Modified double-stranded RNA agents

Abstract

One aspect of the disclosure relates to a dsRNA agent comprising a sense strand and an antisense strand that is sufficiently complementary to at least a portion of a target gene's mRNA, each strand having 14 to 40 nucleotides, and the antisense strand comprising at least one TNA (threose nucleic acid) at position 1, counting from the 5' end. Another aspect of the invention relates to a method of treating a subject with a CNS disorder, the method modulating expression of a target gene in a cell, and the method comprising administering a dsRNA agent to the cell or the subject.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 412,286, filed September 30, 2022, and U.S. Provisional Patent Application No. 63 / 434,690, filed December 22, 2022, both of which are incorporated by reference herein in their entireties.

[0002] Array List This application contains a Sequence Listing which has been submitted in XML format and is hereby incorporated by reference in its entirety. The XML copy created on September 27, 2023, is named 29520_1513-PCT_ALN-501-WO_SL.xml and is 2,546,681 bytes in size.

[0003] The present invention relates generally to the field of RNA interference technology using modified double-stranded RNA agents. [Background technology]

[0004] Chemical modifications of the nucleobase, ribose sugar, and phosphate backbone have been used to improve the drug-like properties of therapeutic oligonucleotides and to confer favorable pharmacological properties to GalNAc-siRNA conjugates in preclinical and clinical development.

[0005] Furthermore, in vivo, the efficient delivery of siRNA agent to cells requires specific targeting and substantial protection from extracellular environment, particularly serum protein.RNAi-based therapy shows promising clinical data for the treatment of liver-related disorders.However, there remains obstacles in the delivery of siRNA to extrahepatic tissues, which limits the use of siRNA-based therapy. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, there is a continuing need for dsRNA agents that improve the nucleose stability and gene silencing efficiency of siRNA gene therapy.In addition, there is a continuing need for new and improved methods for delivering siRNA agents in vivo, so as to achieve and enhance the therapeutic potential of siRNAi agents without using tissue delivery reagents. [Means for solving the problem]

[0007] One aspect of the present invention provides a double-stranded RNA (dsRNA) agent capable of modulating expression of a target gene, the dsRNA agent comprising a sense strand and an antisense strand that is sufficiently complementary to at least a portion of the target gene's mRNA, each strand having 14 to 40 nucleotides, and the antisense strand comprising at least one TNA (threose nucleic acid) at position 1, counting from the 5' end.

[0008] Another aspect of the present invention is a double-stranded RNA (dsRNA) agent capable of modulating expression of a target gene, comprising a sense strand and an antisense strand that is sufficiently complementary to at least a portion of the mRNA of the target gene, each strand having 14 to 40 nucleotides; the sense strand contains at least one 2'-OMe modification at position 1, counting from the 5' end; The antisense strand contains at least one TNA (threose nucleic acid) at position 1, counting from the 5' end. Double-stranded RNA agents are provided.

[0009] In some embodiments, the dsRNA agent of claim 1, wherein the sense strand further comprises at least one 2'-OMe modification at position 2, counting from the 5' end.

[0010] In some embodiments, a dsRNA agent contains one or more ligands conjugated to at least one strand, optionally via a linker or carrier.

[0011] Another aspect of the invention is a double-stranded RNA (dsRNA) agent capable of modulating expression of a target gene, comprising a sense strand and an antisense strand that is sufficiently complementary to at least a portion of the mRNA of the target gene, each strand having 14 to 40 nucleotides; One of the sense or antisense strands is [ka] [In formula: each R is independently optionally substituted alkyl; B is an optionally modified nucleobase; * represents a bond to an H or internucleotide linkage to the next nucleotide] At least one 4'-modified TNA (threose nucleic acid) having the structure Relating to double-stranded RNA agents.

[0012] In some embodiments, a dsRNA agent contains one or more ligands conjugated to at least one strand, optionally via a linker or carrier.

[0013] In some embodiments, at least one ligand is a lipophilic moiety.

[0014] In all of the above aspects of the invention relating to dsRNA agents, in some embodiments, at least one ligand is an ASGPR ligand. The ASGPR ligand can be one or more GalNAc derivatives joined by a bivalent or trivalent branched linker. In one embodiment, the ASGPR ligand is [ka] is.

[0015] Another aspect of the present invention relates to a double-stranded RNA (dsRNA) agent capable of modulating the expression of a target gene, the dsRNA agent comprising a sense strand and an antisense strand sufficiently complementary to at least a portion of the mRNA of the target gene. The antisense strand comprises at least one TNA (threose nucleic acid) at position 1, counting from the 5' end. The dsRNA agent comprises one or more lipophilic moieties conjugated to at least one strand, optionally via a linker or carrier.

[0016] The embodiments discussed below apply to all of the above aspects of the invention relating to dsRNA agents.

[0017] In some embodiments, the octanol-water partition coefficient, log K ow The lipophilic moiety has a log K ow can have.

[0018] In some embodiments, the hydrophobicity of dsRNA agent measured by the unbound fraction in the plasma protein binding assay of dsRNA agent is greater than 0.2.In one embodiment, the plasma protein binding assay determined is electrophoretic mobility shift assay (EMSA) using human serum albumin protein.The hydrophobicity of dsRNA agent measured by the unbound siRNA fraction in binding assay is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45 or greater than 0.5 to enhance the in vivo delivery of siRNA.

[0019] In some embodiments, the lipophilic moiety is an aliphatic, cyclic, such as alicyclic, or polycyclic, such as polycyclic alicyclic, compound, such as a steroid (e.g., sterol) or a linear or branched aliphatic hydrocarbon. Exemplary lipophilic moieties are lipids, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxytrityl, or phenoxazine.

[0020] Suitable lipophilic moieties are saturated or unsaturated C4-C 30 Hydrocarbon chains (e.g., C4 to C 30 In some embodiments, the lipophilic moiety is a saturated or unsaturated C4-C6 functional group. 18 Hydrocarbon chains (e.g., linear C4-C 18 In some embodiments, the lipophilic moiety contains a saturated or unsaturated C-C 18 Hydrocarbon chains (e.g., linear C6-C 18 In one embodiment, the lipophilic moiety contains a saturated or unsaturated C 16 Hydrocarbon chains (e.g., linear C 16 alkyl or alkenyl).

[0021] In some embodiments, the lipophilic moiety is a C4-C 30 Acid or C4-C 18acids (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-docosahexaenoic acid, vitamin A, vitamin E, cholesterol, etc.). In some embodiments, the lipophilic moiety is a C4-C6 30 Alcohol or C4-C 18 Alcohols (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-docosahexanose, retinol, vitamin E, cholesterol, etc.).

[0022] In some embodiments, a ligand (e.g., an ASGPR ligand) or lipophilic moiety can be conjugated to any part of a dsRNA agent, such as a nucleobase, a sugar moiety, or an internucleoside linkage. A ligand (e.g., an ASGPR ligand) or lipophilic moiety can be conjugated to a dsRNA agent via direct conjugation to a nucleobase, a ribosugar, or an internucleoside linkage of the dsRNA agent. Alternatively, a ligand (e.g., an ASGPR ligand) or lipophilic moiety can be conjugated to a dsRNA agent via a non-ribose-substituted unit, such as a linker or carrier.

[0023] In certain embodiments, the ligand (eg, an ASGPR ligand) or lipophilic moiety is conjugated to the dsRNA agent via one or more linkers (tethers).

[0024] In some embodiments, the ligand (e.g., ASGPR ligand) or lipophilic moiety is conjugated to the dsRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide thioether, disulfide, phosphodiester, sulfonamide linkage, the product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate.

[0025] In some embodiments, at least one linker (tether) is a redox-cleavable linker (e.g., a reductively cleavable linker; e.g., a disulfide group), an acid-cleavable linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group), an esterase-cleavable linker (e.g., an ester group), a phosphatase-cleavable linker (e.g., a phosphate group), or a peptidase-cleavable linker (e.g., a peptide bond).

[0026] In other embodiments, at least one linker (tether) is a biocleavable linker selected from the group consisting of DNA, RNA, disulfides, amides, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0027] In certain embodiments, the ligand (e.g., ASGPR ligand) or lipophilic moiety is conjugated to the dsRNA agent via a carrier that replaces one or more nucleotides. 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, pyridazinonyl, tetrahydrofuryl, and decalin. In one embodiment, the acyclic group is a moiety based on a serinol skeleton or a diethanolamine skeleton.

[0028] In one embodiment, the ligand (e.g., ASGPR ligand) or lipophilic moiety is conjugated to one or more internal positions of at least one chain, including all positions from each end of the chain except the terminal 2. In one embodiment, the ligand (e.g., ASGPR ligand) or lipophilic moiety is conjugated to one or more internal positions of at least one chain, including all positions from each end of the chain except the terminal 3.

[0029] In one embodiment, at least one lipophilic moiety is conjugated to one or more positions at at least one end of the duplex region, including all positions within the duplex region, but not including the overhang region or carrier replacing the terminal nucleotide at the 3' end of the sense strand. In one embodiment, at least one lipophilic moiety is conjugated to the sense strand within the first five base pairs at the 5' end of the antisense strand of the duplex region.

[0030] In one embodiment, at least one lipophilic moiety is conjugated to the sense strand within the first four base pairs of the 5' end of the antisense strand of the duplex region.

[0031] In one embodiment, at least one lipophilic moiety is conjugated to the sense strand within the first three base pairs of the 5' end of the antisense strand of the duplex region.

[0032] In one embodiment, at least one lipophilic moiety is conjugated to the sense strand within the first two base pairs of the 5' end of the antisense strand of the duplex region.

[0033] In one embodiment, at least one lipophilic moiety is conjugated to the sense strand at the first base pair at the 5' end of the antisense strand of the duplex region.

[0034] In one embodiment, the lipophilic moiety is conjugated to one or more internal positions of at least one strand, excluding the cleavage site region of the sense strand. For example, the internal positions exclude positions 9-12, counting from the 5'-end of the sense strand. For example, the internal positions exclude positions 9-11, counting from the 5'-end of the sense strand. Alternatively, the internal positions exclude positions 11-13, counting from the 3'-end of the sense strand.

[0035] In one embodiment, the lipophilic moiety is conjugated to one or more internal positions of at least one strand, excluding the cleavage site region of the antisense strand, for example, internal positions excluding positions 12-14 counting from the 5' end of the antisense strand.

[0036] In one embodiment, the lipophilic moiety is conjugated to one or more internal positions of at least one strand, excluding positions 11-13 of the sense strand, counting from the 3' end, and positions 12-14 of the antisense strand, counting from the 5' end.

[0037] In one embodiment, 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.

[0038] In one embodiment, one or more lipophilic moieties are conjugated to one or more of the following internal positions: positions 5, 6, 7, 15, and 17 of the sense strand, and positions 15 and 17 of the antisense strand, counting from the 5' end of each strand.

[0039] In some embodiments, the carrier replaces one or more nucleotide(s) of the dsRNA agent.

[0040] In some embodiments, the carrier replaces one or more nucleotide(s) at an internal position(s) of the dsRNA agent.

[0041] In other embodiments, the carrier replaces a nucleotide at the end of the sense strand or antisense strand. In one embodiment, the carrier replaces the terminal nucleotide at the 3' end of the sense strand, thereby functioning as an end cap to protect the 3' end of the sense strand. In one embodiment, the carrier is a cyclic group having an amine, for example, the carrier can be pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxoleanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, or decalinyl.

[0042] In some embodiments, the lipophilic moiety (or a lipophilic monomer comprising a lipophilic moiety and a carrier and / or linker that conjugates the lipophilic moiety to a dsRNA agent) is [ka] [ka] [ka] [In formula: m is an integer from 0 to 8; n is an integer from 1 to 21; R2' is H, OH, F, OMe, O-methoxyalkyl, O-allyl, O-N-methylacetamido, O-dimethylaminoethoxyethyl, or O-aminopropyl; B is a modified or unmodified nucleobase; W is alkyl; and R and R' are each independently H or alkyl. is selected from the group consisting of:

[0043] The above structures of the lipophilic moieties or lipophilic monomers may contain one or more asymmetric centers and therefore may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures, and all such isomeric forms are expressly included.

[0044] In the above structures of the lipophilic moiety or lipophilic monomer, the alkylene chain may contain one or more unsaturated bonds.

[0045] The integer m is 0 to 8. The integer n is 1 to 21. R2' can be any functional group that is an acceptable 2'-modification of the ribose sugar, such as a 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropanyl) modification, a 2'-O-allyl modification, a 2'-C-allyl modification, a 2'-fluoro modification, a 2'-ON-methylacetamido (2'-O-NMA) modification, a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) modification, a 2'-O-aminopropyl (2'-O-AP) modification, or a 2'-ara-F modification. For example, R2' can be H, OH, F, OMe, O-methoxyalkyl, O-allyl, ON-methylacetamido, O-dimethylaminoethoxyethyl, or O-aminopropyl. B is a modified or unmodified nucleobase. W is an alkyl group, such as a 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).

[0046] In certain embodiments, the lipophilic moiety (or a lipophilic monomer comprising a lipophilic moiety and a carrier and / or linker that conjugates the lipophilic moiety to a dsRNA agent) has the formula: [ka] wherein n is 1 to 21 (e.g., 4 to 14) and B is a modified or unmodified nucleobase. In one embodiment, the lipophilic moiety or lipophilic monomer has the formula [ka] It has.

[0047] In certain embodiments, the lipophilic moiety (or a lipophilic monomer comprising a lipophilic moiety and a carrier and / or linker that conjugates the lipophilic moiety to a dsRNA agent) has the formula: [ka] wherein n is 1 to 21 (e.g., 1 to 13) and B is a modified or unmodified nucleobase. In one embodiment, the lipophilic monomer has the structure [ka] by.

[0048] In certain embodiments, the lipophilic moiety (or a lipophilic monomer comprising a lipophilic moiety and a carrier and / or linker that conjugates the lipophilic moiety to a dsRNA agent) has the formula: [ka] wherein n is 1 to 21 (e.g., 1 to 13), and B is a modified or unmodified nucleobase. It has.

[0049] In some embodiments, the sense strand and antisense strand of the dsRNA agent are each 15-30 nucleotides in length. In one embodiment, the sense strand and antisense strand of the dsRNA agent are each 19-25 nucleotides in length. In one embodiment, the sense strand and antisense strand of the dsRNA agent are each 21-23 nucleotides in length.

[0050] In some embodiments, the dsRNA agent includes a single-stranded overhang on at least one of its ends, e.g., a 3' and / or 5' overhang(s) 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.

[0051] In some embodiments, a dsRNA agent can also have blunt ends. In some embodiments, a dsRNA agent includes a single-stranded overhang on one end and a blunt end on the other end.

[0052] In some embodiments, a dsRNA agent has a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand), or vice versa.

[0053] 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.

[0054] In one embodiment, the dsRNA agent has two blunt ends on either end of the iRNA duplex.

[0055] In one embodiment, the sense strand of the dsRNA agent is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, where the strands form a double-stranded region of 21 contiguous base pairs with a 2-nucleotide long single-stranded overhang at the 3' end.

[0056] The dsRNA agent includes at least one TNA (threose nucleic acid) at position 1 of the antisense strand, counting from the 5' end.

[0057] In some embodiments, the TNA is [ka] wherein B is an optionally modified nucleobase; * represents attachment to the internucleotide linkage to the next nucleotide] It has the following structure.

[0058] In some embodiments, the TNA is [ka] wherein B is an optionally modified nucleobase; * represents the bond to the internucleotide linkage to the next nucleotide, and R is optionally substituted alkyl. In some embodiments, the 4'-modified TNA may be a 4'-modified TNA having the structure: [ka] It has the following structure.

[0059] In some embodiments, the TNA is [ka] wherein each R is independently an optionally substituted alkyl; and B is an optionally modified nucleobase; * represents attachment to the internucleotide linkage to the next nucleotide] In some embodiments, at least one R is a linear or branched C1-C3 alkyl. In some embodiments, each R is independently a linear or branched C1-C3 alkyl. In one embodiment, each R is methyl.

[0060] In some embodiments, the TNA is [ka] wherein X is independently O or S; and each of R1 and R2 is independently H, alkyl (e.g., C1-C3 alkyl such as methyl), alkoxy (e.g., C1-C3 alkoxy), alkenyl (e.g., C2-C4 alkenyl such as vinyl), alkynyl (e.g., C2-C4 alkynyl such as ethynyl), aryl, or alkyl substituted with alkoxy (alkoxyalkyl (e.g., C1-C3 alkoxy C1-C3 alkyl)), alkyl substituted with alkenyl (alkenylalkyl (e.g., C2-C4 alkenyl C1-C3 alkyl such as allyl)), or alkyl substituted with alkynyl (alkynylalkyl (e.g., C2-C4 alkynyl C1-C3 alkyl such as propargyl)). In one embodiment, each X is O. In one embodiment, each X is S. In one embodiment, one X is O and the other two Xs are S. In one embodiment, one X is S and the other two X are O. In some embodiments, each of R1 and R2 is independently H, methyl, alkyl, alkoxyalkyl, vinyl, ethynyl, allyl, propargyl, or aryl. B is a natural nucleobase or has a modification as defined herein. In some embodiments, B has the following structure: [ka] It is one of the following.

[0061] In some embodiments, the dsRNA agent is [ka] [wherein X, B, R1, and R2 are defined as in the formula of the TNA triphosphate derivative above] Further included are TNA triphosphate derivatives having the structure:

[0062] In some embodiments, in all of the above TNA formulas, B is a natural nucleobase, e.g., [ka] is.

[0063] In some embodiments, in all of the above TNA formulas, B is a purine having a modification. An exemplary structure of B is: [ka] is.

[0064] In some embodiments, in all of the above TNA formulas, B is a pyrimidine having a modification. Exemplary structures of B are: [ka] is.

[0065] In some embodiments, in all of the above TNA formulas, B is [ka] It has the structure: R b may contain a lipophilic moiety, carbohydrate, folic acid, or glutamate urea (PUPA). For example, R b is saturated or unsaturated C4-C 30 Carbohydrate chains, e.g., saturated or unsaturated C 16 It can be a lipophilic moiety containing a carbohydrate chain.

[0066] In some embodiments, R b is the following: [ka] [ka] It is one of the following.

[0067] In some embodiments, R b teeth, [ka] [In the formula, s is 1 to 10, and Ln is one of the following: [ka] is one of the It has the formula:

[0068] In some embodiments, in the formula of the 4'-modified TNA above, R is selected from the group consisting of alkenyl, alkynyl, aryl, heteroaryl, OR a , halo, NR'R'', and CR'R''R''', where R', R'', and R''' are each independently H, alkyl, halo, or COR a and R a is H, alkyl, or alkoxyalkyl, where each substituent is optionally substituted with one or more of C(O), N(H), halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, lipophilic moiety, carbohydrate, and / or vitamin.

[0069] In some embodiments, in the formula of the 4'-modified TNA above, R is selected from the group consisting of alkenyl, alkynyl, aryl, heteroaryl, OR a methyl optionally substituted with one or more substituents selected from the group consisting of halo, NR'R'', and CR'R''R''', where R', R'', and R''' are each independently H, alkyl, halo, or COR a and R a is H, alkyl, or alkoxyalkyl, where each substituent is optionally substituted with one or more of C(O), N(H), halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, lipophilic moiety, carbohydrate, and / or vitamin.

[0070] In one embodiment, in the formula for the 4'-modified TNA above, R is methyl and R is in the (R) or (S) configuration. [ka] It has the following structure.

[0071] In some embodiments, in the formula of the 4'-modified TNA above, R is methyl substituted with hydroxyl, alkoxy, or alkoxyalkoxy, and R is in the (R) or (S) configuration. In one embodiment, the 4'-modified TNA is [ka] It has the following structure.

[0072] In some embodiments, in the formula of the 4'-modified TNA above, R is a C1-C3 alkyl substituted with one or more halogen groups, and R is in the (R) or (S) configuration. In one embodiment, the 4'-modified TNA is [ka] It has the following structure.

[0073] In some embodiments, in the formula of the 4'-modified TNA above, R is methyl substituted with alkenyl or alkynyl, and R is in the (R) or (S) configuration. In one embodiment, the 4'-modified TNA is [ka] It has the following structure.

[0074] In some embodiments, in the formula of the 4'-modified TNA above, R is methyl substituted with a triazolyl group, the triazolyl group having an N atom optionally substituted with one or more of C(O), N(H), alkyl, alkenyl, alkynyl, aryl, heteroaryl, lipophilic moiety, carbohydrate, and / or vitamin, and R is in the (R) or (S) configuration. In one embodiment, the 4'-modified TNA is [ka] wherein R1 is: [ka] [ka] [ka] [ka] It is one of the n is 1 to 10. It has the following structure.

[0075] In some embodiments, in the formula for the 4'-modified TNA above, R is methyl substituted with an amino group, optionally substituted with one or more of C(O), N(H), halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, lipophilic moiety, carbohydrate, and / or vitamin, and R is in the (R) or (S) configuration. In one embodiment, the 4'-modified TNA is [ka] [In the formula, R1 is the following: [ka] is one of the It has the following structure.

[0076] In some embodiments, the dsRNA agent further comprises a phosphate or phosphomimetic at the 5'-end of the antisense strand. In one embodiment, the phosphomimetic is a 5'-terminal vinylphosphonate (VP).

[0077] In some embodiments, the 5' end of the antisense strand of a dsRNA agent does not contain a phosphate or phosphate mimetic (eg, a 5' terminal phosphate or a 5' terminal vinylphosphonate (VP)).

[0078] In some embodiments, the dsRNA agent further comprises at least one terminal, chiral phosphorus atom.

[0079] Site-specific chiral modifications to internucleotide linkages can be present at the 5'-end, 3'-end, or both the 5'-end and 3'-end of the strand. This is referred to herein as a "terminal" chiral modification. Terminal modifications can be present at the 3'- or 5'-end position of the terminal region, for example, 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 in the sense strand, the antisense strand, or both the sense and antisense strands. Each chirally pure phosphorus atom can be in either the Rp or Sp configuration, or a combination thereof. Details regarding chiral modifications and chirally modified dsRNA agents can be found in PCT / US18 / 67103, filed December 21, 2018, entitled "Chirally-Modified Double-Stranded RNA Agents," which is incorporated herein by reference in its entirety.

[0080] In some embodiments, the dsRNA agent further comprises a terminal chiral modification at the first internucleotide linkage at the 3' end of the antisense strand having the linking phosphorus atom in the Sp configuration; a terminal chiral modification at the first internucleotide linkage at the 5' end of the antisense strand having the linking phosphorus atom in the Rp configuration; and a terminal chiral modification at the first internucleotide linkage at the 5' end of the sense strand having the linking phosphorus atom in either the Rp or Sp configuration.

[0081] In one embodiment, the dsRNA agent further comprises a terminal chiral modification present in the first and second internucleotide linkages at the 3' end of the antisense strand, the terminal chiral modification having the linking phosphorus atom in the Sp configuration; a terminal chiral modification present in the first internucleotide linkage at the 5' end of the antisense strand, the terminal chiral modification having the linking phosphorus atom in the Rp configuration; and a terminal chiral modification present in the first internucleotide linkage at the 5' end of the sense strand, the terminal chiral modification having the linking phosphorus atom in either the Rp or Sp configuration.

[0082] In one embodiment, the dsRNA agent further comprises a terminal chiral modification present in the first, second, and third internucleotide linkages at the 3' end of the antisense strand having the linking phosphorus atom in the Sp configuration; a terminal chiral modification present in the first internucleotide linkage at the 5' end of the antisense strand having the linking phosphorus atom in the Rp configuration; and a terminal chiral modification present in the first internucleotide linkage at the 5' end of the sense strand having the linking phosphorus atom in either the Rp or Sp configuration.

[0083] In one embodiment, the dsRNA agent further comprises a chiral modification at the ends of the first and second internucleotide linkages at the 3' end of the antisense strand, the linking phosphorus atom being in the Sp configuration; a chiral modification at the third internucleotide linkage at the 3' end of the antisense strand, the linking phosphorus atom being in the Rp configuration; a chiral modification at the first internucleotide linkage at the 5' end of the antisense strand, the linking phosphorus atom being in the Rp configuration; and a chiral modification at the first internucleotide linkage at the 5' end of the sense strand, the linking phosphorus atom being in either the Rp or Sp configuration.

