Modified double-stranded RNA agents

Abstract

To provide novel iRNA duplex agents capable of improving the efficacy of gene silencing in gene therapeutics using siRNA.SOLUTION: The sense strand of the dsRNA agent comprises at least one thermally destabilizing nucleotide, and the at least one thermally destabilizing nucleotide occurring at a site opposite to the seed region (positions 2-8) of the antisense strand; and the antisense strand of the dsRNA agent comprises at least two modified nucleotides that provide the nucleotide a steric bulk that is less than or equal to the steric bulk of a 2'-OMe modification, wherein the modified nucleotides are separated by 11 nucleotides in length. Other aspects of the invention relate to pharmaceutical compositions comprising these dsRNA agents suitable for therapeutic use, and methods of inhibiting the expression of a target gene by administering these dsRNA agents, e.g., for the treatment of various disease conditions.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 093,919, filed December 18, 2014, U.S. Provisional Patent Application No. 62 / 083,744, filed November 24, 2014, and U.S. Provisional Patent Application No. 62 / 039,507, filed August 20, 2014, the entire contents of each of which are incorporated herein by reference.

[0002] The present invention relates to the RNAi duplex agent that contains specific motifs that are advantageous for inhibiting the expression of target genes, and the RNAi composition that is suitable for therapeutic use.In addition, the present invention provides the method of inhibiting the expression of target genes by administering these RNAi duplex agents, for example, for the treatment of various diseases. [Background technology]

[0003] RNA interference or "RNAi" was originally coined by Fire and colleagues to describe the observation that double-stranded RNAi (dsRNA) can block gene expression (Fire et al., 2003). Short dsRNA induces gene-specific post-translational silencing in a variety of organisms, including vertebrates, providing a new tool for the study of gene function. RNAi is mediated by the RNA-induced silencing complex (RISC), a sequence-specific multicomponent nuclease that destroys messenger RNAs that are homologous to the silencing trigger. RISC is known to contain short RNAs (about 22 nucleotides) derived from a double-stranded RNA trigger, but the protein components of this activity are still unknown.

[0004] Double-stranded RNA (dsRNA) molecules with good gene silencing properties are necessary for the development of drugs based on RNA interference (RNAi). The first step of RNAi is the activation of the RNA-induced silencing complex (RISC), which requires the degradation of the sense strand of the dsRNA duplex. It was known that the sense strand serves as the first RISC substrate to be cleaved by Argonaute 2 in the middle of the duplex region. Immediately after the cleaved 5'- and 3'-end fragments of the sense strand are removed by endonuclease Ago2, RISC is activated by the antisense strand (Non-Patent Document 2).

[0005] It was believed that inhibition of sense strand cleavage would prevent endonucleolytic cleavage of target mRNA (Non-Patent Document 3). Leuschner et al. showed that incorporation of 2'-O-Me ribose into the Ago2 cleavage site of the sense strand inhibited RNAi in HeLa cells (Non-Patent Document 4). A similar effect was observed with phosphorothioate modification, indicating that sense strand cleavage is required for efficient RNAi in mammals.

[0006] Morrissey et al. have obtained suitable silencing compared to unmodified siRNA by using siRNA duplexes that contain 2'-F modified residues at Ago2 cleavage site among other sites and modifications (Non-Patent Document 5).However, Morrissey's modifications are not motif-specific, for example, one modification includes 2'-F modification at all pyrimidines without any selectivity if pyrimidine residues are present in both sense and antisense strands; therefore, based on these teachings, it is unclear whether specific motif modifications at the cleavage site of sense strand can have actual effect on gene silencing activity.

[0007] Muhonen et al. used an siRNA duplex containing two 2'-F modified residues at the Ago2 cleavage site of the sense or antisense strand and found that the siRNA duplex was tolerated (Non-Patent Document 6). However, Muhonen's modification is also sequence-specific, e.g., for each particular strand, Muhonen's modification only modifies all pyrimidines or all purines without any selectivity.

[0008] Choung et al. stabilized siRNA in serum against Sur10058 using siRNA duplexes containing selective modifications with 2'-OMe or various combinations of 2'-F, 2'-OMe, and phosphorothioate modifications (Non-Patent Document 7). Choung suggested that the residue at the cleavage site of the antisense strand should not be modified with 2'-OMe to enhance siRNA stability. Summary of the Invention [Problem to be solved by the invention]

[0009] Thus, there is currently a need for iRNA duplexes to improve the gene silencing efficacy of siRNA gene therapy.The present invention addresses this need. [Means for solving the problem]

[0010] The present invention provides effective nucleotide or chemical motifs of dsRNA agents, optionally conjugated with at least one ligand, that are advantageous for inhibiting expression of target genes, and RNAi compositions suitable for therapeutic use.

[0011] In one aspect, the present invention relates to a double-stranded RNA (dsRNA) agent for 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 dsRNA agent is represented by formula (I): [ka]

[0012] In formula (I), B1, B2, B3, B1', B2', B3', and B4' are each independently nucleotides that include 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 include a 2'-OMe modification. In one embodiment, B1, B2, B3, B1', B2', B3', and B4' each include a 2'-OMe modification or a 2'-F modification. In one embodiment, at least one of B1, B2, B3, B1', B2', B3', and B4' includes a 2'-ON-methylacetamide (2'-O-NMA) modification.

[0013] C1 is a heat destabilizing nucleotide at a site 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 at a position in the sense strand that pairs with nucleotides 2-8 of the 5' end of the antisense strand. In one example, C1 is at position 15 of the 5' end of the sense strand. The C1 nucleotide has a heat destabilizing modification that can include an abasic modification; a mismatch with the opposing nucleotide of the duplex; and a sugar modification, e.g., a 2'-deoxy modification or an acyclic nucleotide, e.g., an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA). In one embodiment, C1 has (i) a mismatch with the opposing nucleotide of the antisense strand; (ii) an abasic modification selected from the group consisting of: [ka] and (iii) a sugar modification selected from the group consisting of: [ka] where B is a modified or unmodified nucleobase and R 1 and R 2 are independently H, halogen, OR 3 or alkyl; R 3is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. In one embodiment, the thermally destabilizing 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; optionally, at least one nucleobase in the mismatch pair is a 2'-deoxy-nucleobase. In one example, the thermally destabilizing modification in C1 is GNA or [ka] It is.

