comprises phosphorothioate or methylphosphonate internucleotide linkage modification in overhang region.For example, overhang region comprises two nucleotides with phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides.Internucleotide linkage modification can also be performed to connect overhang nucleotide with the terminal pairing nucleotide in duplex region.For example, at least 2, 3, 4 or all overhang nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkage, and there can be additional phosphorothioate or methylphosphonate internucleotide linkage that connects overhang nucleotide with the pairing nucleotide adjacent to overhang nucleotide.For example, there can be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of which are overhang nucleotides, and the third is the pairing nucleotide adjacent to overhang nucleotide.Preferably, these terminal three nucleotides can be the 3' end of antisense strand.

[0387] In some embodiments, the sense strand of the dsRNA molecule comprises 1 to 10 blocks of 2 to 10 phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is positioned at any position in the oligonucleotide sequence, and the sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0388] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages and an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0389] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0390] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0391] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0392] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of six phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0393] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of seven phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, or 8 phosphate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0394] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, or 6 phosphate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0395] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by one, two, three, or four phosphate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0396] In some embodiments, the dsRNA molecules of this disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within the 1-10 terminal positions of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkages at one or both termini of the sense or antisense strand.

[0397] In some embodiments, dsRNA molecules of the present disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-10 of the internal region of the duplex of each of the sense or antisense strands. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides can be linked by phosphorothioate methylphosphonate internucleotide linkages at positions 8-16 of the duplex region, counting from the 5' end of the sense strand. The dsRNA molecule may further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-10 of the terminal positions.

[0398] In some embodiments, dsRNA molecules of this disclosure further comprise one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-5 of the sense strand and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and one to five within positions 18-23 (counting from the 5' end).

[0399] In some embodiments, dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 and one phosphorothioate or methylphosphonate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0400] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0401] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0402] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the antisense strand.

[0403] In some embodiments, dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0404] In some embodiments, dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 and one within positions 18-23 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the antisense strand.

[0405] In some embodiments, dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 of the sense strand (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end).

[0406] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications (counting from the 5' end) within positions 1-5 of the sense strand and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications (counting from the 5' end) within positions 18-23 of the antisense strand.

[0407] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and one within positions 18-23 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the antisense strand.

[0408] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0409] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0410] In some embodiments, a dsRNA molecule of the present disclosure further comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at position 1 and one at position 21 (counting from the 5' end) of the antisense strand.

[0411] In some embodiments, dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 (counting from the 5' end) of the antisense strand.

[0412] In some embodiments, dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0413] In some embodiments, dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 (counting from the 5' end) of the antisense strand.

[0414] In some embodiments, dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 22 and 23 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0415] In some embodiments, dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 (counting from the 5' end) of the antisense strand.

[0416] In some embodiments, the compounds of the present disclosure comprise a pattern of backbone chiral centers. In some embodiments, the general pattern of backbone chiral centers comprises at least 5 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 6 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 7 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 8 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 9 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 16 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 17 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 18 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 19 internucleotide linkages in the Sp configuration.In some embodiments, the general pattern of backbone chiral centers comprises eight or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises seven or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises six or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises five or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises four or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises three or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises two or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises one or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises eight or fewer non-chiral internucleotide linkages (phosphodiesters, as a non-limiting example). In some embodiments, the general pattern of backbone chiral centers comprises seven or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises six or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises five or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises four or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises three or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises two or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises one or fewer non-chiral internucleotide linkages.In some embodiments, the general pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration and no more than 8 nonchiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration and no more than 7 nonchiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration and no more than 6 nonchiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration and no more than 6 nonchiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration and no more than 5 nonchiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration and no more than 4 nonchiral internucleotide linkages. In some embodiments, the internucleotide linkages in the Sp configuration may be contiguous or non-contiguous. In some embodiments, the internucleotide linkages in the Rp configuration may or may not be contiguous. In some embodiments, the non-chiral internucleotide linkages may or may not be contiguous.

[0417] In some embodiments, compounds of the present disclosure include blocks that are stereochemical blocks. In some embodiments, a block is an Rp block, 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, a block is an Sp block, in that each internucleotide linkage of the block is Sp. In some embodiments, the 5'-block is an Sp block. In some embodiments, the 3'-block is an Sp block. In some embodiments, provided oligonucleotides include both Rp and Sp blocks. In some embodiments, provided oligonucleotides include one or more Rp blocks but do not include Sp blocks. In some embodiments, provided oligonucleotides include one or more Sp blocks but do not include Rp blocks. In some embodiments, provided oligonucleotides include one or more PO blocks in which each internucleotide linkage is a natural phosphate linkage.

[0418] In some embodiments, compounds of the present disclosure include a 5'-block that is an Sp block in which each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each internucleotide linkage is a modified internucleotide linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each internucleotide linkage is a phosphorothioate linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block comprises four or more nucleoside units. In some embodiments, the 5'-block comprises five or more nucleoside units. In some embodiments, the 5'-block comprises six or more nucleoside units. In some embodiments, the 5'-block comprises seven or more nucleoside units. In some embodiments, the 3'-block is an Sp block in which each sugar moiety comprises a 2'-F modification. In some embodiments, the 3'-block is an Sp block in which each internucleotide linkage is a modified internucleotide linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 3'-block is an Sp block in which each of the internucleotide linkages is a phosphorothioate linkage and each sugar moiety includes a 2'-F modification. In some embodiments, the 3'-block includes 4 or more nucleoside units. In some embodiments, the 3'-block includes 5 or more nucleoside units. In some embodiments, the 3'-block includes 6 or more nucleoside units. In some embodiments, the 3'-block includes 7 or more nucleoside units.

[0419] In some embodiments, compounds of the disclosure include a certain type of nucleoside in a region, or an oligonucleotide is followed by a particular type of internucleotide linkage, 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.

[0420] In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23, the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand), and the dsRNA may further have at least one (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 contains 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA contains at least four 2'-fluoro modifications; (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.

[0421] 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 thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand), and the dsRNA may further have at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or all 8) of the following features: (i) the antisense strand has 2, 3, 4, 5, or 6 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; (ii) the sense strand is conjugated to a ligand; (iii) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (iv) the sense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least four 2'-fluoro modifications; (vi) the dsRNA comprises a duplex region 12 to 40 nucleotide pairs in length; (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.

[0422] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., positions 2-9 at the 5' end of the antisense strand), and the dsRNA may further have 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; (ii) the antisense strand comprises two, three, four, five, or six 2'-fluoro modifications; (iii) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iv) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand contains 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA contains at least four 2'-fluoro modifications; (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.

[0423] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, 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 thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand), and the dsRNA has at least one of the following characteristics (e.g., 1, (ii) the sense strand is conjugated to a ligand; (iii) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (iv) the sense strand comprises three, four, or five phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least four 2'-fluoro modifications; (vi) the dsRNA comprises 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.

[0424] In some embodiments, the dsRNA molecule of the present disclosure comprises mismatch(es) or combinations thereof in the double strand with the target. Mismatches can occur in overhang regions or double-stranded regions. Base pairs can be ranked based on their tendency to promote dissociation or melting (for example, the free energy of association or dissociation of a particular pairing; the simplest approach is to examine pairs on an individual basis, but next-neighbor analysis or similar analysis can also be used). In terms of promoting dissociation, A:U is preferred over G:C, G:U is preferred over G:C, and I:C is preferred over G:C (I=inosine). Mismatches, such as non-canonical pairings or those other than canonical pairings (as described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings, and pairings involving universal bases are preferred over canonical pairings.