[0084] In one embodiment, the dsRNA agent further comprises a chiral modification at the first and second internucleotide linkages at the 3' end of the antisense strand, the linking phosphorus atom being in the Sp configuration; a chiral modification at the first and second internucleotide linkages at the 5' end of the antisense strand, the linking phosphorus atom being in the Rp configuration; and a chiral modification at the first internucleotide linkage at the 5' end of the sense strand, the linking phosphorus atom being in either the Rp or Sp configuration.

[0085] In some embodiments, the internucleotide linkage between the 1st and 2nd nucleotides at the 5' end of the antisense strand is not a phosphorothioate linkage.

[0086] In some embodiments, a dsRNA agent has at least two phosphorothioate internucleotide linkages in the first 5, 4, 3, or 2 nucleotides of the antisense strand (counting from the 5' end).

[0087] In some embodiments, a dsRNA agent has at least two phosphorothioate internucleotide linkages in the first 5, 4, 3, or 2 nucleotides of the antisense strand (counting from the 3' end).

[0088] In some embodiments, the antisense strand comprises two blocks of 1, 2, or 3 phosphorothioate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages.

[0089] In some embodiments, the sense strand comprises at least one phosphorothioate linkage at the 3'-end. In some embodiments, the sense strand comprises at least two phosphorothioate linkages at the 3'-end. In some embodiments, one or more lipophilic monomers are located at the 3'-end of the sense strand. In one embodiment, the first phosphorothioate is between the lipophilic monomer and the first nucleotide from the 3'-end of the sense strand.

[0090] In some embodiments, the sense strand comprises at least two phosphorothioate internucleotide linkages within the first 5, 4, 3, or 2 nucleotides, counting from the 5' end of the sense strand.

[0091] In some embodiments, at least one strand comprises one or more phosphorodithioate (PS2) linkages. In some embodiments, the sense strand comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, and up to all) phosphorodithioate (PS2) linkages. In some embodiments, the antisense strand comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, and up to all) phosphorodithioate (PS2) linkages. In some embodiments, both the sense strand and the antisense strand comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, and up to all) phosphorodithioate (PS2) linkages.

[0092] In some embodiments, the antisense strand comprises at least two phosphorodithioate internucleotide linkages within the first 5, 4, 3, or 2 nucleotides of the antisense strand (counting from the 5' end). In some embodiments, the antisense strand comprises at least two phosphorodithioate internucleotide linkages within the first 5, 4, 3, or 2 nucleotides of the antisense strand (counting from the 3' end).

[0093] In some embodiments, the sense strand comprises at least one phosphorodithioate linkage at its 3'-end. In some embodiments, the sense strand comprises at least two phosphorodithioate linkages at its 3'-end. In some embodiments, one or more lipophilic monomers are located at the 3'-end of the sense strand. In one embodiment, the first phosphorodithioate linkage is between the lipophilic monomer and the first nucleotide from the 3'-end of the sense strand. In some embodiments, the sense strand comprises at least two phosphorodithioate internucleotide linkages within the first 5, 4, 3, or 2 nucleotides, counting from the 5'-end of the sense strand.

[0094] 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-related protein (LRP) ligand, bEnd.3 cell-binding ligand, transferrin receptor (TfR) ligand, mannose receptor ligand, glucose transporter protein, and LDL receptor ligand.

[0095] In some embodiments, dsRNA agent further comprises targeting ligand that targets the receptor that mediates delivery to ocular tissue.In one embodiment, targeting ligand is selected from the group consisting of trans-retinol, RGD peptide, LDL receptor ligand and carbohydrate-based ligand.In one embodiment, targeting ligand is RGD peptide, such as H-Gly-Arg-Gly-Asp-Ser-Pro-Lys-Cys-OH (SEQ ID NO: 1) or cyclo(-Arg-Gly-Asp-D-Phe-Cys) (SEQ ID NO: 2).

[0096] In some embodiments, the dsRNA agent further comprises the targeting ligand that targets liver tissue.In some embodiments, the targeting ligand is carbohydrate-based ligand.In one embodiment, the targeting ligand is GalNAc conjugate.

[0097] In some embodiments, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of the antisense or sense strand is modified. For example, if 50% of the strand is modified, then 50% of all nucleotides present in the strand contain a modification as described herein.

[0098] In one embodiment, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or essentially 100% of the nucleotides of the dsRNA agent are independently modified with 2'-O-methyl, 2'-O-allyl, 2'-deoxy, or 2'-fluoro.

[0099] In one embodiment, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or essentially 100% of the nucleotides of the dsRNA agent are independently modified with LNA, CeNA, 2'-methoxyethyl, or 2'-deoxy.

[0100] In some embodiments, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or essentially 100% of the nucleotides of the dsRNA agent are 2'-O-methyl modified nucleotides.

[0101] In some embodiments, the dsRNA agent has fewer than 12, fewer than 10, fewer than 8, fewer than 6, fewer than 4, fewer than 2, or no 2'-F modifications in the sense strand. In some embodiments, the dsRNA agent has fewer than 12, fewer than 10, fewer than 8, fewer than 6, fewer than 4, fewer than 2, or no 2'-F modifications in the antisense strand.

[0102] In some embodiments, a dsRNA agent has one or more 2'-F modifications anywhere in the sense or antisense strand.

[0103] In some embodiments, the dsRNA agent has less than 20%, less than 15%, less than 10%, less than 5% non-natural nucleotides, or is substantially free of 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 abasic sugars, abasic cyclics, and open chain alkyls.

[0104] In some embodiments, the dsRNA agent has 80% or more, 85% or more, 90% or more, 95% or more, or essentially 100% or more natural nucleotides. For these embodiments, natural nucleotides can include 2'-OH, 2'-deoxy, and 2'-OMe.

[0105] In one embodiment, the dsRNA agent has sense and antisense strands each having a length of 15-30 nucleotides; includes at least two phosphorothioate internucleotide linkages in the first five nucleotides of the antisense strand (counting from the 5' end); the duplex region is between 19-25 base pairs (preferably 19, 20, 21, or 22); and the dsRNA agent has less than 20%, less than 15%, less than 10%, less than 5% non-natural nucleotides, or is substantially free of non-natural nucleotides.

[0106] In one embodiment, the dsRNA agent has sense and antisense strands each having a length of 15-30 nucleotides; includes at least two phosphorothioate internucleotide linkages in the first five nucleotides of the antisense strand (counting from the 5' end); the duplex region is between 19-25 base pairs (preferably 19, 20, 21, or 22); and the dsRNA agent has 80% or more, 85% or more, 95% or more, or essentially 100% or more natural nucleotides, e.g., those with 2'-OH, 2'-deoxy, or 2'-OMe.

[0107] In some embodiments, the antisense strand contains at least one unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA) modification, for example, in the seed region of the antisense strand. In one embodiment, the seed region is positions 2-8 (or 5-7) of the 5' end of the antisense strand.

[0108] In some embodiments, the dsRNA agent is a dsRNA agent having at least one sense or antisense strand. [ka] The R group in the formula of the 4'-modified TNA can be in the (R) or (S) configuration. In one embodiment, R is methyl. In one embodiment, the 4'-modified TNA contains at least one 4'-modified TNA having the structure: [ka] It has the following structure.

[0109] In some embodiments, the R group of the formula of the 4'-modified TNA is in the (S) configuration (e.g., [ka] and at least one 4'-modified TNA is at one of positions 1-9 and 12-15 of the sense strand, counting from the 5' end of the sense strand.

[0110] In some embodiments, the R group in the formula of the 4'-modified TNA is in the (S) configuration (e.g., [ka] and at least one 4'-modified TNA is at one of positions 3, 5-11, 14, 16, 18, and 20 of the antisense strand, counting from the 5' end of the antisense strand.

[0111] In some embodiments, the R group in the formula of the 4'-modified TNA is in the (R) configuration (e.g., [ka] and at least one 4'-modified TNA is at one of positions 1-10 and 12-21 of the sense strand, counting from the 5' end of the sense strand.

[0112] In some embodiments, the R group in the formula of the 4'-modified TNA is in the (R) configuration (e.g., [ka] and at least one 4'-modified TNA is at one of positions 2-10, 19-21, and 23 of the antisense strand, counting from the 5' end of the antisense strand.

[0113] Another aspect of the present invention relates to a single-stranded oligonucleotide capable of modulating the expression of a target gene, which comprises at least one TNA (threose nucleic acid) at position 1, counting from the 5' end. The single-stranded oligonucleotide comprises one or more lipophilic moieties conjugated, as appropriate, via a linker or carrier.

[0114] Another aspect of the present invention is a single stranded oligonucleotide capable of modulating expression of a target gene, comprising: [ka] [In formula: each R is independently optionally substituted alkyl; B is an optionally modified nucleobase; * represents a bond to an H or internucleotide linkage to the next nucleotide] At least one 4'-modified TNA (threose nucleic acid) having the structure Concerning single-stranded oligonucleotides.

[0115] In all of the above embodiments, the single-stranded oligonucleotide can be an inhibitory single-stranded oligonucleotide, such as an antisense oligonucleotide (ASO), an antimiR (antagomir) oligonucleotide, a microRNA mimic, a supermir, an aptamer, a U1 adaptor, a triplex-forming oligonucleotide, an RNA activator, an immunostimulatory oligonucleotide, a decoy oligonucleotide, a heteroduplex-forming oligonucleotide, or a single-stranded siRNA (ss-siRNA) oligonucleotide.

[0116] In some embodiments, single-stranded oligonucleotide is antisense oligonucleotide (ASO).As used herein, the term " antisense oligonucleotide " refers to the oligomeric compound that is substantially or 100% complementary to the target sequence of interest.The term " antisense strand " includes both the oligomeric compound that is formed by two separate strands, and the antisense region of the unimolecular oligomeric compound that can form hairpin or dumbbell structure.

[0117] All of the above-described embodiments relating to dsRNA agents, TNA modifications, ligands and lipophilic moieties and their conjugation to dsRNA agents in the above-described aspects of the invention relating to (TNA-modified) dsRNA agents are suitable for these aspects of the invention relating to single-stranded oligonucleotides (e.g., antisense oligonucleotides (ASOs)).

[0118] Another aspect of the invention relates to a method of modulating expression of a target gene in a cell, comprising contacting or administering to the cell a dsRNA agent comprising a sense strand and an antisense strand sufficiently complementary to at least a portion of the mRNA of the target gene, each strand having from 14 to 40 nucleotides, and the antisense strand optionally comprising at least one TNA (threose nucleic acid) at position 1, counting from the 5' end.

[0119] Another aspect of the present invention relates to a method for modulating the expression of a target gene in a cell, comprising contacting or administering to the cell a single-stranded oligonucleotide comprising at least one TNA (threose nucleic acid) at position 1, counting from the 5' end. In some embodiments, the single-stranded oligonucleotide can be an inhibitory single-stranded oligonucleotide, such as an antisense oligonucleotide (ASO), an antimiR (antagomir) oligonucleotide, a microRNA mimic, a supermir, an aptamer, a U1 adaptor, a triplex-forming oligonucleotide, an RNA activator, an immunostimulatory oligonucleotide, a decoy oligonucleotide, a heteroduplex-forming oligonucleotide, or a single-stranded siRNA (ss-siRNA) oligonucleotide. In some embodiments, the single-stranded oligonucleotide is an antisense oligonucleotide (ASO).

[0120] All of the above-described embodiments of dsRNA agents, single-stranded oligonucleotides, TNA modifications, ligands and lipophilic moieties and their conjugation to dsRNA agents or single-stranded oligonucleotides in the above-described aspects of the invention relating to dsRNA agents (modified with TNAs) and single-stranded oligonucleotides (modified with TNAs) are suitable for this aspect of the invention relating to methods of modulating expression of a target gene in a cell.

[0121] In one embodiment, the cells are extrahepatic cells.

[0122] In one embodiment, the cell is a liver cell.

[0123] Another aspect of the invention relates to a method of modulating expression of a target gene in a subject, comprising administering to the subject a dsRNA agent comprising a sense strand and an antisense strand sufficiently complementary to at least a portion of the mRNA of the target gene, each strand having from 14 to 40 nucleotides, and the antisense strand optionally comprising at least one TNA (threose nucleic acid) at position 1, counting from the 5' end.

[0124] Another aspect of the present invention relates to a method for modulating the expression of a target gene in a subject, comprising administering to the subject a single-stranded oligonucleotide comprising at least one TNA (threose nucleic acid), optionally at position 1, counting from the 5' end. In some embodiments, the single-stranded oligonucleotide can be an inhibitory single-stranded oligonucleotide, such as an antisense oligonucleotide (ASO), an antimiR (antagomir) oligonucleotide, a microRNA mimic, a supermir, an aptamer, a U1 adaptor, a triplex-forming oligonucleotide, an RNA activator, an immunostimulatory oligonucleotide, a decoy oligonucleotide, a heteroduplex-forming oligonucleotide, or a single-stranded siRNA (ss-siRNA) oligonucleotide. In some embodiments, the single-stranded oligonucleotide is an antisense oligonucleotide (ASO).

[0125] All of the above-described embodiments relating to dsRNA agents, single-stranded oligonucleotides, TNA modifications, ligands and lipophilic moieties and their conjugation to dsRNA agents or single-stranded oligonucleotides in the above-described aspects of the invention relating to dsRNA agents (modified with TNAs) or single-stranded oligonucleotides (modified with TNAs) are suitable for this aspect of the invention relating to a method of modulating expression of a target gene in a subject.

[0126] In some embodiments, the dsRNA agent is administered extrahepatically.

[0127] In some embodiments, the dsRNA agent is administered to the liver.

[0128] In one embodiment, dsRNA agent is administered intrathecally.By administering dsRNA agent intrathecally, this method can reduce the expression of target gene in brain or spinal tissue, for example, cortex, cerebellum, cervical vertebrae, lumbar vertebrae and thoracic vertebrae.

[0129] In some embodiments, exemplary target genes are APP, ATXN2, C9orf72, TARDBP, MAPT (Tau), HTT, SNCA, FUS, ATXN3, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, TTR, SCN9A, LRRK2, GPR75, APOE, SCD5 and GSK3α.To reduce the expression of these target genes in subject, dsRNA agent can be administered intravitreally.By administering dsRNA agent intravitreally, method can reduce the expression of target genes in ocular tissue.

[0130] Another aspect of the invention relates to a method of treating a subject having a CNS disorder, the method comprising administering to the subject a therapeutically effective amount of a double-stranded RNA agent, thereby treating the subject, the dsRNA agent comprising a sense strand and an antisense strand sufficiently complementary to at least a portion of an mRNA of a target gene, each strand having 14 to 40 nucleotides, and the antisense strand comprising at least one TNA (threose nucleic acid) at position 1, counting from the 5' end.

[0131] Another aspect of the present invention relates to a method for treating a subject with a CNS disorder, comprising administering a therapeutically effective amount of a single-stranded oligonucleotide to the subject, thereby treating the subject. The single-stranded oligonucleotide comprises at least one TNA (threose nucleic acid), optionally at position 1, counting from the 5' end. In some embodiments, the single-stranded oligonucleotide may be an inhibitory single-stranded oligonucleotide, such as an antisense oligonucleotide (ASO), an antimiR (antagomir) oligonucleotide, a microRNA mimic, a supermir, an aptamer, a U1 adaptor, a triplex-forming oligonucleotide, an RNA activator, an immunostimulatory oligonucleotide, a decoy oligonucleotide, a heteroduplex-forming oligonucleotide, or a single-stranded siRNA (ss-siRNA) oligonucleotide. In some embodiments, the single-stranded oligonucleotide is an antisense oligonucleotide (ASO).

[0132] All of the above-described embodiments of dsRNA agents, single-stranded oligonucleotides, TNA modifications, ligands, and lipophilic moieties, and their conjugation to dsRNA agents or single-stranded oligonucleotides, in the above-described aspects of the invention relating to dsRNA agents (modified with TNAs) or single-stranded oligonucleotides (modified with TNAs), are suitable for this aspect of the invention relating to methods for treating a subject with a CNS disorder. Exemplary CNS disorders that can be treated by the methods of the invention include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Huntington's disease, Parkinson's disease, spinocerebellar ataxia, prion, and Lafora. [Brief explanation of the drawings]

[0133]

Figure 1A

Figure 1B

[0134]

Figure 2

[0135]

Figure 3

[0136]

Figure 4

[0137]

Figure 5

[0138]

Figure 6

[0139]

Figure 7

[0140] The present inventors have found that, in particular, TNA modification is well tolerated at the 1st position of the antisense strand of double-stranded RNA (dsRNA) agent, and even when TNA-modified dsRNA agent is loaded into RISC in cells, it provides a modified dsRNA agent with high silencing activity, and usually requires 5'-terminal phosphate or phosphate mimicking modification (for example, 5'-VP).The present inventors have surprisingly found that when TNA is introduced at the 1st position of antisense strand, regardless of whether phosphate group is pre-installed at the 5'-end of antisense strand, the silencing activity of free incorporation is equivalent to that of siRNA with terminal 5'-phosphorylation.This discovery makes TNA-modified dsRNA agent particularly useful in extrahepatic delivery, such as CNS targeting.

[0141] One aspect of the present invention provides a dsRNA agent capable of modulating expression of a target gene, the dsRNA agent comprising a sense strand and an antisense strand that is sufficiently complementary to at least a portion of the mRNA of the target gene, each strand having 14 to 40 nucleotides, and the antisense strand comprising at least one TNA (threose nucleic acid) at position 1, counting from the 5' end.

[0142] Another aspect of the invention is a double-stranded RNA (dsRNA) agent capable of modulating expression of a target gene, comprising a sense strand and an antisense strand that is sufficiently complementary to at least a portion of the mRNA of the target gene, each strand having 14 to 40 nucleotides: the sense strand contains at least one 2'-OMe modification at position 1, counting from the 5' end; The antisense strand contains at least one TNA (threose nucleic acid) at position 1, counting from the 5' end. Double-stranded RNA agents are provided.

[0143] Another aspect of the present invention relates to a double-stranded RNA (dsRNA) agent capable of modulating the expression of a target gene, the double-stranded RNA agent comprising a sense strand and an antisense strand sufficiently complementary to at least a portion of the mRNA of the target gene. The antisense strand comprises at least one TNA (threose nucleic acid) at position 1, counting from the 5' end. The dsRNA agent comprises one or more lipophilic moieties conjugated to at least one strand, optionally via a linker or carrier.

[0144] Another aspect of the invention is a double-stranded RNA (dsRNA) agent capable of modulating expression of a target gene, comprising a sense strand and an antisense strand that is sufficiently complementary to at least a portion of the mRNA of the target gene, each strand having 14 to 40 nucleotides; One of the sense or antisense strands is [ka] [In formula: each R is independently optionally substituted alkyl; B is an optionally modified nucleobase; * represents a bond to an H or internucleotide linkage to the next nucleotide] At least one 4'-modified TNA (threose nucleic acid) having the structure Relating to double-stranded RNA agents.

[0145] TNA modification Alpha-(L)-threofuranosyl nucleic acid (TNA) with a (3'-2') phosphodiester backbone is a nucleic acid surrogate (Schoning et al. Science 290: 1347 (2000), which is incorporated herein by reference in its entirety). The sugar-phosphate backbone of TNA, and for comparison, RNA, is shown in the following scheme: [ka]

[0146] TNAs consist of an unnatural four-carbon threose sugar and possess a unique sugar-phosphate backbone that allows the formation of a stable, antiparallel Watson-Crick duplex structure. TNAs also exhibit efficient cross-pairing with complementary strands of DNA and RNA and exhibit strong nuclease stability under biologically relevant conditions.

[0147] The dsRNA agent includes at least one TNA (threose nucleic acid) at position 1 of the antisense strand, counting from the 5' end.

[0148] In some embodiments, the TNA is [ka] wherein B is an optionally modified nucleobase; * represents attachment to the internucleotide linkage to the next nucleotide] It has the following structure.

[0149] Exemplary TNA monomers are shown below. [Table 1] [Table 2]

[0150] In some embodiments, the TNA is [ka] wherein each X is independently O or S; each of R1 and R2 is independently H, alkyl (e.g., C1-C3 alkyl such as methyl), alkoxy (e.g., C1-C3 alkoxy), alkenyl (e.g., C2-C4 alkenyl such as vinyl), alkynyl (e.g., C2-C4 alkynyl such as ethynyl), aryl, or alkyl substituted with alkoxy (alkoxyalkyl (e.g., C1-C3 alkoxy C1-C3 alkyl), alkyl substituted with alkenyl (alkenylalkyl (e.g., C2-C4 alkenyl C1-C3 alkyl such as allyl), or alkyl substituted with alkynyl (alkynylalkyl In one embodiment, each X is O. In one embodiment, each X is S. In one embodiment, one X is O and the other two Xs are S. In one embodiment, one X is S and the other two Xs are O. In some embodiments, each of R1 and R2 is independently H, methyl, alkyl, alkoxyalkyl, vinyl, ethynyl, allyl, propargyl, or aryl. B is a natural nucleobase or has a modification as defined herein. In some embodiments, B has the following structure: [ka] It is one of the following.

[0151] In some embodiments, the dsRNA agent is [ka] [wherein X, B, R1, and R2 are defined as in the formula of the TNA triphosphate derivative above] Further included are TNA triphosphate derivatives having the structure:

[0152] TNA triphosphate derivatives may be useful in the enzymatic synthesis of oligonucleotides. Enzymatic oligonucleotide synthesis using template-independent or template-dependent polymerases has recently reemerged (see Mathews et al., "Photo-cleavable nucleotides for primer-free enzyme-mediated DNA synthesis." Org. Biomol. Chem. 14: 8278-88 (2016); Mathews et al., "3'-O-Caged 2'-Deoxynucleoside Triphosphates for Light-Mediated, Enzyme-Catalyzed, Template-Independent DNA Synthesis." Curr. Protoc. Nucleic Acid Chem. 71: 13.17.1-13.17.38 (2017); Palluk et al., "De novo DNA synthesis using polymerase-nucleotide conjugates." Nat. Biotechnol. 36: 645-650 (2018), which are incorporated by reference in their entireties). Enzymatic oligonucleotide synthesis methods can offer certain advantages over oligonucleotide synthesis: enzymatic synthesis reactions are carried out under hydrated and mild conditions; coupled with the specificity of the enzyme, enzymatic synthesis reduces the formation of by-products, DNA depurination, and other damage, allowing for the direct synthesis of long oligonucleotides. For example, enzymatic oligonucleotide synthesis can use a polymerase such as terminal deoxynucleotidyl transferase (TdT) to catalyze the stepwise addition of nucleotides to the 3'-OH of an oligonucleotide primer. Therefore, TNA triphosphate derivatives can be useful as polymerases or as part of polymerases that catalyze chain elongation reactions in enzymatic oligonucleotide synthesis.

[0153] In some embodiments, the TNA is [ka] wherein B is an optionally modified nucleobase; * represents the bond to the internucleotide linkage to the next nucleotide, and R is optionally substituted alkyl. In some embodiments, the 4'-modified TNA has the structure: [ka] It has the following structure.

[0154] In some embodiments, the TNA is [ka] wherein each R is independently an optionally substituted alkyl; and B is an optionally modified nucleobase; * represents attachment to the internucleotide linkage to the next nucleotide] In some embodiments, at least one R is a linear or branched C1-C3 alkyl. In some embodiments, each R is independently a linear or branched C1-C3 alkyl. In one embodiment, each R is methyl.

[0155] In some embodiments, in the formula of the 4'-modified TNA above, R is selected from the group consisting of alkenyl, alkynyl, aryl, heteroaryl, OR a , halo, NR'R'', and CR'R''R''', where R', R'', and R''' are each independently H, alkyl, halo, or COR a and R a is H, alkyl, or alkoxyalkyl, where each substituent is optionally substituted with one or more of C(O), N(H), halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, lipophilic moiety, carbohydrate, and / or vitamin.

[0156] In some embodiments, in the formula of the 4'-modified TNA above, R is selected from the group consisting of alkenyl, alkynyl, aryl, heteroaryl, OR a methyl optionally substituted with one or more substituents selected from the group consisting of halo, NR'R'', and CR'R''R''', where R', R'', and R''' are each independently H, alkyl, halo, or COR a and R a is H, alkyl, or alkoxyalkyl, where each substituent is optionally substituted with one or more of C(O), N(H), halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, lipophilic moiety, carbohydrate, and / or vitamin.

