Extrahepatic delivery of double-stranded RNA agents

Abstract

One aspect of the present invention relates to a double-stranded RNA (dsRNA) agent for modulating the expression of a target gene in the central nervous system (CNS), comprising: an antisense strand complementary to the target gene in the CNS; a sense strand complementary to the antisense strand; and one or more saturated or unsaturated C RNAs conjugated onto at least one strand via a linker or carrier as appropriate. 22 The present invention relates to a dsRNA agent comprising one or more lipophilic moieties containing a hydrocarbon chain. Another aspect of the present invention relates to a pharmaceutical composition comprising a dsRNA agent. Another aspect of the present invention relates to a method for modulating the expression of a target gene in CNS cell genes and a method for treating or preventing CNS damage in a subject, comprising administering a therapeutically effective amount of a dsRNA agent to cells or a subject.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 458,788, filed April 12, 2023; U.S. Provisional Patent Application No. 63 / 254,117, filed June 29, 2023; and U.S. Provisional Patent Application No. 63 / 566,106, filed March 15, 2024, all of which are incorporated herein by reference in their entirety.

[0002] Array List This application includes an array list submitted in XML format, which is incorporated herein by reference in its entirety. The XML copy, created on 11 April 2024, is named 29520_1512-PCT_ALN-499-WO_SL.xml and has a size of 2,102,861 bytes. [Background technology]

[0003] Efficient delivery of dsRNA agents to cells in vivo requires specific targeting and substantial protection from the extracellular environment, specifically serum proteins. RNAi-based therapies show promising clinical data for the treatment of liver-related disorders. However, limitations remain in siRNA delivery to extrahepatic tissues, restricting the use of siRNA-based therapies.

[0004] One of the factors limiting the experimental and therapeutic application of dsRNA agents in vivo is their ability to efficiently deliver intact siRNA.

[0005] The delivery of oligonucleotides to the central nervous system (CNS) presents specific challenges due to the blood-brain barrier (BBB), which prevents free oligonucleotides from passing through. One method of delivering oligonucleotides to the CNS is intrathecal delivery. However, oligonucleotides also need to be efficiently translocated into target cells in the CNS to achieve the desired therapeutic effect. Previous studies typically utilize delivery reagents, such as liposomes, cationic lipids, and nanoparticle-forming complexes, to facilitate the intracellular translocation of oligonucleotides into cells of neuronal origin. [Overview of the project] [Problems that the invention aims to solve]

[0006] Therefore, there is a continued need for novel and improved compositions and methods for delivering siRNA molecules in vivo to achieve and enhance the therapeutic potential of dsRNA agents without the use of tissue delivery reagents. [Means for solving the problem]

[0007] One aspect of the present invention provides a compound (for example, an oligonucleotide that may be single-stranded or double-stranded) comprising one or more lipophilic monomers, each containing one or more lipophilic moieties conjugated at one or more positions on at least one chain of the oligonucleotide via a linker or carrier as appropriate.

[0008] Some embodiments of the present invention include an antisense strand complementary to a target gene in the CNS, conjugated at one or more positions on at least one strand via a linker or carrier as appropriate; a sense strand complementary to the antisense strand; and one or more saturated or unsaturated C 22 The present invention provides a compound (e.g., a double-stranded RNA (dsRNA) agent) that modulates the expression of a target gene in the central nervous system (CNS), comprising one or more lipophilic moieties containing a hydrocarbon chain.

[0009] In some embodiments, the target gene in the CNS is selected from the group consisting of APP, SOD1, SCN9A, HTT (HUNTINGTIN), APOE, LRRK2, PRNP, SCD5, GPR75, MAPT, SNCA, ABLIM3, ADRA2A, ATXN1, ATXN2, ATXN3, ELOVL1, FLNA, NOGO-L or NOGO-R, HIF-1α, RHO-A, NAV1.8, CD45, GSK-3, GSK3α, MIG-12, Mgat1, Mgat4, SLC35A1, SLC35A2, GNE, TMPRSS6, complement component C3, APCS, C9orf72, CHI3L1 / YKL-40, EXT1, EXT2, NDST2, RPS25, ALK, and SCD5. In some embodiments, the target gene in the CNS is selected from the group consisting of APP, ATXN2, C9orf72, TARDBP, MAPT(Tau), HTT, SNCA, FUS, ATXN3, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, TTR, SCN9A, LRRK2, GPR75, APOE, SCD5, ELOVL1, FLNA, ALK, CHI3L1(YKL-40), RPS25, α2-AR, and GSK3α.

[0010] In some embodiments, the octanol-water partition coefficient, logK ow The lipophilicity of the lipophilic portion measured by is greater than 0. The lipophilic portion is greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10, logK ow It is possible to possess this.

[0011] In some embodiments, the hydrophobicity of the compound (e.g., a dsRNA agent) as measured by the unbound fraction in a plasma protein binding assay of the compound is greater than 0.2. In one embodiment, the determined plasma protein binding assay is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. The hydrophobicity of the compound as measured by the unbound siRNA fraction in the binding assay is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45, or greater than 0.5, due to the enhancement of siRNA in vivo delivery.

[0012] In some embodiments, one or more lipophilic moieties are aliphatic, cyclic (such as alicyclic), or polycyclic (such as polycyclic alicyclic) compounds, e.g., steroids (e.g., sterols) or linear or branched aliphatic hydrocarbons. Exemplary lipophilic moieties include lipids, cholesterol, retinoic acid, cholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, granyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)litcholic acid, O3-(oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxytrityl, or phenoxazine.

[0013] Suitable lipophilic portions are saturated or unsaturated C4-C4. 30 Hydrocarbon chains (for example, C4~C 30Those containing any functional group selected from the group consisting of alkyl or alkenyl (linear or branched), hydroxyl, amine, carboxylic acid, carboxylic acid ester, sulfonic acid, ether, phosphate, thiol, azide, alkyne (e.g., terminal alkyne), cycloalkyne (e.g., cyclooctyne, dibenzocyclooctyne, or azadibenzocyclooctyne), trans - cyclooctenyl, N - maleimidyl, and 1,2,4,5 - tetrazin - 3 - yl are also included. The functional group is useful for conjugating the lipophilic moiety to the dsRNA agent.

[0014] In some embodiments, one or more lipophilic moieties are saturated or unsaturated C 20 ~C 30 hydrocarbon chains (e.g., linear C 20 ~C 30 alkyl or alkenyl). In one embodiment, one or more lipophilic moieties are saturated or unsaturated C 20 ~C 28 hydrocarbon chains; saturated or unsaturated C 20 ~C 26 hydrocarbon chains; saturated or unsaturated C 20 ~C 24 hydrocarbon chains. In one embodiment, one or more lipophilic moieties are saturated or unsaturated C 20 hydrocarbon chains; saturated or unsaturated C 21 hydrocarbon chains; saturated or unsaturated C 22 hydrocarbon chains; saturated or unsaturated C 23 hydrocarbon chains; or saturated or unsaturated C 24 hydrocarbon chains may be contained.

[0015] In other embodiments, the lipophilic moiety is a C 20 alkyl chain, or a C 21 alkyl chain, or a C 22 alkyl chain, or a C 23 alkyl chain, or a C 24It contains an alkyl chain. In another embodiment, each of the above may be a linear alkyl chain (e.g., n-eicosyl, or n-henicosanyl, n-docosanyl, or n-tricosanyl, or n-tetracosanyl).

[0016] In one embodiment, one or more lipophilic portions are saturated or unsaturated C 22 Hydrocarbon chains (e.g., linear or branched C) 22 It may contain alkyl or alkenyl groups. In some embodiments, one or more lipophilic moieties may contain two or more carbon-carbon double bonds. In some embodiments, one or more lipophilic moieties may contain one or more carbon-carbon double bonds and one or more carbon-carbon triple bonds (e.g., "enine" such as conjugate enine).

[0017] Examples of branched lipophilic moieties include docosan-2-yl, docosan-3-yl, docosan-4-yl, docosan-5-yl, docosan-6-yl, docosan-7-yl, docosan-8-yl, docosan-9-yl, docosan-10-yl, docosan-11-yl, 2-(decyl)dodecane-1-yl, 2-(nonyl)tridecane-1-yl, 2-(octyl)tetradecane-1-yl, 2-(heptyl)pentadecane-1-yl, 2-(hexyl)hexadecane-1-yl, 2-(pentyl)heptadecane-1-yl, and 2-(butyl)octadecane-1-yl. 4-(octyl)tetradecane-1-yl, 2-(propyl)nonadecan-1-yl, 2-(ethyl)eicosan-1-yl, 2-(methyl)heicosan-1-yl, 3-(nonyl)tridecane-1-yl, 3-(octyl)tetradecane-1-yl, 3-(heptyl)pentadecan-1-yl, 3-(hexyl)hexadecan-1-yl, 3-(pentyl)heptadecan-1-yl, 3-(butyl)octadecan-1-yl, 3-(propyl)nonadecan-1-yl, 3-(ethyl)eicosan-1-yl, 3-(methyl)heicosan-1-yl, 4-(octyl)tetradecane-1-yl 4-(heptyl)pentadecan-1-yl, 4-(hexyl)hexadecan-1-yl, 4-(pentyl)heptadecan-1-yl, 4-(butyl)octadecane-1-yl, 4-(propyl)nonadecan-1-yl, 4-(ethyl)eicosan-1-yl, 4-(methyl)henicosan-1-yl, 5-(heptyl)pentadecan-1-yl, 5-(hexyl)hexadecan-1-yl, 5-(pentyl)heptadecan-1-yl, 5-(butyl)octadecane-1-yl, 5-(propyl)nonadecan-1-yl, 5-(ethyl)eicosan-1-yl L, 5-(methyl)henicosane-1-yl, 6-(hexyl)hexadecane-1-yl, 6-(pentyl)heptadecane-1-yl, 6-(butyl)octadecane-1-yl, 6-(propyl)nonadecane-1-yl, 6-(ethyl)eicosane-1-yl, 6-(methyl)henicosane-1-yl, 7-(pentyl)heptadecane-1-yl, 7-(butyl)octadecane-1-yl, 7-(propyl)nonadecane-1-yl, 7-(ethyl)eicosane-1-yl, 7-(methyl)henicosane-1-yl, 8-(butyl)octadecane-1-yl,This includes, but is not limited to, 8-(propyl)nonadecan-1-yl, 8-(ethyl)eicosan-1-yl, 8-(methyl)henicosan-1-yl, 9-(propyl)nonadecan-1-yl, 9-(ethyl)eicosan-1-yl, 9-(methyl)henicosan-1-yl, 10-(ethyl)eicosan-1-yl, 10-(methyl)henicosan-1-yl, and 11-(methyl)henicosan-1-yl.

[0018] In some embodiments, one or more lipophilic moieties may contain one or more carbohydrate chains such that the total number of carbon atoms in the lipophilic moieties is 20 to 30 (e.g., 21 or 22). For example, the lipophilic moiety may be of the formula

[0019] [ka] [In the formula, Q is a branch point (e.g., N or C(H)) and L 0 is a divalent linking group (L 0 (which may be one of the linkers / tethers specified herein), and R1 and R2 are each independently C1-C such that the total number of carbon atoms in R1 and R2 is 20-30 (e.g., 22 or 21 if Q is C(H)). 20 It can be a hydrocarbon chain. In other embodiments, Q is of the formula -N(R3)-[CH2] n -N(R4)-[wherein n is 2 to 10, and R3 and R4 are each independently C1 to C4 where the total number of carbon atoms in R1, R2, R3, and R4 is 20 to 30 (e.g., 22)] 20 It is a hydrocarbon chain. In other embodiments, Q is a hydrocarbon chain.

[0020] [ka] [In the formula, *R1 and R2 are bonded, and the total number of carbon atoms in R1 and R2 is 20 to 30 (e.g., 22). In some embodiments, one or more R1, R2, R3, and R4 may contain two or more carbon-carbon double bonds. In some embodiments, one or more R1, R2, R3, and R4 may contain one or more carbon-carbon double bonds and one or more carbon-carbon triple bonds (e.g., "enine" such as conjugate enine). In some embodiments, one or more R1, R2, R3, and R4 may contain functional groups selected from the group consisting of hydroxyl, amine, carboxylic acid, carboxylic acid ester, sulfonic acid, ether, phosphoric acid, thiol, azide, alkyne (e.g., terminal alkyne), cycloalkyne (e.g., cyclooctenyl, dibenzocyclooctenyl, or azadibenzocyclooctenyl), trans-cyclooctenyl, N-maleimidyl, and 1,2,4,5-tetrazin-3-yl.

[0021] In some embodiments, one or more lipophilic moieties have free terminal carboxylic acid functionalities (e.g., hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, oleic acid, linolenic acid, arachidonic acid, cis-4,7,10,13,16,19-docosahexaenoic acid) C6-C6 30 It could be a part.

[0022] In some embodiments, one or more lipophilic portions are C6-C 30 Acids (e.g., hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, oleic acid, linolenic acid, arachidonic acid, cis-4,7,10,13,16,19-docosahexaenoic acid, vitamin A, vitamin E, cholesterol, etc.), or C6~C 30It may be an alcohol (e.g., hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecanol, hexadecanol, heptadecanol, octadecanol, oleyl alcohol, linoleyl alcohol, arachidonic acid alcohol, cis-4,7,10,13,16,19-docosahexanol, retinol, vitamin E, cholesterol, etc.).

[0023] In some embodiments, two or more lipophilic moieties may be conjugated to a dsRNA agent.

[0024] In some embodiments, one or more saturated or unsaturated C 22 At least one lipophilic moiety containing a hydrocarbon chain is conjugated to the dsRNA agent.

[0025] In some embodiments, at least one C 22 Hydrocarbon chains can be saturated or unsaturated, linear or branched. 22 It is a hydrocarbon chain. One or more C 22 The hydrocarbon chain may contain functional groups selected from the group consisting of hydroxyl, amine, carboxylic acid, carboxylic acid ester, sulfonic acid, ether, phosphoric acid, thiol, azide, alkyne (e.g., terminal alkyne), and cycloalkyne (e.g., cyclooctenyl, dibenzocyclooctenyl, or azadibenzocyclooctenyl), trans-cyclooctenyl, N-maleimidyl, and 1,2,4,5-tetrazin-3-yl. These functional groups are useful for conjugating the lipophilic moiety to the dsRNA agent.

[0026] In some embodiments, one or more lipophilic portions are of formula -G2-R G [In the formula, G2 is saturated or unsaturated C] 20 ~C 30 Hydrocarbon groups (e.g., saturated or unsaturated C) 21 or C 22 (Hydroxide group) and R G[The compound may be selected from the group consisting of hydrogen, hydroxyl, amino, -COOH, and -C(O)NH2.]

[0027] In some embodiments, one or more lipophilic portions are of formula -G2-R G [In the formula, G2 is saturated or unsaturated C] 21 It is a hydrocarbon group, R G It may have -COOH or -C(O)NH2.

[0028] In some embodiments, one or more lipophilic portions are of formula -G2-R G [In the formula, G2 is saturated or unsaturated C] 22 It is a hydrocarbon group, R G It may have -COOH or -C(O)NH2.

[0029] In some embodiments, one or more lipophilic portions are of formula -G2-R G [In the formula, G2 is saturated or unsaturated C] 22 It is a hydrocarbon group, R G It can have -OH.

[0030] In some embodiments, one or more lipophilic portions are of formula -G3-L K -G2-R G [In formula: G3 is saturated or unsaturated C 1~20 Hydrocarbon group (for example, C 1~6 Alkylene; C 2~6 Alkylene; or hexylene) L K These are linking groups, for example, -O-, -N(H)-, -S-, -SS-, -C(O)O-, OC(O)-, -C(O)N(H)-, -N(H)C(O), -OC(O)N(H)-, -N(H)C(O)O-, -S(O)2-, -S(O)2O-, -S(O)2N(H)-, -P(O)(OH)O-, -OP(O)(OH)-, -P(S)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O-, -OP(S)(OH)O-, G2 is saturated or unsaturated C21~ C 22 It is a hydrocarbon group, R G [These are hydrogen, hydroxyl, amino, -COOH, or -C(O)NH2] It may have L K However, if it contains a carbonyl group bonded to G2 (for example, -N(H)C(O)- or -OC(O)-), then G2 is C 21 It is a hydrocarbon group, L K However, if it does not contain a carbonyl bonded to G2, G2 is C 22 It is a hydrocarbon group. In one embodiment, R G is hydrogen. In one embodiment, R G is OH. In one embodiment, R G is COOH. In one embodiment, R G This is CONH2. In one embodiment, R G It is an amino acid.

[0031] In some embodiments, one or more lipophilic portions are of the formula, -L K2 -G3-L K -G2-R G [In formula: G2 is saturated or unsaturated C 21~22 It is a hydrocarbon group, R G is hydrogen, hydroxyl, amino, -COOH, or -C(O)NH2, G3 is saturated or unsaturated C 1~20 Hydrocarbon group (for example, C 1~6 Alkylene; C 2~6 Alkylene; or hexylene) L K and L K2is, independently, a linking group, for example, -O-, -N(H)-, -S-, -S-S-, -C(O)O-, OC(O)-, -C(O)N(H)-, -N(H)C(O), -OC(O)N(H)-, -N(H)C(O)O-, -S(O)2-, -S(O)2O-, -S(O)2N(H)-, -P(O)(OH)O-, -OP(O)(OH)-, -P(S)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O-, or -OP(S)(OH)O- (for example, L K2 is -C(O)-, -S(O)2-, -P(O)(OH)O-, -OP(O)(OH)-, -P(S)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O-, or -OP(S)(OH)O-).] may have. For example, L K when contains a carbonyl (for example, -N(H)C(O)- or -OC(O)-) bonded to G2, G2 is C 21 is a hydrocarbon group, and L K when does not contain a carbonyl bonded to G2, G2 is C 22 is a hydrocarbon group (for example, -N(H)C(O)- or -OC(O)-). In one embodiment, R G is hydrogen. In one embodiment, R G is OH. In one embodiment, R G is COOH. In one embodiment, R G is CONH2. In one embodiment, R G is amino. In one embodiment, L K2 is -P(O)(OH)O-, -OP(O)(OH)-, -P(S)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O-, or -OP(S)(OH)O-. In one embodiment, L K2 is -OP(O)(OH)O-, or -OP(S)(OH)O-. In one embodiment, L K2 is -OP(O)(OH)O. In one embodiment, L K2 is -OP(S)(OH)O-.

[0032] In some embodiments, at least one C22 The hydrocarbon chain is C 22 Contains acid. Example C 22 The acids are docosanic acid, 6-octyltetradecanoic acid, 10-hexylhexadecanoic acid, all-cis-7,10,13,16,19-docosapentaenoic acid, all-cis-4,7,10,13,16,19-docosahexaenoic acid, all-cis-13,16-docosadienoic acid, all-cis-7,10,13,16-docosatetraenoic acid, all-cis-4,7,10,13,16-docosapentaenoic acid, cis-13-docosenoic acid, 22-hydroxydocosanoic acid (

[0033] [ka] ), 16-hydroxyhexadecanoic acid (

[0034] [ka] ), and C6+16-hydroxyhexadecanoic acid (

[0035] [ka] This includes, but is not limited to, these items.

[0036] In some embodiments, one or more lipophilic portions are of formula -G2-R G [In the formula, G2 is saturated or unsaturated C] 22 It is a hydrocarbon group, R G It may contain hydrogen or hydroxyl.

[0037] In some embodiments, at least one C 22 The hydrocarbon chain is C 22 Contains alcohol. Example C 22Alcohols include, but are not limited to, 1-docosanol, 6-octyltetradecane-1-ol, 10-hexylhexadecane-1-ol, cis-13-docosen-1-ol, docosan-9-ol, docosan-2-ol, docosan-10-ol, docosan-11-ol, cis-4,7,10,13,16,19-docosahexanol, 22-hydroxydocosanoic acid, 16-hydroxyhexadecanoic acid, and C6+16-hydroxyhexadecanoic acid.

[0038] In some embodiments, at least one C 22 The hydrocarbon chain is C 22 Contains amide. Exemplary C 22 Amides include, but are not limited to, (E)-Docos-4-enamide, (E)-Docos-5-enamide, (Z)-Docos-9-enamide, (E)-Docos-11-enamide, 12-docosenaamide, (Z)-Docos-13-enamide, (Z)-N-hydroxy-13-docosenaamide, (E)-Docos-14-enamide, 6-cis-docosenaamide, 14-docosenaamide, Docos-11-enamide, (4E,13E)-Docosa-4,13-dienamide, and (5E,13E)-Docosa-5,13-dienamide.

[0039] The lipophilic portion can be conjugated to any part of the dsRNA agent, such as a nucleobase, sugar moiety, or nucleoside linkage. When the lipophilic portion is conjugated to a modified nucleotide linkage, it is understood that the lipophilic portion is "at" the oligonucleotide position if it forms part of the 3' nucleotide linkage of the reference nucleotide.

[0040] The lipophilic moiety can be conjugated to the dsRNA agent via direct joining to the nucleobase, ribosaccharide, or nucleoside linkage of the dsRNA agent. Alternatively, the lipophilic moiety can be conjugated to the dsRNA agent via a non-ribose substitution unit, such as a linker or carrier.

[0041] In some embodiments, the lipophilic portion is conjugated to the ribosaccharide of the dsRNA agent. In one embodiment, the lipophilic portion is conjugated to the 2' position of the ribosaccharide of the dsRNA agent. In one embodiment, the lipophilic portion is conjugated to the 3' position of the ribosaccharide of the dsRNA agent. In one embodiment, the lipophilic portion is conjugated to the 5' position of the ribosaccharide of the dsRNA agent.

[0042] In some embodiments, the lipophilic moiety is conjugated to the 2'-O-position of the ribosaccharide of the dsRNA agent via one or more linkers, as appropriate. For example, the lipophilic moiety is conjugated to the 2' position of the ribosaccharide of the dsRNA as a 2'-OCH2C(O)N(H)-lipophilic moiety.

[0043] In some embodiments, the lipophilic moiety is conjugated to the 3'-position of the ribosaccharide of the dsRNA agent via one or more linkers, as appropriate. For example, the lipophilic moiety is conjugated to the 3'-position of the ribosaccharide of the dsRNA as a 3'-(N-lipophilic moiety) phosphoramidate (e.g., 3'-P(=O)(O)(O)N-lipophilic moiety).

[0044] In a particular embodiment, the lipophilic moiety is conjugated to a dsRNA agent via one or more linkers (tethers).

[0045] In some embodiments, the lipophilic moiety is conjugated to a nucleobase via one or more linkers (tethers) as appropriate. In some embodiments, the lipophilic moiety is conjugated to a sugar moiety via one or more linkers (tethers) as appropriate. In some embodiments, the lipophilic moiety is conjugated to an internucleotide phosphate linker via one or more linkers (tethers) as appropriate.

[0046] In some embodiments, the lipophilic moiety is conjugated to a dsRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimidothioether, disulfide, phosphodiester, sulfonamide linkage, click reaction product (e.g., triazole from azide-alkyne cyclization), or carbamate.

[0047] In some embodiments, at least one linker (tether) is a redox-cleaving linker (e.g., a reductive-cleaving linker; e.g., a disulfide group), an acid-cleaving linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group), an esterase-cleaving linker (e.g., an ester group), a phosphatase-cleaving linker (e.g., a phosphate group), or a peptidase-cleaving linker (e.g., a peptide bond).

[0048] In other embodiments, at least one linker (tether) is a bio-cleavage linker selected from the group consisting of DNA, RNA, disulfides, amides, functionalized monosaccharides or galactosamine oligosaccharides, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0049] In certain embodiments, the lipophilic moiety is conjugated to the dsRNA agent via a non-ribose substitution unit, i.e., a carrier that substitutes one or more nucleotides of the dsRNA agent. The carrier can be a cyclic or acyclic group. In one embodiment, the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuryl, and decalin. In one embodiment, the acyclic group is a moiety based on a serinol skeleton, a glycerol skeleton, or a diethanolamine skeleton.

[0050] In some embodiments, the carrier substitutes one or more nucleotides of the dsRNA agent. In some embodiments, the carrier substitutes one or more nucleotides at one or more internal positions of the dsRNA agent. In other embodiments, the carrier substitutes nucleotides at the end of the sense strand or antisense strand. In one embodiment, the carrier substitutes a terminal nucleotide at the 3' end of the sense strand, thereby functioning as a terminal cap to protect the 3' end of the sense strand. In one embodiment, the carrier is a cyclic group having an amine, and for example, the carrier may be pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanil, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuranil, or dekalinil.

[0051] In some embodiments, the lipophilic portion conjugated via a carrier is given by the following formula:

[0052] [ka] [In formula: J1 and J2 are, independently, O, S, NR N , appropriately substituted alkyl, OC(O)NH, NHC(O)O, C(O)NH, NHC(O), OC(O), C(O)O, OHC(O)O, NHC(O)NH, NHC(S)NH, OC(S)NH, OP(N(R P )2)O, or OP(N(R P )2) and;

[0053] [ka] is a cyclic or acyclic group, R Nis H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted aralkyl, optionally substituted heteroaryl, or amino protecting group; R P Independently, for each occurrence, is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted cycloalkyl, or optionally substituted heteroaryl; L 10 is a substituted or unsubstituted, saturated or unsaturated C3-C8 hydrocarbon (e.g., a C3-C8 alkyl, alkenyl, or alkynyl, or a C3-C8 hydrocarbon containing two or more double bonds); substituents include those already described herein for "substituted" hydrocarbons, alkyls, alkenyls, or alkynyls; L 11 is a substituted or unsubstituted, saturated or unsaturated C6-C26 hydrocarbon (e.g., a C6-C26 alkyl, alkenyl, or alkynyl, or a C3-C8 hydrocarbon containing two or more double bonds); substituents include those already described herein for "substituted" hydrocarbons, alkyls, alkenyls, or alkynyls; Q is a nucleobase in a support, or at least 10% L in vivo. 11 From L 10 If there are no cleavage groups to cleave it, it is absent. It may be represented by one of the following: For example, Q is cleaved in vivo and contains approximately 10-70%, approximately 15-50%, approximately 20-40%, or approximately 20-30%, of the lipophilic monomer L 11 It can be a cleavage-type group capable of cleaving. Exemplary cleavage-type groups include -OC(O)-, -C(O)O-, -SC(O)-, -C(O)S-, -OC(S)-, -C(S)O-, -SS-, -C(R 5 )=N-, -N=C(R 5 )-,-C(R 5 )=NO-, -ON=C(R 5 )-,-C(O)N(R 5 )-,-N(R5 )C(O)-,-C(S)N(R 5 )-,-N(R 5 )C(S)-, -N(R 5 )C(O)N(R 5 )-,-N(R 5 )C(O)C(R 3 )(R 4 )OC(O)-, -C(O)OC(R 3 )(R 4 )C(O)N(R 5 )-, -OC(O)O-,OSi(R 5 )2O-, -C(O)(CR 3 R 4 )C(O)O-, -OC(O)(CR 3 R 4 )C(O)-,

[0054] [ka] Or, including combinations of these, R 11 It is a C2-C8 alkyl or alkenyl. For each occurrence, R 3 , R 4 , and R 5 Each of these is independently either H or a C1-C4 alkyl group.

[0055] In one embodiment, the cleavage potential of Q is determined by the ligand's stability in cerebrospinal fluid (CSF), its stability in plasma, and its stability in brain homogenates or tissue homogenates (e.g., liver).

[0056] Cyclic and acyclic groups include those already described herein.

[0057] In one embodiment, the acyclic group is selinol, glycerol, or a diethanolamine skeleton.

[0058] In one embodiment, the cyclic group is selected from the group consisting of pyrrolidinyl, hydroxyprolinyl, cyclopentyl, cyclohexyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanil, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuranil, and dekalinil.

[0059] In one embodiment, the cyclic group is ribose or a ribose analog. Examples of ribose analogs include arabinose, 4'-thioribose, 2'-O-methylribose, GNA, UNA, and LNA analogs.

[0060] In some embodiments, one or more saturated or unsaturated C 22 The lipophilic portion containing hydrocarbon chains is

[0061] [ka] TIFF2026514045000010.tif232161 TIFF2026514045000011.tif123153[in formula: B is a appropriately modified nucleobase; G is G1 or saturated or unsaturated C 21 A hydrocarbon chain (i.e., G, together with the carbonyl to which it is joined, can form a group having 22 carbons) (for example, G can be linear or branched C 21 (It can be an alkyl group), and G is a halogen, -OR G , -SR G , -N(R G )2, -C(O)OR G -OC(O)R G ,-C(O)N(R G )2, -N(R G )C(O)R G , -N(R G )C(O)OR G , -N(RG )SO2(R G ), or -SO2N(R G ) may be appropriately substituted by one or two groups selected from the group consisting of 2, and each R G These are independently hydrogen or C1-C6 alkyl (e.g., G is -OR) G , -C(O)OR G , or -N(R G )C(O)R G (This is replaced as appropriate by; G1 is saturated or unsaturated C 22 Hydrocarbon chains (for example, G1 is linear or branched C) 22 (It can be an alkyl group) and G 1 is halogen, -OR G1 , -SR G1 , -N(R G1 )2, -C(O)OR G1 -OC(O)R G1 ,-C(O)N(R G1 )2, -N(R G1 )C(O)R G1 , -N(R G1 )C(O)OR G1 , -N(R G1 )SO2(R G1 ), or -SO2N(R G1 ) may be appropriately substituted by one or two groups selected from the group consisting of 2, and each R G1 These are independently hydrogen or C1-C6 alkyl (e.g., G1 is -OR) G1 , -C(O)OR G1 , or -N(R G1 )C(O)R G1 (This is replaced as appropriate by; The integer m is between 0 and 8 (for example, m is 0; or m is between 1 and 8; or m is between 0 and 6; or m is 1; or 2; or 3; or 4; or 5; or 6; or 7; or 8); The integer n is between 1 and 21 (for example, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4, or 1 to 2; or 2, 3, 4, 5, or 6); W is an alkyl group such as C1-C4 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl); R, R', and R'' are each independently H or an alkyl group such as a C1-C4 alkyl (e.g., methyl, ethyl, propyl, isopropyl, t-butyl); R2' or R3' can be any functional group that is an acceptable 2'-modification of ribose sugar. A lipophilic monomer selected from the group consisting of the following. Examples of suitable R2' or R3' groups include, but are not limited to, hydrogen, halogen (e.g., 2'-fluoro), hydroxy, 2'-O-alkyl (e.g., 2'-O-methyl), 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropanyl) modifications, 2'-O-aryl modifications, 2'-C-aryl modifications, 2'-ON-methylacetamide (2'-O-NMA, i.e., -OCH2C(O)N(H)Me) modifications, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) modifications, 2'-O-aminopropyl (2'-O-AP) modifications, or 2'-ara-F modifications. For example, R2' or R3' can be H, OH, F, OMe, O-methoxyalkyl, O-aryl, ON-methylacetamide, O-dimethylaminoethoxyethyl, or O-aminopropyl.