[0425] In some embodiments, the dsRNA molecules of the present disclosure comprise at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex region from the 5' end of the antisense strand, which can be independently selected from the group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical or other than canonical pairings or pairings including universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0426] In some embodiments, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U and dT.Alternatively, at least one of the first 1, 2 or 3 base pairs in the double-stranded region from the 5' end of the antisense strand is an AU base pair.For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0427] It has been found that the introduction of a 4'- or 5'-modified nucleotide at the 3' end of a dinucleotide phosphodiester (PO), phosphorothioate (PS) or phosphorodithioate (PS2) linkage at any position in a single- or double-stranded oligonucleotide can exert a steric effect on the internucleotide linkage, thus protecting it from nucleases and stabilizing it.

[0428] In some embodiments, 5'-modified nucleoside is introduced at the 3' end of dinucleotide at any position in single-stranded or double-stranded siRNA.For example, 5'-alkylated nucleoside can be introduced at the 3' end of dinucleotide at any position in single-stranded or double-stranded siRNA.The alkyl group at the 5' position of ribose sugar can be racemic or chirally pure R or S isomer.Exemplary 5'-alkylated nucleoside is 5'-methyl nucleoside.5'-methyl can be racemic or chirally pure R or S isomer.

[0429] In some embodiments, 4'-modified nucleosides are introduced at the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA. For example, 4'-alkylated nucleosides can be introduced at the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA. The alkyl group at the 5' position of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4'-alkylated nucleoside is 4'-methyl nucleoside. 4'-methyl can be racemic or chirally pure R or S isomer. Alternatively, 4'-O-alkylated nucleosides can be introduced at the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA. The 4'-O-alkyl group at the ribose sugar can be racemic or chirally pure R or S isomer. Exemplary 4'-O-alkylated nucleosides include 4'-O-methyl nucleosides, which can be either racemic or chirally pure R or S isomers.

[0430] In some embodiments, 5'-alkylated nucleoside is introduced at any position of the sense strand or antisense strand of dsRNA, and this modification maintains or improves the efficacy of dsRNA.5'-alkyl can be either racemic or chirally pure R or S isomer.Exemplary 5'-alkylated nucleoside is 5'-methyl nucleoside.5'-methyl can be either racemic or chirally pure R or S isomer.

[0431] In some embodiments, 4'-alkylated nucleoside is introduced at any position of the sense strand or antisense strand of dsRNA, and this modification maintains or improves the efficacy of dsRNA.4'-alkyl can be either racemic or chirally pure R or S isomer.Exemplary 4'-alkylated nucleoside is 4'-methyl nucleoside.4'-methyl can be either racemic or chirally pure R or S isomer.

[0432] In some embodiments, 4'-O-alkylated nucleosides are introduced at any position of the sense strand or antisense strand of dsRNA, and such modifications maintain or improve the efficacy of dsRNA. 5'-Alkyl can be either racemic or chirally pure R or S isomer. Exemplary 4'-O-alkylated nucleosides include 4'-O-methyl nucleosides. 4'-O-methyl can be either racemic or chirally pure R or S isomer.

[0433] In some embodiments, the dsRNA molecules of the present disclosure may contain 2'-5' linkages (having 2'-H, 2'-OH, and 2'-OMe, with P=O or P=S). For example, 2'-5' linkage modifications can be used to promote nuclease resistance or to inhibit binding of the sense strand to the antisense strand, or at the 5' end of the sense strand to prevent sense strand activation by RISC.

[0434] In another embodiment, the dsRNA molecules of the present disclosure can contain L sugars (e.g., L-ribose, L-arabinose, with 2'-H, 2'-OH, and 2'-OMe).For example, these L sugar modifications can be used to promote nuclease resistance or inhibit binding of the sense strand to the antisense strand, or can be used at the 5' end of the sense strand to prevent sense strand activation by RISC.

[0435] Various publications have described multimeric siRNA, and all of them can be used with the dsRNA of the present disclosure.Such publications include WO2007 / 091269, US7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520, all of which are incorporated herein in their entirety.

[0436] As described in more detail below, RNAi agents containing one or more carbohydrate moieties conjugated to them can improve one or more properties of the RNAi agent. In many cases, the carbohydrate moiety is attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, such as a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is attached. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose-replacement modified subunit (RRMS). The cyclic carrier can be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring structure, i.e., one or more ring atoms can be heteroatoms, such as nitrogen, oxygen, or sulfur. The cyclic carrier can be a monocyclic ring structure or can contain two or more rings, such as fused rings. The cyclic carrier can be a fully saturated ring structure or can contain one or more double bonds.

[0437] Ligands can be attached to polynucleotides via carriers. Carriers include (i) at least one "backbone attachment point," preferably two "backbone attachment points," and (ii) at least one "tethering attachment point." As used herein, "backbone attachment point" refers to a functional group, such as a hydroxyl group, or generally a bond available and suitable for incorporation of a carrier into a backbone, such as a phosphate or modified phosphate, e.g., sulfur-containing backbone, of a ribonucleic acid. In some embodiments, a "tethering attachment point" (TAP) refers to a ring atom, such as a carbon atom or heteroatom (other than the atom providing the backbone attachment point), of a cyclic carrier that connects a selected moiety. The moiety can be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. The selected moiety may be connected to the cyclic carrier by an intervening tether. Thus, cyclic carriers often contain a functional group, such as an amino group, or generally provide a bond suitable for incorporation or tethering another chemical entity, such as a ligand, to the ring.

[0438] RNAi agent can be conjugated to ligand through carrier, and carrier can be cyclic or acyclic group.Cyclic group can be selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalinyl.Acyclic group can be serinol backbone or diethanolamine backbone.

[0439] In certain specific embodiments, an RNAi agent for use in the methods of the present disclosure is an agent selected from the group of agents listed in any one of Tables 3 or 4.

[0440] IV. Ligand-Conjugated iRNA Another modification of the iRNA of the invention includes chemically linking the iRNA to one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA, for example, into cells. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), and the like. 20:533-538), fatty chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973) or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0441] In certain embodiments, a ligand alters the distribution, targeting, or lifespan of the iRNA agent into which it is incorporated. In some embodiments, a ligand provides enhanced affinity for a selected target, such as a molecule, cell or cell type, compartment, such as a cellular or organ compartment, tissue, organ, or region of the body, for example, compared to a species in which such a ligand is not present. Conventional ligands do not participate in duplex pairing in double-stranded nucleic acids.

[0442] Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL) or globulins), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid), or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolized) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylamide), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helical peptides.

[0443] Ligands can also include targeting groups, such as cell or tissue targeting agents, such as lectins, glycoproteins, lipids, or proteins, for example, antibodies that bind to specific cell types, such as kidney cells.Targeting groups can be thyroid-stimulating hormone, melanocyte-stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acids, multivalent galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol steroids, bile acid, folic acid, vitamin B12, biotin, or RGD peptide or RGD peptide mimetics.In certain embodiments, the ligand is multivalent galactose, such as N-acetyl-galactosamine.

[0444] 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 phosphate, phosphate-binding proteins ...

[0445] The ligand can be a protein, such as a glycoprotein or peptide, a molecule with specific affinity for the co-ligand, or an antibody, such as an antibody that binds to a specific cell type, 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, p38 MAP kinase activators, or NF-κB activators.