[0157] In some embodiments, in the formula of the 4'-modified TNA above, R is methyl substituted with hydroxyl, alkoxy, or alkoxyalkoxy, and R is in the (R) or (S) configuration. In one embodiment, the 4'-modified TNA is [ka] It has the following structure.

[0158] In some embodiments, in the formula of the 4'-modified TNA above, R is a C1-C3 alkyl substituted with one or more halogen groups, and R is in the (R) or (S) configuration. In one embodiment, the 4'-modified TNA is [ka] It has the following structure.

[0159] In some embodiments, in the formula of the 4'-modified TNA above, R is methyl substituted with alkenyl or alkynyl, and R is in the (R) or (S) configuration. In one embodiment, the 4'-modified TNA is [ka] It has the following structure.

[0160] In some embodiments, in the formula of the 4'-modified TNA above, R is methyl substituted with a triazolyl group, the triazolyl group having an N atom optionally substituted with one or more of C(O), N(H), alkyl, alkenyl, alkynyl, aryl, heteroaryl, lipophilic moiety, carbohydrate, and / or vitamin, and R is in the (R) or (S) configuration. In one embodiment, the 4'-modified TNA is [ka] wherein R1 is: [ka] [ka] [ka] [ka] [ka] It is one of the n is 1 to 10. It has the following structure.

[0161] In some embodiments, in the formula for the 4'-modified TNA above, R is methyl substituted with an amino group, optionally substituted with one or more of C(O), N(H), halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, lipophilic moiety, carbohydrate, and / or vitamin, and R is in the (R) or (S) configuration. In one embodiment, the 4'-modified TNA is [ka] [In the formula, R1 is the following: [ka] is one of the It has the following structure.

[0162] In some embodiments, in all of the above TNA formulas, B is a natural nucleobase, e.g., [ka] is.

[0163] In some embodiments, in all of the above TNA formulas, B is a purine having a modification. An exemplary structure of B is: [ka] is.

[0164] In some embodiments, in all of the above TNA formulas, B is a pyrimidine having a modification. Exemplary structures of B are: [ka] is.

[0165] In some embodiments, in all of the above TNA formulas, B is [ka] It has the structure: R b may contain a lipophilic moiety, carbohydrate, folic acid, or glutamate urea (PUPA). For example, R b is saturated or unsaturated C4-C 30 Carbohydrate chains, e.g., saturated or unsaturated C 16 It can be a lipophilic moiety containing a carbohydrate chain.

[0166] In some embodiments, R b is the following: [ka] [ka] It is one of the following.

[0167] In some embodiments, R b teeth, [ka] [In the formula, s is 1 to 10, and Ln is one of the following: [ka] is one of the It has the formula:

[0168] lipophilic part The terms "lipophilic" or "lipophilic moiety" refer broadly to any compound or chemical moiety that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is by the octanol-water partition coefficient, logK ow By K ow is the ratio of the concentration of a chemical in the octanol phase to its concentration in the aqueous phase of a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it can also be predicted by using coefficients attributed to the structural components of a chemical, calculated using first-principles or experimental methods (see, e.g., Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), the entire contents of which are incorporated herein by reference). It provides a thermodynamic measure of a substance's tendency to prefer non-aqueous or oil-based environments over water (i.e., its hydrophilic / lipophilic balance). In principle, a chemical can be expressed as a function of its logK ow is greater than 0, the property is lipophilic. Typically, a lipophilic moiety has a log K ow For example, the log K of 6-aminohexanol ow For example, the log K of cholesteryl N-(hexan-6-ol) carbamate is predicted to be approximately 0.7. owis predicted to be 10.7.

[0169] The lipophilicity of a molecule can be modified with respect to the functional groups it possesses. For example, the addition of a hydroxyl or amine group to the terminus of a lipophilic moiety can increase the partition coefficient (e.g., logK ow ) value may be increased or decreased.

[0170] Alternatively, the hydrophobicity of the dsRNA agent that is conjugated with one or more lipophilic moieties can be measured by its protein binding properties.For example, the unbound fraction in the plasma protein binding assay of dsRNA agent can be determined to be positively correlated with the relative hydrophobicity of dsRNA agent, and this can be positively correlated with the silencing activity of dsRNA agent.

[0171] In one embodiment, the plasma protein binding assay that is determined is electrophoretic mobility shift assay (EMSA) that uses human serum albumin protein.The exemplary protocol of this binding assay is detailed in Example 14 of WO2019 / 217459, which is incorporated herein by reference in its entirety.The hydrophobicity of dsRNA agent, as measured by the fraction of unbound siRNA in binding assay, is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45 or greater than 0.5, for enhancing the in vivo delivery of siRNA.

[0172] Thus, conjugation of a lipophilic moiety to a dsRNA agent provides optimal hydrophobicity for enhancing in vivo delivery of the siRNA.

[0173] In certain embodiments, the lipophilic moiety is an aliphatic, cyclic, such as an alicyclic, or polycyclic, such as a polycyclic alicyclic, compound, such as a steroid (e.g., a sterol) or a linear or branched aliphatic hydrocarbon. The lipophilic moiety generally comprises a hydrocarbon chain, which may be cyclic or acyclic. The hydrocarbon chain may contain various substituents and / or one or more heteroatoms, such as oxygen or nitrogen atoms. Such lipophilic aliphatic moieties may comprise saturated or unsaturated C4-C6 30 Hydrocarbon chains (e.g., C4 to C 18 hydrocarbons), saturated or unsaturated fatty acids, waxes (e.g., monohydric alcohol esters of fatty acids and fatty diamides), terpenes (e.g., C 10 Terpene, C 15 Sesquiterpene, C 20 Diterpenes, C 30 Triterpenes, and C 40 tetraterpenes) and other polycyclic alicyclic hydrocarbons. For example, the lipophilic moiety may be C4-C 30 Hydrocarbon chains (e.g., C4 to C 30 In some embodiments, the lipophilic moiety may contain a saturated or unsaturated C-C 18 Hydrocarbon chains (e.g., linear C4-C 18 In some embodiments, the lipophilic moiety contains a saturated or unsaturated C-C 18 Hydrocarbon chains (e.g., linear C6-C 18 In one embodiment, the lipophilic moiety contains a saturated or unsaturated C 16 Hydrocarbon chains (e.g., linear C 16 alkyl or alkenyl).

[0174] Lipophilic moieties can be conjugated to dsRNA agents by any method known in the art, including via functional groups already present on the lipophilic moiety or introduced into the iRNA agent, such as hydroxy groups (e.g., -CO-CH2-OH). Functional groups already present on the lipophilic moiety or introduced into the dsRNA agent include, but are not limited to, hydroxyl, amine, carboxylic acid, sulfonic acid, phosphate, thiol, azide, and alkyne.

[0175] The conjugation of a dsRNA agent and a lipophilic moiety can be, for example, by forming an ether or carboxylic acid or carbamoyl ester linkage between a hydroxyl group and an alkyl group R-, an alkanoyl group RCO-, or a substituted carbamoyl group RNHCO-. The alkyl group R can be cyclic (e.g., cyclohexyl) or acyclic (e.g., straight-chain or branched-chain; and saturated or unsaturated). The alkyl group R can be butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, or octadecyl group, etc.

[0176] In some embodiments, the lipophilic moiety is conjugated to the dsRNA agent via a linker, where the linker contains an ether, thioether, urea, carboxylic acid, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or carbamate.

[0177] In some embodiments, the lipophilic moiety is a steroid such as a sterol. Steroids are polycyclic compounds containing a perhydro-1,2-cyclopentanophenanthrene ring system. Steroids include, but are not limited to, 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 compound derived from cholesterol, for example, by substitution, addition, or removal of a substituent.

[0178] In some embodiments, the lipophilic moiety is an aromatic moiety. In this context, the term "aromatic" broadly refers to monocyclic and polycyclic aromatic hydrocarbons. Aromatic groups are C6-C6 groups containing 1-3 aromatic rings, which may be optionally substituted. 14 "Aryl" or "arylalkyl" groups, which comprise an aryl group covalently bonded to an alkyl group, either of which may be independently substituted or unsubstituted as appropriate; and "heteroaryl" groups. As used herein, the term "heteroaryl" refers to groups having 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms; having 6, 10, or 14 pi electrons shared in a cyclic arrangement; and having, in addition to carbon atoms, between one and about three heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and sulfur (S).

[0179] As used herein, a "substituted" alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclic group is one having between 1 and about 4, preferably between 1 and about 3, and more preferably 1 or 2 non-hydrogen substitutions. Suitable 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, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido groups.

[0180] In some embodiments, the lipophilic moiety is an aralkyl group, such as a 2-arylpropanoyl moiety. The structural characteristics of the aralkyl group are selected so that the lipophilic moiety binds to at least one protein in vivo. In certain embodiments, the structural characteristics of the aralkyl group are selected so that the lipophilic moiety binds to serum, blood vessels, or cellular proteins. In certain embodiments, the structural characteristics of the aralkyl group promote binding to albumin, immunoglobulins, lipoproteins, α-2-macroglobulin, or α-1-glycoprotein.

[0181] In certain embodiments, the ligand is naproxen or a structural derivative of naproxen. Synthetic procedures for naproxen can be found in U.S. Pat. Nos. 3,904,682 and 4,009,197, which are incorporated herein by reference in their entireties. Naproxen has the chemical name (S)-6-methoxy-α-methyl-2-naphthaleneacetic acid and the structure [ka] is.

[0182] In certain embodiments, the ligand is ibuprofen or a structural derivative of ibuprofen. The synthesis procedure for ibuprofen can be found in U.S. Pat. No. 3,228,831, the entire contents of which are incorporated herein by reference. The structure of ibuprofen is: [ka] is.

[0183] Further exemplary aralkyl groups are exemplified in US Pat. No. 7,626,014, which is incorporated herein by reference in its entirety.

[0184] In another embodiment, suitable lipophilic moieties include lipids, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl groups, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxytrityl, or phenoxazine.

[0185] In some embodiments, the lipophilic moiety is a C-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, linoleic acid, arachidonic acid, cis-4,7,10,13,16,19-docosahexanoic acid, vitamin A, vitamin E, cholesterol, etc.) or C6 to C 30Alcohols (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.).

[0186] In some embodiments, the lipophilic moiety (or lipophilic monomer comprising the lipophilic moiety and a carrier and / or linker that conjugates the lipophilic moiety to the dsRNA agent) is [ka] [ka] [In formula: m is an integer from 0 to 8; n is an integer from 1 to 21; R2' is H, OH, F, OMe, O-methoxyalkyl, O-allyl, O-N-methylacetamido, O-dimethylaminoethoxyethyl, or O-aminopropyl; B is a modified or unmodified nucleobase; W is alkyl; and R and R' are each independently H or alkyl. is selected from the group consisting of:

[0187] The above structures of the lipophilic moieties or lipophilic monomers may also contain one or more asymmetric centers and therefore may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms are expressly included.

[0188] In the above structures of the lipophilic moiety or lipophilic monomer, the alkylene chain may contain one or more unsaturated bonds.

[0189] The integer m is 0 to 8. The integer n is 1 to 21. R2' can be any functional group that is an acceptable 2'-modification of the ribose sugar, such as a 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropanyl) modification, a 2'-O-allyl modification, a 2'-C-allyl modification, a 2'-fluoro modification, a 2'-ON-methylacetamido (2'-O-NMA) modification, a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) modification, a 2'-O-aminopropyl (2'-O-AP) modification, or a 2'-ara-F modification. For example, R2' can be H, OH, F, OMe, O-methoxyalkyl, O-allyl, ON-methylacetamido, O-dimethylaminoethoxyethyl, or O-aminopropyl. B is a modified or unmodified nucleobase. W is an alkyl group, such as a 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).

[0190] In some embodiments, the lipophilic moiety conjugated to one or more positions of the chain of the compound (or a lipophilic monomer comprising a lipophilic moiety and a carrier and / or linker that conjugates the lipophilic moiety to the dsRNA agent) is [ka] [ka] [ka] In these structures, B is a modified or unmodified nucleobase.

[0191] Particular embodiments of a lipophilic moiety (or a lipophilic monomer comprising a lipophilic moiety and a carrier and / or linker that conjugates the lipophilic moiety to a dsRNA agent) include: [ka] [ka] [ka] In these structures, B is a modified or unmodified nucleobase; and R and R' are each independently H, methyl, ethyl, isopropyl, or t-butyl.

[0192] In some embodiments, the lipophilic moiety is [ka] The compound is conjugated to a strand (a single strand of a single-stranded oligonucleotide; or the sense and / or antisense strand of a double-stranded oligonucleotide) via a carrier of the formula (I). In these embodiments, R is a lipophilic moiety as defined herein. R2' is H, OH, F, OMe, O-methoxyalkyl, O-allyl, O-N-methylacetamido, O-dimethylaminoethoxyethyl, or O-aminopropyl. B is a modified or unmodified nucleobase.

[0193] In some embodiments, the lipophilic moiety is [ka] The compound is conjugated to an internal position of a strand (the single strand of a single-stranded oligonucleotide; or the sense and / or antisense strand of a double-stranded oligonucleotide) via a carrier of the formula (I). In these embodiments, R is a lipophilic moiety as defined herein. n is an integer from 1 to 21. R2' is H, OH, F, OMe, O-methoxyalkyl, O-allyl, O-N-methylacetamido, O-dimethylaminoethoxyethyl, or O-aminopropyl. B is a modified or unmodified nucleobase.

[0194] Further examples of lipophilic monomers can be found in the Examples of WO2021 / 092371, which is incorporated herein by reference in its entirety.

[0195] In certain embodiments, more than one lipophilic moiety can be incorporated into a dsRNA agent, particularly when the lipophilic moiety has low lipophilicity or hydrophobicity. In one embodiment, two or more lipophilic moieties are incorporated into the same strand of a double-stranded iRNA agent. In one embodiment, each strand of a dsRNA agent has one or more lipophilic moieties incorporated therein. In one embodiment, two or more lipophilic moieties are incorporated into the same position of a dsRNA agent (i.e., the same nucleobase, the same sugar moiety, or the same internucleoside linkage). This can be achieved, for example, by conjugating two or more lipophilic moieties via a carrier, and / or by conjugating two or more lipophilic moieties via a branched linker, and / or by conjugating two or more lipophilic moieties via one or more linkers, with one or more linkers connecting the lipophilic moieties consecutively.

[0196] In some embodiments, lipophilic moieties can be conjugated to any part of dsRNA agents, such as nucleobases, sugar moieties, or internucleoside linkages.Lipophilic moieties can be conjugated to dsRNA agents via direct conjugation to nucleobases, ribosugars, or internucleoside linkages of dsRNA agents.Alternatively, lipophilic moieties can be conjugated to dsRNA agents via linkers or carriers.

[0197] In certain embodiments, the lipophilic moiety can be conjugated to the dsRNA agent via one or more linkers (tethers).

[0198] In one embodiment, the lipophilic moiety is conjugated to the dsRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, the product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate.

[0199] Linker / Tether The linker / tether is connected to the lipophilic moiety at a "tethering junction point (TAP)." The linker / tether can be any C1-C 100 Carbon-containing moieties (e.g., C1-C 75 , C1~C 50 , C1~C 20 , C1~C 10 ;C1, C2, C3, C4, C5, C6, C7, C8, C9, or C 10 ) and may have at least one nitrogen atom. In certain embodiments, the nitrogen atom forms part of the terminal amino or amide (NHC(O)-) group of the linker / tether, which may serve as an attachment point for a lipophilic moiety. Non-limiting examples of linkers / tethers (underlined) include TAP- (CH 2 ) n NH- ;TAP- C(O)(CH 2 ) n NH- ;TAP- NR’’’’(CH 2) n NH- , TAP- C(O)-(CH 2 ) n -C(O)- ;TAP- C(O)-(CH 2 ) n -C(O)O- ;TAP- C(O)-O- ;TAP- C(O)-(CH 2 ) n -NH-C(O)- ;TAP- C(O)-(CH 2 ) n - ;TAP- C(O)-NH- ;TAP- C(O)- ;TAP- (CH 2 ) n -C(O)- ;TAP- (CH 2 ) n -C(O)O- ;TAP- (CH 2 ) n - ; or TAP- (CH 2 ) n -NH-C(O)- wherein n is 1-20 (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 C1-C6 alkyl. Preferably, n is 5, 6, or 11. In other embodiments, the nitrogen may form part of a terminal oxyamino group, e.g., -ONH2, or a hydrazino group, -NHNH2. The linker / tether may be substituted, e.g., by hydroxy, alkoxy, perhaloalkyl, and / or may incorporate one or more additional heteroatoms, e.g., N, O, or S. Preferred tethered ligands include, for example, TAP- (CH 2 ) n NH(LIGAND) ;TAP- C(O)(CH 2 ) n NH(LIGAND) ;TAP- NR’’’’(CH 2 )n NH(LIGAND) ;TAP- (CH 2 ) n ONH(LIGAND) ;TAP- C(O)(CH 2 ) n ONH(LIGAND) ;TAP- NR’’’’(CH 2 ) n ONH(LIGAND) ;TAP- (CH 2 ) n NHNH 2 (LIGAND) ,TAP- C(O)(CH 2 ) n NHNH 2 (LIGAND) ;TAP- NR’’’’(CH 2 ) n NHNH 2 (LIGAND) ;TAP- C(O)-(CH 2 ) n -C(O)(LIGAND) ;TAP- C(O)-(CH 2 ) n -C(O)O(LIGAND) ;TAP- C(O)-O(LIGAND) ;TAP- C(O)-(CH 2 ) n -NH-C(O)(LIGAND) ;TAP- C(O)-(CH 2 ) n (LIGAND) ;TAP- C(O)-NH(LIGAND) ;TAP- C(O)(LIGAND) ;TAP- (CH 2 ) n -C(O)(LIGAND) ;TAP- (CH 2 ) n -C(O)O(LIGAND) ;TAP- (CH 2 ) n (LIGAND) ;またはTAP- (CH 2 ) n -NH-C(O)(LIGAND)In some embodiments, the amino-terminated linker / tether (e.g., NH, ONH, NHNH) can form an imino bond (i.e., C=N) with the ligand. In some embodiments, the amino-terminated linker / tether (e.g., NH, ONH, NHNH) can be acylated, for example, with C(O)CF.

[0200] In some embodiments, the linker / tether can be terminated with a mercapto group (i.e., SH) or an olefin (e.g., CH=CH). For example, the tether can be TAP- (CH 2 ) n -SH , TAP- C(O)(CH 2 ) n SH , TAP- (CH 2 ) n -(CH=CH 2 ) , or TAP- C(O)(CH 2 ) n (CH=CH 2 ) where n can be as described elsewhere herein. The tether can be optionally substituted, for example, by hydroxy, alkoxy, perhaloalkyl, and / or can optionally contain one or more additional heteroatoms, for example, N, O, or S. The double bond can be cis or trans, or E or Z.

[0201] In other embodiments, the linker / tether may include an electrophilic moiety, preferably at a terminal position of the linker / tether. Exemplary electrophilic moieties include, for example, an aldehyde, an alkyl halide, a mesylate, a tosylate, a nosylate, or a brosylate, or an activated carboxylic acid ester, for example, an NHS ester, or a pentafluorophenyl ester. A preferred linker / tether (underlined) is TAP- (CH 2 ) n CHO ;TAP- C(O)(CH 2) n CHO ; or TAP- NR’’’’(CH 2 ) n CHO (wherein n is 1 to 6 and R'''' is C1 to C6 alkyl); or TAP- (CH 2 ) n C(O)ONHS ;TAP- C(O)(CH 2 ) n C(O)ONHS ; or TAP- NR’’’’(CH 2 ) n C(O)ONHS (wherein n is 1 to 6 and R'''' is C1 to C6 alkyl); TAP- (CH 2 ) n C(O)OC 6 F 5;TAP- C(O)(CH 2 ) n C(O)OC 6 F 5; or TAP- NR’’’’(CH 2 ) n C(O)OC 6 F 5 (wherein n is 1 to 11 and R'''' is C1 to C6 alkyl); or -(CH 2 ) n CH 2 LG ;TAP- C(O)(CH 2 ) n CH 2 LG ; or TAP- NR’’’’(CH 2 ) n CH 2 LG [where n is as defined elsewhere herein and R"" comprises a C1-C6 alkyl (LG can release a group such as a halide, mesylate, tosylate, nosylate, or brosylate)]. Tethering can be achieved by coupling a nucleophilic group on the ligand, such as a thiol or amino group, with an electrophilic group on the tether.

[0202] In other embodiments, it may be desirable for the monomer to include a phthalimide group (K) at the terminal position of the linker / tether. [ka]

[0203] In other embodiments, other protected amino groups, such as alloc, monomethoxytrityl (MMT), trifluoroacetyl, Fmoc, or arylsulfonyl (e.g., the aryl moiety can be ortho-nitrophenyl, or ortho, para-dinitrophenyl), can be at the terminal position of the linker / tether.

[0204] Any linker / tether described herein may include one or more additional linking groups, such as -O-(CH) n -, -(CH2) n -SS-, -(CH2) n It may further include - or -(CH=CH)-.

[0205] Cleavable Linker / Tether In some embodiments, at least one linker / tether can be a redox-cleavable linker, an acid-cleavable linker, an esterase-cleavable linker, a phosphatase-cleavable linker, or a peptidase-cleavable linker.

[0206] In one embodiment, at least one linker / tether can be a reductively cleavable linker (eg, a disulfide group).

[0207] In one embodiment, at least one linker / tether can be an acid-cleavable linker (eg, a hydrazone group, an ester group, an acetal group, or a ketal group).

[0208] In one embodiment, at least one linker / tether can be an esterase-cleavable linker (eg, an ester group).

[0209] In one embodiment, at least one linker / tether can be a phosphatase-cleavable linker (eg, a phosphate group).

[0210] In one embodiment, at least one linker / tether can be a peptidase-cleavable linker (eg, a peptide bond).

[0211] The cleavable linking group is sensitive to a cleaving agent, such as pH, redox activity, or the presence of a degradable molecule. Generally, cleaving agents are found more frequently, or at higher levels or activity, inside cells than in serum or blood. Examples of such degrading agents include redox agents that are selective for a specific substrate, or redox agents that do not have substrate specificity, such as mercaptans present inside cells that can degrade redox-cleavable linking groups by reduction; esterases; endosomes; or agents that can create an acidic environment, e.g., a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0212] Cleavable linking groups, such as disulfide bonds, can be pH-sensitive. While the pH of human serum is 7.4, the average intracellular pH is somewhat lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some tethers will have linking groups that cleave at a preferred pH, thereby releasing the dsRNA agent from its internal ligand (e.g., a targeting or cell-permeable ligand, such as cholesterol) or into a desired cellular compartment.

[0213] The chemical bond (e.g., linking group) that connects the ligand to the dsRNA agent can include a disulfide bond.When the iRNA agent / ligand complex is taken up into the 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 can be a second therapeutic agent that can complement the therapeutic effect of the targeting ligand or iRNA agent.

[0214] The 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 targeted by the iRNA agent. For example, a dsRNA agent that targets mRNA in liver cells may be conjugated to a tether containing an ester group. Because liver cells are rich in esterases, the tether will be cleaved more efficiently in liver cells than in cell types that are not rich in esterases. Cleavage of the tether may release the dsRNA agent from the ligand attached to the distal end of the tether, thereby enhancing the silencing activity of the iRNA agent. Other cell types rich in esterases include lung, renal cortex, and testicular cells.

[0215] Tethers containing peptide bonds can be conjugated to iRNA agents to target peptidase-rich cell types, such as liver cells and synovial cells. For example, dsRNA agents that target synovial cells, such as for the treatment of inflammatory diseases (e.g., rheumatoid arthritis), can be conjugated to tethers containing peptide bonds.