[0062] In some embodiments, the lipophilic monomer is

[0063] [ka] [In formula: B is a appropriately modified nucleobase; G3 is saturated or unsaturated C 1~20 Hydrocarbon group (for example, C 1~6 Alkylene; C 2~6 Alkylene (or hexylene); L KThese are linking groups, for example, -O-, -N(H)-, -S-, -SS-, -C(O)O-, OC(O)-, -C(O)N(H)-, -N(H)C(O), -OC(O)N(H)-, -N(H)C(O)O-, -S(O)2-, -S(O)2O-, -S(O)2N(H)-, -P(O)(OH)O-, -OP(O)(OH)-, -P(S)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O-, -OP(S)(OH)O-, G2 is saturated or unsaturated C 21~22 It is a hydrocarbon group, R G [These are hydrogen, hydroxyl, amino, -COOH, or -C(O)NH2] It is selected from the group consisting of L. K However, if it contains a carbonyl group bonded to G2 (for example, -N(H)C(O)- or -OC(O)-), then G2 is C 21 It is a hydrocarbon group; L K However, if it does not contain a carbonyl bonded to G2, G2 is C 22 It is a hydrocarbon group. In one embodiment, R G is hydrogen. In another embodiment, R G is OH. In one embodiment, R G In another embodiment, R G This is CONH2. In one embodiment, R G It is an amino acid.

[0064] In the above structure of the lipophilic monomer, the monomer may contain one or more chiral centers and therefore may exist as a racemate and a racemate mixture, a single enantiomer, individual diastereomers and a mixture of diastereomers. All such isomeric forms of the monomer are clearly included. Furthermore, as described above and herein, modified nucleotide linkages are shown together with substituted atoms fully described at the phosphorus atom, for example,

[0065] [ka] When expressed as [wherein C' is the 2'-carbon or 3'-carbon atom of the ribose ring], the oxygen with a broken bond is understood to be the 5'-oxygen of the substituted nucleotide.

[0066] In the above structure of the lipophilic monomer, the alkylene chain may contain one or more unsaturated bonds.

[0067] Saturated or unsaturated C 22 Certain embodiments of lipophilic monomers containing hydrocarbon chains are:

[0068] [ka] TIFF2026514045000015.tif248119 TIFF2026514045000016.tif213156 TIFF2026514045000017.tif230158 TIFF2026514045000018.tif228161 Includes TIFF2026514045000019.tif58138. In these structures, B is a modified or unmodified nucleobase.

[0069] In one embodiment, the lipophilic monomer is

[0070] [ka] [In the formula, B is a modified or unmodified nucleobase.] That is the case.

[0071] In one embodiment, the lipophilic monomer is

[0072] [ka] [In the formula, B is a modified or unmodified nucleobase.] That is the case.

[0073] In one embodiment, the lipophilic monomer is

[0074] [ka] [In the formula, B is a modified or unmodified nucleobase.] That is the case.

[0075] In one embodiment, the lipophilic monomer is

[0076] [ka] [In the formula, B is a modified or unmodified nucleobase.] That is the case.

[0077] In some embodiments, the lipophilic monomer is

[0078] [ka] [In the formula, n is an integer from 1 to 21, for example, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4, or 1 to 2, or 2, 3, 4, 5, or 6; G is a halogen, -OR G , -SR G , -N(R G )2, -C(O)OR G -OC(O)R G ,-C(O)N(R G )2, -N(R G )C(O)R G , -N(R G )C(O)OR G , -N(R G )SO2(R G ), or -SO2N(R G ) may be appropriately substituted by one or two groups selected from the group consisting of 2, and each R G However, independently, C is either hydrogen or a C1-C6 alkyl group. 22It is a hydrocarbon chain; nucleobase B is a modified or unmodified nucleobase. In one embodiment, n is 1. In one embodiment, n is 2-6. In one embodiment, n is 6. In one embodiment, G is C 22 It is an alkyl chain.

[0079] In some embodiments, the lipophilic monomer is

[0080] [ka] [In the formula, n is an integer from 1 to 21, for example, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4, or 1 to 2, or 2, 3, 4, 5, or 6; G is a halogen, -OR G , -SR G , -N(R G )2, -C(O)OR G -OC(O)R G ,-C(O)N(R G )2, -N(R G )C(O)R G , -N(R G )C(O)OR G , -N(R G )SO2(R G ), or -SO2N(R G ) may be appropriately substituted by one or two groups selected from the group consisting of 2, and each R G However, independently, C is either hydrogen or a C1-C6 alkyl group. 22 It is a hydrocarbon chain; nucleobase B is a modified or unmodified nucleobase. In one embodiment, n is 1. In one embodiment, n is 2-6. In one embodiment, n is 6. In one embodiment, G is C 22 It is an alkyl chain.

[0081] In some embodiments, the lipophilic monomer is

[0082] [ka] [In the formula, R2' is H, OH, F, Ome, O-methoxyalkyl, O-aryl, or ON-methylacetamide; nucleobase B is a modified or unmodified nucleobase]. In one embodiment, R2' is H, OH, F, Ome, or O-methoxyalkyl. In one embodiment, G is C 22 It is an alkyl chain.

[0083] In some embodiments, the lipophilic monomer is

[0084] [ka] [In the formula, R3' is H, OH, F, Ome, O-methoxyalkyl, O-aryl, or ON-methylacetamide; nucleobase B is a modified or unmodified nucleobase]. In one embodiment, R3' is H, OH, F, Ome, or O-methoxyalkyl. In one embodiment, G is C 22 It is an alkyl chain.

[0085] In one embodiment, the lipophilic monomer is

[0086] [ka] [In the formula, B is a modified or unmodified nucleobase.] That is the case.

[0087] In one embodiment, the lipophilic monomer is

[0088] [ka] [In the formula, B is a modified or unmodified nucleobase.] That is the case.

[0089] In one embodiment, the lipophilic monomer is

[0090] [ka] [In the formula, B is a modified or unmodified nucleobase.] That is the case.

[0091] In one embodiment, the lipophilic monomer is

[0092] [ka] [In the formula, B is a modified or unmodified nucleobase.] That is the case.

[0093] In one embodiment, the lipophilic monomer is

[0094] [ka] [In the formula, B is a modified or unmodified nucleobase.] That is the case.

[0095] In one embodiment, the lipophilic monomer is

[0096] [ka] [In the formula, B is a modified or unmodified nucleobase.] That is the case.

[0097] In one embodiment, the lipophilic monomer is

[0098] [ka] [In the formula, B is a modified or unmodified nucleobase, and the phosphorus atom in the internucleotide linkage is appropriately concentrated into Sp or Rp isomers, or is a racemic mixture.] That is the case.

[0099] In one embodiment, the lipophilic monomer is

[0100] [ka] [In the formula, B is a modified or unmodified nucleobase, and the phosphorus atom in the internucleotide linkage is appropriately concentrated into Sp or Rp isomers, or is a racemic mixture.] That is the case.

[0101] In some embodiments, one or more saturated or unsaturated C 22 Lipophilic portions containing hydrocarbon chains belong to groups (i), (ii), and (iii): (i)

[0102] [ka] (ii)

[0103] [ka] And, (iii)

[0104] [ka] [In formula: B is a appropriately modified nucleobase; G is G1 or saturated or unsaturated C 21 A hydrocarbon chain (i.e., G, together with the carbonyl to which it is joined, can form a group with 22 carbons), and G is a halogen, -OR G , -SR G , -N(R G )2, -C(O)OR G -OC(O)R G ,-C(O)N(R G )2, -N(R G )C(O)R G , -N(R G )C(O)OR G , -N(R G )SO2(R G ), or -SO2N(R G) may be appropriately substituted by one or two groups selected from the group consisting of 2, and each R G These are independently hydrogen or C1-C6 alkyl (e.g., G is -OR) G , -C(O)OR G , or -N(R G )C(O)R G (This is replaced as appropriate by; G1 is saturated or unsaturated C 22 It is a hydrocarbon chain, G 1 is halogen, -OR G1 , -SR G1 , -N(R G1 )2, -C(O)OR G1 -OC(O)R G1 ,-C(O)N(R G1 )2, -N(R G1 )C(O)R G1 , -N(R G1 )C(O)OR G1 , -N(R G1 )SO2(R G1 ), or -SO2N(R G1 ) may be appropriately substituted by one or two groups selected from the group consisting of 2, and each R G1 These are independently hydrogen or C1-C6 alkyl (e.g., G1 is -OR) G1 , -C(O)OR G1 , or -N(R G1 )C(O)R G1 (This is replaced as appropriate by; R2' or R3' can be any functional group that is an acceptable 2'-modification of ribose sugar. A lipophilic monomer selected from one of the members. Suitable R2' or R3' group examples include, but are not limited to, hydrogen, halogen (e.g., 2'-fluoro), hydroxy, 2'-O-alkyl (e.g., 2'-O-methyl), 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropanyl) modifications, 2'-O-aryl modifications, 2'-C-aryl modifications, 2'-ON-methylacetamide (2'-O-NMA, i.e., -OCH2C(O)N(H)Me) modifications, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) modifications, 2'-O-aminopropyl (2'-O-AP) modifications, or 2'-ara-F modifications. For example, R2' or R3' can be H, OH, F, OMe, O-methoxyalkyl, O-aryl, ON-methylacetamide, O-dimethylaminoethoxyethyl, or O-aminopropyl.

[0105] In some embodiments, one or more saturated or unsaturated C 22 The lipophilic portions containing hydrocarbon chains belong to groups (i'), (ii'), and (iii'): (i')

[0106] [ka] (ii')

[0107] [ka] And, (iii')

[0108] [ka] It is a lipophilic monomer selected from one of the members. In these embodiments, B is a suitably modified nucleobase. 22 is saturated or unsaturated C 22 It is a hydrocarbon chain. For example, R 22 C is linear or branched22 It is an alkyl group. R2' can be any functional group that is an acceptable 2'-modification of ribose sugar. Examples of preferred R2' or R3' groups include, but are not limited to, hydrogen, halogen (e.g., 2'-fluoro), hydroxy, 2'-O-alkyl (e.g., 2'-O-methyl), 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropanyl) modifications, 2'-O-aryl modifications, 2'-C-aryl modifications, 2'-ON-methylacetamide (2'-O-NMA, i.e., -OCH2C(O)N(H)Me) modifications, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) modifications, 2'-O-aminopropyl (2'-O-AP) modifications, or 2'-ara-F modifications. For example, R2' or R3' is H, OH, F, OMe, O-methoxyalkyl, O-aryl, ON-methylacetamide, O-dimethylaminoethoxyethyl, or O-aminopropyl. B is a modified or unmodified nucleobase.

[0109] In some embodiments, the lipophilic portion is conjugated to one of the 3' or 5' ends of the sense or antisense chain via direct bonding or by a carrier or linker. In some embodiments, the lipophilic portion is conjugated to the 3' end of the sense or antisense chain via direct bonding or by a carrier or linker. In some embodiments, the lipophilic portion is conjugated to the 5' end of the sense or antisense chain via direct bonding or by a carrier or linker.

[0110] In some embodiments, the lipophilic portion is of the formula

[0111] [ka] [In the formula, X is O or S (e.g., S); L is a divalent linking group (e.g., C] 1~20 Alkyl or C 1~10 Alkyl-SSC 1~10(Alkyl) or a salt thereof. In one embodiment, the lipophilic portion is of the formula

[0112] [ka] [In the formula, X is either O or S (for example, S)] or a salt thereof. In these embodiments, R リガンド The element is selected from the elements listed in Table R-1.

[0113] [Table 1]

[0114] In one embodiment, the lipophilic moiety is bonded to the 5'-oxygen of the 5'-terminal nucleotide or the 3'-oxygen of the 3'-terminal nucleotide, and formula

[0115] [ka] That is the case.

[0116] In some embodiments, the lipophilic portion is conjugated to one of the 3' or 5' ends of the sense and antisense strands via a carrier or linker, the carrier or linker being an inverted debasalized nucleotide, e.g., an inverted debasalized deoxyribonucleotide or an inverted debasalized ribonucleotide, each connected to the rest of the oligonucleotide via a phosphodiester (PO) linkage or a phosphorothioate (PS) linkage. An example is,

[0117] [ka] This includes, but is not limited to, the following:

[0118] In some embodiments, the lipophilic moiety is bonded to the 5'-oxygen of the 5'-terminal nucleotide or the 3'-oxygen of the 3'-terminal nucleotide, and formula

[0119] [ka] [In the formula, each X is independently either O or S (for example, each is S); R リガンド The group is selected from the groups listed in Table R-1; L is a divalent linking group (e.g., C 1~20 Alkyl or C 1~10 Alkyl-SSC 1~10 (Alkyl) or its salt.

[0120] For example, the lipophilic portion is bonded to the 5'-oxygen of the 5'-terminal nucleotide or the 3'-oxygen of the 3'-terminal nucleotide, and the formula

[0121] [ka] [In the formula, each X is either O or S (for example, each is S) and R リガンド [The base is selected from the bases listed in Table R-1.] or a salt thereof. In some embodiments, R リガンド The element is selected from the elements listed in Table R-2.

[0122] [Table 2]

[0123] In some embodiments, the lipophilic moiety is bonded to the 5'-oxygen of the 5'-terminal nucleotide or the 3'-oxygen of the 3'-terminal nucleotide, and formula

[0124] [ka] [In the formula, each X is independently either O or S (for example, each is S) and R リガンド The group is selected from the groups listed in Table R-1, and L is a divalent linking group (for example, C 1~20 Alkyl or C 1~10 Alkyl-SSC 1~10(Alkyl) or its salt.

[0125] In one embodiment, the lipophilic moiety is bonded to the 5'-oxygen of the 5'-terminal nucleotide or the 3'-oxygen of the 3'-terminal nucleotide, and formula

[0126] [ka] [In the formula, each X is either O or S (for example, each is S) and R リガンド [The base is selected from the bases listed in Table R-1.] or a salt thereof. In some embodiments, R リガンド The element is selected from the elements listed in Table R-2.

[0127] In some embodiments, the dsRNA agent comprises a double-stranded region formed between the sense strand and the antisense strand, and optionally one or two single-stranded non-loop overhangs, with one or more lipophilic moieties conjugated to either the double-stranded region or the non-loop overhangs. In some embodiments, the dsRNA agent does not contain a loop (e.g., stem-loop) region. In some embodiments, the dsRNA agent contains a loop (e.g., stem-loop) region, and one or more lipophilic moieties are not conjugated to the loop (e.g., stem-loop) region.

[0128] In some embodiments, the dsRNA agent comprises a sense strand 10–53 nucleotides long, where the sense strand forms a double-stranded region with the antisense strand. For example, the sense strand may be 10–49, 12–49, 12–45, 12–42, 12–40, 15–49, 15–45, 15–42, 15–40, 15–38, or 15–36 nucleotides long. In some embodiments, the double-stranded region is at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 nucleotides long. In some embodiments, the region complementary to the target sequence is at least 19 consecutive nucleotides long.

[0129] In some embodiments, the sense chain includes a stem loop at its 3' end described as S1-L-S2, where S1 is complementary to S2 and L forms a loop between S1 and S2.

[0130] In some embodiments, the first 17–25 nucleotides counted from the 5' end of the sense strand form a double-stranded region with the antisense strand, and the last 11–28 nucleotides counted from the 5' end of the sense strand form a 3'-terminal stem-loop described as S1-L-S2.

[0131] In some embodiments, the length of the stem loop S1-L-S2 is 11-28, 13-26, or 15-24 nucleotides. In one embodiment, the stem loop S1-L-S2 is 16 nucleotides long. In some embodiments, the stem loop S1-L-S2 contains the sequence GCAGCCGAAAGGCUGC (Sequence ID 1).

[0132] In some embodiments, L is at least 3, 4, or 5 nucleotides long. In some embodiments, L comprises a GAAA sequence.

[0133] In some embodiments, the sense strand is 36 nucleotides long, with the first 20 nucleotides counting from the 5' end of the sense strand forming a double-stranded region with the antisense strand, and the last 16 nucleotides forming a stem-loop S1-L-S2. In one embodiment, the 16-nucleotide stem-loop S1-L-S2 has the sequence GCAGCCGAAAGGCUGC (SEQ ID NO: 2), where L is GAAA.

[0134] In some embodiments, one or more lipophilic portions are conjugated at a non-terminal position of the sense chain.

[0135] In some embodiments, one or more lipophilic moieties are conjugated to one or more nucleotides of the stem-loop S1-L-S2. In some embodiments, one or more lipophilic moieties are conjugated to one or more nucleotides of loop L.

[0136] In some embodiments, S1 and S2 are complementary and contain 4 to 10 nucleotides, for example, S1 and S2 each contain 6 complementary nucleotides.

[0137] In some embodiments, S1 and S2 are complementary and contain 4 to 10 nucleotides, and L is GAAA, for example, S1 and S2 each contain 6 complementary nucleotides, and L is GAAA.

[0138] In some embodiments, one or more saturated or unsaturated C 22 One or more lipophilic moieties containing hydrocarbon chains are conjugated at one or more internal positions on at least one strand of the dsRNA agent.

[0139] In some embodiments, the internal positions include all positions on at least one chain except for the three end positions from each end. In some embodiments, the internal positions include all positions on at least one chain except for the two end positions from each end.

[0140] In some embodiments, the internal position excludes the cleavage region of the sense strand. In some embodiments, the internal position excludes positions 9–11, 9–12, or 11–13, counting from the 5' end of the sense strand.

[0141] In some embodiments, the internal position excludes the cleavage region of the antisense chain. In some embodiments, the internal position excludes positions 12-14 counting from the 5' end of the antisense chain. In some embodiments, the internal position excludes positions 2-5 counting from the 5' end of the antisense chain.

[0142] In some embodiments, one or more saturated or unsaturated C 22 One or more lipophilic moieties containing hydrocarbon chains are conjugated to one or more of the following internal positions: positions 4–8 and 13–18 of the sense chain, and positions 6–10 and 15–18 of the antisense chain, counting from the 5' end of each chain. In some embodiments, one or more lipophilic moieties are conjugated to one or more of the following internal positions: positions 5, 6, 7, 15, 16, and 17 of the sense chain, and positions 15, 16, and 17 of the antisense chain, counting from the 5' end of each chain.

[0143] In some embodiments, one or more lipophilic moieties are conjugated to one or more of the following internal positions: counting from the 5' end of each chain, positions 5, 6, 7, 15, 16, and 17 of the sense chain, and positions 6, 7, 8, 9, 10, 15, 16, and 17 of the antisense chain.

[0144] In some embodiments, one or more saturated or unsaturated C 22 One or more lipophilic moieties containing hydrocarbon chains are conjugated at the 5th position of the sense chain, counted from the 5' end of the sense chain.

[0145] In some embodiments, one or more saturated or unsaturated C 22 One or more lipophilic moieties containing hydrocarbon chains are conjugated at the 6th position of the sense chain, counting from the 5' end of the sense chain.

[0146] In some embodiments, one or more saturated or unsaturated C 22 One or more lipophilic moieties containing hydrocarbon chains are conjugated at position 7 of the sense chain, counting from the 5' end of the sense chain.

[0147] In some embodiments, one or more saturated or unsaturated C 22One or more lipophilic moieties containing hydrocarbon chains are conjugated at the 15th position of the sense chain, counting from the 5' end of the sense chain.

[0148] In some embodiments, one or more saturated or unsaturated C 22 One or more lipophilic moieties containing hydrocarbon chains are conjugated at the 16th position of the sense chain, counted from the 5' end of the sense chain.

[0149] In some embodiments, one or more saturated or unsaturated C 22 One or more lipophilic moieties containing hydrocarbon chains are conjugated at the 17th position of the sense chain, counting from the 5' end of the sense chain.

[0150] In some embodiments, one or more saturated or unsaturated C 22 One or more lipophilic moieties containing hydrocarbon chains are conjugated at the 6th position of the antisense chain, counting from the 5' end of the antisense chain.

[0151] In some embodiments, one or more saturated or unsaturated C 22 One or more lipophilic moieties containing hydrocarbon chains are conjugated at the 7th position of the antisense chain, counting from the 5' end of the antisense chain.

[0152] In some embodiments, one or more saturated or unsaturated C 22 One or more lipophilic moieties containing hydrocarbon chains are conjugated at the 8th position of the antisense chain, counting from the 5' end of the antisense chain.

[0153] In some embodiments, one or more saturated or unsaturated C 22 One or more lipophilic moieties containing hydrocarbon chains are conjugated at the 9-position of the antisense chain, counting from the 5' end of the antisense chain.

[0154] In some embodiments, one or more saturated or unsaturated C 22 One or more lipophilic moieties containing hydrocarbon chains are conjugated at the 10-position of the antisense chain, counting from the 5' end of the antisense chain.

[0155] In some embodiments, one or more saturated or unsaturated C 22 One or more lipophilic moieties containing hydrocarbon chains are conjugated at the 15th position of the antisense chain, counting from the 5' end of the antisense chain.

[0156] In some embodiments, one or more saturated or unsaturated C 22 One or more lipophilic moieties containing hydrocarbon chains are conjugated at the 16th position of the antisense chain, counting from the 5' end of the antisense chain.

[0157] In some embodiments, one or more saturated or unsaturated C 22 One or more lipophilic moieties containing hydrocarbon chains are conjugated at the 17th position of the antisense chain, counting from the 5' end of the antisense chain.

[0158] In some embodiments, the dsRNA agent is at least one lipophilic monomer

[0159] [ka] [wherein B is a modified or unmodified nucleobase] comprises. In one embodiment, this lipophilic monomer is conjugated at position 5 of the sense chain, counting from the 5' end of the sense chain. In one embodiment, this lipophilic monomer is conjugated at position 6 of the sense chain, counting from the 5' end of the sense chain. In one embodiment, this lipophilic monomer is conjugated at position 7 of the sense chain, counting from the 5' end of the sense chain. In one embodiment, this lipophilic monomer is conjugated at position 15 of the sense chain, counting from the 5' end of the sense chain. In one embodiment, this lipophilic monomer is conjugated at position 16 of the sense chain, counting from the 5' end of the sense chain. In one embodiment, this lipophilic monomer is conjugated at position 17 of the sense chain, counting from the 5' end of the sense chain.

[0160] In some embodiments, one or more saturated or unsaturated C 22 One or more lipophilic moieties containing hydrocarbon chains are conjugated at one or more of the following terminal positions: position 1, 2, or 3 of a sense or antisense chain, counting from the 5' or 3' end of each chain. In some embodiments, one or more saturated or unsaturated C 22 At least one lipophilic moiety containing a hydrocarbon chain is conjugated at one or more of the following terminal positions: the 1st, 2nd, or 3rd position of a sense or antisense chain, counting from the 5' end of each chain. In one embodiment, saturated or unsaturated C 22 The lipophilic portion containing hydrocarbon chains is conjugated to the terminal 1 position of the sense or antisense chain, counted from the 5' end of each chain. In some embodiments, one or more saturated or unsaturated C 22 At least one lipophilic moiety containing a hydrocarbon chain is conjugated at one or more of the following terminal positions: the 1st, 2nd, or 3rd position of a sense or antisense chain, counting from the 3' end of each chain. In one embodiment, saturated or unsaturated C 22The lipophilic portion containing hydrocarbon chains is conjugated to the terminal 1 position of the sense or antisense chain, counting from the 3' end of each chain.

[0161] In some embodiments, one or more saturated or unsaturated C 22 At least one lipophilic moiety containing a hydrocarbon chain is conjugated to position 1 of a sense or antisense chain, counting from the 5' end of each chain. In certain embodiments, the conjugation to position 1 is by modification of the 2' position of the sugar moiety of the nucleotide at position 1. In certain embodiments, the conjugation to position 1 is by modification of the 5' position of the sugar moiety of the nucleotide at position 1. In certain embodiments, the conjugation to position 1 is by modification of the 4' position of the sugar moiety of the nucleotide at position 1. In certain embodiments, the conjugation to position 1 is by modification of the nucleobase of the nucleotide at position 1.

[0162] In some embodiments, the sense strand and antisense strand of the dsRNA agent are each 15 to 30 nucleotides long. In one embodiment, the sense strand and antisense strand of the dsRNA agent are each 19 to 25 nucleotides long. In one embodiment, the sense strand and antisense strand of the dsRNA agent are each 21 to 23 nucleotides long.

[0163] In some embodiments, the dsRNA agent includes a single-stranded overhang at at least one of its ends, e.g., a 3' and / or 5' overhang (or more) of 1 to 10 nucleotides in length, e.g., an overhang of 1, 2, 3, 4, 5, or 6 nucleotides. In some embodiments, both strands have at least one stretch of 1 to 5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides in the double-stranded region. In one embodiment, the single-stranded overhang is 1, 2, or 3 nucleotides in length. In some embodiments, the dsRNA agent also has a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand), and vice versa. In one embodiment, the dsRNA agent includes a 3' overhang at the 3' end of the antisense strand and, optionally, a blunt end at the 5' end of the antisense strand. In one embodiment, the dsRNA agent has a 5' overhang at the 5' end of the sense strand and, optionally, a blunt end at the 5' end of the antisense strand. In one embodiment, the dsRNA agent has two blunt ends at both ends of the dsRNA double helix.

[0164] In one embodiment, the sense strand of the dsRNA agent is 21 nucleotides long, and the antisense strand is 23 nucleotides long, where the strands form a double-stranded region of 21 consecutive base pairs with a single-stranded projection 2 nucleotides longer at the 3' end.

[0165] In one embodiment, the dsRNA agent has two blunt ends at both ends of the dsRNA double helix, the sense strand of the dsRNA agent is 19 nucleotides long, and the antisense strand is 19 nucleotides long, where the strands form a double-stranded region of 19 consecutive base pairs.

[0166] In one embodiment, the dsRNA agent has two blunt ends at both ends of the dsRNA double helix, the sense strand of the dsRNA agent is 20 nucleotides long, and the antisense strand is 20 nucleotides long, where the strands form a double-stranded region of 20 consecutive base pairs.

[0167] In one embodiment, the dsRNA agent has two blunt ends at both ends of a dsRNA double helix, the sense strand of the dsRNA agent is 21 nucleotides long, and the antisense strand is 21 nucleotides long, where the strands form a double-stranded region of 21 consecutive base pairs.

[0168] In one embodiment, the dsRNA agent has two blunt ends at both ends of the dsRNA double helix, the sense strand of the dsRNA agent is 22 nucleotides long, and the antisense strand is 22 nucleotides long, where the strands form a double-stranded region of 22 consecutive base pairs.

[0169] In one embodiment, the dsRNA agent has two blunt ends at both ends of the dsRNA double helix, the sense strand of the dsRNA agent is 23 nucleotides long, and the antisense strand is 23 nucleotides long, where the strands form a double-stranded region of 23 consecutive base pairs.

[0170] In some embodiments, the sense chain further comprises at least one phosphorothioate linkage at its 3' end. In some embodiments, the sense chain further comprises at least two phosphorothioate linkages at its 3' end. In some embodiments, one or more lipophilic monomers (e.g., one or more saturated or unsaturated C) 22 The lipophilic moiety containing the hydrocarbon chain is located at the 3' end of the sense chain. In one embodiment, one of the phosphorothioate linkages is located between the lipophilic monomer and the first nucleotide from the 3' end of the sense chain.

[0171] In some embodiments, the sense chain further comprises at least one phosphorothioate linkage at its 5' end. In some embodiments, the sense chain further comprises at least two phosphorothioate linkages at its 5' end. In some embodiments, one or more lipophilic monomers (e.g., one or more saturated or unsaturated C) 22The lipophilic moiety containing the hydrocarbon chain is located at the 5' end of the sense chain. In one embodiment, one of the phosphorothioate linkages is located between the lipophilic monomer and the first nucleotide from the 5' end of the sense chain.

[0172] In some embodiments, the antisense chain further comprises at least one phosphorothioate linkage at its 3' end. In some embodiments, the antisense chain further comprises at least two phosphorothioate linkages at its 3' end. In some embodiments, one or more lipophilic monomers (e.g., one or more saturated or unsaturated C) 22 The lipophilic moiety containing the hydrocarbon chain is located at the 3' end of the antisense chain. In one embodiment, one of the phosphorothioate linkages is located between the lipophilic monomer and the 3' end of the antisense chain and the first nucleotide.

[0173] In some embodiments, the antisense chain further comprises at least one phosphorothioate linkage at its 5' end. In some embodiments, the antisense chain further comprises at least two phosphorothioate linkages at its 5' end. In some embodiments, one or more lipophilic monomers (e.g., one or more saturated or unsaturated C) 22 The lipophilic moiety containing the hydrocarbon chain is located at the 5' end of the antisense chain. In one embodiment, one of the phosphorothioate linkages is located between the lipophilic monomer and the 5' end of the antisense chain and the first nucleotide.

[0174] In some embodiments, the dsRNA agent further comprises a phosphate group or a phosphate mimetic at the 5' end of the sense strand or antisense strand. In one embodiment, the sense strand has a phosphate group or a phosphate mimetic at the 5' end. In one embodiment, the antisense strand has a phosphate group or a phosphate mimetic at the 5' end.

[0175] In some embodiments, the phosphate mimetic is a 5'-terminated phosphorothioate (5'-PS), a 5'-terminated phosphorodithioate (5'-PS2), a 5'-terminated vinylphosphonate (5'-VP), a 5'-terminated methylphosphonate (MePhos), or a 5'-deoxy-5'-C-malonyl (

[0176] [ka] )

[0177] In one embodiment, the phosphate mimetic is 5'-vinylphosphonate (VP). In one embodiment, the phosphate mimetic is a 5'-(E)-vinylphosphonate (VP) isomer (i.e., trans-vinylphosphonate), a 5'-(Z)-VP isomer (i.e., cis-vinylphosphonate), or a mixture thereof.

[0178] In exemplary embodiments, the 5'-vinylphosphonate modified nucleotides of the present disclosure have the following structure:

[0179] [ka] [In formula: X is either O or S; R is hydrogen, hydroxyl, fluoro, or C 1~20 It is an alkoxy (e.g., methoxy or n-hexadecyloxy); R 5’ This is =C(H)-P(O)(OH)2, with C5' carbon and R 5’ The double bond between them is in the E or Z direction (for example, the E direction); B is a nucleobase or a modified nucleobase, and B may be adenine, guanine, cytosine, thymine, or uracil. It has.

[0180] In one embodiment, R 5’ This is =C(H)-P(O)(OH)2, with C5' carbon and R5 ’The double bond between them is in the E direction. In another embodiment, R is methoxy, and R 5’ This is =C(H)-P(O)(OH)2, with C5' carbon and R 5’ The double bond between them is in the E direction. In another embodiment, X is S, R is methoxy, and R 5’ This is =C(H)-P(O)(OH)2, with C5' carbon and R5 ’ The double bond between them is in the E direction.

[0181] In some embodiments, the -CH2OH group at the 4' position of the 5' terminal nucleotide is represented by the formula -O-CH2-P(O)(OR)2[wherein each R is independently hydrogen or C]. 1~4 Substitution with alkyl (for example, one R group is hydrogen and one R group is methyl, or both R groups are hydrogen) phosphate mimetic.