[0446] The ligand can be, for example, a substance, e.g., a drug, that can increase cellular uptake of an iRNA agent by, for example, disrupting the cytoskeleton of a cell, e.g., by disrupting cellular 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.

[0447] In some embodiments, the ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, polyethylene glycol (PEG), vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing several phosphorothioate linkages are also known to bind to serum proteins; therefore, short oligonucleotides, e.g., oligonucleotides of about 5, 10, 15, or 20 bases containing multiple phosphorothioate linkages in the backbone, are also suitable as ligands (e.g., as PK-modulating ligands) for the present invention. Furthermore, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0448] Ligand-conjugated iRNAs of the invention can be synthesized by using oligonucleotides bearing pendant reactive functionalities, e.g., derived from the attachment of a linking molecule onto an oligonucleotide (described below). This reactive oligonucleotide can be reacted directly with commercially available ligands, ligands that have been synthesized with any of a variety of protecting groups, or ligands that have a linking moiety attached to them.

[0449] The oligonucleotides used in the conjugates of the present invention can be conveniently and routinely produced by known techniques of solid phase synthesis.The equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems® (Foster City, California).Any other means for such synthesis known in the art can also or alternatively be used.It is also known to use similar techniques to prepare other oligonucleotides, for example, phosphorothioates and alkylated derivatives.

[0450] For the ligand-conjugated oligonucleotides and molecules having ligand-sequence-specific linked nucleosides of the present invention, the oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer using standard nucleotide or nucleoside precursors or nucleotide or nucleoside conjugate precursors that already have a linking moiety, ligand-nucleotide or nucleoside conjugate precursors that already have a ligand molecule, or non-nucleoside ligand-containing building blocks.

[0451] When using a nucleotide-conjugate precursor that already has a linking moiety, the synthesis of the sequence-specific linked nucleoside is usually completed, and then a ligand molecule is reacted with the linking moiety to form a ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the invention are synthesized by automated synthesizers using phosphoramidites derived from ligand-nucleoside conjugates, in addition to standard and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

[0452] A. Lipid Conjugates In certain embodiments, the ligand or conjugate is lipid or lipid-based molecule.This lipid or lipid-based molecule can usually bind to serum protein, for example, human serum albumin (HSA).HSA binding ligand allows the distribution of conjugate to target tissue in the body, for example, non-renal target tissue.For example, target tissue can be brain or liver.Other molecules that can bind to 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) adjust the binding to serum protein, for example, HSA.

[0453] The lipid-based ligand can be used to modulate, e.g., manage (e.g., inhibit), the binding of the conjugate to the target tissue. For example, a lipid or lipid-based ligand that binds more strongly to HSA is less likely to be targeted to 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 to target the conjugate to the kidney.

[0454] In certain embodiments, lipid-based ligand binds to HSA.For example, the ligand can bind to HSA with sufficient affinity, so that the distribution of conjugate to non-renal tissue is enhanced.However, the affinity is usually not so strong that HSA-ligand binding cannot be reversed.

[0455] In certain embodiments, the lipid-based ligand binds weakly or not at all to HSA, resulting in enhanced distribution of the conjugate to the kidney. Other moieties that target kidney cells can be used instead of or in addition to the lipid-based ligand.

[0456] In another embodiment, the ligand is a moiety, such as a vitamin, that is taken up by target cells, for example, proliferating cells. These are particularly useful for treating disorders characterized by unwanted cell proliferation, for example, malignant or non-malignant types, such as cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low-density lipoprotein (LDL).

[0457] B. Cell-penetrating agents In another aspect, the ligand is a cell-penetrating agent, for example, a helical cell-penetrating agent. In certain embodiments, these cell-penetrating agents are amphipathic. Exemplary cell-penetrating agents include peptides, such as tat or antennopedia. When the agent is a peptide, it may be modified, including peptidyl mimetics, invertomers, non-peptide or pseudopeptide linkages, and the use of D-amino acids. Helical agents are usually α-helical agents and may have a lipophilic and lipophobic phase.

[0458] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules capable of folding into defined three-dimensional structures similar to natural peptides. Attachment of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic distribution of iRNAs, such as by enhancing cellular recognition and uptake. The peptide or peptidomimetic moiety can be about 5 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0459] 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 crosslinked peptide. In another alternative, the peptide moiety can contain a hydrophobic membrane translocation 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, the sequence derived from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 5)) and the sequence derived from the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 6)) have been found 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). Typically, peptides or peptidomimetics tethered to dsRNA agents via incorporated monomer units include cell-targeting peptides, such as arginine-glycine-aspartic acid (RGD) peptides or RGD mimics. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications, for example, to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

[0460] The RGD peptide for use 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 to specific tissue(s).RGD-containing peptides and peptidomimetics can include D-amino acids and synthetic RGD mimics.In addition to RGD, other moieties can be used to target integrin ligands.In certain embodiments, the conjugate of this ligand targets PECAM-1 or VEGF.

[0461] RGD peptide moieties can be used to target specific cell types, such as tumor cells, e.g., endothelial tumor cells or breast cancer tumor cells (Zitzmann et al., Cancer Res., 62:5139-43, 2002). RGD peptides can facilitate targeting of dsRNA agents to tumors in various other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Typically, RGD peptides facilitate targeting of iRNA agents to the kidney. RGD peptides can be linear or cyclic, and can be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). For example, glycosylated RGD peptides can facilitate targeting of iRNA agents to α- V It can be delivered to tumor cells that express β3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

[0462] A "cell-penetrating peptide" is capable of penetrating cells, such as microbial cells, e.g., bacterial or fungal cells, or mammalian cells, e.g., human cells. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or seropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain a nuclear localization signal (NLS). For example, a cell-penetrating peptide can be a bisected amphipathic peptide such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0463] C. Carbohydrate conjugates In some embodiments of the compositions and methods of the present invention, the iRNA further comprises a carbohydrate. Carbohydrate-conjugated iRNA is advantageous for in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic use, as described herein. As used herein, "carbohydrate" refers to a compound that is either a carbohydrate itself, composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom, or a compound that has as part thereof a carbohydrate moiety composed of one or more monosaccharide units, each having at least six carbon atoms (which may be linear, branched, or cyclic), with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide gums. Particular monosaccharides include C5, and above (e.g., C5, C6, C7, or C8) sugars, disaccharides, and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0464] In certain embodiments, the carbohydrate conjugate comprises a monosaccharide.

[0465] In certain embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc).GalNAc conjugates containing one or more N-acetylgalactosamine (GalNAc) derivatives are described, for example, in US 8,106,022, the entire contents of which are incorporated herein by reference.In some embodiments, the GalNAc conjugate serves as a ligand that targets iRNA to specific cells.In some embodiments, the GalNAc conjugate targets iRNA to liver cells, for example, by serving as a ligand for the asialoglycoprotein receptor of liver cells (e.g., hepatocytes).

[0466] In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives can be attached via a linker, e.g., 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., a linker as described herein. In some embodiments, the GalNAc conjugate is conjugated to the 5' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 5' end of the sense strand) via a linker, e.g., a linker as described herein.

[0467] In certain embodiments of the invention, GalNAc or GalNAc derivatives are attached to iRNA agents of the invention via a monovalent linker. In some embodiments, GalNAc or GalNAc derivatives are attached to iRNA agents of the invention via a bivalent linker. In yet other embodiments of the invention, GalNAc or GalNAc derivatives are attached to iRNA agents of the invention via a trivalent linker. In other embodiments of the invention, GalNAc or GalNAc derivatives are attached to iRNA agents of the invention via a tetravalent linker.