[0216] Generally, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degrading agent (or degrading conditions) to cleave the candidate linking group.It may also be desirable to examine the candidate cleavable linking group for its ability to resist cleavage in blood or other non-target tissues, such as the tissues to which the dsRNA agent is exposed when administered to a subject.It may also be desirable to examine the candidate cleavable linking group for its ability to resist cleavage in blood or other non-target tissues.Thus, when the first condition is selected to indicate cleavage in target cells, and the second condition is selected to indicate cleavage in other tissues or body fluids, such as blood or serum, the relative susceptibility to cleavage between the first condition and the second condition can be determined.Evaluation can be carried out in a cell-free system, cells, cell cultures, organ cultures or tissue cultures, or whole animals. It may be useful to perform initial evaluations in cell-free or culture conditions and confirm by further evaluation in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least 2, 4, 10, or 100 times faster in cells (or under in vivo conditions selected to mimic intracellular conditions) than in blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0217] Redox-cleavable linker One class of cleavable linkers is redox-cleavable linkers, which are cleaved upon reduction or oxidation. An example of a reductively cleavable linker is a disulfide linker (-SS-). To determine whether a candidate cleavable linker is a suitable "reductively cleavable linker" or, for example, whether it is suitable for use with a particular iRNA moiety and a particular targeting agent, the methods described herein can be considered. For example, candidate substances can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate observed in cells, e.g., target cells. Candidate substances can also be evaluated under conditions selected to mimic blood or serum conditions. In a preferred embodiment, the candidate compound is cleaved at a maximum of 10% in blood. In preferred embodiments, useful candidate compounds are degraded at least 2, 4, 10, or 100 times faster in cells (or under in vivo conditions selected to mimic intracellular conditions) than in blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic the intracellular media and compared to conditions selected to mimic the extracellular media.

[0218] Phosphate-based cleavable linkers Phosphate-based linking groups are cleaved by agents that degrade or hydrolyze phosphate groups. In cells, examples of agents that cleave phosphate groups include enzymes such as intracellular 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)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, and -OP(S)(Rk)-S-. 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 attachment points can be evaluated using methods similar to those described above.

[0219] Acid-cleavable linking group An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linking group is cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0, or less), or by an agent, such as an enzyme, that can act as a general acid. Within cells, specific low-pH organelles, such as endosomes and lysosomes, provide a cleavage environment for the acid-cleavable linking group. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, ketals, acetals, esters, and esters of amino acids. Acid-cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). A preferred embodiment is one 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.

[0220] Ester-based linking groups Ester-based linking groups are cleaved by enzymes such as intracellular esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester-cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These candidate linking groups can be evaluated using methods similar to those described above.

[0221] Peptide-based cleaving groups Peptide-based linking groups are cleaved by enzymes such as intracellular peptidases and proteases. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to give rise to oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable linking groups do not include 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 give rise to peptides and proteins. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to give rise to peptides and proteins, and do not include the entire amide functionality. Peptide cleavable linking groups have the general formula: -NHCHR 1 C(O)NHCHR 2 C(O)-[wherein, R 1 and 2 are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0222] Biocleavable Linkers / Tethers Linker can also include biocleavable linkers, which are nucleotide and non-nucleotide linkers or combinations thereof, which connect two parts of a molecule, for example, one or both strands of two individual siRNA molecules to generate bis(siRNA).In some embodiments, slight electrostatic or stacking interaction between two individual siRNAs can represent linker.Non-nucleotide linkers include tethers or linkers derived from monosaccharides, disaccharides, oligosaccharides, and their derivatives, aliphatic, alicyclic, heterocyclic, and combinations thereof.

[0223] In some embodiments, at least one linker (tether) is a biocleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, functional mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, and mannose, and combinations thereof.

[0224] In one embodiment, the biocleavable carbohydrate linker can have 1-10 saccharide units with at least one anomeric linkage that can connect two siRNA units. When two or more saccharides are present, these units can be linked via 1-3, 1-4, or 1-6 saccharide linkages or via alkyl chains.

[0225] Exemplary biocleavable linkers include the following endosomally cleavable linkers as well as phosphoramidites: [ka] [ka] [ka] [ka] [ka] Including, but not limited to:

[0226] Further discussion of bio-cleavable linkers can be found in PCT Application No. PCT / US18 / 14213, entitled "Endosomally-cleavable Linkers," filed January 18, 2018, the entire contents of which are incorporated herein by reference.

[0227] Carrier In certain embodiments, a lipophilic moiety is conjugated to a dsRNA agent via a carrier that replaces one or more nucleotide(s).

[0228] The carrier 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, pyridazinonyl, tetrahydrofuryl, and decalin. In one embodiment, the acyclic group is a moiety based on a serinol skeleton or a diethanolamine skeleton.

[0229] In some embodiments, the carrier replaces one or more nucleotide(s) at an internal position(s) of the dsRNA agent.

[0230] In other embodiments, the carrier replaces a nucleotide at the end of the sense strand or antisense strand. In one embodiment, the carrier replaces the terminal nucleotide at the 3' end of the sense strand, thereby functioning as an end cap to protect the 3' end of the sense strand. In one embodiment, the carrier is an amine-containing cyclic group, for example, the carrier can be pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, or decalinyl.

[0231] Herein, a ribonucleotide subunit in which the ribose sugar of the subunit has been replaced in this manner is referred to as a ribose-replacement modified subunit (RRMS). The carrier may be a cyclic or acyclic moiety and may contain two "backbone attachment points" (e.g., hydroxyl groups) and a ligand (e.g., a lipophilic moiety). The lipophilic moiety may be directly attached to the carrier or, as described above, may be indirectly attached to the carrier via an intervening linker / tether. [ka]

[0232] A ligand-conjugated monomeric subunit can be the 5'- or 3'-terminal subunit of an iRNA molecule, i.e., one of the two "W" groups can be a hydroxyl group and the other "W" group can be a chain of two or more unmodified or modified ribonucleotides. Alternatively, a ligand-conjugated monomeric subunit can occupy an internal position, where both "W" groups can be one or more unmodified or modified ribonucleotides. More than one ligand-conjugated monomeric subunit can be present in an iRNA agent.

[0233] Sugar substitution-based monomers, e.g., ligand-conjugated monomers (cyclic) Cyclic sugar substitution-based monomers, e.g., sugar substitution-based ligand-conjugated monomers, are also referred to herein as RRMS monomer compounds. The carriers may have the general formula (LCM-2) provided below (in which the preferred backbone attachment points are 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 may be selected from two backbone junctions, e.g., R 1 and R 4 , or R 4 and R 9 The preferred tethering junction is when X is CH2, R 7 ;R 5 or R 6 Carriers are described below as entities that can be incorporated into the chain. Thus, structures may be provided with one (for terminal positions) or two (for internal positions) of the junctions, e.g., R 1 Or R 2 , R 3 or 4 , or R 9 Or R 10 (Y is CR 9 R10 It is understood that the term "R" also encompasses the situation where the R group is connected to a phosphate or modified phosphate, e.g., a sulfur-containing backbone. For example, one of the R groups named above may be -CH2-, where one bond is connected to the carrier and one is connected to a backbone atom, e.g., linking the oxygen or central phosphorus atom. [ka] [In formula: X is N(CO)R 7 , N.R. 7 or CH2; Y is NR 8 ,O,S,CR 9 R 10 and; Z is CR 11 R 12 is or is not present; R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 At least two of the following are true: OR a and / or (CH2) n OR b If so, each R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 are independently H, OR a and / or (CH2) n OR b and; Each R 5 , R 6 , R 11 , and R 12 are independently a ligand, H, 1 to 3R 13 C1-C6 alkyl optionally substituted by, or C(O)NHR 7 or R 5 and R 11 are both R 14 is a C3-C8 cycloalkyl optionally substituted by R7 may be a ligand, e.g., R 7 is R d or R 7 is, for example, a tethering part, e.g., NR c R d C1~C replaced by 20 Alkyl, or NHC(O)R d C1~C replaced by 20 It may be a ligand indirectly tethered to a carrier via an alkyl; R 8 is H or C1-C6 alkyl; R 13 is hydroxy, C1-C4 alkoxy, or halo; R 14 is NR C R 7 and; R 15 is C1-C6 alkyl or C2-C6 alkenyl optionally substituted by cyano; R 16 is C1~C 10 is alkyl; R 17 is a liquid-phase or solid-phase supported reagent; L is -C(O)(CH2) q C(O)- or -C(O)(CH2) q S- and; R a is a protecting group, such as CAr; (e.g., dimethoxytrityl group) or Si(X 5 ')(X 5 '')(X 5 '''), where (X 5 '), (X 5 ''), and (X 5 ''') are described elsewhere herein, R b is P(O)(O-)H, P(OR 15 )N(R 16 )2 or LR 17 and; R Cis H or C1-C6 alkyl; R d is H or a ligand; Each Ar is independently a C6-C alkoxy group optionally substituted by C1-C4 alkoxy. 10 is aryl, n is 1 to 4; and q is 0 to 4.

[0234] Exemplary carriers include, for example, those in which X is N(CO)R 7 or NR 7 and Y is CR 9 R 10 and Z is absent; or X is N(CO)R 7 or NR 7 and Y is CR 9 R 10 and Z is CR 11 R 12 or X is N(CO)R 7 or NR 7 and Y is O and Z is CR 11 R 12 or X is CH2 and Y is CR 9 R 10 and Z is CR 11 R 12 and R 5 and R 11 together form a C6 cycloalkyl (H, z=2) or an indane ring system, e.g., X is CH2; Y is CR 9 R 10 and Z is CR 11 R 12 and R 5 and R 11 together form a C5 cycloalkyl (H, z=1).

[0235] In certain embodiments, the carrier is a pyrroline ring system or a 4-hydroxypyrroline ring system, e.g., where X is N(CO)R 7 or NR 7 and Y is CR 9 R 10 and can be based on (D) where Z is absent. [ka] OFG 1 is preferably attached to a first carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, attached to one of the carbons of the five-membered ring (CHOFG in D). 1 ). OFG 2 is preferably attached directly to one of the carbons of the five-membered ring (OFG of D 2 ) For pyrroline-based supports, CH2OFG 1 may be joined to C-2, OFG 2 may be attached to C-3; or -CH2OFG 1 may be joined to C-3, OFG 2 may be conjugated to C-4. 1 and OFG 2 may be substituted onto one of the carbons referenced above. In 3-hydroxyproline-based carriers, -CHOFG 1 may be joined to C-2, OFG 2 may be attached to C-4. Pyrroline- and 4-hydroxyproline-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) where bond rotation is restricted by constraints resulting from the presence of certain linkages, e.g., rings. Thus, CH2OFG 1 and OFG 2 can be cis or trans with respect to each other in any of the pairs depicted above. Thus, all cis / trans isomers are expressly included. The monomers may contain one or more asymmetric centers and therefore may occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., CHOFG 1 and OFG 2(The centers having the formula (I) 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. Preferred examples of carrier D include: [ka] Includes.

[0236] In certain embodiments, the carrier may be based on a piperidine ring system (E), e.g., X is N(CO)R 7 or NR 7 and Y is CR 9 R 10 and Z is CR 11 R 12 is. [ka] OFG 1 is preferably attached to a first carbon, e.g., an exocyclic alkylene group, such as a methylene group (n=1) or an ethylene group (n=2), connected to one of the carbons of the six-membered ring (E's -(CH2) n OFG 1 ). OFG 2 is preferably attached directly to one of the carbons of the six-membered ring (-OFG of E) 2 ).-(CH2) n OFG 1 and OFG 2 may be arranged in a paired manner on the ring, i.e., both groups may be attached to the same carbon, e.g., C-2, C-3, or C-4. Alternatively, -(CH) n OFG 1 and OFG 2 may be positioned in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, for example, -(CH) n OFG 1 may be joined to C-2, OFG 2 may be attached to C-3; -(CH2) n OFG 1may be joined to C-3, OFG 2 may be attached to C-2; -(CH) n OFG 1 may be joined to C-3, OFG 2 may be attached to C-4; or -(CH2) n OFG 1 may be joined to C-4, OFG 2 may be attached to C-3. Piperidine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) where bond rotation is restricted by restrictions resulting from the presence of certain linkages, such as rings. Thus, -(CH2) n OFG 1 and OFG 2 can be cis or trans with respect to each other in any of the pairs depicted above. Thus, all cis / trans isomers are expressly included. The monomers may contain one or more asymmetric centers and therefore may occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., CHOFG 1 and OFG 2 (The centers having the formula (I) can both have the R configuration; or both can have the S configuration; or one center can have the R configuration and the other center can have the S configuration, and vice versa.) The tethering junction is preferably nitrogen.

[0237] In certain embodiments, the carrier may be based on a piperidine ring system (F), e.g., X is N(CO)R 7 or NR 7 and Y is NR 8 and Z is CR 11 R 12 or may be based on a morpholine ring system (G), for example, X is N(CO)R 7 or NR 7 and Y is O and Z is CR 11 R 12 is. [ka] OFG 1 is preferably attached to a first carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, attached to one of the carbons of the six-membered ring (-CHOFG of F or G). 1 ). OFG 2 is preferably attached directly to one of the carbons of the six-membered ring (OFG of F or G) 2 ) for both F and G, -CH2OFG 1 may be joined to C-2, OFG 2 may be attached to C-3; or vice versa. In certain embodiments, CH2OFG 1 and OFG 2 may be counter-substituted onto one of the carbons referenced above. Piperazine- and morpholine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) where bond rotation is restricted by restrictions resulting from the presence of certain linkages, e.g., rings. Thus, CH2OFG 1 and OFG 2 can be cis or trans with respect to each other in any of the pairs depicted above. Thus, all cis / trans isomers are expressly included. The monomers may contain one or more asymmetric centers and therefore may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., CHOFG 1 and OFG 2 (The centers having the formula (I) can both have the R configuration; or both can have the S configuration; or one center can have the R configuration and the other center can have the S configuration, and vice versa). R''' can be, for example, a C1-C6 alkyl, preferably CH3. The tethering junction is preferably nitrogen in both F and G.

[0238] In certain embodiments, the carrier is a morpholine ring system, e.g., [ka] For example, such carriers may be based on, but are not limited to, [ka] wherein T is thymine, and [ka] is] can be incorporated into a dsRNA agent by a monomer such as

[0239] In certain embodiments, the aforementioned three are incorporated at the end of the dsRNA agent, for example, at the 5' end of the sense strand. In one embodiment, three IgT3 monomers are incorporated at the 5' end of the sense strand. In another embodiment, the following: [ka] Two monomers independently selected from are incorporated at the 3'-end of the sense strand. In another embodiment, two IT4 monomers are incorporated at the 3'-end of the sense strand. In another embodiment, three IgT3 monomers are incorporated at the 5'-end of the sense strand, and two IT4 monomers are incorporated at the 3'-end of the sense strand. See, for example, WO2019 / 170731, WO2021 / 037972, WO2021 / 044004, and WO2022 / 084331, each of which is incorporated herein by reference.

[0240] In certain embodiments, the carrier may be based on a decalin ring system, e.g., X is CH and Y is CR 9 R 10 and Z is CR 11 R 12 and R 5 and R 11 together form a C6 cycloalkyl (H, z=2) or may be based on an indane ring system, e.g., X is CH2 and Y is CR 9 R 10 and Z is CR 11 R 12 and R5 and R 11 together form a C5 cycloalkyl (H, z=1). [ka] OFG 1 is preferably attached to a primary carbon, e.g., an exocyclic methylene group (n=1) or an ethylene group (n=2), attached to one of C-2, C-3, C-4, or C-5 [H of -(CH2) n OFG 1 ]. OFG 2 is preferably attached directly to one of C-2, C-3, C-4, or C-5 (-OFG of H 2 ).-CH2OFG 1 and OFG 2 may be arranged in a paired manner on the ring, i.e., both groups may be attached to the same carbon, e.g., C-2, C-3, C-4, or C-5. Alternatively, -(CH) n OFG 1 and OFG 2 may be positioned in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, for example, -(CH) n OFG 1 may be joined to C-2, OFG 2 may be attached to C-3; -(CH2) n OFG 1 may be joined to C-3, OFG 2 may be attached to C-2; -(CH) n OFG 1 may be joined to C-3, OFG 2 may be attached to C-4; or -(CH2) n OFG 1 may be joined to C-4, OFG 2 may be attached to C-3; -(CH2) n OFG 1 may be joined to C-4, OFG 2 may be attached to C-5; or -(CH2) n OFG 1may be joined to C-5, OFG 2 may be attached to C-4. Decalin or indane-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) where bond rotation is restricted by restrictions resulting from the presence of certain linkages, e.g., rings. Thus, -(CH2) n OFG 1 and OFG 2 can be cis or trans with respect to each other in any of the pairs depicted above. Thus, all cis / trans isomers are expressly included. The monomers may contain one or more asymmetric centers and therefore may occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., CHOFG 1 and OFG 2 (The centers having the formula (I) 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 preferred embodiments, the substituents at C-1 and C-6 are trans relative to each other. The tethering junction is preferably at C-6 or C-7.

[0241] Other carriers may include those based on 3-hydroxyproline (J). [ka] Therefore, -(CH2) n OFG 1 and OFG 2 can be cis or trans with respect to each other. Thus, all cis / trans isomers are expressly included. The monomers may contain one or more asymmetric centers and therefore may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., CHOFG 1 and OFG 2(The centers having the formula (I) can both have the R configuration; or both can have the S configuration; or one center can have the R configuration and the other center can have the S configuration, and vice versa.) The tethering junction is preferably nitrogen.

[0242] Details regarding more representative cyclic, sugar-substituted based carriers can be found in US Pat. Nos. 7,745,608 and 8,017,762, which are incorporated herein by reference in their entireties.

[0243] Sugar-based monomers (acyclic) Acyclic sugar-substituted based monomers, such as sugar-substituted based ligand-conjugated monomers, are also referred to herein as ribose-substituted monomer subunit (RRMS) monomer compounds. Preferred acyclic carriers are represented by the formula LCM-3 or LCM-4: [ka] may have.

[0244] In some embodiments, each x, y, and z can be, independently of one another, 0, 1, 2, or 3. When y and z are different in formula LCM-3, then the third carbon can have either the R or S configuration. In a preferred embodiment, x is zero, y and z are each 1 in formula LCM-3 (e.g., based on serinol), and y and z are each 1 in formula LCM-3. Each of the following formulas LCM-3 or LCM-4 may be substituted, for example, by hydroxy, alkoxy, or perhaloalkyl.

[0245] Details regarding more representative acyclic, sugar-substituted based carriers can be found in US Pat. Nos. 7,745,608 and 8,017,762, which are incorporated herein by reference in their entireties.

[0246] In some embodiments, the dsRNA agent includes one or more lipophilic moieties conjugated to the 5' end of the sense strand or the 5' end of the antisense strand.

[0247] In certain embodiments, the lipophilic moiety is conjugated to the 5' end of the chain via a carrier and / or a linker. In one embodiment, the lipophilic moiety has the formula: [ka] R is a ligand, such as a lipophilic moiety.

[0248] In some embodiments, the dsRNA agent includes one or more lipophilic moieties conjugated to the 3' end of the sense strand or the 3' end of the antisense strand.

[0249] In certain embodiments, the lipophilic moiety is conjugated to the 3' end of the chain via a carrier and / or a linker. In one embodiment, the lipophilic moiety has the formula: [ka] R is a ligand, such as a lipophilic moiety.

[0250] In some embodiments, the dsRNA agent includes one or more lipophilic moieties conjugated to both ends of the sense strand.

[0251] In some embodiments, the dsRNA agent includes one or more lipophilic moieties conjugated to both ends of the antisense strand.

[0252] 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.

[0253] In some embodiments, the lipophilic moiety is conjugated to the end of the chain via one or more linkers (tethers) and / or carriers.

[0254] In one embodiment, the lipophilic moiety is conjugated to the end of the chain via one or more linkers (tethers).

[0255] In one embodiment, the lipophilic moiety is conjugated to the 5' end of the sense or antisense strand via a cyclic carrier, optionally via one or more intervening linkers (tethers).

[0256] In some embodiments, the lipophilic moiety is conjugated to one or more internal positions on at least one strand. Internal positions on a strand refer to nucleotides at any position on the strand, excluding the terminal positions from the 3' and 5' ends of the strand (e.g., position 2: excluding position 1 counting from the 3' end and position 1 counting from the 5' end).

[0257] In one embodiment, the lipophilic moiety is conjugated to one or more internal positions on at least one chain, including all positions from each end of the chain except the terminal 2 positions (e.g., position 4: excluding positions 1 and 2 counting from the 3' end and positions 1 and 2 counting from the 5' end). In one embodiment, the lipophilic moiety is conjugated to one or more internal positions on at least one chain, including all positions from each end of the chain except the terminal 3 positions (e.g., position 6: excluding positions 1, 2 and 3 counting from the 3' end and positions 1, 2 and 3 counting from the 5' end).

[0258] In one embodiment, the lipophilic moiety is conjugated to one or more internal positions on at least one strand excluding the cleavage site region of the sense strand, e.g., the lipophilic moiety is not conjugated to positions 9-12 counting from the 5'-end of the sense strand, e.g., the lipophilic moiety is not conjugated to positions 9-11 counting from the 5'-end of the sense strand. Alternatively, the internal positions exclude positions 11-13 counting from the 3'-end of the sense strand.

[0259] In one embodiment, the lipophilic moiety is conjugated to one or more internal positions on at least one strand, excluding the cleavage site region of the antisense strand, e.g., internal positions excluding positions 12-14, counting from the 5' end of the antisense strand.

[0260] In one embodiment, the lipophilic moiety is conjugated to one or more internal positions on at least one strand, excluding positions 11-13 of the sense strand, counting from the 3' end, and positions 12-14 of the antisense strand, counting from the 5' end.

[0261] In one embodiment, the 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.

[0262] In one embodiment, the one or more lipophilic moieties are conjugated to one or more of the following internal positions: positions 5, 6, 7, 15, and 17 of the sense strand, and positions 15 and 17 of the antisense strand, counting from the 5' end of each strand.

[0263] In some embodiments, the lipophilic moiety is conjugated to a nucleobase, sugar moiety, or internucleoside linkage of a dsRNA agent.

[0264] definition Unless otherwise specified, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis and chemical analysis. Certain such techniques and procedures can be found, 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," 2002, which are incorporated herein by reference in their entireties for all purposes. nd Edition, Cold Spring Harbor Laboratory Press, 1989. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout this disclosure are incorporated herein by reference in their entirety.

[0265] Unless otherwise stated, the following terms have the following meanings:

[0266] As used herein, the term "target nucleic acid" refers to any nucleic acid molecule whose expression or activity can be modulated by siRNA compounds.Target nucleic acids include, but are not limited to, RNA (including but not limited to pre-mRNA and mRNA or a part thereof) transcribed from DNA encoding target protein, as well as cDNA and miRNA derived from such RNA.For example, target nucleic acids can be cellular genes (or mRNA transcribed from genes) whose expression is associated with a specific disorder or disease state.In some embodiments, target nucleic acids can be nucleic acid molecules derived from infectious pathogens.

[0267] As used herein, the term "iRNA agent" refers to an agent that mediates targeted cleavage of RNA transcripts. These agents associate with a cytoplasmic multiprotein complex known as the RNAi-induced silencing complex (RISC). Agents that are effective in inducing RNA interference are also referred to herein as siRNAs, RNAi agents, or simply RNA agents or dsRNA agents. Thus, these terms can be used interchangeably herein. As used herein, the term dsRNA agent also includes microRNAs and pre-microRNAs. Furthermore, as used herein, the "compound" or "compounds" of the present invention also refer to dsRNA agents and can be used interchangeably with dsRNA agents.

[0268] A dsRNA agent must contain a region having sufficient homology to the target gene and be of sufficient length, in terms of nucleotides, so that the iRNA agent, or a fragment thereof, can mediate down-regulation of the target gene. (For ease of explanation, the terms nucleotide or ribonucleotide may be used herein to refer to one or more monomeric subunits of an iRNA agent. It will be understood that the use of the terms "ribonucleotide" or "nucleotide" herein may also refer to the modified nucleotide or surrogate replacement moiety at one or more positions, in the case of a modified RNA or nucleotide surrogate.) Thus, a dsRNA agent is or contains a region that is at least partially, and in some embodiments, completely complementary to the target RNA. While perfect complementarity between a dsRNA agent and a target is not required, there must be sufficient correspondence to allow the iRNA agent or its cleavage product to direct sequence-specific silencing, such as by RNAi cleavage of the target RNA, e.g., mRNA. Complementarity, or the degree of homology with the target strand, is most important in the antisense strand. While perfect complementarity, particularly in the antisense strand, is often desired, some embodiments can include one or more, e.g., 6, 5, 4, 3, 2, or fewer, mismatches, particularly in the antisense strand (with respect to the target RNA). The sense strand need only be sufficiently complementary to the antisense strand to maintain the overall double-stranded nature of the molecule.