[0014] T1, T1', T2', and T3' each independently represent a nucleotide that includes a modification that provides the nucleotide with a steric bulk equal to or less than that of a 2'-OMe modification. Steric bulk refers to the total steric effect of the modification. Methods for determining the steric effect of a modification of a nucleotide are known to those skilled in the art. The modification may be a modification at the 2' position of the ribose sugar of the nucleotide, or a modification of the backbone of a nucleotide similar or equivalent to the 2' position of a non-ribose nucleotide, acyclic nucleotide, or ribose sugar, and provides the nucleotide with a steric bulk equal to or less than that of a 2'-OMe modification. For example, T1, T1', T2', and T3' each independently are 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.

[0015] n 1 , n 3 , and q 1 are independently 4 to 15 nucleotides in length.

[0016] n 5 , q 3 , and q 7 are independently 1 to 6 nucleotides in length.

[0017] n 4 , q 2 , and q 6 are independently 1 to 3 nucleotides in length; or 4 is 0.

[0018] q 5 are independently 0 to 10 nucleotides in length.

[0019] n 2 and q 4 are independently 0 to 3 nucleotides in length.

[0020] Or, n 4 is 0 to 3 nucleotides in length.

[0021] 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, having two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0022] In one embodiment, n 4 , q 2 , and q 6 are each equal to 1.

[0023] In one embodiment, n 2 , n 4 , q 2 , q 4 , and q 6 are each equal to 1.

[0024] In one embodiment, when the sense strand is 19 to 22 nucleotides in length, C1 is located at positions 14 to 17 of the 5' end of the sense strand; 4 is 1. In one embodiment, C1 is at position 15 of the 5' end of the sense strand.

[0025] In one embodiment, T3' begins at position 2 of the 5' end of the antisense strand. In one example, T3' is at position 2 of the 5' end of the antisense strand and 6 is equal to 1.

[0026] In one embodiment, T1' begins at position 14 of the 5' end of the antisense strand. In one example, T1' is at position 14 of the 5' end of the antisense strand and 2 is equal to 1.

[0027] 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. 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.

[0028] In one embodiment, T1' and T3' are separated by 11 nucleotides (ie, not counting the T1' and T3' nucleotides).

[0029] In one embodiment, T1' is at position 14 of the 5' end of the antisense strand. 2 is equal to 1 and the modification is at the 2' position or at a non-ribose, acyclic, or backbone position that provides less steric bulk than 2'-OMe ribose.

[0030] In one embodiment, T3' is at position 2 of the 5' end of the antisense strand. 6is equal to 1 and the modification is at the 2' position or at a non-ribose, acyclic, or backbone position that provides steric bulk less than or equal to that of 2'-OMe ribose.

[0031] In one embodiment, T1 is at the cleavage site of the sense strand. In one example, when the sense strand is 19-22 nucleotides long, T1 is at position 11 of the 5' end of the sense strand; 2 is 1. In an exemplary embodiment, when the sense strand is 19-22 nucleotides in length, T1 is at the cleavage site of the sense strand at position 11 of the 5' end of the sense strand, and n 2 is 1.

[0032] In one embodiment, T2' begins at position 6 of the 5' end of the antisense strand. In one example, T2' is from position 6 to position 10 of the 5' end of the antisense strand, 4 is 1.

[0033] In an exemplary embodiment, T1 is at the cleavage site of the sense strand, e.g., at position 11 of the 5' end of the sense strand, where the sense strand is 19-22 nucleotides in length, 2 is 1; T1' is at position 14 of the 5' end of the antisense strand; q 2 is equal to 1, and the T1' modification is at the 2' position of the ribose sugar or at a non-ribose, acyclic, or backbone position that provides less steric bulk than 2'-OMe ribose; T2' is located at positions 6-10 of the 5' end of the antisense strand, and q 4 is 1; and T3' is at position 2 of the 5' end of the antisense strand, q 6 is equal to 1, and the T3' modification is at the 2' position or at a non-ribose, acyclic, or backbone position that provides steric bulk less than or equal to that of 2'-OMe ribose.

[0034] In one embodiment, T2' begins at position 8 of the 5' end of the antisense strand. 4 is 2.

[0035] In one embodiment, T2' begins at position 9 of the 5' end of the antisense strand. 4 is 1.

[0036] 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; having two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0037] In one embodiment, n 4 is 0, B3 is 2'-OMe, 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; having two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0038] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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.

[0039] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 q5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0040] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 6, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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.

[0041] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 6, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 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; having two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0042] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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.

[0043] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 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; having two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0044] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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 2 additional TTs at the 3' end of the antisense strand.

[0045] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 having at least two additional TTs at the 3' end of the antisense strand; two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), as well as two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0046] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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.

[0047] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; having two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0048] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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 5is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1.

[0049] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; having two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0050] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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.

[0051] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; having two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0052] The dsRNA agent may include a phosphorus-containing group at the 5' end of the sense or antisense strand. The 5'-terminal phosphorus-containing group may be a 5'-terminal phosphate (5'-P), a 5'-terminal phosphorothioate (5'-PS), a 5'-terminal phosphorodithioate (5'-PS 2 ), 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 phosphate, [ka] ), or the 5'-Z-VP isomer (i.e., cis-vinyl phosphate, [ka] ) or a mixture thereof.

[0053] In one embodiment, the dsRNA agent comprises a phosphorus-containing group at the 5'-end of the sense strand.In one embodiment, the dsRNA agent comprises a phosphorus-containing group at the 5'-end of the antisense strand.

[0054] In one embodiment, the dsRNA agent includes a 5'-P. In one embodiment, the dsRNA agent includes a 5'-P in the antisense strand.