[0468] In certain embodiments, double-stranded RNAi agents of the invention comprise one GalNAc or GalNAc derivative attached to an iRNA agent. In certain embodiments, double-stranded RNAi agents of the invention comprise multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently attached to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers.

[0469] 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 stretch of nucleotides between the 3' end of one strand and the 5' end of each of the other strands, forming a hairpin loop containing multiple unpaired nucleotides, the unpaired nucleotides in the hairpin loop can each independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker. Hairpin loops can also be formed by an extended overhang on one strand of the duplex.

[0470] 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 stretch of nucleotides between the 3' end of one strand and the 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. Hairpin loops can also be formed by an extended overhang on one strand of the duplex.

[0471] In some embodiments, the GalNAc conjugate is

[0472] [ka] is.

[0473] 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:

[0474] [ka]

[0475] In some embodiments, the RNAi agent is conjugated to L96 as defined in Table 2 and shown below:

[0476] [ka]

[0477] In certain embodiments, the carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of:

[0478] [ka] TIFF2025516321000022.tif228110 TIFF2025516321000023.tif22594 TIFF2025516321000024.tif21193 TIFF2025516321000025.tif4060 [wherein Y is O or S, and n is 3 to 6] (Formula XXIV);

[0479] [ka] [wherein Y is O or S, and n is 3 to 6] (Formula XXV);

[0480] [ka] [wherein X is O or S] (Formula XXVII);

[0481] [ka] TIFF2025516321000029.tif172143

[0482] In certain embodiments, the carbohydrate conjugate for use in the compositions and methods of the present invention is a monosaccharide. In certain embodiments, the monosaccharide is N-acetylgalactosamine, e.g.,

[0483] [ka] is.

[0484] Further exemplary carbohydrate conjugates for use in the embodiments described herein include, but are not limited to:

[0485] [ka] [When one of X or Y is an oligonucleotide, the other is hydrogen] Examples include:

[0486] In some embodiments, suitable ligands are those disclosed in WO2019 / 055633, the entire contents of which are incorporated herein by reference. In one embodiment, the ligand has the following structure:

[0487] [ka] Includes:

[0488] In certain embodiments, RNAi agents of the present disclosure may include GalNAc ligands, even though such GalNAc ligands are currently predicted to be of limited value for the intrathecal / CNS delivery route(s) of the present disclosure.

[0489] In certain embodiments of the invention, GalNAc or GalNAc derivatives are attached to iRNA agents of the invention via a monovalent linker. In some embodiments, GalNAc or GalNAc derivatives are attached to iRNA agents of the invention via a bivalent linker. In yet other embodiments of the invention, GalNAc or GalNAc derivatives are attached to iRNA agents of the invention via a trivalent linker.

[0490] In one embodiment, the double-stranded RNAi agent of the present invention comprises one or more GalNAc or GalNAc derivatives attached to the iRNA agent. GalNAc can be attached to any nucleotide via a linker on the sense strand or antisense strand. GalNAc can be attached 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 attached to the 3' end of the sense strand, for example, via a trivalent linker.

[0491] 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 independently attached to each of multiple nucleotides of the double-stranded RNAi agent via multiple linkers, e.g., monovalent linkers.

[0492] In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of each of the other strands, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop can independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker.

[0493] 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.

[0494] Further carbohydrate conjugates and linkers suitable for use in the present invention include those described in WO2014 / 179620 and WO2014 / 179627, the contents of each of which are incorporated herein by reference in their entirety.

[0495] 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.

[0496] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound, e.g., attaches two parts of a compound by a covalent bond.A linker is typically a direct bond or an atom such as oxygen or sulfur, a unit such as NR, C(O), C(O)NH, SO, SO, SONH, or a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, or heteroarylalkynyl, where one or more methylenes may be interrupted or terminated by O, S, S(O), SO, N(R), C(O). ynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkynyl

[0044] In some embodiments, R8 is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc.In 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.

[0497] A cleavable linking group is one that is sufficiently stable outside a cell, but that, upon entry into a target cell, is cleaved to release the two moieties held together by the linker. In another embodiment, the cleavable linking group is cleaved at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or more, or at least about 100-fold faster in the target cell or under a first reference condition (which may, for example, be selected to mimic or represent intracellular conditions) than in the subject's blood or under a second reference condition (which may be selected to mimic or represent conditions found in blood or serum).

[0498] Cleavable linking groups are susceptible to the influence of cleaving agents, such as pH, redox potential, or the presence of degradable molecules.Generally, cleaving agents are more common or found at higher levels or activity inside cells than in serum or blood.Examples of such degrading agents include redox agents that are selected for specific substrates or do not have substrate specificity, such as oxidizing enzymes or reductases or reducing agents present in cells, such as mercaptans, which can degrade redox-cleavable linking groups by reduction, esterases, endosomes, or agents that can create an acidic environment, such as those that produce a pH of 5 or less, general acids, peptidases (can be substrate-specific), and enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as phosphatases.

[0499] Cleavable linking groups, such as disulfide bonds, can be pH sensitive. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers will have a cleavable linking group that is cleaved at a selected pH, thereby releasing the cationic lipid from the ligand into the interior of the cell or into a desired compartment of the cell.

[0500] Linker can comprise a cleavable linking group that can be cleaved by specific enzyme.The type of cleavable linking group incorporated into linker can vary depending on the cell to be targeted.For example, liver targeting ligand can be linked to cationic lipid via a linker that comprises ester group.Liver cell is rich in esterase, therefore, linker is cleaved more efficiently in liver cell than in cell types that are not rich in esterase.Other cell types that are rich in esterase include lung, renal cortex and testis cell.

[0501] When targeting cell types that are rich in peptidases, such as liver cells and synovial cells, linkers containing peptide bonds can be used.

[0502] Generally, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degrading agent (or condition) to cleave the candidate linking group. It may also be desirable to test the candidate cleavable linking group for its ability to resist cleavage when in blood or in contact with other non-target tissues. Thus, the relative susceptibility to cleavage between a first and a second condition can be determined, with the first being selected to exhibit cleavage in target cells, and the second being selected to exhibit cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be carried out in a cell-free system, in cells, in cell culture, in organs or tissue culture, or in whole animals. It may be useful to perform initial evaluation in cell-free or culture conditions and confirm by further evaluation in whole animals. In certain embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0503] 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 by incubating with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate that would be observed in cells, e.g., target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In some cases, candidate compounds are cleaved at a maximum of about 10% in blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic intracellular media compared to conditions selected to mimic extracellular media.

[0504] ii. Phosphate-based cleavable linking groups 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 phosphate groups in cells is an enzyme such as a phosphatase in the cell. Examples of phosphate-based linking groups include -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-, where Rk in each occurrence can independently be C1-C20 alkyl, C1-C20 haloalkyl, C6-C10 aryl, or C7-C12 aralkyl. Additional 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-, -OP(S)(H)-S-, wherein Rk at each occurrence can independently be C1-C20 alkyl, C1-C20 haloalkyl, C6-C10 aryl, or C7-C12 aralkyl. In certain embodiments, the phosphate-based linking group is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0505] 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 one embodiment, the acid-cleavable linking group is cleaved in an acidic environment having a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower) or by an agent, such as an enzyme, that can act as a general acid. In cells, certain low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid-cleavable linking group. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). In another embodiment, 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.