[0269] iRNA agents include molecules that are long enough to induce an interferon response (they can be cleaved by Dicer (Bernstein et al. 2001. Nature, 409:363-366) and enter RISC (RNAi-induced silencing complex)), and molecules that are short enough not to induce an interferon response (these molecules can also be cleaved by Dicer and / or enter RISC), for example, molecules of a size that can enter RISC, for example, molecules similar to the cleavage products of Dicer. Molecules that are short enough not to induce 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 that are short enough not to induce a harmful interferon response in human cells, for example, double-stranded RNA agents or single-stranded agents, for example, having a double-stranded region of less than 60, 50, 40, or 30 nucleotide pairs. The siRNA agent, or its cleavage product, can downregulate a target gene, for example, by inducing RNAi with respect to the target RNA, which may include endogenous or pathogenic target RNA.

[0270] As used herein, a "single-stranded iRNA agent" refers to an iRNA agent that is composed of one molecule. A single-stranded iRNA agent may contain a double-stranded region formed by intrastrand pairing, for example, a hairpin structure or a panhandle structure, or may contain such a structure. A single-stranded iRNA agent may be antisense to a target molecule. A single-stranded iRNA agent may be long enough to enter RISC and participate in RISC-mediated cleavage of a target mRNA. A single-stranded iRNA agent is at least 14 nucleotides in length, and in other embodiments, at least 15, 20, 25, 29, 35, 40, or 50 nucleotides in length. In certain embodiments, the length is less than 200, 100, or 60 nucleotides.

[0271] A loop refers to a region of an iRNA strand that, when base-pairing with another strand or another part of the same strand, is not paired with the opposite nucleotide in the duplex.

[0272] A hairpin iRNA agent will have a duplex 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 duplex region may be 200, 100, or 50 or less in length. In certain embodiments, the duplex region ranges from 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 the 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.

[0273] As used herein, a "double-stranded (ds) RNA agent" includes one or more, optionally two, strands, where interstrand hybridization can form a region of duplex structure.

[0274] As used herein, the terms "siRNA activity" and "RNAi activity" refer to gene silencing by siRNA.

[0275] As used herein, " gene silencing " by RNA interference molecules refers to the mRNA level in cells with target gene is reduced by 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 the mRNA level found in cells in the absence of miRNA or RNA interference molecules.In a preferred embodiment, mRNA level is reduced by 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%.

[0276] As used herein, the term "modulating 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 up-regulated or down-regulated so that it is 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.

[0277] As used herein, gene expression modulation occurs when the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, differs by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, or more from that observed in the absence of an siRNA, e.g., an RNAi agent. The % and / or fold difference is expressed relative to a control or non-control, e.g.,

number

number

[0278] As used herein, the terms "inhibit," "down-regulate," or "reduce" in relation to gene expression mean 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, or the activity of one or more proteins or protein subunits, is reduced below that observed in the absence of a modulator. Gene expression is down-regulated when the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule 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% compared to a corresponding unmodulated control, preferably by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or most preferably 100% (i.e., no gene expression).

[0279] As used herein, the terms "increase" or "up-regulate" in relation to gene expression mean 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, or the activity of one or more proteins or protein subunits, is increased above that observed in the absence of a modulator. Gene expression is up-regulated when the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule 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 a corresponding unmodulated control, and preferably by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 100%, 1.1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold, or more.

[0280] As used herein, the term "increased" or "increase" generally refers to an increase by a statistically significant amount. For the avoidance of doubt, "increased" refers to an increase of at least 10% compared to a reference level, e.g., 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 at least about 90%, or up to a 100% increase, or any increase between 10 and 100% compared to a reference level, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold increase, or any increase between 2-fold and 10-fold or more compared to a reference level.

[0281] As used herein, the term "reduced" or "reducing" generally refers to a statistically significant decrease.However, for the avoidance of doubt, "reduced" refers to a decrease of at least 10% compared to the reference level, for example, 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 non-existent level compared to the reference sample), or any decrease between 10% and 100% compared to the reference level.

[0282] dsRNA comprises two oligonucleotide strands that are sufficiently complementary to hybridize and form a double-stranded structure.Generally, the length of double-stranded structure is between 15 and 30 base pairs, more generally between 18 and 25, even more generally between 19 and 24, and most generally between 19 and 21 base pairs.In some embodiments, the long dsRNA is preferred to be between 25 and 30 base pairs in length.In some embodiments, the short dsRNA is preferred to be between 10 and 15 base pairs in length.In another embodiment, dsRNA is at least 21 nucleotides in length.

[0283] In some embodiments, the dsRNA comprises a sense strand and an antisense strand, wherein the antisense RNA strand has a region of complementarity that is complementary to at least a portion of the target sequence, and the duplex region is 14 to 30 nucleotides in length. Similarly, the region complementary to the target sequence is between 14 and 30, more commonly between 18 and 25, even more commonly between 19 and 24, and most commonly between 19 and 21 nucleotides in length.

[0284] As used herein, the term " antisense strand " refers to the oligomeric compound that is substantially or 100% complementary to the target sequence of interest.The term " antisense strand " includes the oligomeric compound that is formed by two separate strands, and the antisense region of both the single-molecule oligomeric compound that can form hairpin or dumbbell structure.The terms " antisense strand " and " guide strand " are used interchangeably herein.

[0285] The phrase "sense strand" refers to an oligomeric compound having a nucleoside sequence that is the same in whole or in part as a target sequence, such as a messenger RNA or DNA sequence. The terms "sense strand" and "passenger strand" are used interchangeably herein.

[0286] " Specifically hybridizable " and " complementary " mean that nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either conventional Watson-Crick or other non-conventional methods.With respect to the nucleic acid molecule of the present invention, the binding free energy between nucleic acid molecule and its complementary sequence is sufficient to allow the relevant function of nucleic acid to proceed, such as RNAi activity.The determination of the binding free energy of nucleic acid molecule 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). Percent complementarity refers to the percentage of contiguous 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, 10 out of 10 for 50%, 60%, 70%, 80%, 90%, 100% complementarity). "Fully complementary" or 100% complementarity means that all contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. Less than fully complementary refers to a situation in which some, but not all, of the nucleoside units of two strands can hydrogen bond with each other. "Substantial complementarity" refers to polynucleotide strands that exhibit 90% or greater complementarity, excluding regions of the polynucleotide strands that are selected to be non-complementary, such as overhangs. Specific binding requires a degree of complementarity sufficient to avoid non-specific binding of the oligomeric compound to non-target sequences under the conditions in which specific binding is desired, i.e., physiological conditions in the case of in vivo assays or therapeutic treatments, or conditions under which the assay is performed in the case of in vitro assays, which non-target sequences typically differ by at least 5 nucleotides.

[0287] In some embodiments, the double-stranded region of a dsRNA agent 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, 30, or more nucleotide pairs in length.

[0288] In some embodiments, the antisense strand of a dsRNA agent is equal to or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0289] In some embodiments, the sense strand of a dsRNA agent 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.

[0290] In one embodiment, the sense and antisense strands of the dsRNA agent are each 15-30 nucleotides in length.

[0291] In one embodiment, the sense and antisense strands of the dsRNA agent are each 19-25 nucleotides in length.

[0292] In one embodiment, the sense and antisense strands of the dsRNA agent are each 21-23 nucleotides in length.

[0293] In some embodiments, one strand has at least one stretch of 1 to 5 single-stranded nucleotides in the double-stranded region. By "a stretch of single-stranded nucleotides in the double-stranded region" is meant that there is at least one nucleotide base pair on 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 in the double-stranded region. When both strands have a stretch of 1 to 5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides in the double-stranded region, such single-stranded nucleotides can face each other (e.g., a mismatched stretch) or can be positioned such that the second strand does not have a single-stranded nucleotide facing the single-stranded iRNA of the first strand, or vice versa (e.g., a single-stranded loop). In some embodiments, the single-stranded nucleotide is located within 8 nucleotides of either end, e.g., within 8 nucleotides, e.g., 8, 7, 6, 5, 4, 3, or 2 nucleotides, of either the 5' or 3' end of the region of complementarity between the two strands.

[0294] In one embodiment, the dsRNA agent includes a single-stranded overhang on at least one of its termini. In one embodiment, the single-stranded overhang is 1, 2, or 3 nucleotides in length.

[0295] In one embodiment, the sense strand of the dsRNA agent is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, where the strands form a double-stranded region of 21 contiguous base pairs with a 2-nucleotide long single-stranded overhang at the 3' end.

[0296] In some embodiments, each strand of a dsRNA agent has a ZXY structure as described in PCT Publication No. 2004080406, which is incorporated herein by reference in its entirety.

[0297] In certain embodiments, the two strands of a double-stranded oligomeric compound may be linked together. The two strands may be linked to each other at both ends, or at least at one end. Linking at one end means that the 5' end of the first strand is linked to the 3' end of the second strand, or the 3' end of the first strand is linked to the 5' end of the second strand. When the two strands are linked to each other at both ends, the 5' end of the first strand is linked to the 3' end of the second strand, and the 3' end of the first strand is linked to the 5' end of the second strand. The two strands are connected to each other by the following formula: (N) n (where N is independently a modified or unmodified nucleotide, and n is 3 to 23). In some embodiments, n is 3 to 10, e.g., 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 (where 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-pairing interactions with other nucleotides in the linker. The two strands may also be linked together by a non-nucleoside linker, such as the linkers described herein. It will be understood by those skilled in the art that any of the oligonucleotide chemical modifications or variations described herein may be used in the oligonucleotide linker.

[0298] Hairpin and dumbbell-shaped oligomeric compounds will have a duplex 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 duplex region may be 200, 100, or 50 or less in length. In some embodiments, the duplex region ranges from 15-30, 17-23, 19-23, and 19-21 nucleotide pairs in length.

[0299] Hairpin oligomeric compounds may have a single-stranded overhang or terminal unpaired region at the 3' end, in some embodiments, on the antisense side of the hairpin. In some embodiments, the overhang is 1 to 4 nucleotides in length, more typically 2 to 3 nucleotides in length. Hairpin oligomeric compounds capable of inducing RNA interference are also referred to herein as "shRNAs."

[0300] In certain embodiments, two oligomer strands specifically hybridize when there is a sufficient degree of complementarity to avoid nonspecific binding of the antisense compound to non-target nucleic acid sequences under conditions where specific binding is desired, i.e., physiological conditions in the case of in vivo assays or therapeutic treatments, or conditions under which the assay is performed in the case of in vitro assays.

[0301] As used herein, " stringent hybridization conditions " or " stringent conditions " refers to the conditions under which antisense compounds hybridize to their target sequence, but only a minimal number of other sequences hybridize.Stringent conditions depend on sequence and are different in different circumstances, and the " stringent conditions " under which antisense compounds hybridize to target sequence are determined by the nature and composition of antisense compounds and the assays they are studied.

[0302] It is understood in the art that incorporating nucleotide affinity modifications can tolerate more mismatches than unmodified compounds.Similarly, certain oligonucleotide sequences may be more tolerant to mismatches than other oligonucleotide sequences.Those skilled in the art can determine the appropriate number of mismatches between oligonucleotides or between an oligonucleotide and a target nucleic acid, for example, by determining melting temperature (Tm).Tm or ΔTm can be calculated by techniques well known to those skilled in the art.For example, those skilled in the art can evaluate nucleotide modifications for their ability to increase the melting temperature of RNA:DNA duplexes by the technique described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443).

[0303] siRNA design In one embodiment, the dsRNA agent is 19 nucleotides in length and blunt-ended at both 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.

[0304] In one embodiment, the dsRNA agent is 20 nucleotides in length and blunt-ended at both 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.

[0305] In one embodiment, the dsRNA agent is 21 nucleotides in length and blunt-ended at both 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.

[0306] In one embodiment, the dsRNA agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand comprises at least one of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end; the antisense strand comprises at least one of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, and one end of the iRNA is blunt, while the other end comprises a two-nucleotide overhang.Preferably, the two-nucleotide overhang is at the 3' end of the antisense strand.The dsRNA agent may further comprise a ligand (for example, GalNAc3).

[0307] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand is 25 to 30 nucleotide residues in length, starting from the 5'-terminal nucleotide (position 1), and positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense strand is 36 to 66 nucleotide residues in length, starting from the 3'-terminal nucleotide, and comprising at least 8 ribonucleotides at positions 1 to 23 of the sense strand to form a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is unpaired from the sense strand, and up to 6 consecutive nucleotides at the 3' end are unpaired from the sense strand, thereby forming a single-stranded 3' overhang of 1 to 6 nucleotides; and the 5' end of the antisense strand is unpaired from the sense strand, and comprises 10 to 30 consecutive nucleotides. The double-stranded nucleic acid comprises a nucleotide sequence that forms a single-stranded 5'-overhang of 10 to 30 nucleotides; at least the 5'- and 3'-terminal nucleotides of the sense strand base-pair with nucleotides of the antisense strand when the sense strand and the antisense strand are aligned for maximum complementarity, thereby forming a substantially duplexed 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 that, when the double-stranded nucleic acid is introduced into a mammalian cell, it reduces expression of the target gene; the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, where at least one of the motifs is located 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.

[0308] 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 and up to 29 nucleotides in length, and a second strand up to 30 nucleotides in length with at least one 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 having a duplex region at its 3' end that is 1-4 nucleotides longer than the first strand and at least 25 nucleotides in length, and the second strand is sufficiently complementary to a target mRNA over a length of at least 19 nucleotides of the second strand such that the iRNA agent, when introduced into a mammalian cell, reduces expression of the target gene, wherein cleavage of the iRNA by Dicer preferentially results in an siRNA that includes the 3' end of the second strand, thereby reducing expression of the target gene in the mammal. The dsRNA agent can further include a ligand (eg, GalNAc3).

[0309] In one embodiment, the sense strand of the dsRNA agent contains at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs is at the cleavage site in the sense strand. For example, the sense strand may contain at least one motif of three 2'-F modifications on three consecutive nucleotides within positions 7-15 from the 5' end.

[0310] 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, where one of the motifs is located at or near the cleavage site in the antisense strand.For example, the antisense strand may contain at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides within positions 9-15 from the 5' end.

[0311] For dsRNA agents having a duplex region 17-23 nucleotides in length, the cleavage site in the antisense strand is typically near positions 10, 11, or 12 from the 5' end. Thus, the three identical modification motifs can be located at positions 9, 10, or 11; 10, 11, or 12; 11, 12, or 13; 12, 13, or 14; or 13, 14, or 15 of the antisense strand, counting from the first nucleotide at the 5' end of the antisense strand or from the first paired nucleotide within the duplex region at the 5' end of the antisense strand. The cleavage site within the antisense strand can also vary according to the length of the duplex region of the iRNA from the 5' end.

[0312] In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, each having 14-30 nucleotides, wherein the sense strand contains at least two motifs of three identical modifications on three consecutive nucleotides, at least one of which is located at or near the cleavage site within the strand, and at least one of which is 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 of three identical modifications on three consecutive nucleotides, at least one of which is located at or near the cleavage site within the strand.The modification of the motif located at or near the cleavage site of the sense strand is different from the modification of the motif located at or near the cleavage site of the antisense strand.

[0313] 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 located at or near the site of strand cleavage. 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 site of strand cleavage.

[0314] 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 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.

[0315] In one embodiment, the dsRNA agent contains mismatch(es) with the target in the duplex, or a combination thereof. The mismatch may be present in the overhang region or in the duplex region. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., for the free energy of association or dissociation of a particular pairing, the simplest approach is to consider the pair at each base pair, but next-neighbor or similar analysis can also be used). With regard 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, such as non-canonical or non-canonical pairings (described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings; pairings involving universal bases are preferred over canonical pairings.

[0316] In one embodiment, the dsRNA agent includes at least one of the first one, two, three, four, or five base pairs in the duplex region at the 5' end of the antisense strand, which may be independently selected from the group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical or other than canonical pairs, or pairings containing universal bases, so as to promote dissociation of the antisense strand at the 5' end of the duplex.

[0317] In one embodiment, the nucleotide at position 1 in the double-stranded region at the 5' end of antisense strand is selected from the group consisting of A, dA, dU, U and dT. Alternatively, at least one of the first one, two or three base pairs in the double-stranded region at the 5' end of antisense strand is an AU base pair.For example, the first base pair in the double-stranded region at the 5' end of antisense strand is an AU base pair.

[0318] In one aspect, the present invention relates to a double-stranded RNA (dsRNA) agent that inhibits expression of a target gene. The dsRNA agent includes a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The dsRNA agent has a structure represented by formula (I): [ka] is expressed by

[0319] In Formula (I), B1, B2, B3, B1', B2', B3', and B4' are each independently a nucleotide 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.

[0320] C1 is a thermolabile nucleotide located opposite the seed region of the antisense strand (i.e., positions 2-8 of the 5' end of the antisense strand). For example, C1 is located in the sense strand, pairing with the nucleotide at positions 2-8 of 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 possesses a thermolabile modification, which may include an abasic modification; a mismatch with the opposing nucleotide in the duplex; and a sugar modification such as a 2'-deoxy modification, or an acyclic nucleotide, for example, an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA). In one embodiment, C1: i) a mismatch with the opposing nucleotide in the antisense strand; ii) [ka] an abasic modification selected from the group consisting of: [ka] wherein B is a modified or unmodified nucleobase; R 1 and R 2 are independently H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. In one embodiment, the thermolabile modification in C1 is a mismatch 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 thermolabile modification in C1 is GNA or [ka] is.

[0321] In another embodiment, the thermolabile modification in C1 is [ka] is selected from the group consisting of:

[0322] In another embodiment, the thermolabile modification in C1 is [ka] wherein R is OH, F, or OMe (e.g., OH). is.

[0323] T1, T1', T2', and T3 each independently represent a nucleotide containing a modification that provides the nucleotide with steric bulk equal to or less than that of a 2'-OMe modification. Steric bulk refers to the sum of the steric effects of the modifications. Those skilled in the art know methods for determining the steric effect of a nucleotide modification. The modification may be a modification at the 2' position of the ribose sugar of the nucleotide, or a non-ribose nucleotide, acyclic nucleotide, or modification to the backbone of the nucleotide that is similar or equivalent to a modification at the 2' position of the ribose sugar, providing the nucleotide with steric bulk equal to or less than that of a 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.

[0324] n 1 , n 3 , and q 1 are independently 4 to 15 nucleotides in length.

[0325] n 5 , q 3 , and q 7are independently 1 to 6 nucleotide(s) in length.

[0326] n 4 , q 2 , and q 6 are independently 1 to 3 nucleotide(s) in length; alternatively, n 4 is 0. q 5 are independently 0 to 10 nucleotide(s) in length.

[0327] n 2 and q 4 are independently 0 to 3 nucleotide(s) in length.

[0328] Alternatively, n 4 is 0 to 3 nucleotides in length.

[0329] In one embodiment, n 4 can be 0. In one example, n 4 is 0, and q 2 and q 6 is 1. In another example, n 4 is 0, and q 2 and q 6 is 1 with modifications of two phosphorothioate internucleotide linkages at positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate internucleotide linkages at positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0330] In one embodiment, n 4 , q 2 , and q 6 are each 1.

[0331] In one embodiment, n 2 , n 4 , q 2 , q 4, and q 6 are each 1.

[0332] In one embodiment, C1 is at positions 14-17 of the 5' end of the sense strand when the sense strand is 19-22 nucleotides in length; 4 is 1. In one embodiment, C1 is at position 15 of the 5' end of the sense strand.

[0333] In one embodiment, T3' begins at position 2 from the 5' end of the antisense strand. In one example, T3' is at position 2 from the 5' end of the antisense strand, and q 6 is equal to 1.

[0334] In one embodiment, T1' begins at position 14 from the 5' end of the antisense strand. 2 is equal to 1.

[0335] In an exemplary embodiment, T3' begins at position 2 of the 5' end of the antisense strand, and T1' begins at position 14 of the 5' end of the antisense strand. In one example, T3' begins at position 2 of the 5' end of the antisense strand, and q 6 is equal to 1, T1' starts from position 14 of the 5' end of the antisense strand, and q 2 is equal to 1.

[0336] In one embodiment, T1' and T3' are separated by a length of 11 nucleotides (ie, not counting the T1' and T3' nucleotides).

[0337] In one embodiment, T1' is at position 14 from the 5' end of the antisense strand. 2 is equal to 1, and modifications at the 2' position, or at non-ribose, acyclic or backbone positions, provide less steric bulk than 2'-OMe ribose.

[0338] In one embodiment, T3' is at position 2 from the 5' end of the antisense strand. 6 is equal to 1, and modifications at the 2' position, or at non-ribose, acyclic, or backbone positions, provide steric bulk less than 2'-OMe ribose. In one embodiment, T1 is at the cleavage site of the sense strand. In one example, T1 is at position 11 from the 5' end of the sense strand when the sense strand is 19-22 nucleotides in length, and n 2 is 1. In an exemplary embodiment, T1 is at the cleavage site of the sense strand, at position 11 from the 5' end of the sense strand, when the sense strand is 19-22 nucleotides in length, and n 2 is 1.

[0339] In one embodiment, T2' begins at position 6 from the 5' end of the antisense strand. In one example, T2' is at positions 6-10 from the 5' end of the antisense strand, and 4 is 1. In an exemplary embodiment, T1 is located at the cleavage site of the sense strand, e.g., at position 11 from the 5' end of the sense strand, when the sense strand is 19 to 22 nucleotides in length, and n 2 is 1; T1' is at position 14 from the 5' end of the antisense strand; 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 backbone position that is less sterically bulky than 2'-OMe ribose; T2' is at positions 6-10 from the 5' end of the antisense strand, and q 4 is 1; T3' is at the second position from the 5' end of the antisense strand; q 6 is equal to 1, and the modification to T3' is at the 2' position or at a non-ribose, acyclic or backbone position, providing steric bulk less than 2'-OMe ribose.

[0340] In one embodiment, T2' begins at position 8 from the 5' end of the antisense strand. 4In one embodiment, T2' begins at position 9 from the 5' end of the antisense strand. In one example, T2' is at position 9 from the 5' end of the antisense strand, and q 4 is 1. 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 is 1; with modifications by two phosphorothioate internucleotide linkages at positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate internucleotide linkages at positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0341] 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 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 7is 1; with modifications by two phosphorothioate internucleotide linkages at positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate internucleotide linkages at positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0342] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0343] 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 q3 is 4, T2' is 2'-F, and q 4 is 2, 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; with modifications by two phosphorothioate internucleotide linkages at positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate internucleotide linkages at positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0344] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0345] 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 3is 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; with modifications by two phosphorothioate internucleotide linkages at positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate internucleotide linkages at positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0346] 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 7is 1.

[0347] 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 is 1; with modifications by two phosphorothioate internucleotide linkages at positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate internucleotide linkages at positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0348] 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 q3 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; and may be accompanied by at least two additional TTs at the 3' end of the antisense strand.

[0349] 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 7 is 1; optionally with at least two additional TTs at the 3' end of the antisense strand; with a modification with two phosphorothioate internucleotide linkages within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0350] In one embodiment, B1 is 2'-OMe or 2'-F, and n1 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 is 1.

[0351] 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 7is 1; with modification with two phosphorothioate internucleotide linkages in positions 1 to 5 of the sense strand (counting from the 5' end), with modification with two phosphorothioate internucleotide linkages in positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modification with two phosphorothioate internucleotide linkages in positions 18 to 23 of the antisense strand (counting from the 5' end).

[0352] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1.

[0353] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; with modifications by two phosphorothioate internucleotide linkages at positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate internucleotide linkages at positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0354] 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.