[0055] In one embodiment, the dsRNA agent comprises a 5'-PS.In one embodiment, the dsRNA agent comprises a 5'-PS in the antisense strand.

[0056] 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.

[0057] In one embodiment, the dsRNA agent is a 5'-PS 2 In one embodiment, the dsRNA agent comprises a 5'-PS 2 Includes.

[0058] In one embodiment, the dsRNA agent is a 5'-PS 2In one embodiment, the dsRNA agent comprises 5' deoxy 5'-C-malonyl in the antisense strand.

[0059] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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 contains a 5'-PS.

[0060] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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.

[0061] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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.

[0062] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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 is a 5'-PS 2 Also includes.

[0063] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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'-deoxy-5'-C-malonyl.

[0064] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-P.

[0065] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-PS.

[0066] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 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.

[0067] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. 2 Also includes.

[0068] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23. The dsRNA agent also includes a 5'-deoxy-5'-C-malonyl.

[0069] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3is 7, and n 4 is 0, B3 is 2'-OMe, 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.

[0070] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 contains a 5'-PS.

[0071] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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, 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.

[0072] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 is a 5'-PS 2 Also includes.

[0073] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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, 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'-deoxy-5'-C-malonyl.

[0074] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-P.

[0075] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-PS.

[0076] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 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.

[0077] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. 2 Also includes.

[0078] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23. The dsRNA agent also includes a 5'-deoxy-5'-C-malonyl.

[0079] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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.

[0080] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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 contains a 5'-PS.

[0081] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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.

[0082] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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 is a 5'-PS 2 Also includes.

[0083] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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'-deoxy-5'-C-malonyl.

[0084] In one embodiment, B1 is 2'-OMe or 2'-F, and n1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-P.

[0085] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-PS.

[0086] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 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.

[0087] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. 2 Also includes.

[0088] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23. The dsRNA agent also includes a 5'-deoxy-5'-C-malonyl.

[0089] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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.

[0090] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 4is 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 contains a 5'-PS.

[0091] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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.

[0092] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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'-F, and q 7 is 1. The dsRNA agent is a 5'-PS 2 Also includes.

[0093] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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'-deoxy-5'-C-malonyl.

[0094] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 q7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-P.

[0095] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-PS.

[0096] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 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.

[0097] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. 2 Also includes.

[0098] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23. The dsRNA agent also includes a 5'-deoxy-5'-C-malonyl.

[0099] In one embodiment, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of the dsRNA agent of the present invention is modified.For example, when 50% of the dsRNA agent is modified, 50% of all nucleotides present in the dsRNA agent contain modification as described herein.

[0100] In one embodiment, the sense and antisense strands of a dsRNA agent are each 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.

[0101] In one embodiment, each of the sense and antisense strands of a dsRNA agent contains at least two different modifications.

[0102] In one embodiment, the dsRNA agent of formula (I) further comprises a 3' and / or 5' overhang of 1-10 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.

[0103] In one embodiment, a dsRNA agent of the invention does not include any 2'-F modifications.

[0104] 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 one block of two phosphorothioate or methylphosphonate internucleotide linkages. In one example, the antisense strand comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages. For example, the two blocks of phosphorothioate or methylphosphonate internucleotide linkages are separated by 16-18 phosphate internucleotide linkages.

[0105] In one embodiment, each of the sense and antisense strands of the dsRNA agent has between 15 and 30 nucleotides. In one example, the sense strand has between 19 and 22 nucleotides and the antisense strand has between 19 and 25 nucleotides. In another example, the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides.

[0106] In one embodiment, the nucleotide at position 1 of the 5' end of the antisense strand in 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.

[0107] In one embodiment, the antisense strand of the dsRNA agent of the present invention is 100% complementary to target RNA, so as to hybridize with target RNA and inhibit its expression by RNA interference.In another embodiment, the antisense strand of the dsRNA agent of the present invention 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 target RNA.

[0108] In one aspect, the present invention relates to a dsRNA agent as defined herein capable of inhibiting expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides. The sense strand comprises at least one thermolabile nucleotide, at least one of which is located opposite or adjacent to the seed region of the antisense strand (i.e., positions 2-8 of the 5' end of the antisense strand). Each of the embodiments and aspects described herein relating to a dsRNA represented by formula (I) can also be applied to a dsRNA comprising a thermolabile nucleotide.

[0109] The heat destabilizing nucleotide may be present between positions 14 and 17 at the 5' end of the sense strand, for example, when the sense strand is 21 nucleotides long. The antisense strand includes 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 are 11 nucleotides apart. For example, the two modified nucleic acids are at positions 2 and 14 at the 5' end of the antisense strand.

[0110] 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 added to the 3' end of the sense strand.

[0111] For example, the dsRNA agent described herein may include (i) a phosphorus-containing group at the 5'-end of the sense strand or antisense strand; (ii) two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5'-end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand; and (iii) a ligand at the 5'-end or 3'-end of the sense strand or antisense strand, such as an ASGPR ligand (e.g., one or more GalNAc derivatives). For example, the ligand may be at the 3'-end of the sense strand.

[0112] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 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.

[0113] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 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.

[0114] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 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.

[0115] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. 2 and a targeting ligand. 2 is at the 5' end of the antisense strand and the target ligand is at the 3' end of the sense strand.

[0116] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23. 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.

[0117] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 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.

[0118] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 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.

[0119] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 4is 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 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.

[0120] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. 2 and a targeting ligand. 2 is at the 5' end of the antisense strand and the target ligand is at the 3' end of the sense strand.

[0121] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23. 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.

[0122] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 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.

[0123] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 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.

[0124] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 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.

[0125] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. 2 and a targeting ligand. 2is at the 5' end of the antisense strand and the target ligand is at the 3' end of the sense strand.

[0126] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23. 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.

[0127] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 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.

[0128] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 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.

[0129] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 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.

[0130] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. 2 and a targeting ligand. 2 is at the 5' end of the antisense strand and the target ligand is at the 3' end of the sense strand.