[0506] 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 by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester-cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0507] 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 by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids, resulting in oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (—C(O)NH—). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids, resulting in peptides and proteins. Peptide-based cleaving groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids, resulting in peptides and proteins, and do not include the entire amide functionality. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where R and R are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0508] 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:

[0509] [ka] TIFF2025516321000034.tif192142 TIFF2025516321000035.tif50141 [If one of X or Y is an oligonucleotide, the other is hydrogen] Examples include:

[0510] 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.

[0511] 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 (XLV) to (XLVI):

[0512] [ka] [In the formula, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C independently for each occurrence represent 0 to 20, and the repeat units may be the same or different; p 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C is, independently for each occurrence, absent, CO, NH, O, S, OC(O), NHC(O), CH, CHNH, or CHO; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C is, independently for each occurrence, absent, alkylene, substituted alkylene, and one or more methylenes are O, S, S(O), SO, N(R N), C(R')=C(R''), C≡C, or C(O); R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C is independently for each occurrence absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO,

[0513] [ka] or heterocyclyl, L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represents a ligand; i.e., each independently for each occurrence, a monosaccharide (such as GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, or a polysaccharide; R a is H or an amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents to inhibit expression of target genes, such as those of formula (XLIX):

[0514] [ka] [In the formula, L 5A , L 5B and L 5C represents a monosaccharide, e.g., a GalNAc derivative].

[0515] Examples of divalent and trivalent branched linker groups suitable for conjugating GalNAc derivatives include, but are not limited to, the structures listed above, such as Formulas II, VII, XI, X, and XIII.

[0516] Representative United States patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979, 4,948,882, 5,218,105, 5,525,465, 5,541,313, 5,545,730, 5,552,538, 5,578,717, 5,580,731, 5,591,584, 5,109,124, 5,118,802, 5,138,045, 5,414,07 ... 5,486,603, 5,512,439, 5,578,718, 5,608,046, 4,587,044, 4,605,735, 4,667,025, 4,762,779, 4,789,737, 4,824,941, 4,835,263, 4,876,335, 4,904,582, 4,958,013, 5,082,830, 5,112,963, 5,214,136, 5,082,830, No. 5,112,963, No. 5,214,136, No. 5,245,022, No. 5,254,469, No. 5,258,506, No. 5,262,536, No. 5,272,250, No. 5,292,873, No. 5,317,098, No. No. 5,371,241, No. 5,391,723, No. 5,416,203, No. 5,451,463, No. 5,510,475, No. 5,512,667, No. 5,514,785, No. 5,565,552, No. 5,567,810, No. Nos. 5,574,142, 5,585,481, 5,587,371, 5,595,726, 5,597,696, 5,599,923, 5,599,928, 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.

[0517] Not all positions in a given compound need be uniformly modified, and in fact more than one of the above modifications can be incorporated in a single compound, or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0518] In the context of the present 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 composed of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region in which the RNA has been modified to confer increased resistance to nuclease degradation, increased cellular uptake, or increased binding affinity for the target nucleic acid. An additional region of the iRNA can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis, optionally followed by associated nucleic acid hybridization techniques known in the art.

[0519] In certain instances, the RNA of an iRNA can be modified with a non-ligand group. To enhance the activity, cellular distribution, or cellular uptake of an iRNA, several non-ligand molecules have been conjugated to the iRNA, and procedures for performing such conjugation are available in the scientific literature. Such non-ligand moieties include lipid moieties such as cholesterol [Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553], cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), fatty chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969) or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative U.S. patents teaching the preparation of such RNA conjugates are listed above. A typical conjugation protocol involves the synthesis of RNA bearing an amino linker at one or more positions in the sequence. The amino group is then reacted with the molecule being conjugated using an appropriate coupling or activating reagent. The conjugation reaction can be carried out while the RNA is still attached to the solid support or after cleavage of the RNA in solution phase. Purification of the RNA conjugate by HPLC usually yields a pure conjugate.

[0520] V. Delivery of RNAi Agents of the Present Disclosure Delivery of the RNAi agent of the present disclosure to a cell, for example, a cell in a subject, for example, a cell in a human subject (e.g., a subject in need thereof, for example, a subject with an ABLIM3-related disorder, for example, PTSD or age-related memory loss), 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 containing an RNAi agent, for example, a dsRNA, to a 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 alternatives are further described below.

[0521] Generally, any method of delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the RNAi agents of the present disclosure (see, for example, Akhtar S. and Julian RL., (1992) Trends Cell. Biol. 2(5):139-144 and WO94 / 02595, which are incorporated herein by reference in their entirety). For in vivo delivery, factors to consider when delivering RNAi agents include, for example, the biological stability of the delivered agent, prevention of non-specific effects, and accumulation of the delivered agent in target tissues. Non-specific effects of RNAi agents can be minimized by local administration, such as direct injection or implantation into tissues or local administration of preparations. Local administration at the treatment site maximizes the local concentration of the agent, limits exposure of the agent to systemic tissues that may be harmed by or degrade the agent, and allows for a smaller total dose of the administered RNAi agent. Several studies have shown the success of knocking down gene products when RNAi agents are administered locally. For example, intraocular delivery of VEGF dsRNA by intravitreal injection in cynomolgus monkeys [Tolentino, MJ. et al., (2004) Retina 24:132-138] and subretinal injection in mice [Reich, SJ. et al. (2003) Mol. Vis. 9:210-216] have both been shown to prevent neovascularization in experimental models of age-related macular degeneration. In addition, direct intratumoral injection of dsRNA into mice can reduce tumor volume [Pille, J. et al. (2005) Mol. Ther. 11:267-274] and prolong the survival of tumor-bearing mice [Kim, WJ. et al., (2006) Mol. Ther. 14:343-350; Li, S. et al., (2007) Mol. Ther. 15:515-523].RNA interference can be delivered to the CNS by direct injection [Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, PH. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al. (2002) BMC Neurosci. 3:18; Shishkina, GT., et al. (2004) Neuroscience 129:521-528; Thakker, ER., et al. (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya,Y., et al. (2005) J. Neurophysiol. 93 :594-602] and to the lungs by intranasal administration [Howard, KA. et al., (2006) Mol. Ther. 14:476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55], and have also shown success with local delivery. When administering RNAi agents systemically to treat disease, the RNA can be modified or, alternatively, delivered using a drug delivery system; both methods function to prevent the rapid degradation of dsRNA by endonucleases and exonucleases in vivo. Modification of RNA or pharmaceutical carrier can also enable targeting of RNAi agent to target tissue and avoid undesirable off-target effects (for example, without wishing to be bound by theory, it has been identified that the use of GNA described herein destabilizes the seed region of dsRNA, and such off-target effects are significantly weakened by destabilizing such seed region, thereby increasing the priority of such dsRNA for on-target effectiveness compared to off-target effects).RNAi agent 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 a mouse model of prostate cancer [McNamara, JO. et al., (2006) Nat. Biotechnol. 24:1005-1015]. In alternative embodiments, RNAi agents can be delivered using drug delivery systems, such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems promote the binding of molecular RNAi agents (negatively charged) and also enhance their interaction with the negatively charged cell membrane, 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 also prevents the degradation of RNAi agents when administered systemically. The method of creating 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, the entire contents of which are incorporated herein by reference).Some non-limiting examples of drug delivery systems useful for systemic delivery of RNAi agents include DOTAP [Sorensen, DR., et al (2003), supra; Verma, UN. et al., (2003), supra], Oligofectamine, "solid nucleic acid lipid particles" [Zimmermann, TS. et al., (2006) Nature 441:111-114], cardiolipin [Chien, PY. et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091], polyethyleneamine [Bonnet ME. et al., (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659], Arg-Gly-Asp (RGD) peptide [Liu, S. (2006) Mol. Pharm. 3:472-487], and polyamidoamine [Tomalia, DA. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804]. In some embodiments, the RNAi agent is complexed with cyclodextrin for systemic administration. Methods of administration and pharmaceutical compositions of RNAi agents and cyclodextrins can be found in U.S. Patent No. 7,427,605, the entire contents of which are incorporated herein by reference.