[0355] 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 4is 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; with modifications by two phosphorothioate internucleotide linkages at positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate internucleotide linkages at positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0356] A dsRNA agent can include a phosphorus-containing group at the 5'-end of either the sense or antisense strand. The 5'-terminal phosphorus-containing group can be 5'-terminal phosphate (5'-P), 5'-terminal phosphorothioate (5'-PS), 5'-terminal phosphorodithioate (5'-PS2), 5'-terminal vinylphosphonate (5'-VP), 5'-terminal methylphosphonate (MePhos), or 5'-deoxy-5'-C-malonyl ( [ka] When the 5'-terminal phosphorus-containing group is a 5'-terminal vinyl phosphonate (5'-VP), the 5'-VP may be a 5'-E-VP isomer (i.e., trans-vinyl phosphonate, [ka] ), 5'-Z-VP isomer (i.e., cis-vinyl phosphonate, [ka] ), or a mixture thereof. In another example, the phosphorus-containing group at the 5'-terminus can be [ka] or a salt thereof (eg, sodium salt), wherein B may be a modified nucleobase (eg, U).

[0357] In one embodiment, the dsRNA agent includes a phosphorus-containing group at the 5'-end of the sense strand.In one embodiment, the dsRNA agent includes a phosphorus-containing group at the 5'-end of the antisense strand.

[0358] In one embodiment, the dsRNA agent includes a 5'-P. In one embodiment, the dsRNA agent includes a 5'-P in the antisense strand. In one embodiment, the dsRNA agent includes a 5'-PS. In one embodiment, the dsRNA agent includes a 5'-PS in the antisense strand.

[0359] In one embodiment, the dsRNA agent comprises a 5'-VP. In one embodiment, the dsRNA agent comprises a 5'-VP in the antisense strand. In one embodiment, the dsRNA agent comprises a 5'-E-VP in the antisense strand. In one embodiment, the dsRNA agent comprises a 5'-Z-VP in the antisense strand.

[0360] In one embodiment, the dsRNA agent comprises a 5'-PS2. In one embodiment, the dsRNA agent comprises a 5'-PS2 in the antisense strand.

[0361] In one embodiment, the dsRNA agent includes a 5'-PS2. In one embodiment, the dsRNA agent includes, in the antisense strand, 5'-deoxy-5'-C-malonyl.

[0362] 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, T2' is 2'-F, and q 4 is 2, 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. The dsRNA agent also includes a 5'-PS.

[0363] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The dsRNA agent also includes a 5'-P.

[0364] 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, T2' is 2'-F, and q 4 is 2, 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. The dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0365] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The dsRNA agent also includes a 5'-PS2.

[0366] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. dsRNA agents also include 5'-deoxy-5'-C-malonyl.

[0367] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; with modifications by two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 5'-P.

[0368] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; with modifications by two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 5'-PS.

[0369] In one embodiment, B1 is 2'-OMe or 2'-F, and n1 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; with modifications by two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0370] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; with modifications by two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 5'-PS2.

[0371] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; with modifications by two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes 5'-deoxy-5'-C-malonyl.

[0372] 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 is 1. The dsRNA agent also includes a 5'-P.

[0373] 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 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 is 1. The dsRNA agent also includes a 5'-PS.

[0374] 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 is 1. The dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0375] 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 1is 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 is 1. The dsRNA agent also includes a 5'-PS2.

[0376] 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 is 1. dsRNA agents also include 5'-deoxy-5'-C-malonyl.

[0377] 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 1is 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 is 1; with modifications by two phosphorothioate internucleotide linkages at positions 1-5 (counting from the 5' end) of the sense strand, with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and with modifications by two phosphorothioate internucleotide linkages at positions 18-23 (counting from the 5' end) of the antisense strand. The dsRNA agent also includes a 5'-P.

[0378] 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 7is 1; with modifications by two phosphorothioate internucleotide linkages at positions 1-5 (counting from the 5' end) of the sense strand, with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and with modifications by two phosphorothioate internucleotide linkages at positions 18-23 (counting from the 5' end) of the antisense strand. The dsRNA agent also includes a 5'-PS.

[0379] 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 is 1; with modifications by two phosphorothioate internucleotide linkages at positions 1 to 5 (counting from the 5' end) of the sense strand, with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and with modifications by two phosphorothioate internucleotide linkages at positions 18 to 23 (counting from the 5' end) of the antisense strand. The dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0380] In one embodiment, B1 is 2'-OMe or 2'-F, and n1 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 is 1; with modifications by two phosphorothioate internucleotide linkages at positions 1-5 (counting from the 5' end) of the sense strand, with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and with modifications by two phosphorothioate internucleotide linkages at positions 18-23 (counting from the 5' end) of the antisense strand. The dsRNA agent also includes a 5'-PS2.

[0381] 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 5is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; with modifications by two phosphorothioate internucleotide linkages at positions 1-5 (counting from the 5' end) of the sense strand, with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and with modifications by two phosphorothioate internucleotide linkages at positions 18-23 (counting from the 5' end) of the antisense strand. The dsRNA agent also includes 5'-deoxy-5'-C-malonyl.

[0382] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNA agent also includes a 5'-P.

[0383] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNA agent also includes a 5'-PS.

[0384] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0385] 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 4is 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNA agent also includes a 5'-PS2.

[0386] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. dsRNA agents also include 5'-deoxy-5'-C-malonyl.

[0387] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; with modifications by two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 5'-P.

[0388] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q7 is 1; with modifications by two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 5'-PS.

[0389] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; with modifications by two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0390] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; with modifications by two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 5'-PS2.

[0391] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; with modifications by two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes 5'-deoxy-5'-C-malonyl.

[0392] 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. The dsRNA agent also includes a 5'-P.

[0393] 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 is 1. The dsRNA agent also includes a 5'-PS.

[0394] 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. The dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0395] 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 is 1. The dsRNA agent also includes a 5'-PS2.

[0396] 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. dsRNA agents also include 5'-deoxy-5'-C-malonyl.

[0397] 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 is 1; with modifications by two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 5'-P.

[0398] 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 is 1; with modifications by two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 5'-PS.

[0399] 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; with modifications by two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0400] 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; with modifications by two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 5'-PS2.

[0401] 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 is 1; with modifications by two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes 5'-deoxy-5'-C-malonyl.

[0402] In one embodiment, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% dsRNA agent is modified.For example, when 50% dsRNA agent is modified, all 50% of the nucleotides present in dsRNA agent contain the modification described herein.

[0403] In one embodiment, each of the sense and antisense strands of a dsRNA agent 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.

[0404] In one embodiment, the sense and antisense strands of the dsRNA agent each contain at least two different modifications.

[0405] In one embodiment, the dsRNA agent of Formula (I) further comprises 3' and / or 5' overhang(s) of 1 to 10 nucleotides in length. In one example, the dsRNA agent of Formula (I) comprises 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.

[0406] In one embodiment, the dsRNA agent contains no 2'-F modifications.

[0407] In one embodiment, the sense strand and / or antisense strand of the dsRNA agent comprises one or more blocks of phosphorothioate or methylphosphonate internucleotide linkages. In one example, the sense strand comprises two blocks of phosphorothioate or methylphosphonate internucleotide linkages. In one example, the antisense strand comprises two blocks of phosphorothioate or methylphosphonate internucleotide linkages. For example, the two blocks of phosphorothioate or methylphosphonate internucleotide linkages are separated by 16 to 18 phosphate internucleotide linkages.

[0408] In one embodiment, the sense strand and antisense strand of the dsRNA agent each have 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.

[0409] In one embodiment, the nucleotide at position 1 of the 5' end of the antisense strand of the duplex is selected from the group consisting of A, dA, dU, U, and dT. In one embodiment, at least one of the first, second, and third base pairs from the 5' end of the antisense strand is an AU base pair.

[0410] In one embodiment, the antisense strand of the dsRNA agent is 100% complementary to the target RNA, hybridizes thereto, 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.

[0411] In one aspect, the present invention relates to a dsRNA agent as defined herein that is capable of inhibiting expression of a target gene. The dsRNA agent includes a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The sense strand contains at least one thermolabile nucleotide, and at least one of the thermolabile nucleotides is located opposite or near the seed region of the antisense strand (i.e., positions 2 to 8 of the 5' end of the antisense strand). Each of the embodiments and aspects described herein regarding dsRNAs represented by Formula (I) can also be applied to dsRNAs containing thermolabile nucleotides.

[0412] For example, if the sense strand is 21 nucleotides long, the thermolabile nucleotide can be located between positions 14 and 17 at the 5' end of the sense strand. The antisense strand contains at least two modified nucleic acids that are less than sterically demanding 2'-OMe modifications. Preferably, the two modified nucleic acids that are less than sterically demanding 2'-OMe modifications are separated by a length of 11 nucleotides. For example, the two modified nucleic acids are located at positions 2 and 14 at the 5' end of the antisense strand.

[0413] In one embodiment, the dsRNA agent further comprises at least one ASGPR ligand. For example, the ASGPR ligand may be a bivalent or trivalent branched linker, such as: [ka] In one example, the ASGPR ligand is conjugated to the 3' end of the sense strand.

[0414] For example, the dsRNA agent defined herein may comprise: i) a phosphorus-containing group at the 5'-end of the sense strand or antisense strand; ii) two phosphorothioate internucleotide linkages within positions 1-5 of the sense strand (counting from the 5'-end of the sense strand), two phosphorothioate internucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5'-end of the antisense strand), and two phosphorothioate internucleotide linkages within positions 18-23 of the antisense strand (counting from the 5'-end of the antisense strand); and iii) a ligand, such as an ASGPR ligand (e.g., one or more GalNAc derivatives), at the 5'-end or 3'-end of the sense strand or antisense strand. For example, the ligand may be at the 3'-end of the sense strand.

[0415] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; with two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), with two phosphorothioate internucleotide linkage modifications within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 5'-P and a targeting ligand. In one embodiment, the 5'-P is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0416] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; with two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), with two phosphorothioate internucleotide linkage modifications within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0417] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), there is a modification with two phosphorothioate internucleotide linkages; within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), there is a modification with two phosphorothioate internucleotide linkages; and within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand), there is a modification with two phosphorothioate internucleotide linkages. The dsRNA agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0418] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with two phosphorothioate internucleotide linkage modifications within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0419] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; with two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), with two phosphorothioate internucleotide linkage modifications within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0420] 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 7is 1; with a modification by two phosphorothioate internucleotide linkages at positions 1 to 5 (counting from the 5' end) of the sense strand, with a modification by two phosphorothioate internucleotide linkages at positions 1 and 2 (counting from the 5' end) of the antisense strand, and with a modification by two phosphorothioate internucleotide linkages at positions 18 to 23 (counting from the 5' end) of the antisense strand. The dsRNA agent also includes a 5'-P and a targeting ligand. In one embodiment, the 5'-P is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0421] 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 7is 1; with two phosphorothioate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end) of the sense strand, with two phosphorothioate internucleotide linkage modifications within positions 1 and 2 (counting from the 5' end) of the antisense strand, and with two phosphorothioate internucleotide linkage modifications within positions 18 to 23 (counting from the 5' end) of the antisense strand. The dsRNA agent also includes a 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0422] 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 7is 1; with two phosphorothioate internucleotide linkage modifications at positions 1 to 5 (counting from the 5' end) of the sense strand, with two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand, and with two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (counting from the 5' end) of the antisense strand. The dsRNA agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0423] 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 7is 1; with two phosphorothioate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end) of the sense strand, with two phosphorothioate internucleotide linkage modifications within positions 1 and 2 (counting from the 5' end) of the antisense strand, and with two phosphorothioate internucleotide linkage modifications within positions 18 to 23 (counting from the 5' end) of the antisense strand. The dsRNA agent also includes a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0424] 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 7is 1; with two phosphorothioate internucleotide linkage modifications at positions 1 to 5 (counting from the 5' end) of the sense strand, with two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand, and with two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (counting from the 5' end) of the antisense strand. The dsRNA agent also includes 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0425] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; with two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), with two phosphorothioate internucleotide linkage modifications within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 5'-P and a targeting ligand. In one embodiment, the 5'-P is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0426] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; with two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), with two phosphorothioate internucleotide linkage modifications within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0427] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), there is a modification with two phosphorothioate internucleotide linkages; within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), there is a modification with two phosphorothioate internucleotide linkages; and within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand), there is a modification with two phosphorothioate internucleotide linkages. The dsRNA agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0428] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with two phosphorothioate internucleotide linkage modifications within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0429] 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; with two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), with two phosphorothioate internucleotide linkage modifications within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0430] 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 7is 1; with two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), with two phosphorothioate internucleotide linkage modifications within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 5'-P and a targeting ligand. In one embodiment, the 5'-P is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0431] 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 7is 1; with two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), with two phosphorothioate internucleotide linkage modifications within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0432] 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 7is 1; within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), there is a modification with two phosphorothioate internucleotide linkages; within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), there is a modification with two phosphorothioate internucleotide linkages; and within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand), there is a modification with two phosphorothioate internucleotide linkages. The dsRNA agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0433] 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 7is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with two phosphorothioate internucleotide linkage modifications within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0434] 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 7is 1; with two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), with two phosphorothioate internucleotide linkage modifications within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0435] In certain embodiments, a dsRNA agent of the invention comprises: (a) a sense strand, (i) 21 nucleotides in length; (ii) optionally an ASGPR ligand conjugated to the 3' end, the ASGPR ligand comprising three GalNAc derivatives conjugated via a trivalent branched linker; and (iii) 2'-F modifications at positions 1, 3, 5, 7, 9-11, 13, 17, 19, and 21, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14-16, 18, and 20 (counting from the 5' end). a sense strand having: (b) an antisense strand, (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5, 9, 11-13, 15, 17, 19, 21, and 23, and 2'F modifications at positions 2, 4, 6-8, 10, 14, 16, 18, 20, and 22 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23 (counting from the 5' end) and an antisense strand having Includes; In this case, the dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0436] In another specific embodiment, the dsRNA agent is (a) a sense strand, (i) 21 nucleotides in length; (ii) optionally an ASGPR ligand conjugated to the 3' end, the ASGPR ligand comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-F modifications at positions 1, 3, 5, 7, 9-11, 13, 15, 17, 19, and 21, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 (counting from the 5' end); and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end) a sense strand having: (b) an antisense strand, (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11-13, 15, 17, 19, and 21-23, and 2'F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) and an antisense strand having Includes; In this case, the dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0437] In another specific embodiment, the dsRNA agent is (a) a sense strand, (i) 21 nucleotides in length; (ii) optionally an ASGPR ligand conjugated to the 3' end, the ASGPR ligand comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, 10, and 12 to 21, 2'-F modifications at positions 7 and 9, and a deoxynucleotide (e.g., dT) at position 11 (counting from the 5' end); and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end) a sense strand having: (b) an antisense strand, (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 7, 9, 11, 13, 15, 17, and 19-23, and 2'-F modifications at positions 2, 4-6, 8, 10, 12, 14, 16, and 18 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) and an antisense strand having Includes; In this case, the dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0438] In another specific embodiment, the dsRNA agent is (a) a sense strand, (i) 21 nucleotides in length; (ii) optionally an ASGPR ligand conjugated to the 3' end, the ASGPR ligand comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, 10, 12, 14, and 16 to 21, and 2'-F modifications at positions 7, 9, 11, 13, and 15; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end) a sense strand having: (b) an antisense strand, (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 5, 7, 9, 11, 13, 15, 17, 19, and 21-23, and 2'-F modifications at positions 2-4, 6, 8, 10, 12, 14, 16, 18, and 20 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) and an antisense strand having Includes; In this case, the dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0439] In another specific embodiment, the dsRNA agent is (a) a sense strand, (i) 21 nucleotides in length; (ii) optionally an ASGPR ligand conjugated to the 3' end, the ASGPR ligand comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 9 and 12 to 21, and 2'-F modifications at positions 10 and 11; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end) a sense strand having: (b) an antisense strand, (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11-13, 15, 17, 19, and 21-23, and 2'-F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) and an antisense strand having Includes; In this case, the dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0440] In another specific embodiment, the dsRNA agent is (a) a sense strand, (i) 21 nucleotides in length; (ii) optionally an ASGPR ligand conjugated to the 3' end, the ASGPR ligand comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-F modifications at positions 1, 3, 5, 7, 9-11, and 13, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, and 14-21; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end) a sense strand having: (b) an antisense strand, (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5-7, 9, 11-13, 15, 17-19, and 21-23, and 2'-F modifications at positions 2, 4, 8, 10, 14, 16, and 20 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) and an antisense strand having Includes; In this case, the dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0441] In another specific embodiment, the dsRNA agent is (a) a sense strand, (i) 21 nucleotides in length; (ii) optionally an ASGPR ligand conjugated to the 3' end, the ASGPR ligand comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1, 2, 4, 6, 8, 10, 12, 14, 15, 17, and 19-21, and 2'-F modifications at positions 3, 5, 7, 9-11, 13, 16, and 18; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end) a sense strand having: (b) an antisense strand, (i) 25 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 4, 6, 7, 9, 11-13, 15, 17, and 19-23, 2'-F modifications at positions 2, 3, 5, 8, 10, 12, 14, 16, and 18, and deoxynucleotides (e.g., dT) at positions 24 and 25 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) and an antisense strand having Includes; In this case, the dsRNA agent has a four nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0442] In another specific embodiment, the dsRNA agent is (a) a sense strand, (i) 21 nucleotides in length; (ii) optionally an ASGPR ligand conjugated to the 3' end, the ASGPR ligand comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2'-F modifications at positions 7, and 9 to 11; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end) a sense strand having: (b) an antisense strand, (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 8, 10-13, 15, and 17-23, and 2'-F modifications at positions 2, 6, 9, 14, and 16 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) and an antisense strand having Includes; In this case, the dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0443] In another specific embodiment, the dsRNA agent is (a) a sense strand, (i) 21 nucleotides in length; (ii) optionally an ASGPR ligand conjugated to the 3' end, the ASGPR ligand comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2'-F modifications at positions 7, and 9 to 11; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end) a sense strand having: (b) an antisense strand, (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 10-13, 15, and 17-23, and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) and an antisense strand having Includes; In this case, the dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0444] In another specific embodiment, the dsRNA agent is (a) a sense strand, (i) 19 nucleotides in length; (ii) optionally an ASGPR ligand conjugated to the 3' end, the ASGPR ligand comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 4, 6, and 10 to 19, and 2'-F modifications at positions 5, and 7 to 9; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end) a sense strand having: (b) an antisense strand, (i) 21 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 10-13, 15, and 17-21, and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counting from the 5' end) and an antisense strand having Includes; In this case, the dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0445] In one embodiment, the dsRNA agent is (a) a sense strand, (i) length of 18–23 nucleotides; (ii) three consecutive 2′-F modifications at positions 7–15; and a sense strand having (b) an antisense strand, (i) length of 18–23 nucleotides; (ii) at least a 2'-F modification anywhere on the strand; and (iii) at least two phosphorothioate internucleotide linkages in the first five nucleotides (counting from the 5' end) and an antisense strand having Includes; In this case, the dsRNA agent has either one or more lipophilic moieties conjugated to one or more positions on at least one strand; a two-nucleotide overhang 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 duplex.

[0446] In one embodiment, the dsRNA agent is (a) a sense strand, (i) length of 18–23 nucleotides; (ii) less than four 2'-F modifications a sense strand having: (b) an antisense strand, (i) length of 18–23 nucleotides; (ii) fewer than 12 2'-F modifications; and (iii) at least two phosphorothioate internucleotide linkages in the first five nucleotides (counting from the 5' end) and an antisense strand having Includes; In this case, the dsRNA agent has either one or more lipophilic moieties conjugated to one or more positions on at least one strand; a two-nucleotide overhang 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 duplex.

[0447] In one embodiment, the dsRNA agent is (a) a sense strand, (i) length of 19–35 nucleotides; (ii) less than four 2'-F modifications a sense strand having: (b) an antisense strand, (i) length of 19–35 nucleotides; (ii) fewer than 12 2'-F modifications; and (iii) at least two phosphorothioate internucleotide linkages in the first five nucleotides (counting from the 5' end) and an antisense strand having Includes; In this case, the duplex 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 moieties conjugated to one or more positions on at least one strand; a two-nucleotide overhang 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 duplex.

[0448] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand having a length of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages in the first five nucleotides (counting from the 5' end) on the antisense strand; wherein the duplex region is between 19-25 base pairs (preferably 19, 20, 21, or 22); the dsRNA agent has one or more lipophilic moieties conjugated to 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.

[0449] 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, etc.

[0450] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand having a length of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages in the first five nucleotides (counting from the 5' end) on the antisense strand; wherein the duplex region is between 19-25 base pairs (preferably 19, 20, 21, or 22); the dsRNA agent has one or more lipophilic moieties conjugated to one or more positions on at least one strand; and the dsRNA agent has greater than 80%, greater than 85%, and greater than 90% natural nucleotides, such that 2'-OH, 2'-deoxy, and 2'-OMe are natural nucleotides.

[0451] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand having a length of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages in the first five nucleotides (counting from the 5' end) on the antisense strand; wherein the duplex region is between 19-25 base pairs (preferably 19, 20, 21, or 22); the dsRNA agent has one or more lipophilic moieties conjugated to 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.

[0452] Examples of lipophilic moieties include, but are not limited to, lipids (saturated or unsaturated C4-C30 hydrocarbon chains and any functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonic acid, phosphate, thiol, azide, and alkyne), cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl groups, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine.

[0453] In some embodiments, the lipophilic moiety is a C-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, linoleic acid, arachidonic acid, cis-4,7,10,13,16,19-docosahexanoic acid, vitamin A, vitamin E, cholesterol, etc.) or C6 to C 30 Alcohols (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.).

[0454] In one example, the lipophilic moiety is a saturated or unsaturated C4-C 18 It is a hydrocarbon chain.

[0455] In one example, the lipophilic moiety is docosahexaenoic acid.

[0456] In some embodiments, the dsRNA agent includes a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, and the sense strand includes a 2'-fluoro nucleotide at position 10, counting from the 5' end of the sense strand.

[0457] In some embodiments, the sense strand further comprises one or more, for example, 1, 2, 3, 4, or 5, additional 2'-fluoro nucleotides. The additional 2'-fluoro nucleotides can be located anywhere in the sense strand.

[0458] In some embodiments, the sense strand further comprises a 2'-fluoro nucleotide at position 8, 9, 11, or 12, counting from the 5'-end of the sense strand. For example, the sense strand further comprises a 2'-fluoro nucleotide at position 9, counting from the 5'-end of the sense strand. In other words, the sense strand further comprises a 2'-fluoro nucleotide at positions 9 and 10, counting from the 5'-end of the sense strand. In another example, the sense strand further comprises a 2'-fluoro nucleotide at position 11, counting from the 5'-end of the sense strand. For example, the sense strand further comprises a 2'-fluoro nucleotide at positions 10 and 11, counting from the 5'-end of the sense strand.

[0459] In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 9, 10, and 11, counting from the 5' end of the sense strand. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 8, 9, and 10, counting from the 5' end of the sense strand. In some still other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 10, 11, and 12, counting from the 5' end of the sense strand.

[0460] In some embodiments, the sense strand further comprises a 2'-fluoro nucleotide at the 7 position.

[0461] In some embodiments, the sense strand does not include a 2'-fluoro nucleotide at position 7, counting from the 5' end of the sense strand. For example, the sense strand includes a 2'-OMe nucleotide at position 7, counting from the 5' end of the sense strand.

[0462] In some embodiments, any nucleotide in the sense strand that is not a 2'-fluoro nucleotide is a 2'-OMe nucleotide.

[0463] In some embodiments, the antisense strand contains one or more 2'-deoxy, e.g., 2'-H, nucleotides. For example, the antisense strand contains 1, 2, 3, 4, 5, 6, or more 2'-deoxy nucleotides. In some embodiments, the antisense strand contains 2, 3, 4, 5, or 6 2'-deoxy nucleotides. The 2'-deoxy nucleotides can be located anywhere in the antisense strand. For example, the antisense strand contains 2'-deoxy nucleotides at positions 1, 2, 3, 4, 5, or 6, 2, 5, 7, 12, 14, and 16, counting from the 5' end of the antisense strand. In some embodiments, the antisense strand contains 2'-deoxy nucleotides at positions 2 and 12, counting from the 5' end of the antisense strand. In some embodiments, the antisense strand contains 2'-deoxy nucleotides at positions 5 and 7, counting from the 5' end of the antisense strand. In some embodiments, the antisense strand comprises 2'-deoxynucleotides at positions 2, 5, 7, and 12, counting from the 5' end of the antisense strand.