[0131] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 q4 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23. 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.

[0132] In one particular embodiment, the dsRNA agent of the invention: (a) a sense strand: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to its 3' terminus, comprising three GalNAc derivatives attached by a trivalent branched linker; and (iii) a sense strand having 2'-F modifications at positions 1, 3, 5, 7, 9-11, 13, 17, 19, and 21 (counting from the 5' end), and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14-16, 18, and 20; And (b) an antisense strand: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 3, 5, 9, 11-13, 15, 17, 19, 21, and 23 (counting from the 5' end) and 2'F modifications at positions 2, 4, 6-8, 10, 14, 16, 18, 20, and 22; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 (counting from the 5' end) and between nucleotide positions 22 and 23; The dsRNA agent has an antisense strand with a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0133] In another particular embodiment, the dsRNA agent of the invention is: (a) a sense strand: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3′ end, comprising three GalNAc derivatives attached by a trivalent branched linker; (iii) 2'-F modifications at positions 1, 3, 5, 7, 9-11, 13, 15, 17, 19, and 21 (counting from the 5' end) and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20; and (iv) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; And (b) an antisense strand: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11-13, 15, 17, 19, and 21-23 (counting from the 5' end), and 2'-F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; The dsRNA agent comprises an antisense strand having a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0134] In another particular embodiment, the dsRNA agent of the invention is: (a) a sense strand: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3′ end, comprising three GalNAc derivatives attached by a trivalent branched linker; (iii) 2'-OMe modifications at positions 1-6, 8, 10, and 12-21 (counting from the 5' end), 2'-F modifications at positions 7 and 9, and a deoxy-nucleotide (e.g., dT) at position 11; and (iv) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; And (b) an antisense strand: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 3, 7, 9, 11, 13, 15, 17, and 19-23 (counting from the 5' end), and 2'-F modifications at positions 2, 4-6, 8, 10, 12, 14, 16, and 18; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; This 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.

[0135] In another particular embodiment, the dsRNA agent of the invention is: (a) a sense strand: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3′ end, comprising three GalNAc derivatives attached by 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) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; And (b) an antisense strand: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 5, 7, 9, 11, 13, 15, 17, 19, and 21-23 (counting from the 5' end), and 2'-F modifications at positions 2-4, 6, 8, 10, 12, 14, 16, 18, and 20; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; This 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.

[0136] In another particular embodiment, the dsRNA agent of the invention is: (a) a sense strand: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3′ end, comprising three GalNAc derivatives attached by 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) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; And (b) an antisense strand: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 5, 7, 9, 11-13, 15, 17, 19, and 21-23 (counting from the 5' end), and 2'-F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; This 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.

[0137] In another particular embodiment, the dsRNA agent of the invention is: (a) a sense strand: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3′ end, comprising three GalNAc derivatives attached by 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) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; And (b) an antisense strand: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 3, 5-7, 9, 11-13, 15, 17-19, and 21-23 (counting from the 5' end), and 2'-F modifications at positions 2, 4, 8, 10, 14, 16, and 20; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; This 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.

[0138] In another particular embodiment, the dsRNA agent of the invention is: (a) a sense strand: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3′ end, comprising three GalNAc derivatives attached by a trivalent branched linker; (iii) 2'-OMe modifications at positions 1, 2, 4, 6, 8, 12, 14, 15, 17, and 19-21, and 2'-F modifications at positions 3, 5, 7, 9-11, 13, 16, and 18; and (iv) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; And (b) an antisense strand: (i) 25 nucleotides long; (ii) 2'-OMe modifications at positions 1, 4, 6, 7, 9, 11-13, 15, 17, and 19-23 (counting from the 5' end), 2'-F modifications at positions 2, 3, 5, 8, 10, 14, 16, and 18, and deoxy-nucleotides (e.g., dT) at positions 24 and 25; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; This 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.

[0139] In another particular embodiment, the dsRNA agent of the invention is: (a) a sense strand: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3′ end, comprising three GalNAc derivatives attached by 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) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; And (b) an antisense strand: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 8, 10-13, 15, and 17-23 (counting from the 5' end), and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; This 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.

[0140] In another particular embodiment, the dsRNA agent of the invention is: (a) a sense strand: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3′ end, comprising three GalNAc derivatives attached by 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) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; And (b) an antisense strand: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 10-13, 15, and 17-23 (counting from the 5' end), and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; This 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.

[0141] In another particular embodiment, the dsRNA agent of the invention is: (a) a sense strand: (i) 19 nucleotides long; (ii) an ASGPR ligand attached to the 3′ end, comprising three GalNAc derivatives attached by a trivalent branched linker; (iii) 2'-OMe modifications at positions 1-4, 6, and 10-19, and 2'-F modifications at positions 5 and 7-9; and (iv) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; And (b) an antisense strand: (i) 21 nucleotides long; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 10-13, 15, and 17-21 (counting from the 5' end), and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21; This 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.

[0142] In one embodiment, a dsRNA agent described herein further comprises a thermodestabilizing modification at position 7, counting from the 5' end of the antisense strand, position 15, counting from the 5' end of the sense strand, position 21, counting from the 5' end of the sense strand, or a combination of these positions.

[0143] In one aspect, the present invention relates to a dsRNA agent capable of inhibiting expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides. The sense strand comprises at least one thermolabile nucleotide, the at least one thermolabile nucleotide being present at or near the opposite site of the seed region of the antisense strand (i.e., positions 2-8 of the 5' end of the antisense strand). For example, the thermolabile nucleotide is present between positions 14-17 of the 5' end of the sense strand when the sense strand is 21 nucleotides long. The antisense strand comprises two modified nucleic acids that are less than sterically demanding 2'-OMe modified, separated by 11 nucleotides in length. For example, the two modified nucleic acids are at positions 2 and 14 of the 5' end of the antisense strand.