[0522] Certain aspects of the present disclosure relate to a method for reducing the expression of ABLIM3 target genes in cells, comprising contacting cells with the double-stranded RNAi agent of the present disclosure.In one embodiment, the cells are CNS cells, for example, brain cells.In other embodiments, the cells are dentate gyrus granule cells, neurons, astrocytes, or oligodendrocytes.

[0523] Another aspect of the present disclosure relates to a method of reducing expression of an ABLIM3 target gene in a subject, the method comprising administering to the subject a double-stranded RNAi agent of the present disclosure. ...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of ABLIM3, the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region, A dsRNA agent, wherein the sense strand comprises at least 15 contiguous nucleotides which 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 which differ from the nucleotide sequence of SEQ ID NO:2 by no more than 3 nucleotides.

2. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of ABLIM3, the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region, A dsRNA agent, wherein the antisense strand comprises a region of complementarity to an mRNA encoding ABLIM3, wherein the region of complementarity comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:2 by no more than 3 nucleotides.

3. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of ABLIM3, the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region, A dsRNA agent wherein the antisense strand comprises a region of complementarity to an mRNA encoding Ablim3, wherein the region of complementarity comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense nucleotide sequences in Table 3 or 4.

4. The sense strand is selected from the group consisting of nucleotides 82-102, 134-154, 232-252, 276-296, 293-313, 326-346, 351-371, 366-386, 381-401, 414-434, 482-502, 500-520, 542-562, 558-578, 582-602, 622-642, 674-694, 692-712, 713-733, 728-748, 743-763, 779-799, 887-907, 942-962, 983-1003, 1001-1021, 1082-1102, 1102-1122, 1 117-1137, 1177-1197, 1192-1212, 1242-1262, 1269-1289, 1313-1333, 1344-1364, 1359-1379, 1374-1394, 1408-1428, 1437-1457, 1550-1570, 1594-1 614, 1624-1644, 1645-1665, 1674-1694, 1715-1735, 1746-1766, 1767-1787, 1812-1832, 1859-1879, 1884-1904, 1912-1932, 1935-1955, 1971-1991, 2 039-2059, 2096-2116, 2127-2147, 2169-2189, 2184-2204, 2218-2238, 2245-2265, 2273-2293, 2325-2345, 2343-2363, 2385-2405, 2422-2442, 2459- 2479, 2483-2503, 2520-2540, 2535-2555, 2561-2581, 2585-2605, 2667-2687, 2703-2723, 2729-2749, 2764-2784, 2788-2808, 2830-2850, 2861-2881, 2885-2905, 2937-2957, 2958-2978, 2974-2994, 3011-3031, 3044-3064, 3061-3081, 3079-3099, 3110-3130, 3146-3166, 3203-3223, 3220-3240, 3279- 3299, 3303 to 3323, 3321 to 3341, 3338 to 3358, 3388 to 3408, 3407 to 3427, 3422 to 3442, 3437 to 3457, 3452 to 3472, 3532 to 3552, 3548 to 3568, 3579 to 3599, 3594 to 3614,3626-3646, 3644-3664, 3682-3702, 3706-3726, 3721-3741, 3746-3766, 3763-3783, 3796-3816, 3814-3834, 3849-3869, 3866-3886, 3897-3917, 3919-3939, 3986-4006, 4018-4038, 4037-4057, 4058-4078, 4084-4104, 4099-4119, 4131-4151, 4159-4179, 4225-4245, 4241-42 dsRNA agent according to any one of claims 1 to 3, wherein 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: 2, 4276-4296, 4291-4311, 4306-4326, 4336-4356, 4354-4374, 4369-4389, 4387-4407, 4404-4424, or 4421-4441, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:

2.

5. The antisense strand is AD-1807455, AD-1807481, AD-1807539, AD-1807561, AD-1807578, AD-1807591, AD-1807616, AD-1807631, AD-1807646, AD-1807659, AD-1807707, AD-1807725, AD-1807764, AD-1807780, AD-1807804, AD-1807844, AD-1807870, AD-1807888, AD-1807889, AD-1807904, AD-1807919, AD-1807955, AD-1808063, AD-1808098, AD-1808139, AD-1808157, AD-1808238, AD-1808258, AD-1808273, AD-1808312, AD-1808327, AD-1808356, AD-1808383, AD-1808407, AD-1808418, AD-1808433, AD-1808448, AD-1808482, AD-1808511, AD-1808561, AD-1808598, AD-1808628, AD-1808649, AD-1808678, AD-1808698, AD-1808727, AD-1808748, AD-1808773, AD-1808789, AD-1808814, AD-1808842, AD-1808865, AD-1808901, AD-1808941, AD-1808978, AD-1809009, AD-1809051, AD-1809066, AD-1809080, AD-1809107, AD-1809135, AD-1809187, AD-1809205, AD-1809246, AD-1809283, AD-1809293, AD-1809317, AD-1809354, AD-1809369, AD-1809395, AD-1809414, AD-1809456, AD-1809472, AD-1809498, AD-1809511, AD-1809535, AD-1809550, AD-1809561, AD-1809585, AD-1809596, AD-1809617, AD-1809633, AD-1809670, AD-1809702, AD-1809719, AD-1809737, AD-1809757, AD-1809773, AD-1809822, AD-1809839,AD-1809895, AD-1809919, AD-1809937, AD-1809954, AD-1810004, AD-1810023, AD-18100 38, AD-1810053, AD-1810068, AD-1810127, AD-1810143, AD-1810174, AD-1810189, AD-181 0221, AD-1810239, AD-1810255, AD-1810277, AD-1810292, AD-1810296, AD-1810313, AD-1 810345, AD-1810363, AD-1810398, AD-1810415, AD-1810446, AD-1810468, AD-1810494, AD The dsRNA agent according to any one of claims 1 to 4, comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense strand nucleotide sequences of the duplex selected from the group consisting of AD-1810526, AD-1810545, AD-1810566, AD-1810591, AD-1810606, AD-1810638, AD-1810666, AD-1810676, AD-1810692, AD-1810727, AD-1810742, AD-1810757, AD-1810767, AD-1810785, AD-1810800, AD-1810818, AD-1810822, and AD-1810839.

6. 3. The dsRNA agent of claim 1 or 2, wherein the nucleotide sequences of the sense and antisense strands comprise any one of the sense and antisense strand nucleotide sequences in Tables 3 or 4.

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

8. 8. The dsRNA agent of claim 7, wherein the lipophilic moiety is conjugated to one or more internal positions in the double-stranded region of the dsRNA agent.

9. 9. The dsRNA agent of claim 7 or 8, wherein the lipophilic moiety is conjugated via a linker or carrier.

10. logK ｏｗ The dsRNA agent of any one of claims 7 to 9, wherein the lipophilicity of the lipophilic moiety is greater than 0 as measured by:

11. The dsRNA agent of any one of claims 1 to 10, wherein 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.

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

13. The dsRNA agent of any one of claims 1 to 12, wherein the dsRNA agent comprises at least one modified nucleotide.

14. 14. The dsRNA agent of claim 13, wherein no more than five of the sense strand nucleotides and no more than five of the antisense strand nucleotides are unmodified nucleotides.