[0464] In some embodiments, the antisense strand comprises one or more, for example, 1, 2, 3, 4, 5 or more, 2'-fluoro nucleotides. For example, the antisense strand comprises a 2'-fluoro nucleotide at position 14, counting from the 5' end of the antisense strand.

[0465] In some embodiments, the antisense strand comprises, counting from the 5' end of the antisense strand, a 2'-fluoro nucleotide at position 14 and a nucleotide other than 2'-deoxy or 2'-fluoro at position 16. For example, the antisense strand comprises, counting from the 5' end of the antisense strand, a 2'-fluoro nucleotide at position 14 and a nucleotide other than 2'-deoxy or 2'-fluoro at position 16.

[0466] In some embodiments, the antisense strand comprises, counting from the 5' end of the antisense strand, 2'-deoxynucleotides at positions 2 and 12 and 2'-fluoronucleotides at position 14. In some embodiments, counting from the 5' end of the antisense strand, the antisense strand comprises 2'-deoxynucleotides at positions 2 and 12, 2'-fluoronucleotides at position 14, and a nucleotide other than 2'-deoxy and 2'-fluoro at position 16. For example, counting from the 5' end of the antisense strand, the antisense strand comprises 2'-deoxynucleotides at positions 2 and 12, 2'-fluoronucleotides at position 14, and 2'-OMe at position 16.

[0467] In some embodiments, the antisense strand comprises a 2'-deoxynucleotide at position 14, counting from the 5' end of the antisense strand, and the sense strand comprises a non-2'-fluoro nucleotide at position 7, counting from the 5' end of the sense strand. For example, the antisense strand comprises 2'-deoxynucleotides at positions 2, 12, and 14, counting from the 5' end of the antisense strand, and the sense strand comprises a 2'-fluoro nucleotide at position 10 and a non-2'-fluoro nucleotide at position 7, counting from the 5' end of the sense strand.

[0468] In some embodiments, the sense strand comprises a 2'-fluoro nucleotide at position 10, counting from the 5' end of the sense strand, and the antisense strand comprises 2'-deoxy nucleotides at positions 2, 5, 7, and 12, counting from the 5' end of the antisense strand; (i) the antisense strand comprises, counting from the 5' end of the antisense strand, a 2'-fluoro nucleotide at position 14 and a nucleotide other than a 2'-deoxynucleotide or a 2'-fluoro nucleotide at position 16; (ii) the antisense strand contains a 2'-deoxynucleotide at position 14 or 16, counting from the 5' end of the antisense strand, and the sense strand contains a nucleotide other than a 2'-fluoronucleotide at position 7, counting from the 5' end of the sense strand.

[0469] In some embodiments, any nucleotide in the antisense strand that is not a 2'-fluoro nucleotide or a 2'-deoxy nucleotide is a 2'-OMe nucleotide.

[0470] In some embodiments, the dsRNA agent includes a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, and the sense strand sequence is represented 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 0 or 1; p and q are each independently 0 to 6; each N a represents oligonucleotide sequences that independently contain 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b represents an oligonucleotide sequence containing, independently, 1, 2, 3, 4, 5, or 6 modified nucleotides; each n p and each n q independently represent overhanging nucleotides; where N b and Y do not have the same modification; XXX, YYY, and ZZZ each independently represent a motif with three identical modifications on three consecutive nucleotides; the dsRNA agent has one or more lipophilic moieties conjugated to one or more positions on at least one strand; The antisense strand of the dsRNA contains two blocks of one, two, or three phosphorothioate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages. It is expressed by:

[0471] In some embodiments, the dsRNA agent has 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 each independently 0 or 1; p and q are each independently 0 to 6; each N a ' independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p ' and each n q ' independently represent an overhanging nucleotide containing 0 to 6 nucleotides; where N b ' and Y' do not have the same modification; X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a motif with three identical modifications on three consecutive nucleotides. The antisense strand sequence is represented by:

[0472] Further details about the motifs represented by formula (I) and formula (II) above can be found in WO2013 / 074947, which is incorporated herein by reference in its entirety.

[0473] A variety of publications describe multimeric siRNAs, all of which can be used with iRNA agents. Such publications include WO2007 / 091269, U.S. Patent No. 7,858,769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520, which are incorporated herein by reference in their entirety.

[0474] In some embodiments, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of the dsRNA agents are modified.

[0475] In some embodiments, each of the sense and antisense strands of a dsRNA agent 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.

[0476] In some embodiments, the sense and antisense strands of a dsRNA agent each contain at least two different modifications.

[0477] In some embodiments, the dsRNA agent does not contain any 2'-F modifications.

[0478] In some embodiments, the dsRNA agent contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 2'-F modification(s). In one example, the dsRNA agent contains 9 or 10 2'-F modifications.

[0479] dsRNA agent can further comprise at least one phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage.The modification of phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage can be present at any nucleotide of any position of sense strand or antisense strand, or both strands.For example, the modification of internucleotide linkage can be present at every nucleotide on sense strand or antisense strand;The modification of each internucleotide linkage can be present in an alternating pattern on sense strand or antisense strand;Sense strand or antisense strand may contain both internucleotide linkage modifications in an alternating pattern.The alternating pattern of the modification of internucleotide linkage on sense strand can be the same or different from that of antisense strand, and the alternating pattern of the modification of internucleotide linkage on sense strand can have a shift compared with the alternating pattern of the modification of internucleotide linkage on antisense strand.

[0480] In one embodiment, the dsRNA agent comprises the modification of phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage in the protruding region.For example, the protruding region can contain two nucleotides with phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage between the two nucleotides.The modification of internucleotide linkage can also be made so that the protruding nucleotide is connected to the terminal paired nucleotide in the double-stranded region.For example, at least two, three, four or all of the protruding nucleotides can be connected via phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage, but the protruding nucleotide can also be connected to the paired nucleotide adjacent to the protruding nucleotide with additional phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage.For example, there can be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, in which case two of the three nucleotides are protruding nucleotides, and the third nucleotide is the paired nucleotide adjacent to the protruding nucleotide. Preferably, these terminal three nucleotides may be at the 3' end of the antisense strand.

[0481] In some embodiments, the sense strand and / or antisense strand of a dsRNA agent comprises one or more blocks of phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages. In one example, the sense strand comprises two blocks of phosphorothioate internucleotide linkages or one block of methylphosphonate internucleotide linkages. In one example, the antisense strand comprises two blocks of phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages. For example, the two blocks of phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages are separated by 16 to 18 phosphate internucleotide linkages.

[0482] In some embodiments, the antisense strand of the dsRNA agent is 100% complementary to the target RNA, hybridizes thereto, and inhibits its expression by 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.

[0483] In one embodiment, the present invention relates to a dsRNA agent capable of inhibiting expression of a target gene. The dsRNA agent includes a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The sense strand contains at least one thermolabile nucleotide, and at least one of the thermolabile nucleotides is located opposite or near the seed region of the antisense strand (i.e., positions 2 to 8 of the 5' end of the antisense strand). For example, if the sense strand is 21 nucleotides long, the thermolabile nucleotide is located between positions 14 and 17 of the 5' end of the sense strand. The antisense strand contains at least two modified nucleic acids with less than sterically demanding 2'-OMe modifications. Preferably, the two modified nucleic acids with less than sterically demanding 2'-OMe modifications 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.

[0484] In some embodiments, the dsRNA agents disclosed herein are miRNA mimics. In one design, miRNA mimics are double-stranded molecules (e.g., having a double-stranded region between about 16 and about 31 nucleotides in length) that contain one or more sequences that share identity with the mature strand of a given miRNA. Double-stranded miRNA mimics have designs similar to those described above for dsRNAs. In some embodiments, miRNA mimics contain a double-stranded region of between 16 and 31 nucleotides and one or more of the following chemical modification patterns: the sense strand contains 2'-O-methyl modifications of nucleotides 1 and 2 (counting from the 5' end of the sense strand) and all Cs and Us, while modifications of the antisense strand may include 2'F modifications of all Cs and Us, phosphorylation of the 5' end of the oligonucleotide, and stabilized internucleotide linkages associated with two-nucleotide 3' overhangs.

[0485] In some embodiments, the dsRNA agent disclosed herein is an antimir. In some embodiments, the compound comprises at least two antimirs, covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, such as a linker described in the present disclosure, or non-covalently linked to each other. The terms "antimir," "microRNA inhibitor," or "miR inhibitor" are synonymous and refer to oligonucleotides or modified oligonucleotides that inhibit the activity of specific miRNAs. Inhibitors can adopt various configurations, including single-stranded, double-stranded (RNA / RNA or RNA / DNA duplex), and hairpin designs. Generally, microRNA inhibitors comprise one or more sequences or portions of sequences that are complementary or partially complementary to the mature strand (or strands) of the targeted miRNA. In addition, miRNA inhibitors can also comprise additional sequences located 5' and 3' of the sequence that is the reverse complement of the mature miRNA. The additional sequence can be the reverse complement of the sequence adjacent to the mature miRNA in the pri-miRNA from which the mature miRNA is derived, or the additional sequence can be any sequence (having a mixture of A, G, C, U, or dT). In some embodiments, one or both of the additional sequences is any sequence capable of forming a hairpin. Thus, in some embodiments, the sequence that is the reverse complement of the miRNA is flanked by hairpin structures on the 5' and 3' sides. When the microRNA inhibitor is double-stranded, it can contain mismatches between nucleotides on opposite strands. Furthermore, the microRNA inhibitor can be linked to a conjugate moiety to facilitate the uptake of the inhibitor into cells.

[0486] MicroRNA inhibitors, including hairpin miRNA inhibitors, are described in detail in Vermeulen et al., "Double-Stranded Regions Are Essential Design Components of Potent Inhibitors of RISC Function," RNA 13: 723-730 (2007), and WO2007 / 095387 and WO2008 / 036825, the entire contents of each of which are incorporated herein by reference.Those skilled in the art can select the sequence of desired miRNA from the database and design inhibitors useful for the methods disclosed herein.

[0487] In some embodiments, the dsRNA agent disclosed herein is an antagomir. In some embodiments, the dsRNA agent comprises at least two antagomirs, covalently or non-covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, such as a linker described herein. Antagomirs are RNA-like oligonucleotides with various modifications for RNAse protection and pharmacological properties, such as enhanced uptake into tissues and cells. They differ from normal RNA, for example, by complete 2'-O-methylation of the sugars, phosphorothioate intersugar linkages, and, for example, a cholesterol moiety at the 3' end. In a preferred embodiment, the antagomir comprises 2'-O-methyl modifications at all nucleotides, a cholesterol moiety at the 3' end, two phosphorothioate intersugar linkages at the first two positions of the 5' end, and four phosphorothioate linkages at the 3' end of the molecule. Antagomirs can be used to efficiently suppress endogenous miRNAs by forming a duplex containing the antagomir and the endogenous miRNA, thereby preventing miRNA-induced gene silencing. One example of antagomir-mediated miRNA silencing is the silencing of miR-122, which is described in Krutzfeldt et al., Nature, 2005, 438: 685-689, which is expressly incorporated herein by reference in its entirety.

[0488] Recent studies have shown that dsRNA can also activate gene expression, a mechanism known as "small RNA-mediated gene activation" or RNAa (activating RNA). See, for example, Li, LC et al. Proc Natl Acad Sci U S A. (2006), 103(46):17337-42, and Li LC (2008), "Small RNA-Mediated Gene Activation." RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity. Caister Academic Press. ISBN 978-1-904455-25-7. dsRNA targeting gene promoters has been shown to induce potent transcriptional activation of associated genes. Endogenous miRNAs that induce RNAa have also been found in humans. Check E. Nature (2007). 448 (7156): 855-858.

[0489] Another surprising observation is that the gene activation by RNAa lasts for a long time.It has been confirmed that the induction of gene expression lasts for more than 10 days.The long-lasting effect of RNAa may be due to the epigenetic changes at the dsRNA target site.In some embodiments, RNA activators can increase gene expression.In some embodiments, increased gene expression inhibits viability, growth and development, and / or reproduction.

[0490] Therefore, in some embodiments, the dsRNA agent disclosed herein is activator RNA.In some embodiments, dsRNA agent comprises at least two activator RNAs, which are covalently linked together or non-covalently linked together via nucleotide-based linker or non-nucleotide-based linker, for example, the linker described in the present disclosure.

[0491] Therefore, in some embodiments, the dsRNA agent disclosed herein is triplex-forming oligonucleotide (TFO).In some embodiments, the dsRNA agent comprises at least two TFOs, which are covalently linked to each other through nucleotide-based linker or non-nucleotide-based linker, for example, the linker described in the present disclosure, or are non-covalently linked to each other.Recent research has shown that the triplex-forming oligonucleotide can be designed to recognize and bind to the polypurine / polypyrimidine region in double-stranded helix DNA with sequence specificity. These recognition rules are outlined in Maher III, LJ, et al., Science (1989) vol. 245, pp. 725-730; Moser, HE, et al., Science (1987) vol. 238, pp. 645-630; Beal, PA, et al., Science (1992) vol. 251, pp. 1360-1363; Conney, M., et al., Science (1988) vol. 241, pp. 456-459 and Hogan, ME, et al., EP Publication 375408. Modifications of oligonucleotides, such as the introduction of intercalators or substitution of intersugar linkages, and optimization of binding conditions (pH and cation concentration) have helped to overcome inherent obstacles to TFO activity, such as charge repulsion and instability, and it has recently been shown that synthetic oligonucleotides can be targeted to specific sequences (for a recent review, see Seidman and Glazer, J Clin Invest 2003;1 12:487-94). In general, triplex-forming oligonucleotides correspond to the following sequences: Oligo 3'-AGGT Double-stranded 5'-AGCT Double-stranded 3'-TCGA

[0492] However, it has been shown that A-AT and G-GC triplets provide the highest triple helix stability (Reither and Jeltsch, BMC Biochem, 2002, Septl2, Epub). The same authors showed that TFOs designed according to the A-AT and G-GC rules do not form nonspecific triplexes, indicating that triplex formation is indeed sequence-specific.

[0493] Thus, for any given sequence, triplex-forming sequences can be devised. Triplex-forming oligonucleotides are preferably at least 15 nucleotides in length, more preferably 25 nucleotides in length, even more preferably 30 or more nucleotides in length, up to 50 or 100 nucleotides in length.

[0494] The formation of triple helix structures with target DNA induces steric and functional changes, blocking transcription initiation and elongation and allowing the introduction of desired sequence changes into endogenous DNA, resulting in specific downregulation of gene expression. Examples of such gene silencing in TFO-treated cells include knockout of the episomal supFGl and endogenous HPRT genes in mammalian cells (Vasquez et al., Nucl Acids Res. 1999;27:1176-81, and Puri, et al., J Biol Chem, 2001;276:28991-98), sequence- and target-specific downregulation of the expression of the Ets2 transcription factor, which is important in the pathogenesis of prostate cancer (Carbone, et al., Nucl Acids Res. 2003;31:833-43), and the proinflammatory ICAM-I gene (Besch et al., J Biol Chem, 2002;277:32473-79). Furthermore, Vuyisich and Beal recently demonstrated that sequence-specific TFOs bind to dsRNA and inhibit the activity of dsRNA-dependent enzymes such as RNA-dependent kinases (Vuyisich and Beal, Nuc. Acids Res 2000;28:2369-74).

[0495] Furthermore, TFOs designed according to the above principles can induce directed mutagenesis that can effectively repair DNA, resulting in both down- and up-regulation of endogenous gene expression (Seidman and Glazer, J Clin Invest 2003; 112:487-94).Detailed descriptions of the design, synthesis, and administration of effective TFOs can be found in U.S. Patent Applications Nos. 2003-017068 and 2003-0096980 to Froehler et al., U.S. Patent Applications Nos. 2002-0128218 and 2002-0123476 to Emanuele et al., and U.S. Patent No. 5,721,138 to Lawn, the contents of which are incorporated herein by reference in their entirety.

[0496] Nucleic acid modification In some embodiments, dsRNA agent comprises at least one nucleic acid modification as described herein.For example, at least one modification is selected from the group consisting of modified internucleoside linkage, modified nucleobase, modified sugar, and any combination thereof.Without being limited, such modification can be present anywhere in dsRNA agent.For example, modification can be present in one of RNA molecules.

[0497] Nucleic acid modifications (nucleobases) The naturally occurring base moiety of a nucleoside is typically a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. For those nucleosides containing a pentofuranosyl sugar, a phosphate group can be linked to the 2', 3', or 5' hydroxyl moiety of the sugar. In the formation of oligonucleotides, these phosphate groups covalently link adjacent nucleosides to each other to form a linear polymeric compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The naturally occurring linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.

[0498] In addition to "unmodified" or "natural" nucleobases such as the purine nucleobases adenine (A) and guanine (G), as well as the pyrimidine nucleobases thymine (T), cytosine (C), and uracil (U), many modified nucleobases or nucleobase mimics known to those skilled in the art are suitable for use in the compounds described herein. Unmodified or natural nucleobases can be modified or substituted to provide iRNAs with improved properties. For example, nuclease-resistant oligonucleotides can be prepared using these bases, or synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidin), and any one of the oligomer modifications described herein. Alternatively, substituted or modified analogs of any of the above bases and "universal bases" can be used. When natural base is replaced by non-natural and / or universal base, the nucleotide is said to contain modified nucleobase and / or nucleobase modification as described herein.Modified nucleobase and / or nucleobase modification also include natural, non-natural and universal base, and contain conjugated moiety, such as the ligand described herein.Preferred conjugation moiety for conjugation with nucleobase comprises cationic amino group, which can be conjugated to nucleobase via suitable alkyl, alkenyl or amide bond linker.

[0499] The oligomeric compounds described herein may also include nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Exemplary modified nucleobases include other synthetic and natural nucleobases, such as inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 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 cytosine, 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-(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-(guanidiniumalkyl)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-(methoxycarbonylmethyl)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)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted-4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-(aminocarbonylethylenyl)-pseudouracil, 1-(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1-(aminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil Douracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1 ,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy) )-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidin, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazoline Zolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolepyridinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, pripinyl-7-(aza) Indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, 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 pyrimidines, N,2 -substituted purines, N 6 -substituted purines, O 6 -substituted purines, substituted 1,2,4-triazoles, pyrrolo-pyrimidin-2-one-3-yl, 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2- The "universal bases" include, but are not limited to, ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl, 2-oxo-pyridopyrimidin-3-yl, or any O- or N-alkylated derivative thereof. Alternatively, substituted or modified analogs of any of the above bases and "universal bases" can also be used.

[0500] As used herein, a universal nucleobase is any nucleobase that can base pair with all four naturally occurring nucleobases without substantially affecting the melting behavior, recognition by intracellular enzymes, or activity of an iRNA duplex. Some exemplary universal nucleobases include 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methylisocarbostyrilyl, 5-methylisocarbostyrilyl, 3-methyl-7-propynylisocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl, and 9-methyl-imidizopyridinyl. Indolyls include, but are not limited to, indyl, pyrrolepyridinyl, isocarbostyryl, 7-propynylisocarbostyryl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylinolyl, 4,6-dimethylindolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, and structural derivatives thereof (see, e.g., Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

[0501] No. 3,687,808; those disclosed in International Publication No. PCT / US09 / 038425, filed March 26, 2009; those disclosed in Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990; those disclosed by English et al., Angewandte Chemie, International Edition, 1991, 30, 613; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijin, P. Ed. Wiley-VCH, 2008; and those disclosed in Sanghvi, YS, Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Eds., CRC Press, 1993, all of which are incorporated herein by reference.

[0502] In certain embodiments, modified nucleobases are nucleobases that are structurally similar to the parent nucleobase, such as 7-deazapurine, 5-methylcytosine, or G-clamp. In certain embodiments, nucleobase mimics include more complex structures, such as tricyclic phenoxazine nucleobase mimics. It should be noted that the above-mentioned methods for preparing modified nucleobases are well known to those skilled in the art.

[0503] Nucleic acid modification (sugar) Provided herein, dsRNA agents can comprise one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) monomers, including nucleosides or nucleotides with modified sugar moieties.For example, the furanosyl sugar ring of nucleosides can be modified in many ways, including but not limited to, adding a substituent, bridging two non-geminal ring atoms to form a locked nucleic acid or bicyclic nucleic acid.In certain embodiments, oligomeric compounds comprise one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) monomers that are LNA.

[0504] In some embodiments of the locked 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, a 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 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 a protecting group. is connected to the 4' position by a linker selected from

[0505] In some embodiments, each of the linkers of the LNA compound is independently -[C(R1)(R2)] n -, -[C(R1)(R2)] n In another embodiment, each of the linkers is independently 4'-CH-, 4'-(CH)-, 4'-(CH)-, 4'-(CH)-, 4'-CH-O-, 4'-(CH)-O-, 4'-CH-ON(R)-, and 4'-CH-N(R)-O-, wherein each R is independently H, a protecting group, or a C-C alkyl.

[0506] Certain LNAs have been prepared and are disclosed in the patent and scientific literature (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, 10035-10039; Examples of issued US patents and published applications disclosing LNAs 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. Pregrant Publication Nos. 2004-0171570; 2004-0219565; 2004-0014959; 2003-0207841; 2004-0143114; and 20030082807).

[0507] Also provided herein are LNAs 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 to form a bicyclic sugar moiety (reviewed 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. Patent Nos. 6,268,490 and 6,670,461). The linkage can be a methylene (-CH2-) group bridging the 2' oxygen atom and the 4' carbon atom, hence the term methyleneoxy(4'-CH2-O-2')LNA is used for the bicyclic moiety, and when 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 duplex thermal stability (Tm = +3 to +10°C) with complementary DNA and RNA, stability against 3'-exonuclease degradation, and excellent solubility. Potent and non-toxic antisense oligonucleotides, including BNAs, have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638).

[0508] The methyleneoxy(4'-CH2-O-2')LNA isomer under discussion is alpha-L-methyleneoxy(4'-CH2-O-2')LNA, which has been shown to have superior stability against 3' exonucleases. Alpha-L-methyleneoxy(4'-CH2-O-2')LNA has been incorporated into antisense gapmers and chimeras that have demonstrated potent antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0509] The synthesis and preparation of methyleneoxy (4'-CH2-O-2') LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, as well as their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630).These BNAs and their preparation are also described in WO98 / 39352 and WO99 / 14226.

[0510] Analogs of methyleneoxy(4'-CH2-O-2')LNA, such as 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 locked nucleoside analogs containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO99 / 14226). Furthermore, the synthesis of 2'-amino-LNA, a novel structurally restricted, high-affinity oligonucleotide analog, has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Additionally, 2'-amino- and 2'-methylamino-LNAs have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.

[0511] Modified sugar moieties are well known and can be used to alter, typically increase, the affinity of an antisense compound for its 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 with 2'-F, 2'-OCH3, or 2'-O(CH2)2-OCH3 substituents; 4'-thio-modified sugars, 3'-RNA, TNA, and L-nucleotides, inverted nucleotides (5'->5' or 3'->3' linkages), and inverted abasic nucleotides. Sugars can also be replaced with sugar mimetic groups, among others. Methods for the preparation of modified sugars are well known to those of skill in the art. Some representative patents and publications that teach the preparation of such modified sugars are U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591 ,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; 5,700,920; 6,531,584; and 6,600,032; and WO2005 / 121371.

[0512] 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(CH2CHO) n CH2CH2OR, n=1-50; "locked" nucleic acids (LNAs) in which the furanose portion of the nucleoside contains a bridge connecting 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.

[0513] "Deoxy" modifications include hydrogen (i.e., deoxyribose sugars, particularly suitable for single-stranded overhangs); halo (e.g., fluoro); amino (e.g., NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CHCHNH) n Includes 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.

[0514] Other suitable 2' modifications, such as modified MOEs, are described in US Patent Application Publication No. 20130130378, the contents of which are incorporated herein by reference.

[0515] Modifications at the 2' position can be in an arabinose structure. The term "arabinose structure" refers to the placement of the substituent at C2' of the ribose in the same configuration as the 2'-OH of arabinose.