[0182] In one embodiment, the phosphate mimetic is 5'-cyclopropylphosphonate (VP) (i.e., the CH2OH group at the 4' position of the 5' terminal nucleotide is given by formula -Cy-P(O)(OR)2 [wherein Cy is a cyclopropyl ring, and each R is independently hydrogen or C]. 1~4 (Substituted by alkyl groups (for example, one R group is hydrogen or both R groups are hydrogen)).

[0183] In some exemplary embodiments, the 5'-terminated phosphate mimetic is

[0184] [ka] [In the formula, B is a appropriately modified nucleobase (e.g., U)] or its salt (for example, a sodium salt).

[0185] In some embodiments, the 5' terminal phosphate mimetic is part of the modified 5' terminal nucleotide. For example, the phosphate mimetic is structural

[0186] [ka] [In the formula, B is a nucleobase that has been modified as appropriate.] It may be part of a modified 5' terminal nucleotide that has [a specific characteristic].

[0187] In some embodiments, the 5'-terminated phosphate mimetic may also include a 5'-phosphate prodrug or a 5'-phosphonate prodrug. In some embodiments, the 5'-phosphate prodrug or 5'-phosphonate prodrug has the structure of the formula disclosed in WO2022 / 147214, which is incorporated herein by reference. In some exemplary embodiments, the 5'-phosphate prodrug or 5'-phosphonate prodrug is Pmmds(

[0188] [ka] ((4SR,5SR)-3,3,5-trimethyl-1,2-dithiolan-4-ol)phosphodiester); cPmmds(

[0189] [ka] ((4SR,5RS)-3,3,5-trimethyl-1,2-dithiolan-4-ol)phosphodiester (Cis Pmmds));PdAr1s(

[0190] [ka] ((4SR,5RS)-5-phenyl-3,3-dimethyl-1,2-dithiolan-4-ol)phosphodiester);PdAr3s(

[0191] [ka] ((4SR,5RS)-5-(4-methylphenyl)-3,3-dimethyl-1,2-dithiolan-4-ol)phosphodiester);PdAr5s(

[0192] [ka] ((4SR,5RS)-5-(4-methoxyphenyl)-3,3-dimethyl-1,2-dithiolan-4-ol)phosphodiester);PdAr2s(

[0193] [ka] );PdAr4s(

[0194] [ka] );PdAr6s(

[0195] [ka] );Pmmd / Pmmds(

[0196] [ka] );Pmds(

[0197] [ka] );Cymd / Cymds(

[0198] [ka] , X is the OS); or Ptmd / Ptmds(

[0199] [ka] X is the OS), Pd / Pds(

[0200] [ka] (X is the OS).

[0201] In some exemplary embodiments, a 5'-phosphate prodrug or a 5'-phosphonate prodrug is

[0202] [ka] Therefore, an siRNA containing one of the above list of 5'-modified phosphate prodrugs typically has activity comparable to that of an siRNA containing 5'-VP. In some exemplary embodiments, a 5'-phosphate prodrug or 5'-phosphonate prodrug is

[0203] [ka] Therefore, siRNA containing one of the above-listed 5'-modified phosphate prodrugs typically exhibits improved stability and better or comparable activity to siRNA containing 5'-VP.

[0204] In some embodiments, the 5' end of the antisense strand of the dsRNA agent does not contain 5'-vinylphosphonate (VP).

[0205] In some embodiments, the sense chain includes at least two phosphorothioate linkages, one phosphorothioate linkage at the 3' end, and one phosphorothioate linkage at the 5' end. In some embodiments, the sense chain includes a block of at least three phosphorothioate linkages, one phosphorothioate linkage at the 3' end, and two phosphorothioate linkages at the 5' end. In some embodiments, the sense chain includes a block of at least three phosphorothioate linkages, two phosphorothioate linkages at the 3' end, and one phosphorothioate linkage at the 5' end. In some embodiments, the sense chain includes at least four phosphorothioate linkages, a block of two phosphorothioate linkages at the 3' end, and a block of two phosphorothioate linkages at the 5' end. In one embodiment, one or more of these terminal phosphorothioate linkages are internucleotide linkages between the terminal nucleotides at the 3' and / or 5' ends of the sense strand, for example, phosphorothioate internucleotide linkages between the nucleotides at positions 1 and 2, and / or 2 or 3, counting from either the 3' or 5' end of the antisense strand. In one embodiment, one or more of these terminal phosphorothioate linkages connect an inverted debasal nucleotide to the terminal nucleotides at the 3' and / or 5' ends of the sense strand. In one embodiment, at least one of these terminal phosphorothioate linkages is a lipophilic monomer (e.g., one or more saturated or unsaturated C2 as described herein). 22 The sense chain contains a lipophilic portion containing a hydrocarbon chain and is located between the 3' and / or 5' ends of the sense chain and the first nucleotide. In some embodiments, the sense chain further comprises a phosphate, phosphate mimetic, or 5'-phosphate prodrug or 5'-phosphonate prodrug as described herein at its 5' end.

[0206] In some embodiments, the antisense chain includes at least two phosphorothioate linkages, one phosphorothioate linkage at the 3' end, and one phosphorothioate linkage at the 5' end. In some embodiments, the antisense chain includes a block of at least three phosphorothioate linkages, one phosphorothioate linkage at the 3' end, and two phosphorothioate linkages at the 5' end. In some embodiments, the antisense chain includes a block of at least three phosphorothioate linkages, two phosphorothioate linkages at the 3' end, and one phosphorothioate linkage at the 5' end. In some embodiments, the antisense chain includes at least four phosphorothioate linkages, a block of two phosphorothioate linkages at the 3' end, and a block of two phosphorothioate linkages at the 5' end. In one embodiment, one or more of these terminal phosphorothioate linkages are internucleotide linkages between the terminal nucleotides at the 3' and / or 5' ends of the antisense strand, for example, phosphorothioate internucleotide linkages between the nucleotides at positions 1 and 2, and / or 2 or 3, counting from either the 3' or 5' end of the antisense strand. In one embodiment, one or more of these terminal phosphorothioate linkages connect an inverted debasal nucleotide to the terminal nucleotides at the 3' and / or 5' ends of the antisense strand. In one embodiment, at least one of these terminal phosphorothioate linkages is a lipophilic monomer (e.g., one or more saturated or unsaturated C2 as described herein). 22 The antisense chain is located between the 3' and / or 5' ends of the lipophilic portion containing a hydrocarbon chain and the first nucleotide. In some embodiments, the antisense chain further comprises, at its 5' end, a phosphate, a phosphate mimetic, or a 5'-phosphate prodrug or 5'-phosphonate prodrug as described herein.

[0207] In some embodiments, the dsRNA agent comprises at least four phosphorothioate linkages—two phosphorothioate linkages on the sense strand and two phosphorothioate linkages on the antisense strand: one phosphorothioate linkage at each of the 3' and 5' ends of each strand. In some embodiments, the dsRNA agent comprises at least six phosphorothioate linkages—two phosphorothioate linkages on the sense strand and four phosphorothioate linkages on the antisense strand: one phosphorothioate linkage at each of the 3' and 5' ends of the sense strand; and a block of two phosphorothioate linkages at each of the 3' and 5' ends of the antisense strand. In some embodiments, the dsRNA agent has at least six phosphorothioate linkages—four phosphorothioate linkages on the sense strand and two phosphorothioate linkages on the antisense strand: a block of two phosphorothioate linkages at each of the 3' and 5' ends of the sense strand; and a phosphorothioate linkage at each of the 3' and 5' ends of the antisense strand. In some embodiments, the dsRNA agent has at least eight phosphorothioate linkages—four phosphorothioate linkages on the sense strand and four phosphorothioate linkages on the antisense strand: a block of two phosphorothioate linkages at each of the 3' and 5' ends of each strand. In one embodiment, one or more of these terminal phosphorothioate linkages are internucleotide linkages between terminal nucleotides at the 3' and / or 5' ends of the strand, for example, a phosphorothioate internucleotide linkage between the nucleotides at positions 1 and 2, and / or 2 or 3, counting from either the 3' or 5' end of the strand. In one embodiment, one or more of these terminal phosphorothioate links connect inverted debasal nucleotides to the 3' and / or 5' terminal nucleotides of the chain. In one embodiment, at least one of these terminal phosphorothioate links is a lipophilic monomer (e.g., one or more saturated or unsaturated C2 as described herein). 22(including a lipophilic moiety containing a hydrocarbon chain) and located between the 3' and / or 5' ends of the chain and the first nucleotide. In some embodiments, the sense or antisense chain further comprises, at its 5' end, a phosphate, phosphate mimetic, or a 5'-phosphate prodrug or 5'-phosphonate prodrug as described herein.

[0208] In some embodiments, the dsRNA agent further comprises at least one terminal chiral phosphorus atom.

[0209] Site-specific, chiral modifications to nucleotide linkages can be present at the 5' end, 3' end, or both the 5' and 3' ends of the strand. These are referred to herein as “terminal” chiral modifications. Terminal modifications can be located at the 3' or 5' terminal position of the terminal region, e.g., at the terminal nucleotide or within the last 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the strand. Chiral modifications can be present on the sense strand, the antisense strand, or both the sense and antisense strands. Each chiral pure phosphorus atom can be in either an Rp configuration, an Sp configuration, or a combination thereof. Further details regarding chiral modifications and chiral-modified dsRNA agents can be found in PCT / US18 / 67103, filed December 21, 2018, titled “Chirally-Modified Double-Stranded RNA Agents,” which is incorporated herein by reference in its entirety.

[0210] In some embodiments, the dsRNA agent further includes a terminal chiral modification located at the first nucleotide linkage at the 3' end of the antisense strand, having a linked phosphorus atom in the Sp configuration; a terminal chiral modification located at the first nucleotide linkage at the 5' end of the antisense strand, having a linked phosphorus atom in the Rp configuration; and a terminal chiral modification located at the first nucleotide linkage at the 5' end of the sense strand, having a linked phosphorus atom in either the Rp or Sp configuration.

[0211] In one embodiment, the dsRNA agent further comprises: a terminal chiral modification located at the first and second nucleotide linkages at the 3' end of the antisense strand, having a linked phosphorus atom in the Sp configuration; a terminal chiral modification located at the first nucleotide linkage at the 5' end of the antisense strand, having a linked phosphorus atom in the Rp configuration; and a terminal chiral modification located at the first nucleotide linkage at the 5' end of the sense strand, having a linked phosphorus atom in either the Rp or Sp configuration.

[0212] In one embodiment, the dsRNA agent further comprises: a terminal chiral modification located at the first, second, and third nucleotide linkages at the 3' end of the antisense strand, having a linked phosphorus atom in the Sp configuration; a terminal chiral modification located at the first nucleotide linkage at the 5' end of the antisense strand, having a linked phosphorus atom in the Rp configuration; and a terminal chiral modification located at the first nucleotide linkage at the 5' end of the sense strand, having a linked phosphorus atom in either the Rp or Sp configuration.

[0213] In one embodiment, the dsRNA agent further comprises: a chiral modification of a terminal located at the first and second internucleotide linkages at the 3' end of the antisense strand, having a linked phosphorus atom in the Sp configuration; a chiral modification of a terminal located at the third internucleotide linkage at the 3' end of the antisense strand, having a linked phosphorus atom in the Rp configuration; a chiral modification of a terminal located at the first internucleotide linkage at the 5' end of the antisense strand, having a linked phosphorus atom in the Rp configuration; and a chiral modification of a terminal located at the first internucleotide linkage at the 5' end of the sense strand, having a linked phosphorus atom in either the Rp or Sp configuration.

[0214] In one embodiment, the dsRNA agent further comprises: a terminal chiral modification located at the first and second nucleotide linkages at the 3' end of the antisense strand, having a linked phosphorus atom in the Sp configuration; a terminal chiral modification located at the first and second nucleotide linkages at the 5' end of the antisense strand, having a linked phosphorus atom in the Rp configuration; and a terminal chiral modification located at the first nucleotide linkage at the 5' end of the sense strand, having a linked phosphorus atom in either the Rp or Sp configuration.

[0215] In some embodiments, the dsRNA agent has at least two phosphorothioate nucleotide linkages in the first 5, 4, 3, or 2 nucleotides of the antisense strand (counted from the 5' end). In some embodiments, the dsRNA agent has at least two phosphorothioate nucleotide linkages in the first 5, 4, 3, or 2 nucleotides of the antisense strand (counted from the 3' end).

[0216] In some embodiments, the first two nucleotide junctions at the 5' end of the antisense strand are phosphorothioate junctions. In some embodiments, the first three nucleotide junctions at the 5' end of the antisense strand are phosphorothioate junctions.

[0217] In some embodiments, the first two nucleotide junctions at the 5' end of the antisense strand are phosphorothioate junctions; and the last two nucleotide junctions (at the 3' end) of the antisense strand are phosphorothioate junctions.

[0218] In some embodiments, the first three nucleotide junctions at the 5' end of the antisense strand are phosphorothioate junctions; and the last two nucleotide junctions (at the 3' end) of the antisense strand are phosphorothioate junctions.

[0219] In some embodiments, the first three internucleotide junctions at the 5' end of the antisense strand are phosphorothioate junctions; and the last internucleotide junction of the antisense strand (which is at the 3' end) is a phosphorothioate junction.

[0220] In some embodiments, the antisense chain comprises two blocks of one, two, or three phosphorothioate nucleotide linkages separated by phosphate nucleotide linkages of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18.

[0221] In some embodiments, 100%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, or at least 30% of the antisense and sense strands of the dsRNA agent are modified. For example, if 50% of the dsRNA agent is modified, then 50% of all nucleotides present in the dsRNA agent contain the modifications described herein.

[0222] In one embodiment, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or substantially 100% of the nucleotides of the dsRNA agent are independently modified with 2'O-methyl, 2'-O-aryl, 2'-deoxy, or 2'-fluoro.

[0223] In one embodiment, the oligonucleotide is antisense, and at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or substantially 100% of the antisense nucleotide is independently modified with LNA, CeNA, 2'-methoxyethyl, or 2'-deoxy.

[0224] In some embodiments, the sense and antisense strands of the dsRNA agent contain or do not contain 2'-F modified nucleotides less than 12, less than 10, less than 8, less than 6, less than 4, or less than 2. In some embodiments, the dsRNA agent has or does not have 2'-F modifications in the sense strand. In some embodiments, the dsRNA agent has or does not have 2'-F modifications in the antisense strand.

[0225] In some embodiments, the dsRNA agent has one or more 2'-F modifications at any position on the sense strand or antisense strand.

[0226] In some embodiments, the dsRNA agent contains less than 20%, less than 15%, less than 10%, or less than 5% non-natural nucleotides, or substantially no non-natural nucleotides. Examples of non-natural nucleotides include acyclic nucleotides, LNA, HNA, CeNA, 2'O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropanyl), 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), 2'-ara-F, L-nucleoside modifications (e.g., 2'-modified L-nucleosides, e.g., 2'-deoxy-L-nucleosides), BNA, FHNA, debasic sugars, debasic cyclics, and open-chain alkyls.

[0227] In some embodiments, the antisense and sense strands of the dsRNA agent contain at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or virtually 100% 2'O-methyl-modified nucleotides.

[0228] In some embodiments, the dsRNA agent contains 80% or more, 85% or more, 90% or more, 95% or more, or substantially 100% of natural nucleotides. For these embodiments, the natural nucleotides may include 2'-OH, 2'-deoxy, and 2'-OMe.

[0229] In some embodiments, the antisense strand contains, for example, at least one unlocked nucleic acid (UNA) modification in the seed region of the antisense strand. In some embodiments, the antisense strand contains, for example, at least one glycerol nucleic acid (GNA) modification in the seed region of the antisense strand. In one embodiment, the seed region is at positions 2-8 (e.g., positions 5-7) of the 5' end of the antisense strand.

[0230] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand, each having a length of 15 to 30 nucleotides; including at least two phosphorothioate nucleotide linkages in the first 5 nucleotides of the antisense strand (counted from the 5' end); the double-stranded region is between 19 and 25 base pairs (preferably 19, 20, 21, or 22); and the dsRNA agent has less than 20%, less than 15%, less than 10%, or less than 5% non-natural nucleotides, or substantially no non-natural nucleotides.

[0231] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand, each having a length of 15 to 30 nucleotides; including at least two phosphorothioate nucleotide linkages in the first 5 nucleotides of the antisense strand (counted from the 5' end); the double-stranded region is between 19 and 25 base pairs (preferably 19, 20, 21, or 22); and the dsRNA agent comprises 80% or more, 85% or more, 95% or more, or virtually 100% of natural nucleotides, such as those having 2'-OH, 2'-deoxy, or 2'-OMe.

[0232] In some embodiments, the dsRNA agent further comprises a targeting ligand that targets a receptor that mediates delivery to a specific CNS tissue. In one embodiment, the targeting ligand is selected from the group consisting of Angiopep-2, lipoprotein receptor-associated protein (LRP) ligands, bEnd.3 cell-binding ligands, transferrin receptor (TfR) ligands, manose receptor ligands, glucose transporter proteins, and LDL receptor ligands.

[0233] Some embodiments of the present invention provide a dsRNA agent comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate nucleotide interlinks between the first 5 nucleotides counted from the 5' end of the antisense strand; having at least 3, 4, 5, or 6 2'-deoxy modifications of the sense strand and / or antisense strand; the dsRNA agent having a double-stranded region between 19 to 25 base pairs; the dsRNA agent comprising a ligand; and the sense strand not comprising glycol nucleic acid (GNA).

[0234] The antisense strand has sufficient complementarity with the target sequence that mediates RNA interference. In other words, dsRNA agents can inhibit the expression of target genes in the central nervous system (CNS).

[0235] In one embodiment, the dsRNA agent comprises at least three 2'-deoxy modifications. The 2'-deoxy modifications are located at positions 2 and 14 of the antisense strand, counted from the 5' end of the antisense strand, and at position 11 of the sense strand, counted from the 5' end of the sense strand.

[0236] In one embodiment, the dsRNA agent comprises at least five 2'-deoxy modifications. The 2'-deoxy modifications are located at positions 2, 12, and 14 of the antisense strand, counted from the 5' end of the antisense strand, and at positions 9 and 11 of the sense strand, counted from the 5' end of the sense strand.

[0237] In one embodiment, the dsRNA agent comprises at least seven 2'-deoxy modifications. The 2'-deoxy modifications are located at positions 2, 5, 7, 12, and 14 of the antisense strand, counted from the 5' end of the antisense strand, and at positions 9 and 11 of the sense strand, counted from the 5' end of the sense strand.

[0238] In one embodiment, the antisense strand includes at least five 2'-deoxy modifications at positions 2, 5, 7, 12, and 14, counted from the 5' end of the antisense strand. The antisense strand has a length of 18 to 25 nucleotides, or 18 to 23 nucleotides.

[0239] In one embodiment, the dsRNA agent may contain one or more non-natural nucleotides. For example, the dsRNA agent may contain less than 20%, e.g., less than 15%, less than 10%, or less than 5% of non-natural nucleotides, or the dsRNA agent may not contain any non-natural nucleotides. For example, the dsRNA agent may contain all natural nucleotides. Some exemplary non-natural nucleotides include, but are not limited to, acyclic nucleotides, locked nucleic acids (LNA), HNA, CeNA, 2'-methoxyethyl, 2'-O-aryl, 2'-C-aryl, 2'-fluoro, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), and 2'-ara-F.

[0240] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand, each strand independently 15 to 35 nucleotides long; at least two phosphorothioate nucleotide interlinks between the first 5 nucleotides counted from the 5' end of the antisense strand; the sense strand and / or antisense strand have at least 3, 4, 5, or 6 2'-deoxynucleotides; the dsRNA agent has a double-stranded region between 19 to 25 base pairs; the dsRNA agent optionally contains a ligand; the sense strand does not contain glycol nucleic acid (GNA); the dsRNA agent contains less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides, or the dsRNA agent contains all natural nucleotides.

[0241] In one embodiment, at least one sense strand and antisense strand contain at least one, for example, at least two, at least three, at least four, at least five, at least six, or at least seven or more 2'-deoxy modifications in the central region of the sense strand or antisense strand. Thus, in one embodiment, the dsRNA agent comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate nucleotide interlinks between the first five nucleotides counted from the 5' end of the antisense strand; having at least three, four, five, or six 2'-deoxy nucleotides in the sense strand and / or antisense strand; the dsRNA agent having a double-stranded region between 19 to 25 base pairs; the dsRNA agent optionally containing a ligand; and the sense strand and / or antisense strand contain at least one, for example, at least two, at least three, at least four, at least five, at least six, or at least seven or more 2'-deoxy modifications in the central region of the sense strand and / or antisense strand.

[0242] In some embodiments, the sense strand has a length of 18 to 30 nucleotides and contains at least two 2'-deoxy modifications in the central region of the sense strand. For example, the sense strand has a length of 18 to 30 nucleotides and contains at least two 2'-deoxy modifications within positions 7, 8, 9, 10, 11, 12, and 13, counted from the 5' end of the sense strand.

[0243] In one embodiment, the antisense strand has a length of 18 to 30 nucleotides and contains at least two 2'-deoxy modifications in the central region of the antisense strand. For example, the antisense strand has a length of 18 to 30 nucleotides and contains at least two 2'-deoxy modifications within positions 10, 11, 12, 13, 14, 15, and 16, counted from the 5' end of the antisense strand.

[0244] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand; the sense strand has a length of 17 to 30 nucleotides and contains at least one 2'-deoxy modification in the central region of the sense strand; and the antisense strand independently has a length of 17 to 30 nucleotides and contains at least two 2'-deoxy modifications in the central region of the antisense strand.

[0245] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand; the sense strand has a length of 17 to 30 nucleotides and contains at least two 2'-deoxy modifications in the central region of the sense strand; and the antisense strand independently has a length of 17 to 30 nucleotides and contains at least one 2'-deoxy modification in the central region of the antisense strand.

[0246] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate nucleotide interlinks between the first 5 nucleotides counted from the 5' end of the antisense strand; having at least 3, 4, 5, or 6 2'-deoxynucleotides on the sense strand and / or antisense strand; the dsRNA agent having a double-stranded region between 19 to 25 base pairs; the dsRNA agent optionally comprising a ligand; and the sense strand having at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more 2'-deoxy modifications in the central region of the sense strand.

[0247] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate nucleotide interlinks between the first 5 nucleotides counted from the 5' end of the antisense strand; having at least 3, 4, 5, or 6 2'-deoxynucleotides on the sense strand and / or antisense strand; the dsRNA agent having a double-stranded region between 19 to 25 base pairs; the dsRNA agent optionally comprising a ligand; and the antisense strand having at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more 2'-deoxy modifications in the central region of the antisense strand.

[0248] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate nucleotide interlinks between the first 5 nucleotides counted from the 5' end of the antisense strand; having at least 3, 4, 5, or 6 2'-deoxynucleotides on the sense strand and / or antisense strand; the dsRNA agent having a double-stranded region between 19 to 25 base pairs; the dsRNA agent optionally comprising a ligand; the dsRNA agent containing less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides, or the dsRNA agent containing all natural nucleotides, and the sense strand and / or antisense strand containing at least one, e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or more 2'-deoxy modifications in the central region of the sense strand and / or antisense strand.

[0249] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate nucleotide interlinks between the first 5 nucleotides counted from the 5' end of the antisense strand; having at least 3, 4, 5, or 6 2'-deoxynucleotides on the sense strand and / or antisense strand; the dsRNA agent having a double-stranded region between 19 to 25 base pairs; the dsRNA agent optionally comprising a ligand; the dsRNA agent containing less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides, or the dsRNA agent containing all natural nucleotides, and the sense strand containing at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or more 2'-deoxy modifications in the central region of the sense strand.

[0250] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate nucleotide interlinks between the first 5 nucleotides counted from the 5' end of the antisense strand; having at least 3, 4, 5, or 6 2'-deoxynucleotides on the sense strand and / or antisense strand; the dsRNA agent having a double-stranded region between 19 to 25 base pairs; the dsRNA agent comprising a ligand; the dsRNA agent comprising less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-native nucleotides, or the dsRNA agent comprising all native nucleotides, and the antisense strand comprising at least one, e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or more 2'-deoxy modifications in the central region of the antisense strand.

[0251] In one embodiment, if the dsRNA agent contains fewer than 8 non-2'OMe nucleotides, the antisense strand contains at least one DNA. For example, in any embodiment, if the dsRNA agent contains fewer than 8 non-2'OMe nucleotides, the antisense strand may contain at least one DNA.

[0252] In one embodiment, if the antisense contains two deoxynucleotides, the nucleotides are at positions 2 and 14 counted from the 5' end of the antisense strand, and the dsRNA agent contains 8 or fewer non-2'OMe nucleotides (e.g., 8, 7, 6, 5, 4, 3, 2, 1, or 0). For example, in any one embodiment of the present invention, if the antisense contains two deoxynucleotides, the nucleotides are at positions 2 and 14 counted from the 5' end of the antisense strand, and the dsRNA agent contains 0, 1, 2, 3, 4, 5, 6, 7, or 8 non-2'OMe nucleotides.

[0253] Another aspect of the present invention provides cells containing the dsRNA agent described herein.

[0254] Another aspect of the present invention provides a pharmaceutical composition comprising a dsRNA agent as described herein.

[0255] Lipophilic monomer, lipophilic moiety, saturated or unsaturated C 22 All of the above embodiments relating to hydrocarbon chains and their conjugation to dsRNA agents in the first aspect of the present invention relating to dsRNA agents are suitable for these embodiments of the present invention relating to cells and pharmaceutical compositions.

[0256] In another embodiment, the present invention further provides a method for delivering the dsRNA agent of the present invention to a specific target gene in the central nervous system (CNS) of a subject by subcutaneous or intravenous administration. The present invention further provides the dsRNA agent described herein for use in the method of delivering the agent to a specific target in a subject by subcutaneous, intravenous, intrathecal, or intraventricular administration.

[0257] Another aspect of the present invention relates to a method for modulating the expression of a target gene in CNS cells, comprising administering a dsRNA agent described herein to the cells.

[0258] Another aspect of the present invention relates to a method for treating or preventing CNS damage in a subject, comprising administering a therapeutically effective amount of a dsRNA agent described herein to the subject, thereby treating the subject by modulating the expression of a target gene in the subject's CNS.

[0259] In one embodiment, the cells are within the subject. In one embodiment, the subject is a mammal. In one embodiment, the subject is a human.

[0260] Exemplary CNS disorders that can be treated by the methods of the present invention include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Huntington's disease, Parkinson's disease, spinocerebellar disease, prions, and Lafora disease.

[0261] Lipophilic monomer, lipophilic moiety, saturated or unsaturated C 22 All of the above embodiments relating to hydrocarbon chains and their conjugation to dsRNA agents in the first aspect of the present invention relating to dsRNA agents are suitable for these embodiments of the present invention relating to methods for delivering dsRNA agents, methods for modulating the expression of target genes in cells, and methods for treating or preventing CNS damage in subjects.

[0262] In some embodiments, the dsRNA agent is administered extrahepatically.

[0263] In one embodiment, the dsRNA agent is administered intrathecally or intraventricularly. By administering the dsRNA agent intrathecally or intraventricularly, this method can reduce the expression of target genes in brain or spinal tissue, such as the cortex, cerebellum, cervical vertebrae, lumbar vertebrae, and thoracic vertebrae.

[0264] In some embodiments, the CNS target genes are selected from the group consisting of APP, SOD1, SCN9A, HTT (HUNTINGTIN), APOE, LRRK2, PRNP, SCD5, GPR75, MAPT, SNCA, ABLIM3, ADRA2A, ATXN1, ATXN2, ATXN3, ELOVL1, FLNA, MOGO-L or MOGO-R, HIF-1α, RHO-A, NAV1.8, CD45, GSK-3, GSK3α, MIG-12, Mgat1, Mgat4, SLC35A1, SLC35A2, GNE, TMPRSS6, complement component C3, APCS, C9orf72, CHI3L1 / YKL-40, EXT1, EXT2, NDST2, RPS25, ALK, and SCD5. In some embodiments, exemplary target genes include APP, ATXN2, C9orf72, TARDBP, MAPT(Tau), HTT, SNCA, FUS, ATXN3, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, TTR, SCN9A, LRRK2, GPR75, APOE, SCD5, ELOVL1, FLNA, ALK, CHI3L1(YKL-40), RPS25, α2-AR, and GSK3α.