15. 14. The dsRNA agent of claim 13, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

16. At least one of the modified nucleotides is a deoxynucleotide, a 3' terminal deoxythymidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a non-locked 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 ...

16. The dsRNA agent of any one of claims 13-15, wherein the dsRNA agent is selected from the group consisting of: nucleotides modified with a 5'-phosphorothioate group, nucleotides comprising a 5'-methylphosphonate group, nucleotides comprising a 5' phosphate or a 5' phosphate mimic, nucleotides comprising a vinyl phosphonate, glycol nucleic acid (GNA), S-glycol nucleic acid (S-GNA), nucleotides comprising 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides comprising 2'-deoxythymidine-3' phosphate, nucleotides comprising 2'-deoxyguanosine-3'-phosphate, nucleotides comprising a 2' phosphate, and terminal nucleotides linked to a cholesteryl derivative and a dodecanoic acid bisdecylamide group; and combinations thereof.

17. 17. The dsRNA agent of claim 16, wherein the modified nucleotide is selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, 3' terminal deoxythymidine nucleotides (dT), locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and nucleotides containing unnatural bases.

18. 17. The dsRNA agent of claim 16, wherein the modified nucleotides comprise a short sequence of 3' terminal deoxythymidine nucleotides (dT).

19. 17. The dsRNA agent of claim 16, wherein the modifications to the nucleotide are 2'-O-methyl, GNA, and 2'-fluoro modifications.

20. The dsRNA agent of any one of claims 1 to 19, further comprising at least one phosphorothioate internucleotide linkage.

21. The dsRNA agent of claim 20, comprising from 6 to 8 phosphorothioate internucleotide linkages.

22. The dsRNA agent of any one of claims 1-21, wherein each strand is 30 nucleotides or less in length.

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

24. The dsRNA agent of any one of claims 1 to 23, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides.

25. The dsRNA agent of any one of claims 1 to 24, wherein the double-stranded region is 15 to 30 nucleotide pairs in length.

26. 26. The dsRNA agent of claim 25, wherein the double-stranded region is 17 to 23 nucleotide pairs in length.

27. The dsRNA agent of claim 25, wherein the double-stranded region is 17 to 25 nucleotide pairs in length.

28. The dsRNA agent of claim 25, wherein the double-stranded region is 23 to 27 nucleotide pairs in length.

29. 26. The dsRNA agent of claim 25, wherein the double-stranded region is 19 to 21 nucleotide pairs in length.

30. The dsRNA agent of claim 25, wherein the double-stranded region is 21 to 23 nucleotide pairs in length.

31. The dsRNA agent of any one of claims 1 to 30, wherein each strand has from 19 to 30 nucleotides.

32. The dsRNA agent of any one of claims 1 to 30, wherein each strand has 19-23 nucleotides.

33. The dsRNA agent of any one of claims 1 to 30, wherein each strand has 21 to 23 nucleotides.

34. The dsRNA agent of any one of claims 8-33, wherein the one or more lipophilic moieties are conjugated to one or more internal positions in at least one strand.

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

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

37. 36. The dsRNA agent of claim 35, wherein the internal positions include all but the three terminal positions from each end of at least one strand.

38. The dsRNA agent of any one of claims 35-37, wherein the internal position excludes the cleavage site region of the sense strand.

39. 39. The dsRNA agent of claim 38, wherein the internal positions include all positions except positions 9-12, counting from the 5' end of the sense strand.

40. 40. The dsRNA agent of claim 38, wherein the internal positions include all positions except positions 11-13, counting from the 3' end of the sense strand.

41. The dsRNA agent of any one of claims 35-37, wherein the internal position excludes the cleavage site region of the antisense strand.

42. The dsRNA agent of claim 41, wherein the internal positions include all positions except positions 12-14, counting from the 5' end of the antisense strand.

43. The dsRNA agent of any one of claims 35-37, wherein the internal positions include all positions except positions 11-13 counting from the 3' end and positions 12-14 counting from the 5' end.

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

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

46. 9. The dsRNA agent of claim 8, wherein the internal position in the double-stranded region excludes the cleavage site region of the sense strand.

47. 47. The dsRNA agent of any one of claims 7-46, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, and wherein the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, position 7, position 6, or position 2 of the sense strand or position 16 of the antisense strand.

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

49. 48. The dsRNA agent of claim 47, wherein the lipophilic moiety is conjugated to position 21, 20, or 15 of the sense strand.

50. 48. The dsRNA agent of claim 47, wherein the lipophilic moiety is conjugated to position 20 or 15 of the sense strand.

51. 48. The dsRNA agent of claim 47, wherein the lipophilic moiety is conjugated to position 16 of the antisense strand.

52. The dsRNA agent of any one of claims 7-51, wherein the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

53. 53. The dsRNA agent of claim 52, wherein the lipophilic moiety is selected from the group consisting of lipids, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl groups, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, and phenoxazine.

54. 53. The dsRNA agent of claim 52, wherein 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.

55. The dsRNA agent of claim 54, wherein the lipophilic moiety contains a saturated or unsaturated C6 to C18 hydrocarbon chain.

56. 55. The dsRNA agent of claim 54, wherein the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.

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

58. The dsRNA agent of any one of claims 7-57, wherein the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotides at an internal position or in the double-stranded region.

59. 59. The dsRNA agent of claim 58, wherein the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl, or an acyclic moiety based on a serinol backbone or a diethanolamine backbone.

60. The dsRNA agent of any one of claims 7-57, wherein the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker that contains an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide linkage, the product of a click reaction, or a carbamate.

61. The double-stranded iRNA agent of any one of claims 7-60, wherein the lipophilic moiety is conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage.

62. 62. The dsRNA agent of any one of claims 7 to 61, wherein the lipophilic moiety or targeting ligand is conjugated via a biocleavable linker selected from the group consisting of DNA, RNA, disulfides, amides; functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

63. 63. The dsRNA agent of any one of claims 7-62, wherein the 3' end of the sense strand is protected via an end cap that is a cyclic group having an amine, said cyclic group being 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.

64. The dsRNA agent of any one of claims 7 to 61, further comprising a targeting ligand that targets a neuronal cell.

65. The dsRNA agent of any one of claims 7-61, wherein the targeting ligand is a GalNAc conjugate.

66. 66. The dsRNA agent of any one of claims 1-65, further comprising a terminal chiral modification at the first internucleotide linkage at the 3'-end of the antisense strand having a linking phosphorus atom in an Sp configuration, a terminal chiral modification at the first internucleotide linkage at the 5'-end of the antisense strand having a linking phosphorus atom in an Rp configuration, and a terminal chiral modification at the first internucleotide linkage at the 5'-end of the sense strand having a linking phosphorus atom in either an Rp or Sp configuration.