[0144] In one embodiment, the sense strand of the dsRNA agent further comprises an endonuclease-sensitive modified nucleotide at the cleavage site of the sense strand. In one example, the endonuclease-sensitive modified nucleotide is at position 11 of the 5' end of the sense strand.

[0145] In one embodiment, the antisense strand further comprises a third modified nucleotide that provides the nucleotide with steric bulk equal to or less than that of the 2'-OMe modification, and the third modified nucleotide is at position 6-10 of the 5' end of the antisense strand. For example, the third modified nucleotide is at position 10 of the 5' end of the antisense strand.

[0146] The embodiments of the thermally destabilized nucleotide are the same as the various embodiments described above for C1 in formula (I). The embodiments of the modified nucleic acid that are less sterically demanding than the 2'-OMe modification are the same as the various embodiments described above for T1', T2', and T3' in formula (I). The embodiments that describe the length, overhang, additional modifications, and ligand binding for the dsRNA agent of formula I above are suitable here.

[0147] The present invention further relates to the use of a dsRNA agent as defined herein for inhibiting expression of a target gene. In one embodiment, the present invention further relates to the use of a dsRNA agent for inhibiting expression of a target gene in vitro.

[0148] The present invention further relates to the dsRNA agent defined herein, which is used to inhibit the expression of target gene in a subject.The subject can be any animal, preferably mammal, more preferably mouse, rat, sheep, cow, dog, cat or human.

[0149] In one aspect, 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 14-40 nucleotides. The sense strand includes an endonuclease-sensitive modified nucleotide (e.g., DNA, RNA, or 2'-F) near the cleavage site of the sense strand. For example, the endonuclease-sensitive modified nucleotide is at position 11 of the 5' end of the sense strand. An endonuclease-sensitive modification present near the cleavage site can affect the sensitivity of the cleavage site. For example, a thermostabilizing modification near the cleavage site can provide endonuclease sensitivity to that cleavage site. The antisense strand includes two modified nucleic acids that are less than sterically demanding 2'-OMe modifications separated by 11 nucleotides in length. For example, the two modified nucleic acids are at positions 2 and 14 of the 5' end of the antisense strand.

[0150] In another aspect, the present invention further provides a method for delivering a dsRNA agent of the present invention to a specific target in a subject by subcutaneous or intravenous administration.The present invention further provides a dsRNA agent of the present invention for use in a method for delivering said dsRNA agent to a specific target in a subject by subcutaneous or intravenous administration. [Brief description of the drawings]