[0265] In some embodiments, the dsRNA agent is administered at dose levels of 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 1 / 3 or less, 30% or less, and 25% or less of the dose level of the comparative dsRNA agent (Dose 1), and this method achieves the same reduction in target gene expression as the administration of the comparative dsRNA agent (Dose 1). The comparative dsRNA agent has the same sense strand and antisense strand, as well as C 22 They have the same conjugation with the same lipophilic moiety, except that they contain a different hydrocarbon chain having fewer carbon atoms than the hydrocarbon chain (for example, the lipophilic moiety of the comparative dsRNA agent contains a C16 hydrocarbon chain). In a particular embodiment, the dsRNA agent contains a 2'-O-docosanyl modification, and the comparative dsRNA agent contains a 2'-O-hexadecyl modification at the same position. [Brief explanation of the drawing]

[0266] [Figure 1] Figure 1 shows the inhibition of APP gene expression (sAPPα and sAPPβ, respectively) in NHP CSF after IT administration of siRNA double helical cells (6.7 mg and 20 mg) (right) compared with siRNA double helical cells (20 mg and 60 mg) (left) containing a C16 conjugate. [Figure 2] Figure 2A compares the inhibition of APP gene expression (sAPPα and sAPPβ, respectively) in NHP CSF after IT administration of C22 conjugate-containing siRNA double-stranded cells (20 mg) with that of C16 conjugate-containing siRNA double-stranded cells (60 mg) at various time points over 113 days. Figure 2B compares the inhibition of APP gene expression (sAPPα and sAPPβ, respectively) in NHP CSF after IT administration of C22 conjugate-containing siRNA double-stranded cells (6.7 mg) with that of C16 conjugate-containing siRNA double-stranded cells (20 mg) at various time points over 113 days. [Figure 3] Figure 3A compares the overall results of inhibitory intervention (IT) of siRNA duplexes with C22 conjugates (administered at 6.7 mg and 20 mg) with inhibitory intervention (IT) of siRNA duplexes with C16 conjugates (administered at 20 mg and 60 mg) at various time points over 113 days to inhibitory intervention of APP gene expression (sAPPα and sAPPβ, respectively) in NHP CSFs. Figure 3B compares the results of IT) of inhibitory intervention (IT) of siRNA duplexes with C22 conjugates (administered at 20 mg) with inhibitory intervention (IT) of siRNA duplexes with C16 conjugates (administered at 20 mg and 60 mg) at various time points over 29 days to inhibitory intervention of siRNA duplexes with C22 conjugates (administered at 20 mg) to inhibitory intervention of APP gene expression (sAPPα) in NHP CSFs. [Figure 4]Figure 4 shows the results of inhibition of APP gene expression (sAPPα and sAPPβ, respectively) in NHP CSF after IT administration (20 mg) of siRNA double helical cells containing various C16 conjugates: C16 conjugated to N6 of the sense strand (C16, left), C16 conjugated to N1 of the sense strand (SS1 C16, center), and C16 conjugated to N6 of the sense strand with a modified phosphate skeleton (skeleton, right). [Figure 5] Figure 5A shows the inhibition of SOD1 gene expression in mouse CNS tissue after ICV administration (11 μg, 33 μg, 100 μg, or 150 μg) of siRNA double helix with a C22 conjugate at N6 of the sense strand (AD-1427062) compared to D15 with a C16 conjugate at N6 of the sense strand (AD-401824). Figure 5B shows the inhibition of SOD1 gene expression in rat CNS tissue after IT administration (0.03 mg, 0.1 mg, 0.3 mg, and 0.9 mg) of siRNA double helix with a C22 conjugate at N6 of the sense strand (AD-1427062) compared to D15 with a C16 conjugate at N6 of the sense strand (AD-401824). [Figure 6]Figure 6A shows the concentration of C22 conjugate siRNA double-stranded (AD-1427062) in rat frontal cortical tissue after D15 administration of siRNA double-stranded (IT) doses (0.03 mg, 0.1 mg, 0.3 mg, and 0.9 mg), compared to that of C16 conjugate siRNA double-stranded (AD-401824). Figure 6B shows the concentration of C22 conjugate siRNA double-stranded (AD-1427062) in rat vertebral tissue after D15 administration of siRNA double-stranded (IT) doses (0.03 mg, 0.1 mg, 0.3 mg, and 0.9 mg), compared to that of C16 conjugate siRNA double-stranded (AD-401824). Figure 6C shows the results of the concentration ratio of C22 conjugate siRNA double-stranded (AD-1427062) to C16 conjugate siRNA double-stranded (AD-401824) in rat frontal cortical tissue and rat vertebral tissue after IT administration of siRNA double-stranded (0.03 mg, 0.1 mg, 0.3 mg, and 0.9 mg) in D15. [Figure 7] Figure 7A shows the results of inhibition of SOD1 gene expression in rat CNS tissue after IT administration of C22 conjugate siRNA double helix (AD-1427062) (administered at 0.1 mg, 0.3 mg, and 0.6 mg) compared with C16 conjugate siRNA double helix (AD-401824) (administered at 0.3 mg, 0.6 mg, and 0.9 mg) for D30. Figure 7B shows the results of inhibition of SOD1 gene expression in rat CNS tissue after IT administration of C22 conjugate siRNA double helix (AD-1427062) (administered at 0.3 mg and 0.6 mg) compared with C16 conjugate siRNA double helix (AD-401824) (administered at 0.3 mg, 0.6 mg, and 0.9 mg) for D90. [Figure 8]Figures 8A-8D show the inhibition of mRNA expression of rMAP2 (Figure 8A), rMBP (Figure 8B), rGFAP (Figure 8C), and rAif1 (Figure 8D) in rat CNS tissue after IT administration of C22 conjugate siRNA double strand (administered at 0.6 mg) compared to C16 conjugate siRNA (administered at 0.6 mg) on ​​D15. Refer to Table 4 for the corresponding siRNA double strand IDs for the C22 conjugate siRNA double strand and C16 conjugate siRNA for each target referenced in the figures. [Figure 9] Figures 9A–9C show the results of inhibition of CSF APP gene expression after intrathecal (IT) injection of C22 conjugate siRNA double helix (administered at 20 mg and 60 mg on D0; and re-administered on D29) in NHP CNS tissues (prefrontal cortex and putamen in Figure 9A; hippocampus and cerebellum in Figure 9B; and caudate nucleus and lumbar vertebrae in Figure 9C) in D106. [Figure 10] Figure 10 shows the results of 24-hour CSF PK data in NHP CSF after IT administration of C22 conjugate siRNA double helix (administered at 20 mg and 60 mg). [Figure 11] Figure 11 shows the PK / PD correlation results in NHP of all tissues after IT administration of C22-conjugate siRNA double strands. The X-axis shows the concentration of C22-conjugate siRNA double strands in NHP CNS (all tissues) after IT administration of siRNA double strands D85-D105. The Y-axis shows the residual cynoAPP% in NHP CNS (all tissues) after IT administration of siRNA double strands D85-D105. [Figure 12]Figure 12A shows the levels of CSF-soluble APP protein (sAPPβ) after intrathecal (IT) injection of C22-conjugate siRNA double-stranded (AD-2034768) (administered at 20 mg and 60 mg) in NHP CSF at various time points over 85 days, compared with the levels of CSF-soluble APP protein (sAPPβ) after intrathecal (IT) injection of C16-conjugate siRNA (AD-454843) (administered at 72 mg) in NHP CSF at various time points over 78 days. Figure 12B shows the levels of CSF-soluble APP protein (sAPPβ) after intrathecal (IT) injection of C22-conjugate siRNA double-stranded (AD-1956470) (administered at 20 mg and 60 mg) in NHP CSF at various time points over 85 days, compared with the levels of CSF-soluble APP protein (sAPPβ) after intrathecal (IT) injection of C16-conjugate siRNA (AD-454842) (administered at 72 mg) in NHP CSF at various time points over 78 days. Figure 12C shows the levels of CSF-soluble APP protein (sAPPβ) after intrathecal (IT) injection of C22-conjugate siRNA double-stranded (AD-2034769) (administered at 20 mg and 60 mg) in NHP CSF at various time points over 85 days, compared with the levels of CSF-soluble APP protein (sAPPβ) after intrathecal (IT) injection of C16-conjugate siRNA (AD-454972) (administered at 72 mg) in NHP CSF at various time points over 78 days. [Figure 13]Figures 13A-13B show the inhibition of SOD1 gene expression in mouse CNS tissue (right hemisphere) and peripheral tissues (heart, liver) after ICV administration (100 μg) of siRNA double hemisphere containing a lipophilic portion with a C22 hydrocarbon chain having various chemical modifications to the lipophilic portion of D15. In Figure 13A, the data for each tissue, from left to right, represent PBS, AS-401824, AD-1427062, AD-1623136, AD-1962193, AD-1962191, and AD-1962192, respectively. In Figure 13B, the data for each tissue, from left to right, represent PBS, AS-401824, AD-1427062, AD-1623136, AD-1962193, AD-1949272, and AD-1949273, respectively. [Figure 14] Figure 14A is a graph showing the positional effect of C22 conjugation across the sense strand siRNA sequence, evaluated in rodent Neuro2a cells using exemplary siRNA targeting SOD1 containing a C22 hydrocarbon chain at various doses (0.1 nM, 1 nM, and 10 nM, respectively). Figure 14B is a graph showing the positional effect of C22 conjugation across the sense strand siRNA sequence, evaluated in human Be2C cells using exemplary siRNA targeting SOD1 containing a C22 hydrocarbon chain at various doses (0.1 nM, 1 nM, and 10 nM, respectively). See Table 7 for the corresponding siRNA duplex IDs for the various positions referenced in the figures. [Figure 15]Figure 15A is a graph showing the positional effect of C22 conjugation across the sense strand siRNA sequence, evaluated in rodent Neuro2a cells using exemplary siRNA targeting APP containing a C22 hydrocarbon chain at various doses (0.1 nM, 1 nM, and 10 nM, respectively). Figure 15B is a graph showing the positional effect of C22 conjugation across the sense strand siRNA sequence, evaluated in human Be2C cells using exemplary siRNA targeting SOD1 containing a C22 hydrocarbon chain at various doses (0.1 nM, 1 nM, and 10 nM, respectively). See Table 8 for the corresponding siRNA duplex IDs for the various positions referenced in the figures. [Figure 16] Figure 16A is a graph showing the positional effect of C22 conjugation across the antisense strand siRNA sequence, evaluated in rodent Neuro2a cells using exemplary siRNA targeting SOD1 containing a C22 hydrocarbon chain at various doses (0.1 nM, 1 nM, and 10 nM, respectively). Figure 16B is a graph showing the positional effect of C22 conjugation across the antisense strand siRNA sequence, evaluated in human Be2C cells using exemplary siRNA targeting SOD1 containing a C22 hydrocarbon chain at various doses (0.1 nM, 1 nM, and 10 nM, respectively). [Figure 17] Figure 17A is a graph showing the positional effect of C22 conjugation across the antisense strand siRNA sequence, evaluated in rodent Neuro2a cells using exemplary siRNA targeting APP containing a C22 hydrocarbon chain at various doses (0.1 nM, 1 nM, and 10 nM, respectively). Figure 17B is a graph showing the positional effect of C22 conjugation across the antisense strand siRNA sequence, evaluated in human Be2C cells using exemplary siRNA targeting SOD1 containing a C22 hydrocarbon chain at various doses (0.1 nM, 1 nM, and 10 nM, respectively). [Figure 18]Figure 18 shows the results of inhibiting SOD1 gene expression in mouse CNS tissue (right hemisphere) after ICV administration (150 μg) of SOD1-targeting siRNA duplexes containing a C22 hydrocarbon chain conjugated at various positions on the sense or antisense strand of the D7 strand. The control group includes aCSF without siRNA, siRNA duplexes without lipophilic conjugation (AD-1964624, unconjugated), and siRNA duplexes containing a C16 hydrocarbon chain conjugated at N6 of the sense strand (AD-890098, SS6-C16). See Table 11 for the siRNA duplex IDs and corresponding positions referenced in the figure. [Figure 19] Figure 19A shows the inhibition of SOD1 gene expression in right hemisphere brain tissue (striatum, frontal cortex, cerebellum, and hippocampus) and spinal tissue (thoracic spinal cord) after IT administration (0.6 mg in 30 μl of aCSF) of various siRNA duplexes targeting SOD1 containing the internal conjugation of the lipophilic moiety containing the C22 hydrocarbon chain (position 6 of the sense chain) of rat CNS:D14. Figure 19B shows the inhibition of SOD1 gene expression in rat peripheral tissue (liver, heart) after IT administration (0.6 mg in 30 μl of aCSF) of various siRNA duplexes targeting SOD1 containing the internal conjugation of the lipophilic moiety containing the C22 hydrocarbon chain (position 6 of the sense chain) of D14. The controls include siRNA, siRNA double helix containing a C16 hydrocarbon chain conjugated to N6 of the sense strand, and aCSF without the siRNA double helix containing a C6-C16-OH moiety conjugated to N6 of the sense strand. In both figures, the data for each tissue, from left to right, represent aCSF, SS6 C16 (AD-401824), SS6 C22 (AD-1427062), and SS6 C6-C16-OH (AD-2700143), respectively. [Figure 20]Figure 20A shows the results of inhibition of SOD1 gene expression in right hemisphere brain tissue (striatum, frontal cortex, cerebellum, and hippocampus) and spinal tissue (thoracic spinal cord) after IT administration (0.6 mg in 30 μl of aCSF) of various SOD1-targeting siRNA double helixs containing 3' or 5' terminal conjugations of the lipophilic portion (C16 or C22) of rat CNS:D14. Figure 20B shows the results of inhibition of SOD1 gene expression in rat peripheral tissue (liver, heart) after IT administration (0.6 mg in 30 μl of aCSF) of various SOD1-targeting siRNA double helixs containing 3' or 5' terminal conjugations of the lipophilic portion (C16 or C22) of D14. In Figure 20B, various lipod conjugations include L54, L321, Q447, Q448, Q466, Q478, Q483, and the internal C16 control, respectively. [Modes for carrying out the invention]

[0267] The present invention relates to adding C to at least one strand of the dsRNA agent (for example, at position 6 of the sense strand, counting from the 5' end). 22 Conjugating the lipophilic portion allows for the same sense and antisense chains, as well as C 22 Except for containing different hydrocarbon chains having fewer carbon atoms than the hydrocarbon chain, the same lipophilic portion (for example, C 16 This discovery is based on the remarkable finding that, compared to dsRNA agents having the same conjugation as the lipophilic portion, it provides remarkably high in vivo silencing efficacy to CNS target genes at relatively low dose levels. Therefore, this specification describes a C-modulatory agent that modulates the expression of target genes in the central nervous system (CNS) and can provide desired silencing activity in the CNS at significantly lower dose levels. 22 We disclose a double-stranded siRNA containing a conjugate.

[0268] One aspect of the present invention is an antisense strand complementary to a target gene of the central nervous system (CNS); a sense strand complementary to the antisense strand; and one or more saturated or unsaturated C chains conjugated at one or more positions on at least one of the strands, optionally via a linker or carrier. 22 The present invention provides a double-stranded RNA (dsRNA) agent that modulates the expression of a target gene in the CNS, comprising one or more lipophilic moieties containing a hydrocarbon chain.

[0269] The term "lipophilic" or "lipophilic moiety" broadly refers to any compound or chemical moiety that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is by its octanol-water partition coefficient, logK ow Therefore, K ow The logK of a two-phase system at equilibrium is the ratio of the concentration of a chemical in the octanol phase to its concentration in the aqueous phase. 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 the chemical, calculated using first-principles or experimental methods (see, for example, Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), the full contents of which are incorporated herein by reference). It provides a thermodynamic measurement of a substance's tendency to prefer non-aqueous or oily environments to water (i.e., its hydrophilic / lipophilic balance). In principle, a chemical substance has a logK of 100. ow If logK is greater than 0, the property is lipophilic. Typically, the lipophilic portion is greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10. ow It has. For example, the logK of 6-aminohexanol ow For example, it is predicted to be approximately 0.7. Using the same method, the logK of cholesteryl N-(hexane-6-ol) carbamate ow It is predicted to be 10.7.

[0270] The lipophilicity of a molecule can be altered with respect to its functional groups. For example, the addition of a hydroxyl group or amine group to the terminus of a lipophilic moiety can change the partition coefficient (e.g., logK) of the lipophilic moiety. ow The value can be increased or decreased.

[0271] Alternatively, the hydrophobicity of a compound (e.g., a dsRNA agent) conjugated to one or more lipophilic moieties can be measured by its protein-binding properties. For example, the unbound fraction in a plasma protein-binding assay of a compound can be determined to positively correlate with the relative hydrophobicity of the dsRNA agent, which may positively correlate with the silencing activity of the dsRNA agent.

[0272] In one embodiment, the determined plasma protein binding assay is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. The hydrophobicity of the dsRNA agent, as measured by the unbound siRNA fraction in the binding assay, is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45, or greater than 0.5, for enhanced in vivo delivery of dsRNA.

[0273] Therefore, conjugation of the lipophilic moiety to the dsRNA agent provides optimal hydrophobicity to enhance the in vivo delivery of the dsRNA.

[0274] In certain embodiments, one or more lipophilic moieties may be aliphatic, cyclic (such as alicyclic), or polycyclic compounds (such as polycyclic alicyclic), e.g., steroids (e.g., sterols) or linear or branched aliphatic hydrocarbons. One or more lipophilic moieties may generally include hydrocarbon chains that may be cyclic or acyclic. The hydrocarbon chains may include various substituents and / or one or more heteroatoms, e.g., oxygen or nitrogen atoms. Such lipophilic aliphatic moieties may be saturated or unsaturated C4-C 30 Hydrocarbon chains (for example, C6~C 18Hydrocarbons), saturated or unsaturated fatty acids, waxes (e.g., monohydric alcohol esters and fatty diamides of fatty acids), terpenes (e.g., C 10 Terpenes, C 15 Sesquiterpenes, C 20 Diterpenes, C 30 Triterpenes, and C 40 This includes, but is not limited to, tetraterpenes and other polycyclic alicyclic hydrocarbons. For example, one or more lipophilic moieties are C4-C 30 Hydrocarbon chains (for example, C4~C 30 It may contain alkyl or alkenyl compounds. In some embodiments, one or more lipophilic moieties may be saturated or unsaturated C6-C6 compounds. 18 Hydrocarbon chains (e.g., linear C6~C) 18 It may contain alkyl or alkenyl compounds.

[0275] In some embodiments, two or more lipophilic moieties may be conjugated to the dsRNA agent. In some embodiments, only one lipophilic moiety may be conjugated to the dsRNA agent.

[0276] In one embodiment, at least one lipophilic portion is saturated or unsaturated C 22 Hydrocarbon chains (e.g., linear or branched C) 22 Contains alkyl or alkenyl compounds.

[0277] The lipophilic moiety may be conjugated to the dsRNA agent by any method known in the art, including by methods via a functional group already present in the lipophilic monomer or introduced into the dsRNA agent, such as a hydroxyl group (e.g., -CO-CH2-OH). Functional groups already present in the lipophilic monomer or introduced into the dsRNA agent include, but are not limited to, hydroxyl, amine, carboxylic acid, sulfonic acid, phosphoric acid, thiol, azide, and alkyne.

[0278] Conjugation of the dsRNA agent with the lipophilic moiety may occur, for example, by the formation of an ether or carboxylic acid or carbamoyl ester linkage between the hydroxyl group and an alkyl group R-, an alkanoyl group RCO-, or a substituted carbamoyl group RNHCO-. The alkyl group R can be cyclic (e.g., cyclohexyl) or acyclic (e.g., linear or branched; and saturated or unsaturated). The alkyl group R may be butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl or octadecyl, eicosanyl, docosanyl groups, etc.

[0279] In some embodiments, the lipophilic portion is conjugated to a dsRNA agent via a linker, the linker containing an ether, thioether, urea, carboxylic acid, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, click reaction product (e.g., triazole from azido-alkyne cycloaddition), or carbamate.

[0280] In some embodiments, one of the lipophilic moieties may be a steroid, such as a sterol. The steroid is a polycyclic compound containing a perhydro-1,2-cyclopentanophenanthrene ring system. Steroids include, but are not limited to, bile acids (e.g., cholic acid, deoxycholic acid, and dehydrocholic acid), cortisone, digoxigenin, testosterone, cholesterol, and cationic steroids, such as cortisone. "Cholesterol derivative" refers to a cholesterol-derived compound, for example, by substitution, addition, or removal of substituents.

[0281] In some embodiments, one of the lipophilic moieties may be an aromatic moiety. In this context, the term “aromatic” broadly refers to monocyclic and polycyclic aromatic hydrocarbons. The aromatic group is a C6-C6 group containing 1-3 aromatic rings, which may be substituted as appropriate. 14The term "heteroaryl" includes, but is not limited to, aryl groups; and "aralkyl" or "arylalkyl" groups, which include an aryl group covalently bonded to an alkyl group, either of which may independently be substituted or unsubstituted as appropriate; and "heteroaryl" groups. As used herein, the term "heteroaryl" refers to a group having 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms; having 6, 10, or 14π electrons shared in a cyclic arrangement; and having, in addition to carbon atoms, heteroatoms between 1 and about 3 selected from the group consisting of nitrogen (N), oxygen (O), and sulfur (S).

[0282] The “substituted” alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclic groups used herein have non-hydrogen substitutions between 1 and about 4, preferably between 1 and about 3, more preferably 1 or 2. Preferred substitutions include, but are not limited to, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamide, arenesulfonamide, aralkylsulfonamide, alkylcarbonyl, acyloxy, cyano, and ureido groups.

[0283] In some embodiments, one of the lipophilic moieties may be an aralkyl group, for example, a 2-arylpropanoyl moiety. The structural features of the aralkyl group are selected so that the lipophilic moiety binds to at least one protein in vivo. In certain embodiments, the structural features of the aralkyl group are selected so that the lipophilic moiety binds to serum, blood vessels, or cellular proteins. In certain embodiments, the structural features of the aralkyl group facilitate binding to albumin, immunoglobulins, lipoproteins, α-2-macroglobulins, or α-1-glycoproteins.

[0284] In certain embodiments, the ligand is naproxene or a structural derivative of naproxene. The synthesis procedure for naproxene can be found in U.S. Patents 3,904,682 and 4,009,197, which are incorporated herein by reference in their entirety. Naproxene has the chemical name (S)-6-methoxy-α-methyl-2-naphthaleneacetic acid, and its structure is

[0285] [ka] That is the case.

[0286] In certain embodiments, the ligand is ibuprofen or a structural derivative of ibuprofen. A procedure for synthesizing ibuprofen can be found in U.S. Patent No. 3,228,831, which is incorporated herein by reference in its entirety. The structure of ibuprofen is:

[0287] [ka] That is the case.

[0288] Further exemplary aralkyl groups are exemplified in U.S. Patent No. 7,626,014, which is incorporated herein by reference in whole.

[0289] In another embodiment, a preferred lipophilic moiety includes lipids, cholesterol, retinoic acid, cholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxytrityl, or phenoxazine.

[0290] In some embodiments, one of the lipophilic portions is C6~C 30Acids (e.g., hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, oleic acid, linoleic acid, arachidonic acid, cis-4,7,10,13,16,19-docosahexanoic acid, vitamin A, vitamin E, cholesterol, etc.) or C6~C 30 It may be an alcohol (e.g., hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecanol, hexadecanol, heptadecanol, octadecanol, oleyl alcohol, linoleyl alcohol, arachidonic acid alcohol, cis-4,7,10,13,16,19-docosahexanol, retinol, vitamin E, cholesterol, etc.).

[0291] In certain embodiments, more than one lipophilic moiety may be incorporated into the dsRNA agent, particularly if the lipophilic moiety has low lipophilicity or hydrophobicity. In one embodiment, two or more lipophilic moieties are incorporated into the same strand of the dsRNA agent. In one embodiment, each strand of the dsRNA agent has one or more incorporated lipophilic moieties. In one embodiment, two or more lipophilic moieties are incorporated into the same position (i.e., the same nucleobase, the same sugar moiety, or the same internucleoside linkage) of the dsRNA agent. This can be achieved, for example, by using a lipophilic monomer containing a carrier capable of linking two or more lipophilic moieties, and / or a branched linker, and / or one or more linkers.

[0292] The lipophilic moiety can be conjugated to the dsRNA agent via direct joining to the nucleobase, ribosaccharide, or nucleoside linkage of the dsRNA agent. Alternatively, the lipophilic moiety can be conjugated to the dsRNA agent via a non-ribose substitution unit, such as a linker or carrier.

[0293] In a particular embodiment, the lipophilic moiety is conjugated to a dsRNA agent via one or more linkers (tethers).

[0294] In one embodiment, the lipophilic moiety is conjugated to a dsRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, click reaction product (e.g., triazole from azido-alkyne cycloaddition), or carbamate.

[0295] Linker / Tether The linker / tether connects to the lipophilic portion at the "tethering junction (TAP)". The linker / tether connects to any C1~C 100 Carbon-containing portion (for example, C1~C 75 , C1~C 50 , C1~C 20 , C1~C 10 ;C1, C2, C3, C4, C5, C6, C7, C8, C9, or C 10 ) may contain at least one nitrogen atom. In certain embodiments, the nitrogen atom may form a terminal amino or amide (NHC(O)-) group portion of the linker / tether and be used as a connection point for the lipophilic portion. Non-limiting examples of linkers / tethers (underlined) include TAP-(CH2) n NH-;TAP-C(O)(CH2) n NH-;TAP-NR''''(CH2) n NH-, TAP-C(O)-(CH2) n -C(O)-;TAP-C(O)-(CH2) n -C(O)O-;TAP-C(O)-O-;TAP-C(O)-(CH2) n -NH-C(O)-;TAP-C(O)-(CH2) n -;TAP-C(O)-NH-;TAP-C(O)-;TAP-(CH2) n -C(O)-;TAP-(CH2) n -C(O)O-;TAP-(CH2) n -; or TAP-(CH2)n It contains -NH-C(O)-, where n is 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) and R'''' is a C1 to C6 alkyl group. Preferably, n is 5, 6, or 11. In other embodiments, nitrogen may form part of a terminal oxyamino group, e.g., -ONH2, or a hydrazino group, -NHNH2. The linker / tether may be substituted with, for example, hydroxy, alkoxy, or perhaloalkyl groups, and / or may be inserted with one or more further heteroatoms, e.g., N, O, or S. A preferred tethered ligand is, for example, TAP-(CH2) n NH(LIGAND);TAP-C(O)(CH2) n NH(LIGAND);TAP-NR''''(CH2) n NH(LIGAND);TAP-(CH2) n ONH(LIGAND);TAP-C(O)(CH2) n ONH(LIGAND);TAP-NR''''(CH2) n ONH(LIGAND);TAP-(CH2) n NHNH2(LIGAND), TAP-C(O)(CH2) n NHNH2(LIGAND);TAP-NR''''(CH2) n NHNH2(LIGAND);TAP-C(O)-(CH2) n -C(O)(LIGAND);TAP-C(O)-(CH2) n -C(O)O(LIGAND);TAP-C(O)-O(LIGAND);TAP-C(O)-(CH2) n -NH-C(O)(LIGAND);TAP-C(O)-(CH2) n (LIGAND);TAP-C(O)-NH(LIGAND);TAP-C(O)(LIGAND);TAP-(CH2) n -C(O)(LIGAND);TAP-(CH2) n -C(O)O(LIGAND);TAP-(CH2) n (LIGAND); or TAP-(CH2)n -NH-C(O)(LIGAND) may be included. In some embodiments, the amino-terminal linker / tether (e.g., NH2, ONH2, NH2NH2) may form an imino bond (i.e., C=N) with the ligand. In some embodiments, the amino-terminal linker / tether (e.g., NH2, ONH2, NH2NH2) may be acylated by, for example, C(O)CF3.

[0296] In some embodiments, the linker / tether may be terminated by a mercapto group (i.e., SH) or an olefin (e.g., CH=CH2). For example, the tether may be TAP-(CH2) n -SH, TAP-C(O)(CH2) n SH, TAP-(CH2) n -(CH=CH2), or TAP-C(O)(CH2) n It may also be (CH=CH2), where n may be as described elsewhere in this specification. The tether may be optionally substituted with, for example, hydroxy, alkoxy, or perhaloalkyl, and / or may be optionally inserted with one or more further heteroatoms, such as N, O, or S. The double bond may be cis or trans, or E or Z.

[0297] In other embodiments, the linker / tether may include an electrophilic moiety, preferably at the terminal position of the linker / tether. Exemplary electrophilic moieties include, for example, aldehydes, alkyl halides, mesylates, tosylates, nosylates, or brosilates, or activated carboxylic acid esters, such as NHS esters or pentafluorophenyl esters. A preferred linker / tether (underlined) is TAP-(CH2) n CHO;TAP-C(O)(CH2) n CHO; or TAP-NR''''(CH2) n CHO(where n is 1-6 and R'''' is a C1-C6 alkyl group); or TAP-(CH2) n C(O)ONHS;TAP-C(O)(CH2) nC(O)ONHS; or TAP-NR''''(CH2) n C(O)ONHS(where n is 1-6 and R'''' is a C1-C6 alkyl); TAP-(CH2) n C(O)OC6F5;TAP-C(O)(CH2) n C(O)OC6F5; or TAP-NR''''(CH2) n C(O)OC6F5 (where n is 1 to 11 and R'''' is a C1 to C6 alkyl group); or -(CH2) n CH2LG;TAP-C(O)(CH2) n CH2LG; or TAP-NR''''(CH2) n CH2LG [wherein n is described elsewhere herein, and R'''' is a C1-C6 alkyl group (LG may release a group, e.g., a halide, mesylate, tosylate, nosylate, or brosilate)]. Tethering may be performed by coupling a nucleophilic group of the ligand, e.g., a thiol or amino group, with an electrophilic group of the tether.

[0298] In other embodiments, it may be desirable for the monomer to contain a phthalimide group (K) at the terminal position of the linker / tether.

[0299] [ka]

[0300] In other embodiments, other protected amino groups, such as alloc, monomethoxytrityl (MMT), trifluoroacetyl, Fmoc, or arylsulfonyl (for example, the aryl portion may be orthonitrophenyl or ortho,para-dinitrophenyl), may be located at the terminal position of the linker / tether.

[0301] Any linker / tether described herein may have one or more further linking groups, e.g., -O-(CH2) n -,-(CH2) n -SS-, -(CH2)n -, or -(CH=CH)- may further be included.

[0302] Cutting-type linker / tether In some embodiments, at least one linker / tether may be a redox-cleaving linker, an acid-cleaving linker, an esterase-cleaving linker, a phosphatase-cleaving linker, or a peptidase-cleaving linker.

[0303] In one embodiment, at least one linker / tether may be a reductive cleavage type linker (e.g., a disulfide group).

[0304] In one embodiment, at least one linker / tether may be an acid-cleaving linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group).

[0305] In one embodiment, at least one linker / tether may be an esterase-cleaving linker (e.g., an ester group).

[0306] In one embodiment, at least one linker / tether may be a phosphatase-cleaving linker (e.g., a phosphate group).

[0307] In one embodiment, at least one linker / tether may be a peptidase-cleaving linker (e.g., a peptide bond).

[0308] Severable linkers are sensitive to the presence of severing agents, such as pH, redox activity, or degradable molecules. Generally, severing agents are found more frequently, at higher levels, or with higher activity inside cells than in serum or blood. Examples of such degrading agents include redox agents selected for specific substrates, or redox agents without substrate specificity, which include, for example, mercaptans present in cells that can degrade redox-severable linkers by reduction; esterases; endosomes; or agents that can create an acidic environment, such as an acidic environment resulting in a pH of 5 or less; and redox agents containing oxidases or reductases or reducing agents, such as general acids, peptidases (which may be substrate-specific), and enzymes that can hydrolyze or degrade acid-severable linkers by acting as phosphatases.

[0309] Scleavable linking groups, such as disulfide bonds, can be pH-sensitive. While the pH of human serum is 7.4, the average intracellular pH is somewhat lower, ranging from approximately 7.1 to 7.3. Endosomes have a highly acidic pH in the range of 5.5 to 6.0, and lysosomes have a still highly acidic pH of approximately 5.0. Some tethers may be cleaved at a favorable pH, thereby possessing linking groups that release dsRNA agents from intracellular ligands (e.g., targeting or cell-permeable ligands, e.g., cholesterol) or into desired cellular compartments.

[0310] The chemical linkage (e.g., linking group) that connects the ligand to the dsRNA agent may include a disulfide bond. When the dsRNA agent / ligand complex is taken up into a cell by endocytosis, the acidic environment of the endosome causes the disulfide bond to break, thereby releasing the dsRNA agent from the ligand (Quintana et al., Pharm Res. 19:1310-1316, 2002; Patri et al., Curr. Opin. Curr. Biol. 6:466-471, 2002). The ligand may be a secondary therapeutic agent that can complement the therapeutic effect of the targeting ligand or dsRNA agent.

[0311] A tether may contain a linking group that is cleaved by a specific enzyme. The type of linking group incorporated into the tether may depend on the cell being targeted by the dsRNA agent. For example, a dsRNA agent that targets mRNA in liver cells may be conjugated to a tether containing an ester group. Since liver cells are rich in esterases, the tether will likely be cleaved more efficiently in liver cells than in esterase-unriched cell types. Cleavage of the tether may release the dsRNA agent from the ligand conjugated to the distal end of the tether, thereby enhancing the silencing activity of the dsRNA agent. Other esterase-rich cell types include lung, renal cortical, and testicular cells.

[0312] A tether containing a peptide bond can be conjugated to a dsRNA agent and targeted to peptidase-rich cell types, such as liver cells and synovial cells. For example, a dsRNA agent that targets synovial cells, such as for the treatment of inflammatory diseases (e.g., rheumatoid arthritis), can be conjugated to a tether containing a peptide bond.