67. A terminal chiral modification occurring at the first and second internucleotide linkages at the 3' end of the antisense strand, with the linking phosphorus atom in the Sp configuration. a terminal chiral modification occurring at a first internucleotide linkage at the 5′-end of the antisense strand, the linking phosphorus atom being in an Rp configuration; and A terminal chiral modification occurs at the first internucleotide linkage at the 5' end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration. The dsRNA agent of any one of claims 1 to 65, further comprising:

68. Terminal chiral modifications occurring at the first, second, and third internucleotide linkages at the 3' end of the antisense strand, with the linking phosphorus atom in the Sp configuration. a terminal chiral modification occurring at a first internucleotide linkage at the 5′-end of the antisense strand, the linking phosphorus atom being in an Rp configuration; and A terminal chiral modification occurs at the first internucleotide linkage at the 5' end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration. The dsRNA agent of any one of claims 1 to 65, further comprising:

69. A terminal chiral modification occurring at the first and second internucleotide linkages at the 3' end of the antisense strand, with the linking phosphorus atom in the Sp configuration. A terminal chiral modification occurs at the third internucleotide linkage at the 3' end of the antisense strand, with the linking phosphorus atom in the Rp configuration. a terminal chiral modification occurring at a first internucleotide linkage at the 5′-end of the antisense strand, the linking phosphorus atom being in an Rp configuration; and A terminal chiral modification occurs at the first internucleotide linkage at the 5' end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration. The dsRNA agent of any one of claims 1 to 65, further comprising:

70. A terminal chiral modification occurring at the first and second internucleotide linkages at the 3' end of the antisense strand, with the linking phosphorus atom in the Sp configuration. a terminal chiral modification occurring at a first and a second internucleotide linkage at the 5′ end of the antisense strand having a linking phosphorus atom in an Rp configuration; and A terminal chiral modification occurs at the first internucleotide linkage at the 5' end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration. The dsRNA agent of any one of claims 1 to 65, further comprising:

71. The dsRNA agent of any one of claims 1-70, further comprising a phosphate or a phosphate mimic at the 5'-end of the antisense strand.

72. 72. The dsRNA agent of claim 71, wherein the phosphate mimic is a 5'-vinylphosphonate (VP).

73. The dsRNA agent of any one of claims 1 to 70, wherein the base pair at one position on the 5' end of the antisense strand of the duplex is an AU base pair.

74. The dsRNA agent of any one of claims 1 to 70, wherein the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

75. The dsRNA of any one of claims 1 to 74, wherein the dsRNA agent targets a hotspot region of the mRNA encoding ABLIM3.

76. 76. The dsRNA agent of claim 75, wherein the hotspot region comprises nucleotides 3042-3081, 3420-3457, 4223-4261, 726-763, 1080-1137, 1342-1379, 1910-1955, 2167-2204, 480-520, 1080-1122, 1622-1665, 1744-1787, 2323-2363, 540-578, 1175-1212, 274-313, 349-401, or 2518-2555 of SEQ ID NO:

1.

77. The dsRNA agent is AD-1809702, AD-1809719, AD-1810038, AD-1810053, AD-1810676, AD-1810692, AD-1807904, AD-1807919, AD-1808238, A D-1808258, AD-1808273, AD-1808418, AD-1808433, AD-1808842, AD-1808865, AD-1809051, AD-1809066, AD-1807707, AD-1807725, 77. The dsRNA agent of claim 76, selected from the group consisting of AD-1808628, AD-1808649, AD-1808727, AD-1808748, AD-1809187, AD-1809205, AD-1807764, AD-1807780, AD-1808312, AD-1808327, AD-1807561, AD-1807578, AD-1807616, AD-1807631, AD-1807646, AD-1809354, and AD-1809369.

78. A dsRNA agent that targets a hotspot region of actin-binding LIM protein 3 (ABLIM3) mRNA.

79. A cell comprising the dsRNA agent of any one of claims 1-78.

80. A pharmaceutical composition for inhibiting expression of a gene encoding ABLIM3, comprising a dsRNA agent according to any one of claims 1 to 78.

81. 79. A pharmaceutical composition comprising the dsRNA agent of any one of claims 1 to 78 and a lipid formulation.

82. 82. The pharmaceutical composition of claim 80 or 81, wherein the dsRNA agent is in an unbuffered solution.

83. 83. The pharmaceutical composition of claim 82, wherein the unbuffered solution is saline or water.

84. 82. The pharmaceutical composition of claim 80 or 81, wherein the dsRNA agent is in a buffer solution.

85. 85. The pharmaceutical composition of claim 84, wherein the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.

86. 85. The pharmaceutical composition of claim 84, wherein the buffer solution is phosphate buffered saline (PBS).

87. 11. A method of inhibiting expression of the ABLIM3 gene in a cell, comprising contacting the cell with a dsRNA agent according to any one of claims 1 to 78 or a pharmaceutical composition according to any one of claims 80 to 86, thereby inhibiting expression of ABLIM3 in the cell.

88. 88. The method of claim 87, wherein the cell is within a subject.

89. 89. The method of claim 88, wherein the subject is a human.

90. 90. The method of claim 89, wherein the subject has an ABLIM3-associated disorder.

91. 91. The method of claim 90, wherein the ABLIM3-associated disorder is PTSD.

92. 91. The method of claim 90, wherein the ABLIM3-associated disorder is age-related memory loss.

93. 93. The method of any one of claims 87-92, wherein contacting the cell with the dsRNA agent inhibits expression of ABLIM3 by at least 30%.

94. 94. The method of any one of claims 87-93, wherein inhibiting expression of ABLIM3 reduces ABLIM3 protein levels in serum of the subject by at least 30%.

95. A method of treating a subject having a disorder in which reduced ABLIM3 expression would be beneficial, comprising administering to the subject a therapeutically effective amount of a dsRNA agent of any one of claims 1-78 or a pharmaceutical composition of any one of claims 80-86, thereby treating the subject having a disorder in which reduced ABLIM3 expression would be beneficial.

96. A method of preventing at least one symptom or sign in a subject having a disorder in which reduced ABLIM3 expression would be beneficial, comprising administering to the subject a prophylactically effective amount of a dsRNA agent of any one of claims 1-78 or a pharmaceutical composition of any one of claims 80-86, thereby preventing at least one symptom or sign in a subject having a disorder in which reduced ABLIM3 expression would be beneficial.

97. 97. The method of claim 95 or 96, wherein the disorder is an ABLIM3-associated disorder.

98. 98. The method of claim 97, wherein the ABLIM3-associated disorder is PTSD.

99. 98. The method of claim 97, wherein the ABLIM3-associated disorder is age-related memory loss.

100. The method of any one of claims 95 to 99, wherein the subject is a human.

101. 101. The method of any one of claims 95 to 100, wherein administration of the agent to a subject results in improved memory, or a reduction in anxiety, depression, fear, restlessness, hostility, and / or distress.

102. 102. The method of any one of claims 95-101, wherein the dsRNA agent is administered to the subject at a dose of from about 0.01 mg / kg to about 50 mg / kg.

103. 103. The method of any one of claims 95-102, wherein the dsRNA agent is administered to the subject intrathecally.

104. The method of any one of claims 95 to 103, further comprising determining the level of ABLIM3 in a sample from the subject.

105. The method of claim 104, wherein the level of ABLIM3 in the subject sample is the ABLIM3 protein level in a blood, serum, or cerebrospinal fluid sample.

106. 106. The method of any one of claims 95 to 105, further comprising administering to the subject an additional therapeutic agent.

107. A kit comprising a dsRNA agent according to any one of claims 1 to 78 or a pharmaceutical composition according to any one of claims 80 to 86.

108. A vial comprising a dsRNA agent according to any one of claims 1 to 78 or a pharmaceutical composition according to any one of claims 80 to 86.

109. A syringe comprising a dsRNA agent according to any one of claims 1 to 78 or a pharmaceutical composition according to any one of claims 80 to 86.

110. An intrathecal pump comprising a dsRNA agent according to any one of claims 1 to 78 or a pharmaceutical composition according to any one of claims 80 to 86.

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