[0151] [Figure 1]Figures 1A-1C are charts showing the effect of different modifications at position 17 of the sense strand on in vitro efficacy assessed at concentrations of 10 nM and 0.1 nM: (A) siRNA targeting mTTR with a non-F sense strand paired with a parent AS strand; (B) siRNA targeting mTTR with a non-F sense strand paired with a non-F AS strand; (C) siRNA targeting ANG, ApoC3, and TTRSC with a non-F sense strand paired with a parent AS strand. [Diagram 2] FIG. 2 is a chart showing the effect of position on the in vitro potency of heat-destabilizing GNA modifications spanning positions 16-18 of the sense strand, evaluated at concentrations of 10 nM and 0.1 nM. [Diagram 3] FIG. 3 is a chart showing the effect of modifications at position 2 of the antisense strand on the in vitro efficacy of siRNAs targeting mTTR, ApoC3, TTRSC, and TMP, assessed at concentrations of 10 nM and 0.1 nM. [Figure 4] FIG. 4 is a chart showing the effect of modifications at position 14 of the antisense strand on the in vitro efficacy of siRNAs targeting mTTR, ApoC3, and TTRSC, assessed at concentrations of 10 nM and 0.1 nM. [Diagram 5] FIG. 5 is a graph showing mTTR silencing in mice following a single SC dose of 2.5 mg / kg. [Figure 6] FIG. 6 is a chart showing dose response of non-F siRNAs AD-61398 and AD-64273 to the parental 2PS (AD-43527) and 6PS (AD-57727): single SC dose, protein levels measured 96 hours post dose. [Figure 7] FIG. 7 is a chart showing the reduction of mTTR protein in plasma following QW SC administration of 1 mg / kg siRNA in mice, comparing non-F AD-61398 with the parent motif: AD-57727. [Figure 8]FIG. 8 is a chart showing non-F designs compared to the parent motif: AD-60490: silencing of TMPRSS6 mRNA after a single SC dose of 3 mg / kg in mice (n=3 / group). [Figure 9] FIG. 9 compares non-F designs with the parent motif: AD-60490: TMPRSS6 mRNA silencing 7 days after a single SC dose of 3 mg / kg in mice (n=3 / group). [Figure 10] FIG. 10 shows the in vitro activity results of the activity of two motifs, motif 1 and motif 2, compared to the parent compound AD-57727. [Figure 11] FIG. 11 shows the in vivo evaluation of the silencing activity of siRNAs targeting mTTR. [Figure 12] FIG. 12 shows enhanced activity with Stability Enhanced Conjugate Chemistry (SEC-C), where liver was assessed for activity (mRNA) 7 days after dosing. [Figure 13] FIG. 13 shows a chart demonstrating the approximately 4-fold improvement in activity with the new motifs (motifs 1 and 2) compared to the parent compound. [Figure 14] FIG. 14 shows a chart showing the significantly improved duration of motif 1 and motif 2 in all three sequences. [Figure 15] FIG. 15 shows a graph depicting the results of ApoC3-GalNAc3 SAR with a single SC dose of hAAV 1×10 11 GC / mouse, 3 mg / kg. [Figure 16] Figure 16 illustrates a schematic of Ago2-tagged siRNA and the modified phosphate that mimics the stable phosphate, 5'-vinylphosphonate (5'-VP), which is added by the cytoplasmic Clp1 kinase and serves as a key anchor for Ago2 attachment. [Figure 17]Figure 17 shows a chart showing how the presence of 5'-VP generally improves in vivo activity. Four different ApoB sequences were evaluated. LDL levels 7 days after a single SC dose of 3 mg / kg were analyzed for the four conjugates (with or without the 5'-VP modification). [Figure 18] FIG. 18 shows different chemical modifications that can replace the PS bond and provide a more stable chemistry, including phosphorodithioate (PS2) and methylphosphonate (MePhos), which promote endogenous phosphorylation. [Figure 19] Figure 19 shows a chart of the in vitro evaluation of terminal modifications including 2'-OMe-MePhos, 2'-OMe-PS, dN(PS2), and 2'F-PS. Transfection of mouse primary hepatocytes at 10 nM and 0.1 nM (n=4) was performed with the two ApoB conjugates. [Figure 20] Figure 20 shows two charts that show how subtle changes in the antisense 5' end can dramatically improve in vivo efficacy. The chart on the left shows that a 2'F-PS at position 1 of the antisense strand can improve the activity of a 5'P-dependent sequence (F9 activity measured on day 3, single SC dose of 3 mg / kg). The chart on the right shows the approximately 3-fold improved potency by dN(PS)2 versus the parent, as well as VP (LDL measured on day 3 for ApoB, single SC dose of 10 mg / kg). [Figure 21] Figure 21A and Figure 21B show SAR analysis comparing the in vitro and in vivo activity of ApoB siRNA containing 5'-OH modification (at the 5' end of the antisense strand) and ApoB siRNA containing 5'-E-VP modification. Figure 21A shows the results in hepatocytes of in vitro transfected mice. Figure 21B shows the level of LDL 3 days after a single dose (SC administration). [Figure 22]Figure 22 shows the results of a comparison of the in vitro potency of 5'-E-VP and 5'-Z-VP modifications on mTTR and F9 siRNA-GalNAc conjugates from in vitro transfected primary mouse hepatocytes. [Diagram 23] FIG. 23 shows the results of an in vivo comparison of 5'-E-VP and 5'-Z-VP modifications on F9 siRNA-GalNAc conjugates (single SC administration). [Figure 24] Figures 24-24C are graphs showing dose-response curves for (A) 5'-OH, (B) 5'-C-malonyl, and (C) 5'-phosphate PTEN siRNA in mouse primary stem cells in an in vitro PTEN silencing assay. All values ​​are from triplicate experiments. [Diagram 25] Figure 25 shows the results of enzyme stability of 5'-OH, 5'-C-malonyl, and 5'-phosphate siRNA incubated with rat liver trichosomes. siRNA target sequences are shown in Table 10. Data was normalized to untreated controls. [Figure 26] Figure 26 shows the results of RISC addition of 5'-OH, 5'-C-malonyl, and 5'-phosphate siRNA (5' modification of antisense strand) determined by immunoprecipitation of Ago2 from mouse primary stem cells and by RT-PCR amplification of Ago2-added single strand. The level of endogenous miR122 was determined as a control. The siRNA target sequences are shown in Table 10. [Figure 27] Figure 27 is a graph showing the in vitro knockdown of TTR using siRNA modified with one (S)-GNA nucleotide. The level of TTR mRNA was measured after 24 hours of incubation with 10 nM siRNA in mouse primary stem cells. TTR mRNA was assessed using RT-qPCR and normalized to PBS-treated cells. All data points are the average of four measurements. [Figure 28]Figure 28A is a graph showing the in vitro knockdown of TTR using siRNA modified with one (S)-GNA base pair. The level of TTR mRNA was measured after 24 hours of incubation with 10 nM siRNA in mouse primary stem cells. TTR mRNA was evaluated using RT-qPCR and normalized to PBS-treated cells. All data points are the average of four measurements. Figure 28B shows a mix and match duplex in which the sense and antisense strands containing one (S)-GNA nucleotide are paired here as GNA:RNA-base pair. [Figure 29] Figure 29 is a graph showing the in vivo levels of TTR in mouse serum. Animals were administered a single dose of 2.5mg / kg siRNA. Before or at the indicated times after administration, animals were bled and serum samples were measured by sandwich ELISA assay using HRP-conjugated antibody and 3,3',5,5'-tetramethylbenzidine for reading at 450nm. All samples were measured in duplicate, and each data point is the average of mice in each cohort (n=3). [Diagram 30] Figure 30 is a graph showing in vivo quantification of TTR mRNA levels. Animals were administered a single dose of 2.5 mg / kg siRNA. RNA extraction was performed on total liver homogenates at the indicated times after administration. TTR mRNA was assessed by RT-qPCR as described above with the ΔΔCt method using GAPDH as a control transcript and normalized to PBS-treated animals. Dark bars show results at day 21; white bars show results at day 7. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0152] The inventors found that having 2'-OMe modifications at nucleotide positions 2 and 14 at the 5' end of the antisense strand suppressed the gene silencing activity of a dsRNA agent. By introducing chemical modifications at the 2' or equivalent positions in the backbone that are non-ribose, acyclic, or less sterically bulky than the 2'-OMe modifications at certain positions in the antisense and / or sense strand, the dsRNA agent was able to regain gene silencing activity. The inventors also determined that introducing a heat destabilized nucleotide into the sense strand at a site opposite the seed region of the antisense strand (i.e., positions 2-8 at the 5' end of the antisense strand) improved gene silencing activity.

[0153] The sense strand and antisense strand of dsRNA agent can be completely modified.The dsRNA agent is optionally conjugated with, for example, asialoglycoprotein receptor (ASGPR) ligand on the sense strand.The resulting dsRNA agent shows effective in vivo gene silencing activity.

[0154] Thus, the present invention provides double-stranded RNAi (dsRNA) agents capable of inhibiting expression of a target gene. The dsRNA agent includes a sense strand and an antisense strand. Each strand of the dsRNA agent can range from 12 to 40 nucleotides in length. For example, each strand can range from 14 to 40 nucleotides in length, 17 to 37 nucleotides in length, 25 to 37 nucleotides in length, 27 to 30 nucleotides in length, 17 to 23 nucleotides in length, 17 to 21 nucleotides in length, 17 to 19 nucleotides in length, 19 to 25 nucleotides in length, 19 to 23 nucleotides in length, 19 to 21 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 23 nucleotides in length.