[0313] Generally, the suitability of a candidate cleavage ligator can be evaluated by testing the ability of a degrading agent (or degradation conditions) to cleave the candidate ligator. It may also be desirable to investigate the candidate cleavage ligator's resistance to cleavage in the blood or in contact with other non-target tissues, such as tissues to which the dsRNA agent would be exposed if administered to a subject. It may also be desirable to investigate the candidate cleavage ligator's resistance to cleavage in the blood or in contact with other non-target tissues. Therefore, when the first condition is selected to indicate cleavage within target cells, and the second condition is selected to indicate cleavage in other tissues or in body fluids, such as blood or serum, the relative sensitivity to cleavage between the first and second conditions can be determined. The evaluation may be performed in cell-free systems, cells, cell cultures, organ cultures or tissue cultures, or in whole animals. Initial evaluation under cell-free or culture conditions may be useful, followed by further evaluation in the whole animal for confirmation. In a preferred embodiment, a useful candidate compound is cleaved at a rate of at least 2, 4, 10, or 100 times in cells (or under in vivo conditions selected to mimic intracellular conditions) compared to blood or serum [or under in vitro conditions selected to mimic extracellular conditions].

[0314] Redox-cut type connector One class of cleavage-type linkers is the redox-cleavage-type linker, which is cleaved upon reduction or oxidation. An example of a reductive-cleavage-type linker is the disulfide linker (-SS-). Methods described herein can be considered to determine whether a candidate cleavage-type linker is a suitable “reductive-cleavage-type linker” or whether it is suitable for use with, for example, a specific iRNA moiety and a specific targeting agent. For example, a candidate substance may be evaluated by incubation with dithiothreitol (DTT) or other reducing agents known in the art that mimic the cleavage rate observed intracellularly, for example, in a target cell. The candidate substance may also be evaluated under conditions selected to mimic blood or serum conditions. In a preferred embodiment, the candidate compound is cleaved to a maximum of 10% in blood. In a preferred embodiment, a useful candidate compound is degraded at a rate of at least 2, 4, 10, or 100 times in cells (or in vivo conditions selected to mimic intracellular conditions) compared to in blood (or in in vitro conditions selected to mimic extracellular conditions). The cleavage rate of the candidate compound may be determined using a standard enzyme kinetic assay under conditions selected to mimic an intracellular medium and compared to conditions selected to mimic an extracellular medium.

[0315] Phosphate-based cleavage-type linking groups Phosphate-based linking groups are cleaved by agents that break down or hydrolyze the phosphate group. In cells, examples of agents that cleave the phosphate group include intracellular enzymes such as phosphatases. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-O-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(O)(Rk)-O-, -SP(O)(Rk)-S-, and -OP(S)(Rk)-S-. Preferred embodiments are -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-. A preferred embodiment is -OP(O)(OH)-O-. These candidate linkages can be evaluated using methods similar to those described above.

[0316] Acid-cleavable linking group Acid-cleaved linking groups are linking groups that are cleaved under acidic conditions. In preferred embodiments, acid-cleaved linking groups are cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0, or less), or by agents such as enzymes that can act as general acids. Within cells, specific low-pH organelles, such as endosomes and lysosomes, provide a cleavage environment for acid-cleaved linking groups. Examples of acid-cleaved linking groups include, but are not limited to, hydrazones, ketals, acetals, esters, and amino acid esters. Acid-cleaved groups may have the general formula -C=NN-, C(O)O, or -OC(O). Preferred embodiments are those in which 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 candidate linking groups can be evaluated using methods similar to those described above.

[0317] Ester-based linking groups Ester-based linking groups are cleaved by enzymes such as esterases and amidases within cells. Examples of ester-based cleaving linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester-cleaving linking groups have the general formulas C(O)O- or -OC(O)-. These candidate linking groups can be evaluated using methods similar to those described above.

[0318] Peptide-based cleavage groups Peptide-based linkers are cleaved by enzymes such as peptidases and proteases within cells. Peptide-based cleavage linkers are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavage linkers do not contain amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. Peptide bonds are a special type of amide bond formed between amino acids to yield peptides and proteins. Peptide-based cleavage linkers are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to yield peptides and proteins, and do not include the entire amide functional group. The general formula for a peptide cleavage linker is -NHCHR 1 C(O)NHCHR 2 C(O)-[wherein, R 1 and 2 [These have the R groups of two adjacent amino acids.] These candidates can be evaluated using methods similar to those described above.

[0319] Biological cutting linker / tether Linkers may also include biocleavage linkers, which are nucleotide and non-nucleotide linkers or combinations thereof that connect the chains of one or both of the two individual siRNA molecules that make up bis(siRNA), for example, bis(siRNA). In some embodiments, a slight electrostatic or stacking interaction between the two individual siRNAs may represent a linker. Non-nucleotide linkers include tethers or linkers derived from monosaccharides, disaccharides, oligosaccharides and their derivatives, aliphatic, alicyclic, heterocyclic, and combinations thereof.

[0320] In some embodiments, at least one linker (tether) is a biological cleavage linker selected from the group consisting of DNA, RNA, disulfides, amides, galactosamine functional monosaccharides or oligosaccharides, glucosamine, glucose, galactose, and mannose, as well as combinations thereof.

[0321] In one embodiment, the bio-cleavage type carbohydrate linker may have 1 to 10 sugar units, each having at least one anomeric linkage capable of connecting two siRNA units. If two or more sugar units are present, these units may be linked via 1 to 3, 1 to 4, or 1 to 6 sugar linkages, or via alkyl chains.

[0322] Exemplary biological cleavage linkers include the following endosomal cleavage linkers and phosphoramidites:

[0323] [ka] TIFF2026514045000076.tif233161 TIFF2026514045000077.tif202161 TIFF2026514045000078.tif214161 This includes, but is not limited to, TIFF2026514045000079.tif124161.

[0324] Further discussion of bio-cleavage linkers can be found in PCT application PCT / US18 / 14213, filed on 18 January 2018, titled “Endosome-cleavage linkers,” the full contents of which are incorporated herein by reference.

[0325] Carrier In a particular embodiment, the lipophilic moiety is conjugated to the dsRNA agent via a non-ribose substitution unit, i.e., a carrier that substitutes one or more nucleotides.

[0326] The support may be a cyclic or acyclic group. In one embodiment, the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinol, tetrahydrofuryl, and decalin. In one embodiment, the acyclic group is a portion based on a serinol skeleton or a diethanolamine skeleton.

[0327] The carrier may substitute one or more nucleotides of the dsRNA agent.

[0328] In some embodiments, the carrier substitutes one or more nucleotides at one or more internal positions of the dsRNA agent.

[0329] In other embodiments, the support substitutes a nucleotide at the end of the sense or antisense strand. In one embodiment, the support substitutes a terminal nucleotide at the 3' end of the sense strand, thereby functioning as a terminal cap that protects the 3' end of the sense strand. In one embodiment, the support is a cyclic group having an amine, and for example, the support may be pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanil, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuranil, and dekalinyl.

[0330] In this specification, a ribonucleotide subunit in which the ribose sugar of the subunit is replaced in this manner is referred to as a ribose substitution modified subunit (RRMS). The carrier may be cyclic or acyclic and may comprise two "skeletal junctions" (e.g., hydroxyl groups) and a ligand (e.g., a lipophilic moiety). The lipophilic moiety may be directly attached to the carrier or indirectly attached to the carrier by an intervening linker / tether, as described above.

[0331] [ka]

[0332] The ligand-conjugate monomer subunit may be the 5' or 3' terminal subunit of the iRNA molecule, i.e., one of the two "W" groups may be a hydroxyl group and the other "W" group may be a chain of two or more unmodified or modified ribonucleotides. Alternatively, the ligand-conjugate monomer subunit may occupy an internal position, and both "W" groups may be one or more unmodified or modified ribonucleotides. More than one ligand-conjugate monomer subunit may be present in the dsRNA agent.

[0333] Sugar substitution-based monomers, e.g., ligand-conjugate monomers (cyclic) Cyclic sugar-substituted monomers, such as sugar-substituted ligand-conjugate monomers, are also referred to herein as RRMS monomer compounds. The support may have the general formula (LCM-2) provided below (in its structure, the preferred skeletal junction is R 1 Or R 2 , R 3 Or R 4 , or Y is CR 9 R 10 If R 9 and R 10 (The two positions can be selected from the two skeletal junctions, e.g., R) 1 and R 4 , or R4 and R 9 The preferred tethering junction is R when X is CH2. 7 ;R 5 or R 6 This includes. The carrier is described below as an entity that can be incorporated into the chain. Thus, the structure has one (in the case of terminal positions) or two (in the case of internal positions) junctions, for example, R 1 Or R 2 , R 3 or 4 , or R 9 Or R 10 (Y is CR 9 R 10 In this case, it is understood that this also includes situations where the R group is connected to a phosphate group or a modified phosphate group, for example, a sulfur atom containing a skeleton. For example, one of the R groups named above may be -CH2-, where one bond is connected to a support and the other to a skeleton atom, for example, oxygen or a central phosphorus atom.

[0334] [ka] [In formula: X is N(CO)R 7 , NR 7 or CH2; Y is NR 8 O, S, CR 9 R 10 and; Z is CR 11 R 12 or not; R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 At least two of these are OR a and / or (CH2) n Ure b If so, each R 1 , R 2 , R 3 , R 4 , R 9, and R 10 H, OR a and / or (CH2) n Ure b And; Each R 5 , R 6 , R 11 , and R 12 These are, independently, ligand, H, 1-3R 13 C1-C6 alkyl or C(O)NHR which may be substituted by 7 is; or R 5 and R 11 Both are R 14 A C3-C8 cycloalkyl group which may be substituted by; R 7 R may be a ligand, for example, 7 R d Is it okay, or R 7 For example, the tethering part, for example, NR c R d C1~C substituted by 20 Alkyl, or NHC(O)R d C1~C substituted by 20 The ligand may also be indirectly tethered to the carrier via an alkyl group; R 8 is H or C1-C6 alkyl; R 13 These are hydroxyl, C1-C4 alkoxy, or halo; R 14 , NR C R 7 and; R 15 is a C1-C6 alkyl or C2-C6 alkenyl which may be substituted with cyano; R 16 C1~C 10 It is alkyl; R 17 It is a liquid-phase or solid-phase supported reagent; L is -C(O)(CH2) q C(O)-, or -C(O)(CH2) q It is S-; R a This refers to a protecting group, for example, CAr3 (e.g., dimethoxytrityl group) or Si(X 5 ')(X 5 '')(X 5 ''') and here, (X 5 '), (X 5 ''), and (X 5 ''') is described elsewhere in this specification, R b P(O)(O-)H, P(OR 15 )N(R 16 )2 or LR 17 and; R C is H or C1-C6 alkyl; R d is H or a ligand; Each Ar may be independently substituted with a C1-C4 alkoxy, or C6-C4 alkoxy. 10 It is Ariel, n is between 1 and 4; q is between 0 and 4.

[0335] An example of a carrier is, for example, X is N(CO)R 7 or NR 7 And Y is CR 9 R 10 And Z does not exist; or X is N(CO)R 7 or NR 7 And Y is CR 9 R 10 And Z is CR 11 R 12 Either X is N(CO)R 7 or NR 7 And Y is O and Z is CR 11 R 12 Either X is CH2 and Y is CR 9 R 10 And Z is CR 11 R 12 And R 5 and R 11 They together form a C6 cycloalkyl (H, z=2) or an indane ring system, for example, X is CH2; Y is CR9 R 10 And; Z is CR 11 R 12 And R 5 and R 11 This includes those that together form a C5 cycloalkyl (H, z=1) molecule.

[0336] In certain embodiments, the carrier is a pyrroline ring system or a 4-hydroxypyrroline ring system, for example, X is N(CO)R 7 or NR 7 And Y is CR 9 R 10 This can be based on the fact that Z does not exist (D).

[0337] [ka] OFG 1 Preferably, it is bonded to one of the carbon atoms of the five-membered ring, to the first carbon, for example, an extra-ring alkylene group, for example, a methylene group (D's CH2OFG 1 OFG 2 Preferably, it is directly bonded to one of the five-membered ring carbons (D OFG 2 ). In pyrroline-based carriers, CH2OFG 1 It may be joined to C-2, OFG 2 It may be joined to C-3; or -CH2OFG 1 It may be joined to C-3, OFG 2 It may be joined to C-4. In a particular embodiment, CH2OFG 1 and OFG 2 This may be pair-substituted with one of the carbons referenced above. In 3-hydroxyproline-based supports, -CH2OFG 1 It may be joined to C-2, OFG 2 It may be joined to C-4. Pyrroline- and 4-hydroxyproline-based monomers may therefore contain bond rotations that are restricted by certain linkages, such as carbon-carbon bonds, which are limited by the presence of a ring. Thus, CH2OFG 1 and OFG2 The isomers can be cis or trans with respect to each other in any of the pairs described above. Therefore, all cis / trans isomers are clearly included. Monomers may contain one or more chiral centers and therefore may arise as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomer mixtures. All such isomeric forms of monomers are clearly included (e.g., CH2OFG 1 and OFG 2 The centers having the configuration may both have the R configuration; or both may have the S configuration; or one center may have the R configuration and the other center may have the S configuration, and vice versa). The tethering junction is preferably nitrogen. A preferred example of the carrier D is as follows:

[0338] [ka] Includes.

[0339] In certain embodiments, the support may be based on a piperidine ring system (E), for example, X is N(CO)R 7 or NR 7 And Y is CR 9 R 10 And Z is CR 11 R 12 That is the case.

[0340] [ka] OFG 1 Preferably, it is bonded to one of the carbon atoms of the six-membered ring, to the first carbon, for example, an extra-ring alkylene group, for example, a methylene group (n=1) or an ethylene group (n=2) (E-(CH2) n OFG 1 OFG 2 Preferably, it is directly bonded to one of the carbon atoms of the six-membered ring (E-OFG 2 ) -(CH2) n OFG 1 and OFG 2The groups may be arranged in a paired manner on the ring, that is, both groups may be bonded to the same carbon, for example, C-2, C-3, or C-4. Alternatively, -(CH2) n OFG 1 and OFG 2 They may be arranged adjacent to the ring, that is, both groups may be bonded to adjacent ring carbon atoms, for example, -(CH2) n OFG 1 It may be joined to C-2, OFG 2 It may also be joined to C-3;-(CH2) n OFG 1 It may be joined to C-3, OFG 2 It may also be joined to C-2;-(CH2) n OFG 1 It may be joined to C-3, OFG 2 It may be joined to C-4; or -(CH2) n OFG 1 It may be joined to C-4, OFG 2 It may be joined to C-3. Piperidine-based monomers may therefore contain bond rotations that are restricted by certain linkages, such as carbon-carbon bonds, which are limited by the presence of a ring. Thus, -(CH2) n OFG 1 and OFG 2 The isomers can be cis or trans with respect to each other in any of the pairs described above. Therefore, all cis / trans isomers are clearly included. Monomers may contain one or more chiral centers and therefore may arise as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomer mixtures. All such isomeric forms of monomers are clearly included (e.g., CH2OFG 1 and OFG 2 The centers having the configuration may both have the R configuration; or both may have the S configuration; or one center may have the R configuration and the other center may have the S configuration, and vice versa. The tethering junction is preferably nitrogen.

[0341] In certain embodiments, the support may be based on a piperidine ring system (F), for example, X is N(CO)R 7 or NR 7 And Y is NR 8 And Z is CR 11 R 12 Alternatively, it may be based on a morpholine ring system (G), for example, X is N(CO)R 7 or NR 7 Y is O, and Z is CR 11 R 12 That is the case.

[0342] [ka] OFG 1 Preferably, it is bonded to one of the carbon atoms of the six-membered ring, to the first carbon, for example, an extra-ring alkylene group, for example, a methylene group (F or G -CH2OFG). 1 OFG 2 Preferably, it is directly bonded to one of the carbon atoms of the six-membered ring (OFG of F or G). 2 ). In both F and G, -CH2OFG 1 It may be joined to C-2, OFG 2 It may be joined to C-3; or vice versa. In a particular embodiment, CH2OFG 1 and OFG 2 This may be pair-substituted with one of the carbons referenced above. Piperazine- and morpholine-based monomers may therefore contain bond rotations that are restricted by certain linkages, such as carbon-carbon bonds, resulting from the presence of a ring. Thus, CH2OFG 1 and OFG 2The isomers can be cis or trans with respect to each other in any of the pairs described above. Therefore, all cis / trans isomers are clearly included. Monomers may contain one or more chiral centers and therefore may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomer mixtures. All such isomeric forms of monomers are clearly included (e.g., CH2OFG 1 and OFG 2 The centers having R may both have the R configuration; or both may both have the S configuration; or one center may have the R configuration and the other center may have the S configuration, and vice versa). R''' may be, for example, a C1-C6 alkyl group, preferably CH3. The tethering junction is preferably nitrogen at both F and G.

[0343] In certain embodiments, the support may be based on a decalin ring system, for example, where X is CH2 and Y is CR 9 R 10 And Z is CR 11 R 12 And R 5 and R 11 Both may form a C6 cycloalkyl (H, z=2) or be based on an indane ring system, for example, if X is CH2 and Y is CR 9 R 10 And Z is CR 11 R 12 And R 5 and R 11 Both form a C5 cycloalkyl (H, z=1) structure.

[0344] [ka] OFG 1 Preferably, the first carbon atom is bonded to one of C-2, C-3, C-4, or C-5, for example, an extra-ring methylene group (n=1) or an ethylene group (n=2) [H-(CH2) n OFG 1 OFG 2Preferably, it is directly bonded to one of C-2, C-3, C-4, or C-5 (H-OFG 2 ) -CH2OFG 1 and OFG 2 The groups may be arranged in a paired manner on the ring, that is, both groups may be bonded to the same carbon, for example, C-2, C-3, C-4, or C-5. Alternatively, -(CH2) n OFG 1 and OFG 2 They may be arranged adjacent to the ring, that is, both groups may be bonded to adjacent ring carbon atoms, for example, -(CH2) n OFG 1 It may be joined to C-2, OFG 2 It may also be joined to C-3;-(CH2) n OFG 1 It may be joined to C-3, OFG 2 It may also be joined to C-2;-(CH2) n OFG 1 It may be joined to C-3, OFG 2 It may be joined to C-4; or -(CH2) n OFG 1 It may be joined to C-4, OFG 2 It may also be joined to C-3;-(CH2) n OFG 1 It may be joined to C-4, OFG 2 It may be joined to C-5; or -(CH2) n OFG 1 It may be joined to C-5, OFG 2 It may be joined to C-4. Decalin or indan-based monomers may therefore contain bond rotations that are restricted by certain linkages, such as carbon-carbon bonds, which are limited by the presence of a ring. Thus, -(CH2) n OFG 1 and OFG 2The isomers can be cis or trans with respect to each other in any of the pairs described above. Therefore, all cis / trans isomers are clearly included. Monomers may contain one or more chiral centers and therefore may arise as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomer mixtures. All such isomeric forms of monomers are clearly included (e.g., CH2OFG 1 and OFG 2 The centers having C-1 and C-6 may both have the R configuration; or both may have the S configuration; or one center may have the R configuration and the other center may have the S configuration, and vice versa. In a preferred embodiment, the substituents at C-1 and C-6 are trans with respect to each other. The tethering junction is preferably C-6 or C-7.

[0345] Other carriers may include those based on 3-hydroxyproline (J).

[0346] [ka] Therefore, -(CH2) n OFG 1 and OFG 2 These can be cis or trans with respect to each other. Therefore, all cis / trans isomers are clearly included. Monomers may contain one or more chiral centers and therefore may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomer mixtures. All such isomeric forms of monomers are clearly included (e.g., CH2OFG 1 and OFG 2 The centers having the configuration may both have the R configuration; or both may have the S configuration; or one center may have the R configuration and the other center may have the S configuration, and vice versa. The tethering junction is preferably nitrogen.

[0347] Further details regarding more representative cyclic, sugar-substituted base carriers can be found in U.S. Patents 7,745,608 and 8,017,762, which are incorporated herein by reference in their entirety.

[0348] Sugar-substituted monomers (acyclic) Acyclic sugar-substituted monomers, such as sugar-substituted ligand-conjugate monomers, are also referred to herein as ribose-substituted monomer subunit (RRMS) monomer compounds. Preferred acyclic carriers are those of formula LCM-3 or LCM-4:

[0349] [ka] It may have.

[0350] In some embodiments, each x, y, and z can be independently 0, 1, 2, or 3. In formula LCM-3, if y and z are different, then the third carbon can have either an R configuration or an S configuration. In preferred embodiments, x is zero, and y and z are each 1 in formula LCM-3 (e.g., based on selinol), and y and z are each 1 in formula LCM-3. Each of the following formulas LCM-3 or LCM-4 may be substituted with, for example, hydroxy, alkoxy, or perhaloalkyl.

[0351] More details on more representative acyclic, sugar-substituted base carriers can be found in U.S. Patents 7,745,608 and 8,017,762, which are incorporated herein by reference in their entirety.

[0352] In some embodiments, the dsRNA agent comprises one or more lipophilic moieties conjugated to the 5' end of the sense strand or the 5' end of the antisense strand.

[0353] In a particular embodiment, the lipophilic portion is conjugated to the 5' end of the chain via a carrier and / or linker. In one embodiment, the lipophilic portion is of formula:

[0354] [ka] (In the formula, R is a ligand such as a lipophilic moiety.) It is conjugated to the 5' end of the chain via a carrier. In one embodiment, R is a halogen, -OR G , -SR G , -N(R G )2, -C(O)OR G ,-OC(O)R G ,-C(O)N(R G )2, -N(R G )C(O)R G , -N(R G )C(O)OR G , -N(R G )SO2(R G ), or -SO2N(R G ) may be appropriately substituted by one or two groups selected from the group consisting of 2, and each R G However, independently, hydrogen or C1-C6 alkyl (for example, R is -OR) G , -C(O)OR G , or -N(R G )C(O)R G The above saturated or unsaturated C (which is appropriately substituted by) 22 It is a hydrocarbon chain. In one embodiment, R can form a group having 22 carbons together with the carbonyl to which it is joined (for example, R can be linear or branched C 21 Saturated or unsaturated C such as alkyl groups 21 (Possibly a hydrocarbon chain), halogen, -OR G , -SR G , -N(R G )2, -C(O)OR G ,-OC(O)R G ,-C(O)N(R G )2, -N(R G )C(O)R G , -N(RG )C(O)OR G , -N(R G )SO2(R G ), or -SO2N(R G ) may be appropriately substituted by one or two groups selected from the group consisting of 2, and each R G These are independently hydrogen or C1-C6 alkyl (for example, R is -OR). G , -C(O)OR G , or -N(R G )C(O)R G (This is appropriately substituted by...). In some embodiments, R is substituted by OH or COOH.

[0355] In some embodiments, the dsRNA agent comprises one or more lipophilic moieties conjugated to the 3' end of the sense strand or the 3' end of the antisense strand.

[0356] In a particular embodiment, the lipophilic portion is conjugated to the 3' end of the chain via a carrier and / or linker. In one embodiment, the lipophilic portion is of formula:

[0357] [ka] (In the formula, R is a ligand such as a lipophilic moiety.) It is conjugated to the 3' end of the chain via a carrier. In one embodiment, R is a halogen, -OR G , -SR G , -N(R G )2, -C(O)OR G ,-OC(O)R G ,-C(O)N(R G )2, -N(R G )C(O)R G , -N(R G )C(O)OR G , -N(R G )SO2(R G ), or -SO2N(R G ) may be appropriately substituted by one or two groups selected from the group consisting of 2, and each RG However, independently, hydrogen or C1-C6 alkyl (for example, R is -OR) G , -C(O)OR G , or -N(R G )C(O)R G The above saturated or unsaturated C (which is appropriately substituted by) 22 It is a hydrocarbon chain. In one embodiment, R can form a group having 22 carbons together with the carbonyl to which it is joined (for example, R can be linear or branched C 21 Saturated or unsaturated C such as alkyl groups 21 (Possibly a hydrocarbon chain), halogen, -OR G , -SR G , -N(R G )2, -C(O)OR G ,-OC(O)R G ,-C(O)N(R G )2, -N(R G )C(O)R G , -N(R G )C(O)OR G , -N(R G )SO2(R G ), or -SO2N(R G ) may be appropriately substituted by one or two groups selected from the group consisting of 2, and each R G These are independently hydrogen or C1-C6 alkyl (for example, R is -OR). G , -C(O)OR G , or -N(R G )C(O)R G (This is appropriately substituted by...). In some embodiments, R is substituted by OH or COOH.

[0358] In a particular embodiment, the lipophilic portion is conjugated to an internal position of the chain via a carrier and / or linker. In one embodiment, the lipophilic portion is of formula:

[0359] [ka] [In the formula, R is a ligand such as a lipophilic moiety.] It is conjugated at an internal position of the chain via a carrier. In one embodiment, R is the saturated or unsaturated C mentioned above. 22 It is a hydrocarbon chain.

[0360] In some embodiments, the dsRNA agent includes one or more lipophilic moieties conjugated to both ends of the sense strand.

[0361] In some embodiments, the dsRNA agent includes one or more lipophilic moieties conjugated to both ends of the antisense strand.

[0362] In some embodiments, the dsRNA agent comprises one or more lipophilic moieties conjugated to an internal position on the sense strand or antisense strand. In some embodiments, one or more lipophilic moieties are conjugated to ribose, nucleobases, and / or internucleotide links. In some embodiments, one or more lipophilic moieties are conjugated to ribose at its 2', 3', 4', and / or 5' positions. In some embodiments, one or more lipophilic moieties are conjugated to a natural nucleobase (e.g., A, T, G, C, or U) or a modified nucleobase as defined herein. In some embodiments, one or more lipophilic moieties are conjugated to a phosphate group or a modified phosphate group as defined herein.

[0363] In some embodiments, the dsRNA agent includes one or more lipophilic moieties conjugated to the 5' or 3' end of the sense strand, and one or more lipophilic moieties conjugated to the 5' or 3' end of the antisense strand.

[0364] In some embodiments, the dsRNA agent comprises a lipophilic moiety conjugated to the end of the chain via one or more linkers (tethers) and / or carriers.

[0365] In one embodiment, the dsRNA agent includes a lipophilic moiety conjugated to the end of the strand via one or more linkers (tethers).

[0366] In one embodiment, the dsRNA agent includes a lipophilic moiety conjugated to the 5' end of a sense strand or antisense strand via a cyclic carrier, and optionally via one or more intervening linkers (tethers).

[0367] In some embodiments, at least one lipophilic moiety is located at one or more terminal positions of the sense chain or antisense chain. In one embodiment, at least one lipophilic moiety is located at the 3' or 5' end of the sense chain. In one embodiment, at least one lipophilic moiety is located at the 3' or 5' end of the antisense chain.

[0368] In some embodiments, at least one lipophilic moiety is conjugated to one or more internal positions on at least one chain. An internal position on a chain refers to any nucleotide at any position on the chain, excluding terminal positions from the 3' and 5' ends (e.g., position 2: excluding position 1 when counting from the 3' end and position 1 when counting from the 5' end).

[0369] In one embodiment, at least one lipophilic portion is located at one or more internal positions on at least one chain and includes all positions from each end of the chain except for terminal 2 (e.g., excluding 4: positions 1 and 2 counting from the 3' end, and positions 1 and 2 counting from the 5' end). In one embodiment, at least one lipophilic portion is located at one or more internal positions on at least one chain and includes all positions from each end of the chain except for terminal 3 (e.g., excluding 6: positions 1, 2 and 3 counting from the 3' end, and positions 1, 2 and 3 counting from the 5' end).

[0370] In one embodiment, at least one lipophilic moiety is located at one or more positions at at least one end of the double-stranded region and includes all positions within the double-stranded region, but does not include an overhang region or a carrier that substitutes the terminal nucleotide at the 3' end of the sense strand.

[0371] In one embodiment, at least one lipophilic moiety is located on the sense strand within the first 5, 4, 3, 2, or first base pair at the 5' end of the antisense strand of the double-stranded region.

[0372] In one embodiment, at least one lipophilic moiety is located at one or more internal positions on at least one chain, excluding the cleavage region of the sense chain. For example, the lipophilic moiety is not located at positions 9-12 counting from the 5' end of the sense chain. Alternatively, the internal positions exclude positions 11-13 counting from the 3' end of the sense chain.

[0373] In one embodiment, at least one lipophilic portion is located at one or more internal positions on at least one chain, excluding the cleavage region of the antisense chain. For example, internal positions exclude positions 12-14, counting from the 5' end of the antisense chain.

[0374] In one embodiment, at least one lipophilic moiety is located at one or more internal positions on at least one chain, excluding positions 11-13 of the sense chain (counted from the 3' end) and positions 12-14 of the antisense chain (counted from the 5' end).

[0375] In one embodiment, one or more lipophilic moieties are located at one or more of the following internal positions: counting from the 5' end of each chain, positions 4–8 and 13–18 of the sense chain, and positions 6–10 and 15–18 of the antisense chain.

[0376] In one embodiment, one or more lipophilic moieties are located at one or more of the following internal positions: counting from the 5' end of each chain, positions 5, 6, 7, 15, and 17 of the sense chain, and positions 15 and 17 of the antisense chain.

[0377] definition Unless otherwise noted, the nomenclature, procedures, and techniques used in connection with analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein are well-known and commonly used in the art. Standard techniques may be used for chemical synthesis and chemical analysis. Certain such techniques and procedures are incorporated herein in their entirety by reference for all purposes, for example, in “Carbohydrate Modifications in Antisense Research” Edited by Sangvi and Cook, American Chemical Society, Washington DC, 1994; “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 18th edition, 1990; and “Antisense Drug Technology, Principles, Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A Laboratory Manual,” 2 nd See Edition, Cold Spring Harbor Laboratory Press, 1989. Where permitted, all patents, applications, published applications and other publications and other data referenced throughout this disclosure are incorporated herein by reference in their entirety.

[0378] As used herein, the term “target nucleic acid” refers to any nucleic acid molecule whose expression or activity can be modulated by an siRNA compound. Target nucleic acids include, but are not limited to, RNA transcribed from DNA encoding a target protein (including, but not limited to, pre-mRNA and mRNA or portions thereof), as well as cDNA and miRNA derived from such RNA. For example, a target nucleic acid may be a cellular gene (or mRNA transcribed from a gene) whose expression is associated with a particular disorder or disease condition, e.g., a CNS disorder or disease condition.

[0379] As used herein, the term “iRNA” refers to agents that mediate targeted cleavage of RNA transcripts. These agents associate with a cytoplasmic multiprotein complex known as the RNAi-inducible silencing complex (RISC). Agents effective in inducing RNA interference are also referred herein to as siRNA, RNAi agents, or iRNA agents. These terms may be used interchangeably herein. As used herein, the term iRNA includes microRNA and premicroRNA. Furthermore, as used herein, “compound” or “compounds” of the present invention also refers to iRNA agents and may be used interchangeably with iRNA agents.