[0516] A sugar can contain two different modifications, e.g., gem modifications, at the same carbon of the sugar. The sugar group can also contain one or more carbons with the opposite stereochemical configuration to that of the corresponding carbon in ribose. Thus, an oligomeric compound can contain one or more monomers containing, for example, arabinose as the sugar. The monomer can have an alpha linkage at the 1-position of the sugar, e.g., an alpha-nucleoside. The monomer can also have the opposite configuration at the 4'-position, e.g., C5' and H4', or the substituents replacing them are interchanged. When C5' and H4', or the substituents replacing them, are interchanged, the sugar is said to be modified at the 4'-position.

[0517] The double-stranded RNA agent disclosed herein can also contain abasic sugars, i.e., sugars that lack a nucleobase at C-1' or have other chemical groups in place of a nucleobase at C1'.See, for example, U.S. Patent No. 5,998,203, the entire contents of which are incorporated herein.These abasic sugars can also contain modifications in one or more of the constituent sugar atoms.The dsRNA agent can also contain one or more sugars that are L-isomers, for example, L-nucleosides.Modifications to the sugar group can also include replacing 4'-O with sulfur, optionally substituted nitrogen, or a CH2 group.In some embodiments, the bond between C1' and the nucleobase is in the α-configuration.

[0518] Sugar modifications may also include acyclic nucleotides, in which the C-C bond between the ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', C1'-O4') is absent and / or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4'), independently or in combination, is present in the nucleotide. In some embodiments, an acyclic nucleotide is [ka] wherein B is a modified or unmodified nucleobase, R and R are independently H, halogen, OR, or alkyl; and R is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. is.

[0519] 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 with gem 2'-OMe / 2'-OMe in the arabinose structure.

[0520] When a particular nucleotide is linked to the next nucleotide through its 2' position, it is understood that the sugar modifications described herein can be placed at the 3' position of the sugar for that particular nucleotide, for example, a nucleotide linked through its 2' position. The modification at the 3' position can be present in the xylose structure. The term "xylose structure" refers to the placement of the substituent at the C3' of the ribose in the same structure as the 3'-OH of the xylose sugar.

[0521] The hydrogen bonded to C4' and / or C1' may be replaced by a straight or branched chain optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and the alkyl, alkenyl, and alkynyl backbone may contain one or more of O, S, S(O), SO2, N(R'), C(O), N(R')C(O)O, OC(O)N(R'), CH(Z'), phosphate-containing linkages, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycle, or optionally substituted cycloalkyl, wherein R' is hydrogen, acyl, or optionally substituted aliphatic, and Z' is OR 11 , C.O.R. 11 , CO2R 11 , [ka] , N.R. 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 R 51 , SO2R11 , SOR 11 , S.R. 11 and substituted or unsubstituted heterocycles; R 21 and R 31 is independently, for each occurrence, hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR 11 , C.O.R. 11 , CO2R 11 , or NR 11 R 11 ' or R 21 and R 31 together with the atoms to which they are attached form a heterocyclic ring; R 41 and R 51 is independently, for each occurrence, hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR 11 , C.O.R. 11 , or CO2R 11 , or NR 11 R 11 ' and R 11 and R 11 ’ is independently hydrogen, aliphatic, substituted aliphatic, aryl, heteroaryl, or heterocyclic. In some embodiments, the hydrogen attached to C4' of the 5'-terminal nucleotide is substituted.

[0522] In some embodiments, C4' and C5' together preferably form an optionally substituted heterocycle containing at least one -PX(Y)-, where X is H, OH, OM, SH, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted alkylamino, or optionally substituted dialkylamino; M is independently, for each occurrence, an alkali metal or transition metal with an overall charge of +1; and Y is O, S, or NR', where R' is hydrogen, optionally substituted aromatic. Preferably, this modification is at the 5' end of the iRNA.

[0523] In certain embodiments, the LNA has the formula: [ka] [In formula: Bx is a heterocyclic base moiety; T1 is H or a hydroxyl protecting group; T2 is H, a hydroxyl protecting group or a reactive phosphate group; Z is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, or substituted amido. The bicyclic nucleosides include those having the formula:

[0524] In some embodiments, each substituent is independently mono- or polysubstituted with an appropriately protected substituent independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 and CN, and each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O, S, or NJ1.

[0525] In certain such embodiments, each substituent is independently mono- or polysubstituted with substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, and NJ3C(=X)NJ1J2, and each J1, J2, and J3 is independently H, C1-C6 alkyl, or substituted C1-C6 alkyl, and X is O or NJ1.

[0526] In certain embodiments, the Z group is C-C alkyl substituted with one or X, where each X is independently OJ, NJJ, SJ, N, OC(=X)J, OC(=X)NJJ, NJC(=X)NJJ, or CN; each J, J, and J is independently H or C-C alkyl, and X is O, S, or NJ. In another embodiment, the Z group is C-C alkyl substituted with one or X, where each X is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO—), substituted alkoxy, or azido.

[0527] In certain embodiments, the Z group is -CH2Xx, where Xx is OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, or CN; each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O, S, or NJ1. In another embodiment, the Z group is -CH2Xx, where Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO-), or azido.

[0528] In certain such embodiments, the Z group is in the (R)-configuration: [ka] is.

[0529] In certain such embodiments, the Z group is in the (S)-configuration: [ka] is.

[0530] In certain embodiments, each T1 and T2 is a hydroxyl protecting group. A preferred list of hydroxyl protecting groups includes benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, mesylate, tosylate, dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthin-9-yl (MOX). In certain embodiments, T1 is a hydroxyl protecting group selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl and dimethoxytrityl, where the more preferred hydroxyl protecting group T1 is 4,4'-dimethoxytrityl.

[0531] In certain embodiments, T2 is a reactive phosphorus group, and preferred reactive phosphorus groups include diisopropylcyanoethoxyphosphoramidite and H-phosphonate. In certain embodiments, T1 is 4,4'-dimethoxytrityl and T2 is diisopropylcyanoethoxyphosphoramidite.

[0532] In certain embodiments, the dsRNA agent has the formula: [ka] or the expression: [ka] or the expression: [ka] [In formula: Bx is a heterocyclic base moiety; T3 is H, a hydroxyl protecting group, a linked conjugate group, or an internucleoside linking group attached to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomeric subunit, or oligomeric compound; T4 is H, a hydroxyl protecting group, a linked conjugate group, or an internucleoside linking group attached to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomeric subunit, or oligomeric compound; wherein at least one of T3 and T4 is an internucleoside linking group attached to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomeric subunit, or oligomeric compound; and Z is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, or substituted amido. It contains at least one monomer of

[0533] In some embodiments, each substituent is independently mono- or polysubstituted with an appropriately protected substituent independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 and CN, and each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O, S, or NJ1.

[0534] In some embodiments, each substituent is independently mono- or polysubstituted with substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, and NJ3C(=X)NJ1J2, and each J1, J2, and J3 is independently H, C1-C6 alkyl, and X is O or NJ1.

[0535] In certain such embodiments, at least one Z is C1-C6 alkyl or substituted C1-C6 alkyl. In certain embodiments, each Z is independently C1-C6 alkyl or substituted C1-C6 alkyl. In certain embodiments, at least one Z is C1-C6 alkyl. In certain embodiments, each Z is independently C1-C6 alkyl. In certain embodiments, at least one Z is methyl. In certain embodiments, each Z is methyl. In certain embodiments, at least one Z is ethyl. In certain embodiments, each Z is ethyl. In certain embodiments, at least one Z is substituted C1-C6 alkyl. In certain embodiments, each Z is independently substituted C1-C6 alkyl. In certain embodiments, at least one Z is substituted methyl. In certain embodiments, each Z is substituted methyl. In certain embodiments, at least one Z is substituted ethyl. In certain embodiments, each Z is substituted ethyl.

[0536] In certain embodiments, at least one substituent is C1-C6 alkoxy (e.g., at least one Z is C1-C6 alkyl substituted with one or more C1-C6 alkoxy). In other embodiments, each substituent is independently C1-C6 alkoxy (e.g., each Z is independently C1-C6 alkyl substituted with one or more C1-C6 alkoxy).

[0537] In certain embodiments, at least one C1-C6 alkoxy substituent is CHO- (e.g., at least one Z is CHOCH-). In other embodiments, each C1-C6 alkoxy substituent is CHO- (e.g., each Z is CHOCH-).

[0538] In certain embodiments, at least one substituent is halogen (e.g., at least one Z is C-C alkyl substituted with one or more halogens). In certain embodiments, each substituent is independently halogen (e.g., each Z is independently C-C alkyl substituted with one or more halogens). In certain embodiments, at least one halogen substituent is fluoro (e.g., at least one Z is CHFCH-, CHFCH-, or CFCH-). In certain embodiments, each halo substituent is fluoro (e.g., each Z is independently CHFCH-, CHFCH-, or CFCH-).

[0539] In certain embodiments, at least one substituent is hydroxyl (e.g., at least one Z is C1-C6 alkyl substituted with one or more hydroxyl). In certain embodiments, each substituent is independently hydroxyl (e.g., each Z is independently C1-C6 alkyl substituted with one or more hydroxyl). In certain embodiments, at least one Z is HOCH2-. In other embodiments, each Z is HOCH2-.

[0540] In certain embodiments, at least one Z is CH3-, CH3CH2-, CHOCH3-, CH2F-, or HOCH2-. In certain embodiments, each Z is independently CH3-, CH3CH2-, CHOCH3-, CH2F-, or HOCH2-.

[0541] In certain embodiments, at least one Z group is C-C alkyl substituted with one or more X, where each X is independently OJ, NJJ, SJ, N, OC(=X)J, OC(=X)NJJ, NJC(=X)NJJ, or CN; each J, J, and J is independently H or C-C alkyl, and X is O, S, or NJ. In another embodiment, at least one Z group is C-C alkyl substituted with one or more X, where each X is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO—), or azido.

[0542] In certain embodiments, each Z group is independently C-C alkyl substituted with one or more X, where each X is independently OJ, NJJ, SJ, N, OC(=X)J, OC(=X)NJJ, NJC(=X)NJJ, or CN; each J, J, and J is ind...

Claims

1. A double-stranded RNA (dsRNA) agent capable of modulating expression of a target gene, comprising a sense strand and an antisense strand that is sufficiently complementary to at least a portion of the mRNA of the target gene, each strand having 14 to 40 nucleotides; the sense strand contains at least one 2'-OMe modification at position 1, counting from the 5' end; The antisense strand contains at least one TNA (threose nucleic acid) at position 1, counting from the 5' end; Double-stranded RNA agents.

2. 10. The dsRNA agent of claim 1, wherein the sense strand further comprises at least one 2'-OMe modification at position 2, counting from the 5' end.

3. 10. The dsRNA agent of claim 1, wherein the dsRNA agent contains one or more ligands conjugated to at least one strand, optionally via a linker or carrier.

4. A double-stranded RNA (dsRNA) agent capable of modulating expression of a target gene, comprising a sense strand and an antisense strand that is sufficiently complementary to at least a portion of the mRNA of the target gene, each strand having 14 to 40 nucleotides; One of the sense or antisense strands is 【Chemical 1】 [In the formula: each R is independently optionally substituted alkyl; B is an optionally modified nucleobase; ＊ represents an H or a bond to the internucleotide linkage to the next nucleotide] At least one 4'-modified TNA (threose nucleic acid) having the structure Double-stranded RNA agents.

5. 5. The dsRNA agent of claim 4, wherein the dsRNA agent contains one or more ligands conjugated to at least one strand, optionally via a linker or carrier.

6. 6. The dsRNA agent of claim 5, wherein at least one ligand is a lipophilic moiety.

7. 6. The dsRNA agent of claim 3 or 5, wherein at least one ligand is an ASGPR ligand.

8. 8. The dsRNA agent of claim 7, wherein the ASGPR ligand is one or more GalNAc derivatives conjugated by a bivalent or trivalent branched linker.

9. The ASGPR ligand is 【Chemistry 2】 9. The dsRNA agent of claim 8, wherein:

10. A double-stranded RNA (dsRNA) agent capable of modulating expression of a target gene, comprising a sense strand and an antisense strand that is sufficiently complementary to at least a portion of the mRNA of the target gene, each strand having 14 to 40 nucleotides; the antisense strand contains at least one TNA (threose nucleic acid) at position 1, counting from the 5' end; one or more lipophilic moieties are conjugated to at least one chain, optionally via a linker or carrier; Double-stranded RNA agents.

11. logK ｏｗ 11. The dsRNA agent of claim 6 or 10, wherein the lipophilicity of the lipophilic moiety as measured by is greater than 0.

12. 11. The dsRNA agent of claim 6 or 10, wherein the hydrophobicity of the dsRNA agent is greater than 0.2 as measured by the unbound fraction in a plasma protein binding assay of the dsRNA agent.

13. 11. The dsRNA agent of claim 6 or 10, wherein the lipophilic moiety is an aliphatic, alicyclic, or heterocyclic compound.

14. 14. The dsRNA agent of claim 13, wherein the lipophilic moiety is a lipid, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, a heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine.

15. The lipophilic moiety is a saturated or unsaturated C ４ ~C ３０ 14. The dsRNA agent of claim 13, containing a hydrocarbon chain and any functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonic acid, phosphate, thiol, azide, and alkyne.

16. The lipophilic moiety is a saturated or unsaturated C ４ ~C １８ 16. The dsRNA agent of claim 15, wherein the dsRNA agent contains a hydrocarbon chain and 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.

17. The lipophilic part is 【Chemistry 3】 【Chemistry 4】 【Chemistry 5】 [In the formula: m is an integer from 0 to 8; n is an integer from 1 to 21; R ２ ' is H, OH, F, OMe, O-methoxyalkyl, O-aryl, O-N-methylacetamido, O-dimethylaminoethoxyethyl, or O-aminopropyl; B is a modified or unmodified nucleobase; W is alkyl; and R and R' are each independently H or alkyl.

11. The dsRNA agent of claim 6 or 10, selected from the group consisting of:

18. 11. The dsRNA agent of claim 6 or 10, further comprising a targeting ligand that targets a receptor that mediates delivery to CNS tissue.

19. 19. The dsRNA agent of claim 18, wherein the targeting ligand is selected from the group consisting of Angiopep-2, lipoprotein receptor-related protein (LRP) ligand, bEnd.3 cell-binding ligand, transferrin receptor (TfR) ligand, mannose receptor ligand, glucose transporter protein, and LDL receptor ligand.

20. 11. The dsRNA agent of claim 6 or 10, further comprising a targeting ligand that targets a receptor that mediates delivery to ocular tissue.

21. 21. The dsRNA agent of claim 20, wherein the targeting ligand is selected from the group consisting of trans-retinol, RGD peptide, LDL receptor ligand, and carbohydrate-based ligand.

22. TNA, 【Chemistry 6】 wherein B is an optionally modified nucleobase; ＊ represents the attachment to the internucleotide linkage to the next nucleotide] 11. The dsRNA agent of claim 1 or 10, having the structure:

23. TNA, 【Chemistry 7】 wherein R is an optionally substituted alkyl and B is an optionally modified nucleobase; ＊ represents the attachment to the internucleotide linkage to the next nucleotide] 11. The dsRNA agent of claim 1, 4, or 10, which is a 4'-modified TNA having the structure:

24. 4'-modified TNA 【Chemistry 8】 24. The dsRNA agent of claim 23 having the structure:

25. TNA, 【Chemistry 9】 wherein each R is independently an optionally substituted alkyl; and B is an optionally modified nucleobase; ＊ represents the attachment to the internucleotide linkage to the next nucleotide] 11. The dsRNA agent of claim 1, 4, or 10, which is a 4'-modified TNA having the structure:

26. At least one R is a linear or branched C １ ~C ３ 26. The dsRNA agent of claim 25, which is alkyl.

27. Each R is independently a linear or branched C １ ~C ３ 26. The dsRNA agent of claim 25, which is alkyl.

28. 28. The dsRNA agent of claim 27, wherein each R is methyl.

29. R is alkenyl, alkynyl, aryl, heteroaryl, OR ａ , optionally substituted by one or more substituents selected from the group consisting of halo, NR′R″, and CR′R″R′″ １ ~C ６ alkyl, where R', R'', and R''' are each independently H, alkyl, halo, or COR ａ and R ａ is H, alkyl, or alkoxyalkyl, wherein each substituent is optionally substituted with one or more of C(O), N(H), halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, lipophilic moiety, carbohydrate, and / or vitamin.

30. R is alkenyl, alkynyl, aryl, heteroaryl, OR ａ methyl optionally substituted with one or more substituents selected from the group consisting of halo, NR'R'', and CR'R''R''', where R', R'', and R''' are each independently H, alkyl, halo, or COR ａ and R ａ 30. The dsRNA agent of claim 29, wherein is H, alkyl, or alkoxyalkyl.

31. B is the following: 【Chemistry 10】 26. The dsRNA agent of claim 22, 23, 24, or 25, which is one of:

32. B is the following: 【Chemistry 11】 26. The dsRNA agent of claim 22, 23, 24, or 25, which is one of:

33. B, 【Chemistry 12】 [In the formula, R ｂ is a lipophilic moiety, a carbohydrate, folic acid, or glutamate urea (PUPA).

26. The dsRNA agent of claim 22, 23, 24, or 25, wherein

34. R ｂ is saturated or unsaturated C ４ ~C ３０ 34. The dsRNA agent of claim 33, wherein the lipophilic moiety comprises a carbohydrate chain.

35. R ｂ but the following: 【Chemistry 13】 【Chemistry 14】 or R ｂ but, 【Chemistry 15】 [In the formula, s is 1 to 10, and Ln is as follows: 【Chemistry 16】 34. The dsRNA agent of claim 33, having the formula:

36. 24. The dsRNA agent of claim 23, wherein R is methyl and R is in the (R) or (S) configuration.

37. 24. The dsRNA agent of claim 23, wherein R is methyl substituted with hydroxyl, alkoxy, or alkoxyalkoxy, and R is in the (R) or (S) configuration.

38. 4'-modified TNA 【Chemistry 17】 38. The dsRNA agent of claim 37, having the structure:

39. R is C substituted by one or more halogen groups １ ~C ３ 24. The dsRNA agent of claim 23, wherein R is alkyl and R is in the (R) or (S) configuration.

40. 4'-modified TNA 【Chemistry 18】 40. The dsRNA agent of claim 39, having the structure:

41. 24. The dsRNA agent of claim 23, wherein R is methyl substituted with alkenyl or alkynyl, and R is in the (R) or (S) configuration.

42. 4'-modified TNA 【Chemistry 19】 42. The dsRNA agent of claim 41, having the structure:

43. 24. The dsRNA agent of claim 23, wherein R is methyl substituted with a triazolyl group, the triazolyl group having an N atom optionally substituted with one or more of C(O), N(H), alkyl, alkenyl, alkynyl, aryl, heteroaryl, lipophilic moiety, carbohydrate, and / or vitamin, and R is in the (R) or (S) configuration.

44. 4'-modified TNA 【Chemistry 20】 [In the formula, R １ Below: 【Chemical 21】 【Chemical 22】 【Chemical 23】 【Chemistry 24】 and n is 1 to 10.

44. The dsRNA agent of claim 43, having the structure:

45. 24. The dsRNA agent of claim 23, wherein R is methyl substituted by an amino group, optionally substituted by one or more of C(O), N(H), halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, lipophilic moiety, carbohydrate, and / or vitamin, and R is in the (R) or (S) configuration.

46. 4'-modified TNA 【Chemistry 25】 [In the formula, R １ Below: 【Chemical 26】 is one of the 46. ​​The dsRNA agent of claim 45, having the structure:

47. At least one 4'-modified TNA is 【Chemical 27】 5. The dsRNA agent of claim 4, having the structure:

48. 48. The dsRNA agent of claim 47, wherein R is methyl and R is in the (R) or (S) configuration.

49. 49. The dsRNA agent of claim 48, wherein R is in the (S) configuration and the at least one 4'-modified TNA is at one of positions 1-9 and 12-15 of the sense strand, counting from the 5' end of the sense strand.

50. 49. The dsRNA agent of claim 48, wherein R is in the (S) configuration and the at least one 4'-modified TNA is at one of positions 3, 5-11, 14, 16, 18, and 20 of the antisense strand, counting from the 5' end of the antisense strand.

51. 49. The dsRNA agent of claim 48, wherein R is in the (R) configuration and the at least one 4'-modified TNA is at one of positions 1-10 and 12-21 of the sense strand, counting from the 5' end of the sense strand.

52. 49. The dsRNA agent of claim 48, wherein R is in the (R) configuration and the at least one 4'-modified TNA is at one of positions 2-10, 19-21, and 23 of the antisense strand, counting from the 5' end of the antisense strand.

53. 11. The dsRNA agent of claim 1, 4, or 10, wherein the internucleotide linkage between the 1st and 2nd nucleotides at the 5' end of the antisense strand is not a phosphorothioate linkage.

54. 11. The dsRNA agent of claim 1, 4, or 10, wherein the 5' end of the antisense strand does not contain a phosphate or phosphomimetic.

55. 11. The dsRNA agent of claim 1, 4, or 10, wherein the sense strand and the antisense strand are each 19 to 25 nucleotides in length.

56. 56. The dsRNA agent of claim 55, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, wherein the strands form a double-stranded region of 21 contiguous base pairs with a 2 nucleotide-long single-stranded overhang at the 3' end.

57. 11. The dsRNA agent of claim 1, 4, or 10, wherein the dsRNA agent comprises a single-stranded overhang on one end and a blunt end on the other end.

58. 58. The dsRNA agent of claim 57, wherein the single-stranded overhang is 1, 2, or 3 nucleotides in length.

59. 11. The dsRNA agent of claim 1, 4, or 10, wherein the sense strand comprises at least two phosphorothioate internucleotide linkages within the first five nucleotides counting from the 5' end of the sense strand.

60. 11. The dsRNA agent of claim 1, 4, or 10, wherein the antisense strand comprises at least two phosphorothioate internucleotide linkages within the first five nucleotides counting from the 3' end of the antisense strand.

61. 11. The dsRNA agent of claim 1, 4, or 10, wherein the antisense strand comprises at least two phosphorothioate internucleotide linkages within the first five nucleotides counting from the 5' end of the antisense strand.

62. At least one chain contains one or more phosphorothioates (PS ２ 11. The dsRNA agent of claim 1, 4, or 10, comprising a .

63. 11. The dsRNA agent of claim 3, 5, or 10, wherein the ligand or lipophilic moiety is conjugated via a biocleavable linker selected from the group consisting of DNA, RNA, disulfides, amides, functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

64. 11. The dsRNA agent of claim 3, 5, or 10, wherein the ligand or lipophilic moiety is conjugated via a carrier selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl, or is an acyclic moiety based on a serinol or diethanolamine backbone.

65. 13. A method of modulating expression of a target gene in a cell, comprising administering to the cell the dsRNA agent of claim 1, 4, or 10.

66. 66. The method of claim 65, wherein the cell is in a subject.

67. 67. The method of claim 65 or 66, wherein the cell is a liver cell.

68. 67. The method of claim 65 or 66, wherein the cell is an extrahepatic cell.

69. 67. The method of claim 66, wherein the dsRNA agent is administered intrathecally.

70. 70. The method of claim 69, wherein the method reduces expression of the target gene in brain or spinal tissue.

71. 71. The method of claim 70, wherein the brain or spinal tissue is selected from the group consisting of cortex, cerebellum, cervical vertebrae, lumbar vertebrae, and thoracic vertebrae.

72. 70. The method of claim 69, 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, and GSK3α.

73. 69. The method of claim 68, wherein the dsRNA agent is administered intravitreally.

74. 74. The method of claim 73, wherein the method reduces expression of a target gene in ocular tissue.

75. 1. A method of treating a subject having a CNS disorder, comprising: administering to the subject a therapeutically effective amount of a double-stranded RNA (dsRNA) agent capable of modulating expression of a target gene, thereby treating the subject; A method wherein the dsRNA agent comprises a sense strand and an antisense strand that is sufficiently complementary to at least a portion of the mRNA of the target gene, each strand having 14-40 nucleotides, and the antisense strand comprises at least one TNA (threose nucleic acid) at position 1, counting from the 5' end.

76. 76. The method of claim 75, 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 ataxia, prion, and Lafora.

77. 76. The method of claim 75, wherein the dsRNA agent is administered extrahepatically.

78. 78. The method of claim 77, wherein the dsRNA agent is administered intrathecally.

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