[0155] The sense strand and the antisense strand form a double-stranded dsRNA. The double-stranded region of the dsRNA agent can be 12-40 nucleotide pairs in length. For example, the double-stranded region can be 14-40 nucleotide pairs in length, 17-30 nucleotide pairs in length, 25-35 nucleotide pairs in length, 27-35 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the double-stranded region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotide pairs in length.

[0156] In one embodiment, the dsRNA agent of the invention comprises one or more overhang regions and / or capping groups at the 3' end, 5' end, or both ends of the strand of the dsRNA agent. The overhang can be 1-10 nucleotides long, 1-6 nucleotides long, e.g., 2-6 nucleotides long, 1-5 nucleotides long, 2-5 nucleotides long, 1-4 nucleotides long, 2-4 nucleotides long, 1-3 nucleotides long, 2-3 nucleotides long, or 1-2 nucleotides long. The overhang can be the result of one strand being longer than the other, or the result of two strands of the same length being shifted. The overhang can form a mismatch with the target mRNA, or the overhang can be complementary to the targeted gene sequence, or it can be some other sequence. The first and second strands can be connected, for example, by additional bases forming a hairpin, or by other non-basic linkers.

[0157] In one embodiment, each nucleotide in the overhang region of the dsRNA agent of the present invention can be independently modified or unmodified, including but not limited to 2'-sugar modification, such as 2-F, 2'-O methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof.For example, TT can be the overhang sequence at either end of either strand.The overhang can form a mismatch with the target mRNA, or the overhang can be complementary to the targeted gene sequence, or it can be other sequences.

[0158] The 5' or 3' overhang on the sense strand, antisense strand, or both strands of the dsRNA agent of the present invention may be phosphorylated. In some embodiments, the overhang region comprises two nucleotides with phosphorothioate between them, and the two nucleotides may be the same or different. In one embodiment, the overhang is present at the 3' end of the sense strand, antisense strand, or both strands. In one embodiment, the 3' overhang is present on the antisense strand. In one embodiment, the 3' overhang is present on the sense strand.

[0159] The dsRNA agent of the present invention may contain only one overhang, which can enhance the interference activity of dsRNA without affecting the overall stability. For example, the single-stranded overhang is located at the 3' end of the sense strand or the 3' end of the antisense strand. The dsRNA may have a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand), or vice versa. In general, the antisense strand of the dsRNA has a 3'-end nucleotide overhang and the 5' end is blunt. Without being bound by theory, the asymmetric 5'-end blunt end of the antisense strand and the 3'-end overhang of the antisense strand favor the guide strand added in the RISC process. For example, the single overhang has a length of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0160] In one embodiment, a dsRNA agent of the invention can also have two blunt ends on either end of the dsRNA duplex.

[0161] In one embodiment, a dsRNA agent of the invention is a 19 nucleotide long double ended bluntmer, wherein the sense strand comprises at least one heat destabilized nucleotide, the at least one heat destabilized nucleotide being located at or near the site opposite the seed region of the antisense strand (i.e., positions 2-8 of the 5' end of the antisense strand). For example, the heat destabilized nucleotide is located between positions 14-17 of the 5' end of the sense strand. The antisense strand comprises at least two modified nucleic acids smaller than sterically demanding 2'-OMe; preferably, the two modified nucleic acids smaller than sterically demanding 2'-OMe are located at positions 2 and 14 of the 5' end of the antisense strand.

[0162] In one embodiment, the dsRNA agent of the invention is a 20 nucleotide long blunt-ended duplex, the sense strand comprising at least one heat destabilized nucleotide, the at least one heat destabilized nucleotide being located at or near the site opposite the seed region of the antisense strand (i.e., positions 2-8 of the 5' end of the antisense strand). For example, the heat destabilized nucleotide is located between positions 14-17 of the 5' end of the sense strand. The antisense strand comprises at least two modified nucleic acids smaller than sterically demanding 2'-OMe; preferably, the two modified nucleic acids smaller than sterically demanding 2'-OMe are located at positions 2 and 14 of the 5' end of the antisense strand.

[0163] In one embodiment, the dsRNA agent of the invention is a 21 nucleotide long blunt-ended duplex, the sense strand comprising at least one heat destabilizing nucleotide, the at least one heat destabilizing nucleotide being located at or near the site opposite the seed region of the antisense strand (i.e., positions 2-8 of the 5' end of the antisense strand). For example, the heat destabilizing nucleotide is located between positions 14-17 of the 5' end of the sense strand. The antisense strand comprises at least two modified nucleic acids smaller than sterically demanding 2'-OMe; preferably, the two modified nucleic acids smaller than sterically demanding 2'-OMe are located at positions 2 and 14 of the 5' end of the antisense strand.

[0164] In one embodiment, the dsRNA agent of the invention comprises a 21 nucleotide (nt) sense strand and a 23 nucleotide (nt) antisense strand, the sense strand comprising at least one heat destabilizing nucleotide, the at least one heat destabilizing nucleotide being present at or near the opposite site of the seed region of the antisense strand (i.e., positions 2-8 of the 5' end of the antisense strand). For example, the heat destabilizing nucleotide is present between positions 14-17 of the 5' end of the sense strand when the sense strand is 21 nucleotides long. The antisense strand comprises at least two modified nucleic acids smaller than sterically demanding 2'-OMe; preferably, the two modified nucleic acids smaller than sterically demanding 2'-OMe are at positions 2 and 14 of the 5' end of the antisense strand, and one end of the dsRNA 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. Optionally, the dsRNA further comprises a ligand (preferably a receptor ligand, ie, an ASGPR ligand).