[0380] The dsRNA agent must contain a region with sufficient homology to the target gene, be of sufficient length with respect to nucleotides, and the iRNA agent or a fragment thereof must be capable of mediating the downregulation of the target gene. (For ease of explanation, the terms nucleotide or ribonucleotide may be used herein in reference to one or more monomeric subunits of the iRNA agent. It should be understood herein that the use of the terms “ribonucleotide” or “nucleotide” may also refer to a modified nucleotide or substitute substitution region at one or more positions in the case of modified RNA or nucleotide substitutes.) Therefore, the iRNA agent is or contains a region that is at least partially, and in some embodiments, fully complementary to the target RNA. While complete complementarity between the iRNA agent and the target is not required, there must be sufficient correspondence to enable the iRNA agent or its cleavage products to direct sequence-specific silencing by RNAi cleavage of the target RNA, e.g., mRNA. The degree of complementarity, or homology to the target strand, is most important in the antisense strand. While complete complementarity is often desired, particularly in the antisense strand, some embodiments may include one or more mismatches, such as 6, 5, 4, 3, 2, or fewer, especially in the antisense strand (with respect to the target RNA). The sense strand only needs to be sufficiently complementary to the antisense strand in order to maintain the overall double-stranded nature of the molecule.

[0381] iRNA agents include molecules long enough to trigger an interferon response [which can be cleaved by Dicer (Bernstein et al. 2001. Nature, 409:363-366) and enter RISC (RNAi-induced silencing complex)], and molecules short enough not to trigger an interferon response (which can also be cleaved by Dicer and / or enter RISC), e.g., molecules small enough to enter RISC, e.g., molecules similar to the cleavage products by Dicer. Molecules short enough not to trigger an interferon response are referred to herein as siRNA agents or shorter iRNA agents. As used herein, “siRNA agents or shorter iRNA agents” refers to iRNA agents short enough not to induce a harmful interferon response in human cells, e.g., double-stranded RNA agents or single-stranded RNA agents having a double-stranded region of less than 60, 50, 40, or 30 nucleotide pairs, e.g. siRNA agents, or their cleavage products, can downregulate target genes, for example, by inducing RNAi with respect to target RNA, and the target may include endogenous or pathogenic target RNA.

[0382] As used herein, “single-stranded RNA agent” is an RNA agent consisting of one molecule. The single-stranded RNA agent may contain a double-stranded region formed by intra-strand pairing, which may be, for example, a hairpin structure or a panhandle structure, or may contain such a structure. The single-stranded RNA agent may be antisense against the target molecule. The single-stranded RNA agent may be long enough to enter RISC and participate in RISC-mediated cleavage of the target mRNA. The single-stranded RNA agent is at least 14 nucleotides long, and in other embodiments at least 15, 20, 25, 29, 35, 40, or 50 nucleotides long. In certain embodiments, the length is less than 200, 100, or 60 nucleotides.

[0383] A loop refers to a region of an RNA strand that is not paired with an opposing nucleotide in a double helix when a portion of the RNA strand forms a base pair with another strand or another portion of the same strand.

[0384] The hairpin RNA agent will have a double-stranded region of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs or at least 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The double-stranded region may be 200, 100, or 50 or less in length. In certain embodiments, the range of the double-stranded region is 15-30, 17-23, 19-23, and 19-21 nucleotide pairs in length. The hairpin may have a single-stranded overhang or terminal unpaired region at its 3' end in some embodiments, and at the antisense side of the hairpin in certain embodiments. In some embodiments, the overhang is 2-3 nucleotides in length.

[0385] As used herein, “double-stranded (ds) RNA agent” refers to an RNA agent containing one or more strands, possibly two, in which interstrand hybridization can form regions of a double-strand structure.

[0386] As used herein, the terms "siRNA activity" and "RNAi activity" refer to gene silencing by siRNA.

[0387] As used herein, “gene silencing” by RNA interference molecules means that the mRNA level in a cell containing the target gene is reduced to at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, up to 100%, and any integer between 5% and 100%. In a preferred embodiment, the mRNA level is reduced to at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, up to 100%, and any integer between 5% and 100%.

[0388] As used herein, the term “modulate gene expression” means that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, is upregulated or downregulated to be greater or less than the expression, level, or activity observed in the absence of the modulator. For example, the term “modulate” can mean “inhibit,” but the use of the term “modulate” is not limited to this definition.

[0389] Gene expression modulation as used herein occurs when the level of gene expression, or RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, differs from that observed in the absence of siRNA, e.g., RNAi agents, by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2x, 3x, 4x, or 5x. The percentage and / or multiplier of this difference are relative to a control or non-control, e.g., [Expression in the presence of siRNA - Expression in the absence of siRNA] Difference % = ----------------------------- Expression in the absence of siRNA or [Expression in the presence of siRNA - Expression in the absence of siRNA] Difference % = ----------------------------- Expression in the absence of siRNA It can be calculated as follows.

[0390] As used herein, the terms “inhibit,” “downregulate,” or “reduce” in relation to gene expression mean that the level of gene expression, or the level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, is reduced to what is observed in the absence of the modulator. Downregulation of gene expression occurs when the level of gene expression, or the level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, is reduced by at least 10%, preferably 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or most preferably 100% (i.e., no gene expression).

[0391] As used herein, the terms “increased” or “upregulated” in relation to gene expression mean that the level of gene expression, or the level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, is increased compared to what is observed in the absence of the modulator. Upregulated gene expression occurs when the level of gene expression, or the level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, is increased by at least 10% compared to the corresponding unmodulated control, preferably by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 100%, 1.1x, 1.25x, 1.5x, 1.75x, 2x, 3x, 4x, 5x, 10x, 50x, 100x or more.

[0392] As used herein, the terms “increased” or “increasing” generally mean an increase of a statistically significant amount. To avoid any doubt, “increased” means an increase of at least 10% compared to a baseline level, for example, an increase of at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or an increase of at least about 90%, or up to 100%, or any increase between 10% and 100% compared to a baseline level, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times compared to a baseline level, or any increase between 2 times and 10 times or more compared to a baseline level.

[0393] As used herein, the terms “reduced” or “decrease” generally mean a statistically significant decrease. However, to avoid any doubt, “reduced” means a decrease of at least 10% compared to a reference level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100% (i.e., a level that does not exist compared to a reference sample), or any decrease between 10% and 100% compared to a reference level.

[0394] Double-stranded RNA contains two oligonucleotide chains that are sufficiently complementary to hybridize to form a double-stranded structure. Generally, the double-stranded structure has a base pair length between 15 and 30, more generally between 18 and 25, even more generally between 19 and 24, and most commonly between 19 and 21. In some embodiments, long dsRNA with a base pair length between 25 and 30 is preferred. In some embodiments, short dsRNA with a base pair length between 10 and 15 is preferred. In another embodiment, the dsRNA is at least 21 nucleotides long.

[0395] In some embodiments, the double-stranded iRNA comprises a sense strand and an antisense strand, the antisense RNA strand having a complementary region that is complementary to at least a portion of the target sequence, and the double-stranded region is 14 to 30 nucleotides long. Similarly, the region complementary to the target sequence is between 14 and 30 nucleotides long, more commonly between 18 and 25, even more commonly between 19 and 24, and most commonly between 19 and 21.

[0396] As used herein, the term “compound” refers to an oligomeric compound that may be an oligonucleotide, antisense, or iRNA agent such as siRNA.

[0397] As used herein, the term “antisense chain” refers to an oligomeric compound that is substantially or 100% complementary to the target sequence of interest. The term “antisense chain” includes the antisense regions of both oligomeric compounds formed from two separate chains and monomolecular oligomeric compounds that can form hairpin or dumbbell-shaped structures. The terms “antisense chain” and “guide chain” are used interchangeably herein.

[0398] The term "sense strand" refers to an oligomeric compound that has a nucleoside sequence that is all or part identical to a target sequence, such as a messenger RNA or DNA sequence. The terms "sense strand" and "passenger strand" are used interchangeably herein.

[0399] "Specifically hybridizable" and "complementary" mean that a nucleic acid can form hydrogen bonds (or more) with another nucleic acid sequence in either a conventional Watson-Crick configuration or a non-conventional configuration. With respect to the nucleic acid molecules of the present invention, the binding free energy between the nucleic acid molecule and its complementary sequence is sufficient to enable the relevant function of the nucleic acid, such as RNAi activity. The determination of the binding free energy of nucleic acid molecules is well known in the art (see, for example, Turner et al, 1987, CSH Symp. Quant. Biol. LII pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, / . Am. Chem. Soc. 109:3783-3785). Complementarity percentage refers to the percentage of consecutive residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairs) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, and 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Full complementarity" or 100% complementarity means that all consecutive residues in a nucleic acid sequence can form hydrogen bonds with the same number of consecutive residues in the second nucleic acid sequence. Less than full complementarity refers to a situation where some, but not all, nucleoside units in the two strands can form hydrogen bonds with each other. "Substantial complementarity" refers to polynucleotide chains that exhibit 90% or more complementarity, excluding regions of the polynucleotide chain that are selected to be non-complementary, such as overhangs. Specific binding requires a degree of complementarity sufficient to avoid nonspecific binding of the oligomeric compound to the non-target sequence under the conditions under which specific binding is desired, i.e., physiological conditions in the case of in vivo assays or therapeutic procedures, or under the conditions under which the assay is performed in the case of in vitro assays. The non-target sequence is typically at least 5 nucleotides different.

[0400] In some embodiments, the double-stranded region of the dsRNA is equal to the length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotide pairs, or at least the length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotide pairs.

[0401] In some embodiments, the antisense strand of the dsRNA is equal in length to 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0402] In some embodiments, the sense strand of the dsRNA is equal to or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0403] In one embodiment, the sense strand and antisense strand of the dsRNA are each 15 to 30 nucleotides long.

[0404] In one embodiment, the sense strand and antisense strand of the dsRNA are each 19 to 25 nucleotides long.

[0405] In one embodiment, the sense strand and antisense strand of the dsRNA are each 21 to 23 nucleotides long.

[0406] In some embodiments, one strand has at least one stretch of 1 to 5 single-stranded nucleotides within the double-stranded region. “Stretch of single-stranded nucleotides within the double-stranded region” means that there is at least one nucleotide base pair at both ends of the single-stranded stretch. In some embodiments, both strands have at least one stretch of 1 to 5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides within the double-stranded region. If both strands have stretches of 1 to 5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides within the double-stranded region, such single-stranded nucleotides may be arranged so that they face each other (e.g., mismatched stretches), or the second strand does not have single-stranded nucleotides facing the single-stranded RNA of the first strand, or vice versa (e.g., single-stranded loops). In some embodiments, single-stranded nucleotides are located within 8 nucleotides from either end, for example, within 8 nucleotides from either the 5' or 3' end of the complementary region between the two strands, for example, within 8, 7, 6, 5, 4, 3, or 2 nucleotides.

[0407] In one embodiment, the dsRNA includes a single-stranded overhang at at least one of its ends. In one embodiment, the single-stranded overhang is 1, 2, or 3 nucleotides long.

[0408] In one embodiment, the sense strand of the dsRNA agent is 21 nucleotides long, and the antisense strand is 23 nucleotides long, where the strands form a double-stranded region of 21 consecutive base pairs with a single-stranded projection 2 nucleotides longer at the 3' end.

[0409] In some embodiments, each strand of dsRNA has a ZXY structure as described in PCT Publication No. 2004080406, which is incorporated herein by reference in its entirety.

[0410] In certain embodiments, the two chains of a double-stranded oligomer compound may be linked together. The two chains may be linked at both ends or at least at one end. Linking at one end means that the 5' end of the first chain is linked to the 3' end of the second chain, or the 3' end of the first chain is linked to the 5' end of the second chain. When the two chains are linked at both ends, the 5' end of the first chain is linked to the 3' end of the second chain, and the 3' end of the first chain is linked to the 5' end of the second chain. The two chains are (N) n The two chains may be linked together by an oligonucleotide linker, which includes, but is not limited to, a modified nucleotide or an unmodified nucleotide (wherein N is independently a modified or unmodified nucleotide, and n is 3 to 23). In some embodiments, n is 3 to 10, for example, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the oligonucleotide linker is selected from the group consisting of GNRA, (G)4, (U)4, and (dT)4 (wherein N is a modified or unmodified nucleotide, and R is a modified or unmodified purine nucleotide). Some of the nucleotides in the linker may be involved in base-pair interactions with other nucleotides in the linker. The two chains may be linked together by a non-nucleoside linker, for example, the linkers described herein. It will be understood by those skilled in the art that any oligonucleotide chemical modification or variation described herein may be used for the oligonucleotide linker.

[0411] Hairpin and dumbbell-shaped oligomeric compounds may have a double-stranded region of 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs or at least 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The double-stranded region may be 200, 100, or 50 or less in length. In some embodiments, the double-stranded region ranges in length from 15 to 30, 17 to 23, 19 to 23, and 19 to 21 nucleotide pairs.

[0412] The hairpin oligomer compound may have a single-stranded overhang or terminal unpaired region at the 3' end in some embodiments, and at the antisense side of the hairpin in some embodiments. In some embodiments, the overhang is 1 to 4 nucleotides long, more generally 2 to 3 nucleotides. The hairpin oligomer compound capable of inducing RNA interference is also referred to herein as “shRNA”.

[0413] In certain embodiments, the two oligomeric chains specifically hybridize if there is sufficient complementarity to avoid nonspecific binding of the antisense compound to a non-target nucleic acid sequence under conditions where specific binding is desired, i.e., under physiological conditions in the case of an in vivo assay or therapeutic treatment, or under the conditions under which the assay is performed in the case of an in vitro assay.

[0414] As used herein, “strict hybridization conditions” or “strict conditions” refer to conditions under which an antisense compound hybridizes to its target sequence but to a minimum number of other sequences. Strict conditions are sequence-dependent and vary in different situations, and the “strict conditions” under which an antisense compound hybridizes to its target sequence are determined by the properties and composition of the antisense compound, as well as the assay in which they are studied.

[0415] It is understood in the art that the incorporation of nucleotide affinity modifications can tolerate more mismatches compared to unmodified compounds. Similarly, certain oligonucleotide sequences may be more tolerant of mismatches than other oligonucleotide sequences. Those skilled in the art can determine the appropriate number of mismatches between oligonucleotides, or between oligonucleotides and target nucleic acids, for example, by determining the melting temperature (Tm). Tm or ΔTm can be calculated by techniques well known to those skilled in the art. For example, the technique described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443) allows those skilled in the art to evaluate the ability of nucleotide modifications to increase the melting temperature of RNA:DNA double helix.

[0416] siRNA design In one embodiment, the dsRNA agent is 19 nucleotides long with both blunt ends, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 7, 8, and 9 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0417] In one embodiment, the dsRNA agent is 20 nucleotides long with both blunt ends, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0418] In one embodiment, the dsRNA agent is 21 nucleotides long with both blunt ends, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0419] In one embodiment, the dsRNA agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end; and the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, with one end of the dsRNA being a blunt end and the other end containing a 2-nucleotide overhang. Preferably, the 2-nucleotide overhang is at the 3' end of the antisense strand.

[0420] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand, the sense strand being 25-30 nucleotides long, starting with a 5' terminal nucleotide (position 1), and positions 1-23 of the first strand containing at least 8 ribonucleotides; the antisense strand being 36-66 nucleotides long, starting with a 3' terminal nucleotide, and containing at least 8 ribonucleotides so as to form a double helix at positions 1-23 of the sense strand; in this case, at least 3' terminal nucleotides of the antisense strand are unpaired with the sense strand, and at the 3' end, up to 6 consecutive nucleotides are unpaired with the sense strand, thereby forming a single-stranded 3' overhang of 1-6 nucleotides; the 5' end of the antisense strand contains 10-30 consecutive nucleotides that are unpaired with the sense strand. The sense strand contains a rheotide, thereby forming a single-stranded 5' overhang of 10-30 nucleotides; at least the 5' and 3' terminal nucleotides of the sense strand base-pair with the nucleotides of the antisense strand when the sense strand and the antisense strand are aligned for maximum complementarity, thereby forming a substantial double-stranded region between the sense strand and the antisense strand; the antisense strand is sufficiently complementary to the target RNA over a length of at least 19 ribonucleotides of the antisense strand so as to reduce the expression of the target gene when the double-stranded nucleic acid is introduced into mammalian cells; the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, in which case at least one of the motifs is present at or near the cleavage site; the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0421] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand, the dsRNA agent comprising a first strand at least 25 nucleotides long and at most 29 nucleotides long, and a second strand at most 30 nucleotides long, with at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end; the 3' end of the first strand and the 5' end of the second strand form a blunt end, the second strand has a double-stranded region at its 3' end that is 1 to 4 nucleotides longer than the first strand and at least 25 nucleotides long, and the second strand is sufficiently complementary to the target mRNA over a length of at least 19 nucleotides of the second strand so that when the dsRNA agent is introduced into mammalian cells, it reduces the expression of the target gene, in which case cleavage of the dsRNA by Dicer preferentially results in siRNA including the 3' end of the second strand, thereby reducing the expression of the target gene in mammals.

[0422] In one embodiment, the sense strand of the dsRNA agent contains at least one motif from three identical modifications on three consecutive nucleotides, in which case one of the motifs is located at a cleavage site within the sense strand. For example, the sense strand may contain at least one motif from three 2'-F modifications on three consecutive nucleotides between positions 7 and 15 from the 5' end.

[0423] In one embodiment, the antisense strand of the dsRNA agent may also contain at least one motif of three identical modifications on three consecutive nucleotides, in which case one of the motifs is located at or near a cleavage site within the antisense strand. For example, the antisense strand may contain at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides between positions 9 and 15 from the 5' end.

[0424] For dsRNA agents with a double-stranded region of 17–23 nucleotides in length, the cleavage sites on the antisense strand are typically near positions 10, 11, and 12 from the 5' end. Therefore, the three identical modification motifs can be located at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 on the antisense strand, depending on the count from the first nucleotide at the 5' end of the antisense strand, or from the count from the first paired nucleotide within the double-stranded region at the 5' end of the antisense strand. The cleavage sites within the antisense strand can also vary depending on the length from the 5' end of the double-stranded region of the dsRNA.

[0425] In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, each having 14 to 30 nucleotides, wherein the sense strand contains at least two motifs from three identical modifications on three consecutive nucleotides, at least one of the motifs located at or near a cleavage site within the strand, and at least one of the motifs located in another part of the strand separated from the motif at the cleavage site by at least one nucleotide. In one embodiment, the antisense strand also contains at least one motif from three identical modifications on three consecutive nucleotides, at least one of the motifs located at or near a cleavage site within the strand. The modifications of the motifs located at or near the cleavage site of the sense strand are different from the modifications of the motifs located at or near the cleavage site of the antisense strand.

[0426] In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, each having 14 to 30 nucleotides, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, at least one of which is present at or near the cleavage site of the strand. In one embodiment, the antisense strand also contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0427] In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, each having 14 to 30 nucleotides, wherein the sense strand contains at least one motif from three consecutive nucleotides with 2'-F modifications at positions 9, 10, and 11 from the 5' end, and the antisense strand contains at least one motif from three consecutive nucleotides with 2'-O-methyl modifications at positions 11, 12, and 13 from the 5' end.

[0428] In one embodiment, the dsRNA agent includes mismatch(s) with a target within the double helix, or a combination thereof. Mismatches may be present in the protruding region or within the double helix region. Base pairs may be ranked based on their tendency to promote dissociation or dissolution [for example, regarding the free energy of association or dissociation of a particular pair, the simplest method is to consider the pair in individual pair bases, but the next-neighbor method or similar analysis may also be used]. With respect to promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or non-canonical pairings (described elsewhere in this specification) are preferred over canonical pairings (A:T, A:U, G:C); pairings containing universal bases are preferred over canonical pairings.

[0429] In one embodiment, the dsRNA agent comprises at least one of the first one, two, three, four, or five base pairs in the 5'-terminal duplex region of the antisense strand, which may be independently selected from a group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical pairings or pairings other than canonical pairings, or pairings containing universal bases to promote the dissociation of the antisense strand at the 5'-terminus of the duplex.

[0430] In one embodiment, the nucleotide at position 1 in the 5'-terminal double-stranded region of the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first one, two, or three base pairs in the 5'-terminal double-stranded region of the antisense strand is an AU base pair. For example, the first base pair in the 5'-terminal double-stranded region of the antisense strand is an AU base pair.

[0431] In one embodiment, the present invention relates to a double-stranded RNA (dsRNA) agent that inhibits the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each having 14 to 40 nucleotides. The dsRNA agent is of formula (I)

[0432] [ka] It is represented by [this].

[0433] In formula (I), B1, B2, B3, B1', B2', B3', and B4' are each independently nucleotides containing a modification selected from the group consisting of 2'-O-alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halo, ENA, and BNA / LNA. In one embodiment, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe modification. In one embodiment, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe or 2'-F modification. In one embodiment, at least one of B1, B2, B3, B1', B2', B3', and B4' contains a 2'-ON-methylacetamide (2'-O-NMA) modification.

[0434] C1 is a thermally unstable nucleotide located opposite the seed region of the antisense strand (i.e., positions 2-8 at the 5' end of the antisense strand). For example, C1 is located on the sense strand, pairing with a nucleotide at positions 2-8 at the 5' end of the antisense strand. In one example, C1 is located at position 15 from the 5' end of the sense strand. The C1 nucleotide may harbor thermally unstable modifications, including debasing; mismatches with opposing nucleotides in the double helix; and sugar modifications such as 2'-deoxy modification, or acyclic nucleotides, such as unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA). In one embodiment, C1 may i) mismatch with opposing nucleotides in the antisense strand; ii)

[0435] [ka] Debase modification selected from the group consisting of; and iii)

[0436] [ka] [In the formula, B is a modified nucleobase or an unmodified nucleobase, and R 1 and R 2R3 is independently H, halogen, OR3, or alkyl; R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. The C1 has a thermally unstable modification selected from the group consisting of sugar modifications selected from the group consisting of G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, and U:T; at least one nucleobase in the mismatch pair may be a 2'-deoxynucleobase. In one example, the thermally unstable modification in C1 is GNA or

[0437] [ka] That is the case.

[0438] T1, T1', T2', and T3' each independently represent a nucleotide containing a modification that imparts a steric bulk to the nucleotide that is less than or equal to the steric bulk of the 2'-OMe modification. Steric bulk refers to the sum of the steric effects of the modifications. Methods for determining the steric effects of nucleotide modifications are known to those skilled in the art. The modifications may be modifications at the 2' position of the ribose sugar of the nucleotide, or modifications to a non-ribose nucleotide, acyclic nucleotide, or the nucleotide backbone, which are similar to or equivalent to modifications at the 2' position of the ribose sugar, and impart a steric bulk to the nucleotide that is less than or equal to the steric bulk of the 2'-OMe modification. For example, T1, T1', T2', and T3' are each independently selected from DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl. In one embodiment, T1 is DNA. In one embodiment, T1' is DNA, RNA, or LNA. In one embodiment, T2' is DNA or RNA. In one embodiment, T3' is DNA or RNA.

[0439] n 1 , n 3 , and q 1 These are independently 4 to 15 nucleotides in length.

[0440] n 5 , q 3 , and q 7 Each nucleotide is independently 1 to 6 nucleotides long.

[0441] n 4 , q 2 , and q 6 Each is independently 1 to 3 nucleotides in length; alternatively, n 4 q is 0. 5 Each nucleotide is independently 0 to 10 nucleotides in length.

[0442] n 2 and q 4 Each nucleotide is independently 0 to 3 nucleotides long.

[0443] Alternatively, n 4 Its length is between 0 and 3 nucleotides.

[0444] In one embodiment, n 4 n can be 0. In one example, n 4 is 0, and q 2 and q 6 In another example, n 4 is 0, and q 2 and q 6 It is 1, with modifications by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modifications by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0445] In one embodiment, n 4 , q 2 , and q 6 Each of these values ​​is 1.

[0446] In one embodiment, n 2 , n 4 , q 2 , q 4 , and q 6 Each of these values ​​is 1.

[0447] In one embodiment, C1 is located at the 14th to 17th position from the 5' end of the sense strand, when the sense strand is 19 to 22 nucleotides long. 4 In one embodiment, C1 is located at the 15th position of the 5' end of the sense strand.

[0448] In one embodiment, T3' begins at position 2 from the 5' end of the antisense chain. In one example, T3' is located at position 2 from the 5' end of the antisense chain, q 6 It is equal to 1.

[0449] In one embodiment, T1' begins at position 14 from the 5' end of the antisense chain. In one example, T1' is located at position 14 from the 5' end of the antisense chain, q 2 It is equal to 1.

[0450] In an exemplary embodiment, T3' starts at position 2 of the 5' end of the antisense chain, and T1' starts at position 14 of the 5' end of the antisense chain. In one example, T3' starts at position 2 of the 5' end of the antisense chain, q 6 is equal to 1, T1' starts at position 14 of the 5' end of the antisense chain, q 2 It is equal to 1.

[0451] In one embodiment, T1' and T3' are separated by a length of 11 nucleotides (i.e., T1' and T3' nucleotides are not counted).

[0452] In one embodiment, T1' is located at position 14 from the 5' end of the antisense chain. In one example, T1' is located at position 14 from the 5' end of the antisense chain, q 2Modifications at the 2' position, or at non-ribose, acyclic, or skeletal positions, result in a steric bulk of less than 2'-OMe-ribose.

[0453] In one embodiment, T3' is located at position 2 from the 5' end of the antisense chain. In one example, T3' is located at position 2 from the 5' end of the antisense chain, q 6 Modifications at the 2' position, or at non-ribose, acyclic, or skeletal positions, result in a steric bulk of 2'-OMe-ribose or less.

[0454] In one embodiment, T1 is located at the cleavage site of the sense strand. In one example, when the sense strand is 19-22 nucleotides long, T1 is located at position 11 from the 5' end of the sense strand, n 2 is 1. In an exemplary embodiment, T1 is located at the sense strand cleavage site at position 11 from the 5' end of the sense strand, when the sense strand is 19 to 22 nucleotides long, and n 2 The value is 1.

[0455] In one embodiment, T2' begins at position 6 from the 5' end of the antisense chain. In one example, T2' is located at positions 6-10 from the 5' end of the antisense chain, q 4 is 1. In an exemplary embodiment, T1 is a cleavage site of the sense strand, for example, at position 11 from the 5' end of the sense strand, when the sense strand is 19 to 22 nucleotides long. 2 is 1; T1' is at position 14 from the 5' end of the antisense chain, q 2 is equal to 1, the modification to T1' is at the 2' position of the ribose sugar, or at a non-ribose, acyclic or skeletal position, and is not as sterically bulky as 2'-OMe-ribose; T2' is at the 6-10 position from the 5' end of the antisense chain, q 4 is 1; T3' is at position 2 from the 5' end of the antisense strand, q 6This is equal to 1, and the modification to T3' is at the 2' position, or in a non-ribose, acyclic, or skeletal position, resulting in a steric bulk of 2'-OMe-ribose or less.

[0456] In one embodiment, T2' begins at position 8 from the 5' end of the antisense chain. In one example, T2' begins at position 8 from the 5' end of the antisense chain, q 4 is 2. In one embodiment, T2' begins at position 9 from the 5' end of the antisense chain. In one example, T2' is at position 9 from the 5' end of the antisense chain, and q 4 The value is 1.

[0457] In one embodiment, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; the sense strand has modifications by two phosphorothioate nucleotide linkages within positions 1-5 (counting from the 5' end of the sense strand), the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 (counting from the 5' end of the antisense strand), and the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 18-23 (counting from the 5' end of the antisense strand).

[0458] In one embodiment, n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; the sense strand has modifications by two phosphorothioate nucleotide linkages within positions 1-5 (counting from the 5' end of the sense strand), the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 (counting from the 5' end of the antisense strand), and the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 18-23 (counting from the 5' end of the antisense strand).

[0459] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1.

[0460] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; the sense strand has modifications by two phosphorothioate nucleotide linkages within positions 1-5 (counting from the 5' end of the sense strand), the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 (counting from the 5' end of the antisense strand), and the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 18-23 (counting from the 5' end of the antisense strand).

[0461] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 6, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 7, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6is 1, B4' is 2'-OMe, and q 7 The value is 1.

[0462] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 6, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 7, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; the sense strand has modifications by two phosphorothioate nucleotide linkages within positions 1-5 (counting from the 5' end of the sense strand), the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 (counting from the 5' end of the antisense strand), and the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 18-23 (counting from the 5' end of the antisense strand).

[0463] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1.

[0464] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; the sense strand has modifications by two phosphorothioate nucleotide linkages within positions 1-5 (counting from the 5' end of the sense strand), the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 (counting from the 5' end of the antisense strand), and the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 18-23 (counting from the 5' end of the antisense strand).

[0465] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2is 3, B2 is 2’-OMe, and n 3 is 7, and n 4 is 0, B3 is 2’OMe, and n 5 is 3, B1’ is 2’-OMe or 2’-F, and q 1 is 9, T1’ is 2’-F, and q 2 is 1, B2’ is 2’-OMe or 2’-F, and q 3 is 5, T2’ is 2’-F, and q 4 is 1, B3’ is 2’-OMe or 2’-F, and q 5 is 5, T3’ is 2’-F, and q 6 is 1, B4’ is 2’-OMe, and q 7 is 1; at the 3’ end of the antisense strand, it may be accompanied by at least two additional TT.

[0466] In one embodiment, B1 is 2’-OMe or 2’-F, and n 1 is 8, T1 is 2’F, and n 2 is 3, B2 is 2’-OMe, and n 3 is 7, and n 4 is 0, B3 is 2’-OMe, and n 5 is 3, B1’ is 2’-OMe or 2’-F, and q 1 is 9, T1’ is 2’-F, and q 2 is 1, B2’ is 2’-OMe or 2’-F, and q 3 is 5, T2’ is 2’-F, and q 4 is 1, B3’ is 2’-OMe or 2’-F, and q 5 is 5, T3’ is 2’-F, and q 6 is 1, B4’ is 2’-OMe, and q 7is 1; may have at least two additional TTs at the 3' end of the antisense strand; has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand); has two phosphorothioate nucleotide linkage modifications within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand); and has two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0467] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1.

[0468] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; with modifications by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end), with modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end), and with modifications by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end).

[0469] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1.

[0470] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1; the sense strand has modifications by two phosphorothioate nucleotide linkages within positions 1-5 (counting from the 5' end of the sense strand), the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 (counting from the 5' end of the antisense strand), and the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 18-23 (counting from the 5' end of the antisense strand).

[0471] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1.

[0472] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; within positions 1 to 5 of the sense strand (counting from the 5'-end of the sense strand), there is a modification with a phosphorothioate internucleotide linkage, at positions 1 and 2 of the antisense strand (counting from the 5'-end of the antisense strand), there is a modification with a phosphorothioate internucleotide linkage, and within positions 18 to 23 of the antisense strand (counting from the 5'-end of the antisense strand), there is a modification with a phosphorothioate internucleotide linkage.

[0473] The dsRNA agent may contain a phosphorus-containing group at the 5'-end of the sense strand or the antisense strand. The phosphorus-containing group at the 5'-end is 5'-terminal phosphate (5'-P), 5'-terminal phosphorothioate (5'-PS), 5'-terminal phosphorodithioate (5'-PS2), 5'-terminal vinylphosphonate (5'-VP), 5'-terminal methylphosphonate (MePhos), or 5'-deoxy-5'-C-malonyl(

[0474]

Chemical formula

[0475] [ka] ), 5'-Z-VP isomer (i.e., cis-vinyl phosphonate,

[0476] [ka] ), or a mixture thereof.

[0477] In one embodiment, the dsRNA agent contains a phosphorus-containing group at the 5' end of the sense strand. In another embodiment, the dsRNA agent contains a phosphorus-containing group at the 5' end of the antisense strand.

[0478] In one embodiment, the dsRNA agent contains 5'-P. In one embodiment, the dsRNA agent contains 5'-P in the antisense strand. In one embodiment, the dsRNA agent contains 5'-PS. In one embodiment, the dsRNA agent contains 5'-PS in the antisense strand.

[0479] In one embodiment, the dsRNA agent contains 5'-VP. In one embodiment, the dsRNA agent contains 5'-VP in the antisense strand. In one embodiment, the dsRNA agent contains 5'-E-VP in the antisense strand. In one embodiment, the dsRNA agent contains 5'-Z-VP in the antisense strand.

[0480] In one embodiment, the dsRNA agent contains 5'-PS2. In one embodiment, the dsRNA agent contains 5'-PS2 in the antisense strand.

[0481] In one embodiment, the dsRNA agent contains 5'-PS2. In one embodiment, the dsRNA agent contains 5'-deoxy-5'-C-malonyl in the antisense strand.

[0482] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. dsRNA agents also include 5'-PS.

[0483] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q6 is 1, B4' is 2'-OMe, and q 7 It is 1. dsRNA agents also contain 5'-P.

[0484] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. dsRNA agents also include 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination of these.

[0485] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. dsRNA agents also contain 5'-PS2.

[0486] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. dsRNA agents also include 5'-deoxy-5'-C-malonyl.

[0487] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; the sense strand has modifications by two phosphorothioate nucleotide linkages within positions 1-5 (counting from the 5' end of the sense strand), the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 (counting from the 5' end of the antisense strand), and the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 18-23 (counting from the 5' end of the antisense strand). dsRNA agents also include 5'-P.

[0488] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7It is 1; the sense strand has modifications by two phosphorothioate nucleotide linkages within positions 1-5 (counting from the 5' end of the sense strand), the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 (counting from the 5' end of the antisense strand), and the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 18-23 (counting from the 5' end of the antisense strand). dsRNA agents also include 5'-PS.

[0489] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; with modifications by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). dsRNA agents also include 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0490] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; the sense strand has modifications by two phosphorothioate nucleotide linkages within positions 1-5 (counting from the 5' end of the sense strand), the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 (counting from the 5' end of the antisense strand), and the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 18-23 (counting from the 5' end of the antisense strand). The dsRNA agent also contains 5'-PS2.

[0491] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also contains 5'-deoxy-5'-C-malonyl.

[0492] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. dsRNA agents also include 5'-P.

[0493] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. dsRNA agents also include 5'-PS.

[0494] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. dsRNA agents also include 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination of these.

[0495] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. dsRNA agents also include 5'-PS2.

[0496] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. dsRNA agents also include 5'-deoxy-5'-C-malonyl.

[0497] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; the sense strand has modifications by two phosphorothioate nucleotide linkages within positions 1-5 (counting from the 5' end), the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 (counting from the 5' end), and the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 18-23 (counting from the 5' end). dsRNA agents also include 5'-P.

[0498] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q7 It is 1; the sense strand has modifications by two phosphorothioate nucleotide linkages within positions 1-5 (counting from the 5' end), the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 (counting from the 5' end), and the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 18-23 (counting from the 5' end). dsRNA agents also include 5'-PS.

[0499] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; the sense strand has modifications by two phosphorothioate nucleotide linkages within positions 1-5 (counting from the 5' end), the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 (counting from the 5' end), and the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 18-23 (counting from the 5' end). dsRNA agents also include 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0500] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; the sense strand has modifications by two phosphorothioate nucleotide linkages within positions 1-5 (counting from the 5' end), the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 (counting from the 5' end), and the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 18-23 (counting from the 5' end). dsRNA agents also include 5'-PS2.

[0501] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q6 is 1, B4' is 2'-OMe, and q 7 It is 1; the sense strand has modifications by two phosphorothioate nucleotide linkages within positions 1-5 (counting from the 5' end), the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 (counting from the 5' end), and the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 18-23 (counting from the 5' end). The dsRNA agent also contains 5'-deoxy-5'-C-malonyl.

[0502] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1. dsRNA agents also contain 5'-P.

[0503] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. dsRNA agents also include 5'-PS.

[0504] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. dsRNA agents also include 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination of these.

[0505] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. dsRNA agents also contain 5'-PS2.

[0506] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. dsRNA agents also include 5'-deoxy-5'-C-malonyl.

[0507] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1; with modifications by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). dsRNA agents also contain 5'-P.

[0508] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7It is 1; with modifications by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). dsRNA agents also contain 5'-PS.

[0509] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1; with modifications by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). dsRNA agents also include 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0510] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). dsRNA agents also contain 5'-PS2.

[0511] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also contains 5'-deoxy-5'-C-malonyl.

[0512] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1. dsRNA agents also contain 5'-P.

[0513] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. dsRNA agents also include 5'-PS.

[0514] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. dsRNA agents also include 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination of these.

[0515] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. dsRNA agents also contain 5'-PS2.

[0516] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. dsRNA agents also include 5'-deoxy-5'-C-malonyl.

[0517] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1; with modifications by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). dsRNA agents also contain 5'-P.

[0518] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6is 1, B4' is 2'-F, and q 7 It is 1; with modifications by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). dsRNA agents also contain 5'-PS.

[0519] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1; with modifications by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). dsRNA agents also include 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0520] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1; the sense strand has modifications by two phosphorothioate nucleotide linkages within positions 1-5 (counting from the 5' end of the sense strand), the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 (counting from the 5' end of the antisense strand), and the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 18-23 (counting from the 5' end of the antisense strand). The dsRNA agent also contains 5'-PS2.

[0521] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1; the sense strand has modifications by two phosphorothioate nucleotide linkages within positions 1-5 (counting from the 5' end of the sense strand), the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 (counting from the 5' end of the antisense strand), and the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 18-23 (counting from the 5' end of the antisense strand). The dsRNA agent also contains 5'-deoxy-5'-C-malonyl.

[0522] In one embodiment, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of the dsRNA agent is modified. For example, if 50% of the dsRNA agent is modified, all 50% of the nucleotides present in the dsRNA agent contain the modifications described herein.

[0523] In one embodiment, each of the sense and antisense strands is independently modified with an acyclic nucleotide, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.

[0524] In one embodiment, each of the sense strand and antisense strand of the dsRNA agent contains at least two different modifications.

[0525] In one embodiment, the dsRNA agent of formula (I) further comprises 3' and / or 5' overhangs (or multiple overhangs) of length 1 to 10 nucleotides. In one example, the dsRNA agent of formula (I) includes a 3' overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand. In another example, the dsRNA agent has a 5' overhang at the 5' end of the sense strand.

[0526] In one embodiment, the dsRNA agent does not contain 2'-F modification.

[0527] In one embodiment, the sense strand and / or antisense strand of the dsRNA agent comprises one or more blocks of phosphorothioate or methylphosphonate nucleotide linkages. In one example, the sense strand comprises one block of two phosphorothioate or methylphosphonate nucleotide linkages. In one example, the antisense strand comprises two blocks of two phosphorothioate or methylphosphonate nucleotide linkages. For example, the two blocks of phosphorothioate or methylphosphonate nucleotide linkages are separated by 16 to 18 phosphate nucleotide linkages.

[0528] In one embodiment, each of the sense strand and antisense strand of the dsRNA agent has 15 to 30 nucleotides. In one example, the sense strand has 19 to 22 nucleotides and the antisense strand has 19 to 25 nucleotides. In another example, the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides.

[0529] In one embodiment, the nucleotide at position 1 of the 5' end of the double-stranded antisense strand is selected from the group consisting of A, dA, dU, U, and dT. In one embodiment, at least one of the 1st, 2nd, and 3rd base pairs from the 5' end of the antisense strand is an AU base pair.

[0530] In one embodiment, the antisense strand of the dsRNA agent is 100% complementary to the target RNA, hybridizes with it, and inhibits its expression by RNA interference. In another embodiment, the antisense strand of the dsRNA agent is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% complementary to the target RNA.

[0531] In one embodiment, the present invention relates to a dsRNA agent as defined herein that can inhibit the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each having 14 to 40 nucleotides. The sense strand contains at least one thermally unstable nucleotide, at least one of which is located opposite or near the seed region of the antisense strand (i.e., positions 2 to 8 at the 5' end of the antisense strand). Each of the embodiments and aspects described herein relating to the dsRNA represented by formula (I) may also be applied to dsRNA containing thermally unstable nucleotides.

[0532] Thermally unstable nucleotides may be located between positions 14 and 17 of the 5' end of the sense strand, for example, if the sense strand is 21 nucleotides long. The antisense strand contains at least two modified nucleic acids smaller than the sterically required 2'-OMe modification. Preferably, the two modified nucleic acids smaller than the sterically required 2'-OMe are separated by a length of 11 nucleotides. For example, the two modified nucleic acids are located at positions 2 and 14 of the 5' end of the antisense strand.

[0533] In one embodiment, the dsRNA agent is (a) a sense chain, (i) Length of 18-23 nucleotides; (ii) Three consecutive 2'-F modifications in positions 7-15; A sense chain having, (b) an antisense chain, (i) Length of 18-23 nucleotides; (ii) At least 2'-F modification at any location on the chain; and (iii) At least two phosphorothioate nucleotide ligations in the first five nucleotides (counting from the 5' end) Antisense chain having Includes; In this case, the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated at one or more positions on at least one strand; two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand; or blunt ends at both ends of the double helix.

[0534] In one embodiment, the dsRNA agent is (a) a sense chain, (i) Length of 18-23 nucleotides; (ii) 2'-F modification less than 4 A sense chain having; (b) an antisense chain, (i) Length of 18-23 nucleotides; (ii) 2'-F modification less than 12; and (iii) At least two phosphorothioate nucleotide ligations in the first five nucleotides (counting from the 5' end) Antisense chain having Includes; In this case, the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated at one or more positions on at least one strand; two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand; or blunt ends at both ends of the double helix.

[0535] In one embodiment, the dsRNA agent is (a) a sense chain, (i) Length of 19-35 nucleotides; (ii) 2'-F modification less than 4 A sense chain having; (b) an antisense chain, (i) Length of 19-35 nucleotides; (ii) 2'-F modification less than 12; and (iii) At least two phosphorothioate nucleotide ligations in the first five nucleotides (counting from the 5' end) Antisense chain having Includes; In this case, the double-stranded region is between 19 and 25 base pairs (preferably 19, 20, 21, or 22); in this case, the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated at one or more positions on at least one strand; and has either two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand; or blunt ends at both ends of the double helix.

[0536] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand having a length of 15 to 30 nucleotides; includes at least two phosphorothioate nucleotide linkages in the first 5 nucleotides (counted from the 5' end) of the antisense strand; in this case, the double-stranded region is between 19 and 25 base pairs (preferably 19, 20, 21, or 22); the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated at one or more positions on at least one strand; and the dsRNA agent has less than 20%, less than 15%, and less than 10% non-natural nucleotides.

[0537] Examples of non-natural nucleotides include acyclic nucleotides, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.

[0538] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand having a length of 15 to 30 nucleotides; includes at least two phosphorothioate nucleotide linkages in the first 5 nucleotides (counted from the 5' end) on the antisense strand; in this case, the double-stranded region is between 19 and 25 base pairs (preferably 19, 20, 21, or 22); the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated at one or more positions on at least one strand; and the dsRNA agent has more than 80%, more than 85%, and more than 90% natural nucleotides such that 2'-OH, 2'-deoxy, and 2'-OMe are natural nucleotides.

[0539] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand having a length of 15 to 30 nucleotides; includes at least two phosphorothioate nucleotide linkages in the first 5 nucleotides (counted from the 5' end) on the antisense strand; in this case, the double-stranded region is between 19 and 25 base pairs (preferably 19, 20, 21, or 22); the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated at one or more positions on at least one strand; and the dsRNA agent has 100% natural nucleotides such that 2'-OH, 2'-deoxy, and 2'-OMe are natural nucleotides.

[0540] In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, each having a length of 15 to 35 nucleotides independently, wherein the sense strand contains a 2'-fluoronucleotide at position 10, counted from the 5' end of the sense strand.

[0541] In some embodiments, the sense strand further comprises one or more, for example, 1, 2, 3, 4, or 5 further 2'-fluoronucleotides. The further 2'-fluoronucleotides may be located anywhere on the sense strand.

[0542] In some embodiments, the sense strand further contains a 2'-fluoronucleotide at position 10, counting from the 5' end of the sense strand. In some embodiments, the sense strand further contains a 2'-fluoronucleotide at one or more of positions 8, 9, 11, and 12, counting from the 5' end of the sense strand. For example, the sense strand further contains a 2'-fluoronucleotide at position 9, counting from the 5' end of the sense strand; in other words, the sense strand contains a 2'-fluoronucleotide at positions 9 and 10, counting from the 5' end of the sense strand. In another example, the sense strand further contains a 2'-fluoronucleotide at position 11, counting from the 5' end of the sense strand; that is, the sense strand contains a 2'-fluoronucleotide at positions 10 and 11, counting from the 5' end of the sense strand.

[0543] In some embodiments, the sense strand contains 2'-fluoronucleotides at positions 9, 10, and 11, counted from the 5' end of the sense strand. In some embodiments, the sense strand contains 2'-fluoronucleotides at positions 7, 8, and 9, counted from the 5' end of the sense strand. In some other embodiments, the sense strand contains 2'-fluoronucleotides at positions 8, 9, and 10, counted from the 5' end of the sense strand. In some embodiments, the sense strand contains 2'-fluoronucleotides at positions 10, 11, and 12, counted from the 5' end of the sense strand.

[0544] In some embodiments, the sense strand contains a 2'-fluoronucleotide at one or more positions opposite to positions 11, 12, and 13 of the antisense strand, counting from the 5' end of the antisense strand. "Opposite to" means that the sense strand and antisense strand form a double helix, and the position of a particular nucleotide on the sense strand is counted based on the position of a nucleotide on the antisense strand that has a base pair with that particular nucleotide on the sense strand.

[0545] In some embodiments, the sense strand does not contain a 2'-fluoronucleotide at position 7, counted from the 5' end of the sense strand. For example, the sense strand contains a 2'-OMe nucleotide at position 7, counted from the 5' end of the sense strand.

[0546] In some embodiments, any nucleotide in the sense strand that is not a 2'-fluoronucleotide is a 2'-OMe nucleotide.

[0547] In some embodiments, the antisense strand contains one or more 2'-deoxynucleotides, for example, 2'-H nucleotides. For example, the antisense strand contains 1, 2, 3, 4, 5, 6, or more 2'-deoxynucleotides. In some embodiments, the antisense strand contains 2, 3, 4, 5, 6 2'-deoxynucleotides. The 2'-deoxynucleotides can be located anywhere on the antisense strand. For example, the antisense strand contains 2'-deoxynucleotides at positions 1, 2, 3, 4, 5, or 6, counting from the 5' end of the antisense strand, at positions 2, 5, 7, 12, 14, and 16. In some embodiments, the antisense strand contains 2'-deoxynucleotides at positions 2 and 12, counting from the 5' end of the antisense strand. In some embodiments, the antisense strand contains 2'-deoxynucleotides at positions 5 and 7, counting from the 5' end of the antisense strand. In some embodiments, the antisense includes 2'-deoxynucleotides at positions 2, 5, 7, and 12, counted from the 5' end of the antisense strand.

[0548] In some embodiments, the antisense strand contains one or more 2'-fluoronucleotides, for example, 1, 2, 3, 4, 5 or more. For example, the antisense strand contains a 2'-fluoronucleotide at position 14, counted from the 5' end of the antisense strand.

[0549] In some embodiments, the antisense strand contains a 2'-fluoronucleotide at position 14 and a 2'-deoxy or non-2'-fluoro nucleotide at position 16, counted from the 5' end of the antisense strand. For example, the antisense strand contains a 2'-fluoronucleotide at position 14 and a 2'-OMe at position 16, counted from the 5' end of the antisense strand.

[0550] In some embodiments, the antisense strand contains 2'-deoxynucleotides at positions 2 and 12 and 2'-fluoronucleotides at position 14, counted from the 5' end of the antisense strand. In some embodiments, the antisense strand contains 2'-deoxynucleotides at positions 2 and 12, 2'-fluoronucleotides at position 14, and nucleotides other than 2'-deoxy and 2'-fluoro at position 16, counted from the 5' end of the antisense strand. For example, the antisense strand contains 2'-deoxynucleotides at positions 2 and 12, 2'-fluoronucleotides at position 14, and 2'-OMe at position 16, counted from the 5' end of the antisense strand.

[0551] In some embodiments, the antisense strand contains a 2'-deoxynucleotide at position 14, counted from the 5' end of the antisense strand, and the sense strand contains a non-2'-fluoro nucleotide at position 7, counted from the 5' end of the sense strand. For example, the antisense strand contains 2'-deoxynucleotides at positions 2, 12, and 14, counted from the 5' end of the antisense strand, and the sense strand contains a 2'-fluoro nucleotide at position 10 and a non-2'-fluoro nucleotide at position 7, counted from the 5' end of the sense strand.

[0552] In some embodiments, the sense strand contains a 2'-fluoronucleotide at position 10, counted from the 5' end of the sense strand, and the antisense strand contains a 2'-deoxynucleotide at positions 2, 5, 7, and 12, counted from the 5' end of the antisense strand. (i) The antisense strand, counting from the 5' end of the antisense strand, contains a 2'-fluoronucleotide at position 14 and a nucleotide other than a 2'-deoxynucleotide or 2'-fluoronucleotide at position 16. (ii) The antisense strand contains a 2'-deoxynucleotide at position 14 or 16, counted from the 5' end of the antisense strand, and the sense strand contains a nucleotide other than a 2'-fluoronucleotide at position 7, counted from the 5' end of the sense strand.

[0553] In some embodiments, any nucleotide in the antisense chain that is neither a 2'-fluoronucleotide nor a 2'-deoxynucleotide is a 2'-OMe nucleotide.

[0554] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand, each having 14 to 40 nucleotides, and the sense strand sequence is given by formula (I): 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3'(I) [In formula: i and j are each independently either 0 or 1; p and q are each independently between 0 and 6; each N a Each sequence independently contains 0 to 25 modified nucleotides, and each sequence represents an oligonucleotide sequence containing at least two nucleotides with different modifications; each N b This independently represents an oligonucleotide sequence containing 1, 2, 3, 4, 5, or 6 modified nucleotides; each n p and each n q Each represents an overhanging nucleotide independently; Here, N b and Y do not have the same modifier; XXX, YYY, and ZZZ each independently represent a single motif consisting of three identical modifications on three consecutive nucleotides. The dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated at one or more positions on at least one strand, The antisense strand of dsRNA contains two blocks of one, two, or three phosphorothioate nucleotide ligatures, separated by phosphate nucleotide ligatures of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18. It is represented by [this].

[0555] In some embodiments, the dsRNA agent is given by formula (II): 5'n q '-N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p '3'(II) [In formula: k and l are, independently, either 0 or 1; p and q are each independently between 0 and 6; each N a ' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two nucleotides with different modifications; each N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p 'and each n q ' independently represents an overhanging nucleotide containing 0-6 nucleotides; Here, N b 'and Y' do not have the same modifier; X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif consisting of three identical modifications on three consecutive nucleotides. It includes an antisense strand sequence represented by .

[0556] Further details regarding the motifs represented by formulas (I) and (II) above can be found in WO2013 / 074947, which is incorporated herein by reference in its entirety.

[0557] A variety of publications describe multimeric siRNAs, all of which can be used in conjunction with the iRNA of the present invention. Such publications include WO2007 / 091269, U.S. Patent No. 7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887, and WO2011 / 031520, which are incorporated herein by reference in their entirety.

[0558] In some embodiments, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of the dsRNA agent is modified with 2'-OMe.

[0559] In some embodiments, the sense strand and antisense strand of the dsRNA agent are independently modified with acyclic nucleotides, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.

[0560] In some embodiments, each of the sense strand and antisense strand of the dsRNA agent contains at least two different modifications.

[0561] In some embodiments, the dsRNA agent of the present invention does not contain any 2'-F modifications.

[0562] In some embodiments, the dsRNA agent of the present invention contains 2'-F modifications (multiple) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. For example, the dsRNA agent of the present invention contains 2'-F modifications of 9 or 10.

[0563] A dsRNA agent may further contain at least one phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage. Modifications by phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages may be present at any nucleotide at any position on the sense strand, the antisense strand, or both. For example, modifications by internucleotide linkages may be present at any nucleotide on the sense strand or the antisense strand; each modification by internucleotide linkage may be present in an alternating pattern on the sense strand or the antisense strand; or the sense strand or antisense strand may contain both modifications by internucleotide linkages in an alternating pattern. The alternating pattern of modifications by internucleotide linkages on the sense strand may be the same as or different from that on the antisense strand, and the alternating pattern of modifications by internucleotide linkages on the sense strand may have a shift compared to the alternating pattern of modifications by internucleotide linkages on the antisense strand.

[0564] In one embodiment, the dsRNA includes modifications within the overhang region by phosphorothioate nucleotide linkages or methylphosphonate nucleotide linkages. For example, the overhang region may contain two nucleotides having a phosphorothioate nucleotide linkage or methylphosphonate nucleotide linkage between the two nucleotides. The nucleotide linkage modification may also be made to link the overhang nucleotide to the terminal paired nucleotide within the double-stranded region. For example, at least two, three, four, or all of the overhang nucleotides may be linked via phosphorothioate nucleotide linkages or methylphosphonate nucleotide linkages, but further phosphorothioate nucleotide linkages or methylphosphonate nucleotide linkages may be made to link the overhang nucleotide to the paired nucleotide adjacent to the overhang nucleotide. For example, there may be at least two phosphorothioate nucleotide linkages between the three terminal nucleotides, in which case two of the three nucleotides are overhang nucleotides and the third nucleotide is the paired nucleotide adjacent to the overhang nucleotide. Preferably, these three terminal nucleotides may be at the 3' end of the antisense strand.

[0565] In some embodiments, the sense strand and / or antisense strand of the dsRNA agent comprises one or more blocks of phosphorothioate nucleotide linkages or methylphosphonate nucleotide linkages. In one example, the sense strand comprises one block of two phosphorothioate nucleotide linkages or methylphosphonate nucleotide linkages. In one example, the antisense strand comprises two blocks of two phosphorothioate nucleotide linkages or methylphosphonate nucleotide linkages. For example, the two blocks of phosphorothioate nucleotide linkages or methylphosphonate nucleotide linkages are separated by 16 to 18 phosphate nucleotide linkages.

[0566] In some embodiments, the antisense strand of the dsRNA agent is 100% complementary to the target RNA, hybridizes with it, and inhibits its expression through RNA interference. In other embodiments, the antisense strand of the dsRNA agent is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% complementary to the target RNA.

[0567] Nucleic acid modification In some embodiments, the dsRNA agent comprises at least one nucleic acid modification as described herein. For example, the at least one modification is selected from the group consisting of modified nucleoside linkages, modified nucleobases, modified sugars, and any combination thereof. Without limitation, such modifications may be present anywhere in the dsRNA agent. For example, the modification may be present on one of the RNA molecules.

[0568] Nucleic acid modification (nucleobase) The naturally occurring base moieties of nucleosides are typically heterocyclic bases. Two of the most common classes of such heterocyclic bases are purines and pyrimidines. For these nucleosides, including pentofuranosyl sugars, phosphate groups can be linked to the 2', 3', or 5' hydroxyl moieties of the sugar. In oligonucleotide formation, these phosphate groups covalently bond adjacent nucleosides to each other, forming linear polymer compounds. Within oligonucleotides, the phosphate groups are generally said to form the internucleoside backbone of the oligonucleotide. The naturally occurring bond or backbone of RNA and DNA is a 3'-to-5' phosphodiester bond.

[0569] In addition to “unmodified” or “natural” nucleobases such as the purine nucleobases adenine (A) and guanine (G), and the pyrimidine nucleobases thymine (T), cytosine (C), and uracil (U), many modified nucleobases or nucleobase mimetic compounds known to those skilled in the art are suitable for the compounds described herein. Unmodified or natural nucleobases can be modified or substituted to provide dsRNAs with improved properties. For example, nuclease-resistant oligonucleotides can be prepared by these bases, or by synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine), and any one of the oligomeric modifications described herein. Alternatively, substitutions or modified analogs of any of the above bases and “universal bases” may be used. When a natural base is substituted with a non-natural and / or universal base, the nucleotide is said to contain a modified nucleobase and / or a nucleobase modification as described herein. Modified nucleobases and / or nucleobase modifications include natural, non-natural, and universal bases and contain a conjugated moiety, for example, a ligand as described herein. Preferred conjugated moieties for conjugation with a nucleobase contain a cationic amino group that can be conjugated to the nucleobase via a linker having a suitable alkyl, alkenyl, or amide bond.

[0570] The oligomeric compounds described herein may also include nucleobase (often simply referred to as "bases" in the art) modification or substitution. The "unmodified" or "natural" nucleobases used herein include the purine bases adenine (A) and guanine (G), as well as the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Exemplary modified nucleobases include other synthetic and natural nucleobases such as inosine, xanthine, hypoxanthine, nubularin, isoguanisine, tubercidine, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, and 2-(methylthio)-N 6 -(isopentenyl)adenine, 6-(alkyl)adenine, 6-(methyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, N 6 -(isopentyl)adenine, N 6 -(methyl)adenine, N 6 ,N 6 -(dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine Nin, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N 4-(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(aminoallyl)uracil , 5-(aminoalkyl)uracil, 5-(guanidinium alkyl)uracil, 5-(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N 3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudracil, 4-(thio)pseudracil, 2,4-(dithio)pseudracil, 5-(alkyl)pseudracil, 5-(methyl)pseudracil, 5-(alkyl)-2-(thio)pseudracil, 5-(methyl)-2-(thio)pseudracil, 5-(alkyl)-4-(thio)pseudracil, 5-(methyl)-4-(thio)pseudracil, 5-(alkyl)-2,4-(dithio)pseudracil Douracil, 5-(methyl)-2,4-(dithio)pseudracil, 1-substituted pseudouracil, 1-substituted 2(thio)pseudracil, 1-substituted 4-(thio)pseudracil, 1-substituted 2,4-(dithio)pseudracil, 1-(aminocarbonylethylenyl)pseudracil, 1-(aminocarbonylethylenyl)-2(thio)pseudracil, 1-(aminocarbonylethylenyl)-4-(thio)pseudracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudracil Douracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudracil, 1-(aminoalkylaminocarbonylethylenyl)-2(thio)-pseudracil, 1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudracil, 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 1 ,3-(diaza)-2-(oxo)-phenthiadin-1-yl,1-(aza)-2-(thio)-3-(aza)-phenthiadin-1-yl,7-substituted 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl,7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl,7-substituted 1,3-(diaza)-2-(oxo)-phenthiadin-1-yl,7-substituted 1-(aza)-2-(thio)-3-(aza)-phenthiadin-1-yl,7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxadin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxadin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiadin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiadin-1-yl, 7-(guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxadin-1-yl, 7-(guanidinium alkylhydroxy )-1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 7-(guanidinium alkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiadin-1-yl, 7-(guanidinium alkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiadin-1-yl, 1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nubralin, tubercidine, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimida Zolyl, Nitropyrazolyl, Nitrobenzimidazolyl, Nitroindazolyl, Aminoindolyl, Pyrrolopyrimidinyl, 3-(methyl)isocarbostyrillyl, 5-(methyl)isocarbostyrillyl, 3-(methyl)-7-(propynyl)isocarbostyrillyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, Imidizopyridinyl, 9-(methyl)-imidizopyridinyl, Pyrrolopyridinyl, Isocarbostyrillyl, 7-(propynyl)isocarbostyrillyl, Pripynyl-7-(aza) Indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenylon, tetracerenyl, pentaceryl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidine, N,2 - Substitution purine, N 6 - Substitution purine, O 6 -substituted purines, substituted 1,2,4-triazoles, pyrrolo-pyrimidine-2-on-3-yl, 6-phenyl-pyrrolo-pyrimidine-2-on-3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidine-2-on-3-yl, ortho-substituted-6-phenyl-pyrrolo-pyrimidine-2-on-3-yl, bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidine-2-on-3-yl, para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidine-2- This includes, but is not limited to, on-3-yl, ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidine-2-on-3-yl, bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidine-2-on-3-yl, pyridopyrimidine-3-yl, 2-oxo-7-amino-pyridopyrimidine-3-yl, 2-oxo-pyridopyrimidine-3-yl, or any O-alkylated or N-alkylated derivative thereof. Alternatively, any substitution or modification analogue of any of the above bases and “universal bases” may also be used.

[0571] As used herein, universal nucleobases are any nucleobases that can base pair with all four naturally occurring nucleobases without substantially affecting fusion behavior, intracellular enzymatic recognition, or dsRNA double-strand activity. Some exemplary universal nucleobases are 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methylisocarbostyrylyl, 5-methylisocarbostyrylyl, 3-methyl-7-propynylisocarbostyrylyl, 7-azaindryl, 6-methyl-7-azaindryl, imidizopyridinyl, and 9-methylimidizopyridyl. This includes, but is not limited to, dinyl, pyrrolepyridinyl, isocarbostyrylyl, 7-propynylisocarbostyrylyl, propynyl-7-azaindryl, 2,4,5-trimethylphenyl, 4-methylinolyl, 4,6-dimethylindolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracerenyl, pentacerenyl, and their structural derivatives (see, for example, Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

[0572] Further nucleobases are disclosed in U.S. Patent No. 3,687,808; in International Publication No. PCT / US09 / 038425, filed March 26, 2009; in Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990; in English et al., Angewandte Chemie, International Edition, 1991, 30, 613; in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijin, P.Ed. Wiley-VCH, 2008; and in Sanghvi, YS, Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Eds., CRC Press. Including what is disclosed in 1993. All of the above is incorporated herein by reference.

[0573] In certain embodiments, the modified nucleobase is a nucleobase that is structurally quite similar to the parent nucleobase, such as 7-deazaprine, 5-methylcytosine, or G-clamp. In certain embodiments, the nucleobase mimetic includes more complex structures, such as tricyclic phenoxazine nucleobase mimetics. Methods for preparing the above-mentioned modified nucleobases are well known to those skilled in the art.

[0574] Nucleic acid modification (sugar)

[0575] The compounds of the present invention provided herein may comprise one or more monomers (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) comprising a nucleoside or nucleotide having a modified sugar moiety. For example, the furanosyl sugar ring of a nucleoside can be modified in many ways, including, but not limited to, the addition of substituents, or the bridging of two non-geminal ring atoms to form a lock nucleic acid or bicyclic nucleic acid. In certain embodiments, the oligomeric compound comprises one or more monomers (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) that are LNAs.

[0576] In some embodiments of the loc nucleic acid, the 2' position of the furanosyl is independently -[C(R1)(R2)] n -,-[C(R1)(R2)] n -O-, -[C(R1)(R2)] n -N(R1)-, -[C(R1)(R2)] n -N(R1)-O-, -[C(R1R2)] n -ON(R1)-, -C(R1)=C(R2)-O-, -C(R1)=N-, -C(R1)=NO-, -C(=NR1)-, -C(=NR1)- O-, -C(=O)-, -C(=O)O-, -C(=S)-, -C(=S)O-, -C(=S)S-, -O-, -Si(R1)2-, -S(=O) x -, and -N(R1)-; [In formula: x is 0, 1, or 2; n is 1, 2, 3, or 4; Each R1 and R2 is independently H, protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, C5-C7 acyclic radical, substituted C5-C7 acyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1); and Each of J1 and J2 is independently H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or protecting group. The linker selected from the options will connect to position 4'.

[0577] In some embodiments, each linker of the LNA compound is independently -[C(R1)(R2)] n -,-[C(R1)(R2)] n The linkers are -O-, -C(R1R2)-N(R1)-O-, or -C(R1R2)-ON(R1)-. In another embodiment, each of the linkers is independently 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2', 4'-(CH2)2-O-2', 4'-CH2-ON(R1)-2', and 4'-CH2-N(R1)-O-2'- [wherein each R1 is independently H, a protecting group, or a C1-C12 alkyl group].

[0578] Certain LNAs have been prepared and disclosed in patent documents and scientific papers (Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; WO94 / 14226; WO2005 / 021570; Singh et al., J. Org. Chem., 1998, 63, Examples of obtained U.S. patents and published applications disclosing 10035-10039;LNA include, for example, U.S. Patent Nos. 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; and 6,525,191; and U.S. Pre-Grant Publication Nos. 2004-0171570; 2004-0219565; 2004-0014959; 2003-0207841; 2004-0143114; and 20030082807).

[0579] In this specification, LNAs are also provided in which the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring, thereby forming a methyleneoxy(4'-CH2-O-2') bond and creating a bicyclic sugar moiety (as outlined in Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S. Patents 6,268,490 and 6,670,461). The bond may be a methylene(-CH2-) group bridging the 2' oxygen atom and the 4' carbon atom; therefore, the term methyleneoxy(4'-CH2-O-2')LNA is used for the bicyclic portion, and if this position is an ethylene group, the term ethyleneoxy(4'-CH2CH2-O-2')LNA is used (Singh et al., Chem. Commun., 1998, 4, 455-456; Morita et al., Bioorganic Medicinal Chemistry, 2003, 11, 2211-2226). Methyleneoxy(4'-CH2-O-2')LNA and other bicyclic sugar analogs exhibit very high double-chain thermal stability (Tm=+3~+10℃) by complementary DNA and RNA, stability against 3'-exonuclease degradation, and excellent solubility. Strong and non-toxic antisense oligonucleotides containing BNA have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638).

[0580] The isomer of methyleneoxy(4'-CH2-O-2')LNA being discussed is alpha-L-methyleneoxy(4'-CH2-O-2')LNA, which has been shown to have excellent stability against 3'-exonucleases. Alpha-L-methyleneoxy(4'-CH2-O-2')LNA has been incorporated into antisense gapmers and chimeras exhibiting potent antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0581] The methyleneoxy(4'-CH2-O-2')LNA monomers adenine, cytosine, guanine, 5-methylcytosine, thymine, and uracil, along with their oligomerization, synthesis and preparation, and nucleic acid recognition properties, have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). Their BNAs and preparations are also described in WO98 / 39352 and WO99 / 14226.

[0582] Analogues of methyleneoxy(4'-CH2-O-2')LNA, phosphorothioate-methyleneoxy(4'-CH2-O-2')LNA and 2'-thio-LNA have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of loc nucleoside analogues containing oligodeoxyribonucleotide double helixes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO99 / 14226). Furthermore, the synthesis of 2'-amino-LNA and novel structurally restricted high-affinity oligonucleotide analogues has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Furthermore, 2'-amino- and 2'-methylamino-LNAs have been prepared, and the thermal stability of their double helix with complementary RNA and DNA strands has been previously reported.

[0583] Modified sugar moieties are well known and can be used to alter, typically increase, the affinity of antisense compounds for their target, and / or increase nuclease resistance. A representative list of preferred modified sugars includes, but is not limited to, bicyclic modified sugars, including methyleneoxy(4'-CH2-O-2')LNA and ethyleneoxy(4'-(CH2)2-O-2' bridged)ENA; substituted sugars, particularly 2'-substituted sugars having 2'-F, 2'-OCH3, or 2'-O(CH2)2-OCH3 substituents; and 4'-thio modified sugars. Sugars can also be replaced with sugar mimetic groups, among others. Methods for preparing modified sugars are well known to those skilled in the art. Some representative patents and publications teaching the preparation of such modified sugars include U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; and 5,591 This includes, but is not limited to, Nos. 722; Nos. 5,597,909; Nos. 5,610,300; Nos. 5,627,053; Nos. 5,639,873; Nos. 5,646,265; Nos. 5,658,873; Nos. 5,670,633; Nos. 5,792,747; Nos. 5,700,920; Nos. 6,531,584; and Nos. 6,600,032; as well as WO2005 / 121371.

[0584] Examples of "oxy"-2'hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), O(CH2CH2O) n CH2CH2OR, n=1~50; "Locked" nucleic acid (LNA) in which the furanose portion of the nucleoside contains a bridge connecting the two carbon atoms of the furanose ring, thereby forming a bicyclic ring system; O-AMINE or O-(CH2) nAMINE (n=1~10, AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, ethylenediamine or polyamino); and O-CH2CH2(NCH2CH2NMe2)2.

[0585] The "deoxy" modification includes hydrogen (i.e., deoxyribose sugars particularly suitable for single-chain overhangs); halo (e.g., fluoro); amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH2CH2NH) n CH2CH2-AMINE(AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino);-NHC(O)R(R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; thioalkyl; alkyl; cycloalkyl; aryl; alkenyl and alkynyl, which may be substituted, for example, by amino functionality.

[0586] Other suitable 2' modifications, such as Modified MOE, are described in U.S. Patent Application Publication No. 20130130378, the contents of which are incorporated herein by reference.

[0587] Modifications at the 2' position can be present in the arabinose structure. The term "arabinose structure" refers to the arrangement of substituents at the C2' position of ribose within the same configuration as the 2'-OH group of arabinose.

[0588] A sugar can contain two different modifications at the same carbon atom, such as gem modifications. The sugar group can also contain one or more carbon atoms with the opposite stereochemical configuration to that of the corresponding carbon in ribose. Thus, an oligomeric compound can contain one or more monomers as a sugar, for example, arabinose. The monomer may have an alpha bond at the 1 position of the sugar, such as an alpha-nucleoside. The monomer may also have an opposite configuration at the 4' position, for example, C5' and H4', or substituents that replace them are exchanged with each other. When C5' and H4', or substituents that replace them, are exchanged with each other, the sugar is said to be modified at the 4' position.

[0589] The compounds of the present invention disclosed herein may also include debasic sugars, i.e., sugars lacking a nucleobase at C-1' or having other chemical groups instead of a nucleobase at C1'. See, for example, U.S. Patent No. 5,998,203, the full text of which is incorporated herein by reference. These debasic sugars may also contain further modifications in one or more constituent sugar atoms. The dsRNA agents of the present invention may also contain one or more sugars that are L-isomers, e.g., L-nucleosides. Modifications to the sugar group may include 4'-O substitution by sulfur, optionally substituted nitrogen, or a CH2 group. In some embodiments, the bond between C1' and the nucleobase is an α structure.

[0590] Sugar modification refers to any nucleotide having an acyclic ribose sugar, and may also include "acyclic nucleotides," where there are no CC bonds between ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', C1'-O4'), and / or at least one of the ribose carbons or oxygen atoms (e.g., C1', C2', C3', C4', or O4') is absent in the nucleotide, either independently or in combination. In some embodiments, acyclic nucleotides are,

[0591] [ka] [In the formula, B is a modified or unmodified nucleobase, R1 and R2 are independently H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar.] That is the case.

[0592] In some embodiments, the sugar modification is selected from the group consisting of 2'-H, 2'-O-Me (2'-O-methyl), 2'-O-MOE (2'-O-methoxyethyl), 2'-F, 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), 2'-S-methyl, 2'-O-CH2-(4'-C)(LNA), 2'-O-CH2CH2-(4'-C)(ENA), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), and 2'F having gem2'-OMe / 2'-OMe in the arabinose structure.

[0593] When a particular nucleotide is linked to the next nucleotide via its 2' position, it is understood that the sugar modifications described herein may be located at the 3' position of the sugar because of the particular nucleotide, for example, the nucleotide linked via its 2' position. Modifications at the 3' position may be present in xylose structures. The term "xylose structure" refers to the arrangement of substituents at the C3' position of ribose in the same structure as the 3'-OH of a xylose sugar.

[0594] The hydrogens bonded to C4' and / or C1' may be substituted with linear or branched alkyls, alkenyls, or alkynyls as appropriate, and the alkyl, alkenyl, and alkynyl skeletons may contain one or more O, S, S(O), SO2, N(R'), C(O), N(R')C(O)O, O(O)N(R'), CH(Z'), phosphate-containing bonds, substituted aryls, heteroaryls, heterocyclics, or cycloalkyls, where R' is hydrogen, acyl, or a substituted aliphatic, and Z' is OR 11 COR 11 CO2R 11 ,

[0595] [ka] , NR 21 R 31 CONR 21 R 31 CON(H)NR 21 R 31 , ONR 21 R 31 CON(H)N=CR 41 R 51 , N(R 21 )C(=NR 31 )NR 21 R 31 , N(R 21 )C(O)NR 21 R 31 , N(R 21 )C(S)NR 21 R 31 , OC(O)NR 21 R 31 SC(O)NR 21 R 31 , N(R 21 )C(S)OR 11 , N(R 21 )C(O)OR 11 , N(R 21 )C(O)SR 11 , N(R 21 )N=CR 41 R 51 , ON=CR 41 R51 SO2R 11 SOR 11 , SR 11 , and selected from the group consisting of substituted or unsubstituted heteroalgebras; R 21 and R 31 Independently, for each occurrence, hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR 11 COR 11 CO2R 11 , or NR 11 R 11 ' is; or R 21 and R 31 They, together with the atoms to which they are joined, form a heterocyclic ring; R 41 and R 51 Independently, for each occurrence, hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR 11 COR 11 , or CO2R 11 , or NR 11 R 11 'and; as well as R 11 and R 11 ’ These are independently hydrogen, aliphatic, substituted aliphatic, aryl, heteroaryl, or heterocyclic. In some embodiments, the hydrogen bonded to the C4' of the 5' terminal nucleotide is substituted.

[0596] In some embodiments, C4' and C5' preferably together form a suitably substituted heterocycle comprising at least one -PX(Y)-[wherein X is H, OH, OM, SH, suitably substituted alkyl, suitably substituted alkoxy, suitably substituted alkylthio, suitably substituted alkylamino or suitably substituted dialkylamino, M is independently an alkali metal or transition metal with an overall charge of +1 for each occurrence; Y is O, S, or NR' (wherein R' is hydrogen, suitably substituted aromatic)]. Preferably, this modification is at the 5' end of the dsRNA.

[0597] In certain embodiments, the dsRNA agent of the present invention comprises at least two regions of at least two consecutive monomers of the above formula. In certain embodiments, the dsRNA agent of the present invention comprises a gap motif. In certain embodiments, the dsRNA agent of the present invention comprises at least one region from about 8 to about 14 consecutive β-D-2'-deoxyribofuranosyl nucleosides. In certain embodiments, the dsRNA agent of the present invention comprises at least one region from about 9 to about 12 consecutive β-D-2'-deoxyribofuranosyl nucleosides.

[0598] In a particular embodiment, the dsRNA agent of the present invention is a compound of at least one formula (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more):

[0599] [ka] (In the formula, Bx is the heterocyclic base portion.) It contains the (S)-cEt monomer.

[0600] In certain embodiments, the monomers include sugar mimetic compounds. In certain such embodiments, the mimetic compounds are used in place of sugars or sugar-nucleoside linkages, and the nucleobase is maintained for hybridization to a selected target. Typical examples of sugar mimetic compounds include, but are not limited to, cyclohexenyl or morpholino. Typical examples of sugar-nucleoside linkage mimetic combinations include, but are not limited to, peptide nucleic acids (PNAs) and morpholino groups linked by uncharged achiral linkages. In some examples, the mimetic compounds are used in place of nucleobases. Typical nucleobase mimetic compounds are well known in the art and include, but are not limited to, tricyclic phenoxazine analogs and universal bases (Berger et al., Nuc Acid Res. 2000, 28:2911-14, incorporated herein by reference). Methods for the synthesis of sugars, nucleosides, and nucleobase mimetic compounds are well known to those skilled in the art.

[0601] Nucleic acid modification (sugar bond) This specification describes linking groups that link monomers (including, but not limited to, modified and unmodified nucleosides and nucleotides) together to form oligomeric compounds, such as oligonucleotides. Such linking groups are also referred to as intersugar links. Two main classes of linking groups are defined by the presence or absence of a phosphorus atom. Typical phosphorus-containing links include, but are not limited to, phosphodiesters (P=O), phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (P=S). Typical non-phosphorus-containing linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters (-OC(O)-S-), thionocarbamates (-OC(O)(NH)-S-); siloxanes (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Compared to natural phosphodiester bonds, modified bonds can be used to alter, typically increase, the nuclease resistance of oligonucleotides. In certain embodiments, bonds containing chiral atoms can be prepared as racemic mixtures or separate enantiomers. Typical chiral bonds include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing bonds are well known to those skilled in the art.

[0602] The phosphate group in the linking group can be modified by substituting one of its oxygen atoms with a different substituent. One result of this modification may increase the resistance of the oligonucleotide to nucleolysis. Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and triesters of phosphate. In some embodiments, one of the oxygen atoms of the unlinked phosphate in the linking group may be substituted with any of the following: S, Se, BR3 (where R is hydrogen, alkyl, or aryl), C (i.e., alkyl, aryl group, etc.), H, NR2 (where R is hydrogen, optionally substituted alkyl, or aryl), or (where R is optionally substituted alkyl or aryl). The phosphorus atom in the unmodified phosphate group is achiral. However, by substituting one of the non-crosslinked oxygen atoms with one of the atoms or groups of atoms mentioned above, the phosphorus atom is made chiral, or in other words, the phosphorus atom in the phosphate group modified in this way is a chiral center. The phosphorus atom of the chiral center can have either an "R" configuration (Rp, as specified herein) or an "S" configuration (Sp, as specified herein).

[0603] Phosphothioates have both sulfur-substituted and non-crosslinked oxygen atoms. The phosphorus center of a phosphorothioate is achiral and prevents the formation of oligonucleotide diastereomers. Therefore, stating without wishing to be bound by theory, modification of both the chiral center and the non-crosslinked oxygen atoms, except for phosphorothioate formation, may be desirable, as they cannot produce diastereomer mixtures. Thus, the non-crosslinked oxygen atoms can independently be any one of O, S, Se, B, C, H,...

Claims

1. A double-stranded RNA (dsRNA) agent that modulates the expression of a target gene in the central nervous system (CNS), The antisense strand complementary to the target gene in the CNS. A sense chain that is complementary to the antisense chain; and One or more saturated or unsaturated C atoms are conjugated onto at least one chain via a linker or carrier as appropriate. ２２ One or more lipophilic parts containing hydrocarbon chains A dsRNA agent containing this agent.

2. at least one C ２２ Hydrocarbon chains are saturated or unsaturated, linear or branched. ２２ The dsRNA agent according to claim 1, wherein the hydrocarbon chain is present.

3. One or more C ２２ The dsRNA agent according to claim 1, wherein the hydrocarbon chain contains a functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, carboxylic acid ester, sulfonic acid, ether, phosphoric acid, thiol, azide, alkyne, cycloalkyne, trans-cyclooctenyl, N-maleimidyl, and 1,2,4,5-tetrazin-3-yl.

4. at least one C ２２ The hydrocarbon chain is C ２２ acid, C ２２ Alcohol, or C ２２ The dsRNA agent according to claim 3, which is an amide.

5. At least one C ２２ The hydrocarbon chain is C selected from the group consisting of docosanic acid, 6-octyltetradecanoic acid, 10-hexylhexadecanoic acid, all-cis-7,10,13,16,19-docosapentaenoic acid, all-cis-4,7,10,13,16,19-docosahexaenoic acid, all-cis-13,16-docosadienoic acid, all-cis-7,10,13,16-docosatetraenoic acid, all-cis-4,7,10,13,16-docosapentaenoic acid, cis-13-docosenoic acid, 22-hydroxydocosanoic acid, 16-hydroxyhexadecanoic acid, and C6+16-hydroxyhexadecanoic acid. ２２ acid; A C selected from the group consisting of 1-docosanol, 6-octyltetradecane-1-ol, 10-hexylhexadecane-1-ol, cis-13-docosen-1-ol, docosan-9-ol, docosan-2-ol, docosan-10-ol, docosan-11-ol, and cis-4,7,10,13,16,19-docosahexanol, 22-hydroxydocosanoic acid, 16-hydroxyhexadecanoic acid, and C6+16-hydroxyhexadecanoic acid. ２２ Alcohol; or C selected from the group consisting of (E)-Docos-4-enamide, (E)-Docos-5-enamide, (Z)-Docos-9-enamide, (E)-Docos-11-enamide, 12-docosenamide, (Z)-Docos-13-enamide, (Z)-N-hydroxy-13-docosenamide, (E)-Docos-14-enamide, 6-cis-docosenamide, 14-docosenamide, Docos-11-enamide, (4E,13E)-Docosa-4,13-dienamide, and (5E,13E)-Docosa-5,13-dienamide ２２ Amido The dsRNA agent according to claim 4.

6. The dsRNA agent according to any one of claims 1 to 5, wherein the lipophilic portion is conjugated via a carrier that substitutes one or more nucleotides of the dsRNA agent.

7. The dsRNA agent according to claim 6, wherein the carrier is a cyclic group selected from the group consisting of pyrrolidinil, pyrazolinil, pyrazolidinil, imidazolinil, imidazolidinil, piperidinil, piperazinil, [1,3]dioxolanil, oxazolidinil, isoxazolidinil, morpholinil, thiazolidinil, isothiazolidinil, quinoxalinil, pyridadinil, tetrahydrofuranil, and dekalinil, or an acyclic portion based on a selinol skeleton or a diethanolamine skeleton.

8. The dsRNA agent according to any one of claims 1 to 5, wherein the lipophilic portion is conjugated via an internucleotide phosphate linker, or a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimidothioether, disulfide, phosphodiester, sulfonamide linkage, click reaction product, or carbamate.

9. The lipophilic part, 【Chemistry 1】 【change】 【change】 [In the formula, G is G １ or saturated or unsaturated C ２１ It is a hydrocarbon chain, and G is a halogen, -OR Ｇ , -SR Ｇ , -N(R Ｇ ) ２ , -C(O)OR Ｇ , -OC(O)R Ｇ , -C(O)N(R Ｇ ) ２ , -N(R Ｇ ) C(O)R Ｇ , -N(R Ｇ ) C(O)OR Ｇ , -N(R Ｇ ) SO ２ (R Ｇ ), or -SO ２ N(R) Ｇ ) ２ It is appropriately substituted with one or more groups selected from the group consisting of each R Ｇ These are, independently, hydrogen or C １ ~C ６ It is alkyl; G １ is saturated or unsaturated C ２２ It is a hydrocarbon chain, G １ is halogen, -OR Ｇ１ , -SR Ｇ１ , -N(R Ｇ１ ) ２ , -C(O)OR Ｇ１ , -OC(O)R Ｇ１ , -C(O)N(R Ｇ１ ) ２ , -N(R Ｇ１ ) C(O)R Ｇ１ , -N(R Ｇ１ ) C(O)OR Ｇ１ , -N(R Ｇ１ ) SO ２ (R Ｇ１ ), or -SO ２ N(R) Ｇ１ ) ２ It is appropriately substituted by one or two groups selected from the group consisting of the following, and each R Ｇ１ These are, independently, hydrogen or C １ ~C ６ It is alkyl; m is an integer between 0 and 8; n is an integer between 1 and 21; R ２ 'and R ３ ' is independently H, OH, F, OMe, O-methoxyalkyl, O-aryl, O-N-methylacetamide, O-dimethylaminoethoxyethyl, or O-aminopropyl; B is a modified or unmodified nucleobase; W is an alkyl group; R and R' are each independently H or alkyl group. The dsRNA agent according to claim 1, which is a lipophilic monomer selected from the group consisting of the following.

10. The lipophilic portions are group (i), group (ii), and group (iii): (i) 【Chemistry 2】 (ii) 【Transformation 3】 And, (iii) 【Chemistry 4】 [In the formula: G is G １ or saturated or unsaturated C ２１ It is a hydrocarbon chain, and G is a halogen, -OR Ｇ , -SR Ｇ , -N(R Ｇ ) ２ , -C(O)OR Ｇ , -OC(O)R Ｇ , -C(O)N(R Ｇ ) ２ , -N(R Ｇ ) C(O)R Ｇ , -N(R Ｇ ) C(O)OR Ｇ , -N(R Ｇ ) SO ２ (R Ｇ ), or -SO ２ N(R) Ｇ ) ２ It is appropriately substituted by one or two groups selected from the group consisting of the following, and each R Ｇ These are, independently, hydrogen or C １ ~C ６ It is alkyl; G １ is a saturated or unsaturated C ２２ hydrocarbon chain, and G １ is optionally substituted by one or two groups selected from the group consisting of halogen, -OR Ｇ１ , -SR Ｇ１ , -N(R Ｇ１ ), -C(O)OR ２ , -OC(O)R Ｇ１ , -C(O)N(R Ｇ１ ), -N(R Ｇ１ ), -N(R ２ ), -N(R Ｇ１ )C(O)R Ｇ１ , -N(R Ｇ１ )C(O)OR Ｇ１ , -N(R Ｇ１ )SO ２ (R Ｇ１ ), or -SO​​​​​​​​​​​​ R ２ 'and R ３ ' is independently H, OH, F, OMe, O-methoxyalkyl, O-aryl, O-N-methylacetamide, O-dimethylaminoethoxyethyl, or O-aminopropyl; B is a modified or unmodified nucleobase. The dsRNA agent according to claim 1, which is a lipophilic monomer selected from one of the members.

11. Lipophilic monomers, 【Transformation 5】 [In the formula, B is a modified or unmodified nucleobase.] The dsRNA agent according to claim 1.

12. Lipophilic monomers, 【Transformation 6】 【change】 【change】 【change】 【change】 【change】 [In the formula, B is a modified or unmodified nucleobase.] The dsRNA agent according to claim 1.

13. The dsRNA agent according to any one of claims 1 to 12, comprising a double-stranded region formed between a sense strand and an antisense strand, and optionally one or two single-stranded non-loop overhangs, wherein one or more lipophilic portions are conjugated to either the double-stranded region or the non-loop overhangs.

14. A dsRNA agent according to any one of claims 1 to 13, wherein one or more lipophilic moieties are conjugated at one or more internal positions on at least one strand.

15. The dsRNA agent according to claim 14, wherein the internal position includes all positions except two or three terminal positions from each end of at least one strand.

16. The dsRNA agent according to claim 14, wherein the internal position excludes the sense strand cleavage site region.

17. The dsRNA agent according to claim 16, wherein the internal position excludes positions 9-12 or 11-13 when counted from the 5' end of the sense strand.

18. The dsRNA agent according to claim 16, wherein the internal position excludes the antisense strand cleavage site region.

19. The dsRNA agent according to claim 16, wherein the internal position excludes positions 12 to 14 when counted from the 5' end of the antisense strand.

20. The dsRNA agent according to claim 14, wherein one or more lipophilic moieties are conjugated to one or more of the following internal positions: positions 4-8 and 13-18 of the sense strand, and positions 6-10 and 15-18 of the antisense strand, counting from the 5' end of each strand.

21. The dsRNA agent according to claim 20, wherein one or more lipophilic moieties are conjugated to one or more of the following internal positions: positions 5, 6, 7, 15, 16, and 17 of the sense strand and positions 10, 15, 16, and 17 of the antisense strand, counting from the 5' end of each strand.

22. The dsRNA agent according to claim 21, wherein one or more lipophilic moieties are conjugated at the 6th position of the sense strand, counted from the 5' end of the sense strand.

23. A dsRNA agent according to any one of claims 1 to 3, wherein one or more lipophilic moieties are conjugated to one or more terminal positions: counting from the 5' or 3' end of each strand, at positions 1, 2, or 3 of the sense strand or antisense strand.

24. The dsRNA agent according to claim 23, wherein at least one lipophilic moiety is conjugated to position 1 of the sense strand or antisense strand, counted from the 5' end of each strand, by modification of the sugar moiety of the nucleotide at position 1 at the 2', 5', or 4' position, or by modification of the nucleobase.

25. The dsRNA agent according to any one of claims 1 to 24, wherein the dsRNA agent comprises at least one single-stranded protrusion at at least one end.

26. The dsRNA agent according to claim 25, wherein the single-stranded overhang is 1, 2, or 3 nucleotides long.

27. The dsRNA agent according to any one of claims 1 to 26, wherein the sense strand and the antisense strand are each independently 15 to 30 nucleotides long, 19 to 25 nucleotides long, or 21 to 23 nucleotides long.

28. The dsRNA agent according to claim 27, wherein the sense strand is 21 nucleotides long and the antisense strand is 23 nucleotides long, and the strands form a double-stranded region of 21 consecutive base pairs having a single-stranded projection of 2 nucleotides long at the 3' end.

29. The dsRNA agent according to claim 27, wherein the dsRNA agent has two blunt ends at both ends of the strand, and the strand forms a double-stranded region of 19 to 23 consecutive base pairs.

30. The dsRNA agent according to any one of claims 1 to 26, wherein the sense strand is 12 to 40 nucleotides in length, and the sense strand forms a double-stranded region with the antisense strand.

31. The sense strand has S at its 3' end. １ -L-S ２ The dsRNA agent according to claim 30, comprising a stem loop described as such, wherein S1 is complementary to S2 and L forms the loop.

32. The dsRNA agent according to claim 31, wherein L contains the sequence GAAA.

33. At least one lipophilic portion is S １ -L-S ２ The dsRNA agent according to claim 31, which is conjugated to a nucleotide.

34. The dsRNA agent according to claim 33, wherein at least one lipophilic moiety is conjugated to a nucleotide of L.

35. A dsRNA agent according to any one of claims 1 to 34, wherein the sense strand and antisense strand each contain less than 10 2'-fluoromodified nucleotides.

36. A dsRNA agent according to any one of claims 1 to 34, wherein the sense strand and the antisense strand each contain at least 50%, at least 60%, or at least 70% of 2'-OMe modified nucleotides.

37. A dsRNA agent according to any one of claims 1 to 34, wherein the sense strand comprises at least one or two phosphorothioate linkages at its 3' or 5' end.

38. A dsRNA agent according to any one of claims 1 to 34, wherein the antisense strand comprises a phosphate group or a phosphate mimetic at its 5' end.

39. The dsRNA agent according to claim 38, wherein the phosphate mimetic is 5'-vinylphosphonate (VP).

40. A dsRNA agent according to any one of claims 1 to 39, wherein the antisense strand comprises at least one GNA in its seed region.

41. The dsRNA agent according to claim 40, wherein the seed region is located at the 5th to 7th position from the 5' end of the antisense strand.

42. A dsRNA agent according to any one of claims 1 to 41, further comprising a targeting ligand that targets a receptor that mediates delivery to CNS tissue.

43. The dsRNA agent according to claim 42, wherein the targeting ligand is selected from the group consisting of Angiopep-2, lipoprotein receptor-associated protein (LRP) ligand, bEnd.3 cell-binding ligand, transferrin receptor (TfR) ligand, manose receptor ligand, glucose transporter protein, and LDL receptor ligand.

44. A dsRNA agent according to any one of claims 1 to 43, further comprising a targeting ligand for targeting liver tissue.

45. A dsRNA agent according to any one of claims 1 to 44, wherein the target gene is selected from the group consisting of APP, ATXN2, C9orf72, TARDBP, MAPT (Tau), HTT, SNCA, FUS, ATXN3, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, TTR, SCN9A, LRRK2, GPR75, APOE, SCD5, ELOVL1, FLNA, ALK, CHI3L1 (YKL-40), RPS25, α2-AR, and GSK3α.

46. Cells containing the dsRNA agent according to any one of claims 1 to 45.

47. A pharmaceutical composition comprising a dsRNA agent according to any one of claims 1 to 45.

48. A method for modulating the expression of a target gene in CNS cells, comprising administering a dsRNA agent according to any one of claims 1 to 45 to the cells.

49. The method according to claim 48, wherein the cells are within the scope.

50. A method for treating or preventing CNS damage in a subject, comprising administering a therapeutically effective amount of a dsRNA agent according to any one of claims 1 to 45 to the subject, thereby treating the subject by modulating the expression of a target gene in the subject's CNS.

51. The method according to claim 50, wherein the CNS disorder is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Huntington's disease, Parkinson's disease, spinocerebellar disease, prions, and Lafora disease.

52. The method according to claim 48 or 50, wherein the dsRNA agent is administered intrathecally or intraventricularly.

53. The method according to claim 48 or 50, wherein the target gene is selected from the group consisting of APP, ATXN2, C9orf72, TARDBP, MAPT (Tau), HTT, SNCA, FUS, ATXN3, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, TTR, SCN9A, LRRK2, GPR75, APOE, SCD5, ELOVL1, FLNA, ALK, CHI3L1 (YKL-40), RPS25, α2-AR, and GSK3α.

54. dsRNA agents use the same sense strand and antisense strand, as well as C ２２ Except for containing a different hydrocarbon chain with fewer carbon atoms than the hydrocarbon chain, it is administered at a dose level of 50% or less of Dose 1, which is the dose level of a comparative dsRNA agent having the same conjugation with the same lipophilic moiety. The method achieves the same reduction in target gene expression as Dose 1 comparative dsRNA drug administration. The method according to claim 48 or 50.

55. dsRNA agents use the same sense strand and antisense strand, as well as C ２２ Except for containing a different hydrocarbon chain with fewer carbon atoms than the hydrocarbon chain, it is administered at a dose level of 35% or less of Dose 1, which is the dose level of a comparative dsRNA agent having the same conjugation with the same lipophilic moiety. The method achieves the same reduction in target gene expression as Dose 1 comparative dsRNA drug administration. The method according to claim 48 or 50.

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