[0165] In one embodiment, the dsRNA agent of the invention comprises a sense strand and an antisense strand: the sense strand is 25-30 nucleotide residues long, starting with the 5'-terminal nucleotide (position 1), and positions 1-23 of the sense strand comprise at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues long, starting with the 3'-terminal nucleotide, and at least 8 ribonucleotides are positioned to pair with positions 1-23 of the sense strand to form a duplex; at least the 3'-terminal nucleotide of the antisense strand is not paired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are not paired with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides; the 5'-end of the antisense strand comprises 10-30 consecutive nucleotides that are not paired with the sense strand, thereby forming a duplex of 10-30 consecutive nucleotides. a single-stranded 5' overhang of 30 nucleotides is formed; at least the 5' and 3' terminal nucleotides of the sense strand base pair with the nucleotides of the antisense strand when the sense strand and the antisense strand are aligned for maximum complementarity, thereby forming a substantial duplex region between the sense strand and the antisense strand; and the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the length of the antisense strand such that expression of the target gene is reduced when the double-stranded nucleic acid is introduced into a mammalian cell; and the sense strand contains at least one heat-destabilizing nucleotide, the at least one heat-destabilizing nucleotide being located at or near the site opposite the seed region of the antisense strand (i.e., positions 2-8 of the 5' end of the antisense strand). For example, the heat-destabilizing nucleotide is located between positions 14-17 of the 5' end of the sense strand. The antisense strand comprises at least two modified nucleic acids smaller than sterically demanding 2'-OMe; preferably, the two modified nucleic acids smaller than sterically demanding 2'-OMe are at positions 2 and 14 of the 5' end of the antisense strand.

[0166] In one embodiment, a dsRNA agent of the invention comprises a sense strand and an antisense strand, said dsRNA agent comprising a sense strand having a length of at least 25 and at most 29 nucleotides, and an antisense strand having a length of at most 30 nucleotides, wherein the sense strand comprises a modified nucleotide at position 11 of the 5' end that is susceptible to enzymatic degradation. the antisense strand comprises two modified nucleic acids smaller than sterically demanding 2'-OMe, the two modified nucleic acids being at positions 2 and 14 at the 5' end of the antisense strand; the 3' end of the sense strand and the 5' end of the antisense strand form a blunt end, the antisense strand is 1-4 nucleotides longer than the sense strand at its 3' end, the duplex region is at least 25 nucleotides in length, the antisense strand is sufficiently complementary to a target mRNA along at least 19 nucleotides of the length of the antisense strand such that when the dsRNA agent is introduced into a mammalian cell, expression of the target gene is reduced, and Dicer cleavage of the dsRNA occurs preferentially at siRNAs that include the 3' end of the antisense strand, thereby reducing expression of the target gene in the mammal. Optionally, the dsRNA agent further comprises a ligand.

[0167] In one embodiment, the sense strand comprises an enzymatically susceptible modified nucleotide at the 5' end at position 11. The antisense strand comprises two modified nucleic acids smaller than the sterically demanding 2'-OMe, the two modified nucleic acids being at positions 2 and 14 at the 5' end of the antisense strand.

[0168] In one embodiment, the antisense strand comprises two modified nucleic acids that are less than sterically demanding 2'-OMe, the two modified nucleic acids being at positions 2 and 14 of the 5' end of the antisense strand.

[0169] In one embodiment, each nucleotide in the sense and antisense strands of a dsRNA agent may be modified. Each nucleotide may be modified with the same or different modifications, which may include one or more changes to one or both of the non-linked phosphate oxygen and / or one or more linked phosphate oxygens; changes to a component of the ribose sugar, such as the 2' hydroxyl of the ribose sugar; bulk replacement of the phosphate moiety with a "dephospho" linker; modifications or replacements of natural bases; and replacements or modifications of the ribose-phosphate backbone.

[0170] Since nucleic acids are polymers of subunits, many modifications, such as modifications of bases, phosphate moieties, or non-linked O of phosphate moieties, are present at repeated positions within the nucleic acid. In some cases, modifications are present at all desired positions within the nucleic acid, but in many cases, they are not. As an example, modifications may be present only at the 3' or 5' terminal positions, or only at the terminal regions, e.g., at the terminal nucleotide positions or the last two, three, four, five, or ten nucleotides of the chain. Modifications may be present in double-stranded regions, single-stranded regions, or both. Modifications may be present only in the double-stranded regions of the RNA, or only in the single-stranded regions of the RNA. For example, phosphorothioate modifications at non-linked oxygen positions may be present only at one or both ends, or only at the terminal regions, e.g., at the terminal nucleotide positions or the last two, three, four, five, or ten nucleotides of the chain, or in the double-stranded and single-stranded regions, especially at the ends. The 5' end or both ends may be phosphorylated.

[0171] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in the single-stranded overhang, e.g., the 5' or 3' overhang, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' or 5' overhang may be modified, e.g., with modifications described herein. Modifications may include, for example, the use of modifications at the 2' position of the ribose sugar, e.g., the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F), or 2'-O-methyl modifications rather than ribosugars of the nucleobases, and modifications of the phosphate group, e.g., phosphothioate modifications. The overhang need not be homologous to the target sequence.

[0172] In one embodiment, each residue of sense strand and antisense strand is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy or 2'-fluoro.Sense strand and antisense strand can contain two or more modifications.In one embodiment, each residue of sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

[0173] At least two different modifications are typically present in the sense and antisense strands. These two modifications may be 2'-deoxy, 2'-O-methyl or 2'-fluoro, acyclic nucleic acids, or other.

[0174] In one embodiment, the sense and antisense strands each comprise two differently modified nucleotides selected from 2'-O-methyl or 2'-deoxy.

[0175] In one embodiment, each residue in the sense and antisense strands is independently modified with 2'-O-methyl nucleotides, 2'-deoxy nucleotides, 2'-deoxyfluoro nucleotides, 2'-ON-methylacetamide (2'-O-NMA) nucleotides, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotides, 2'-O-aminopropyl (2'-O-AP) nucleotides, or 2'-ara-F nucleotides.

[0176] In one embodiment, the dsRNA agent of the present invention comprises an alternating pattern of modifications, particularly in the B1, B2, B3, B1', B2', B3', B4' regions, as shown in formula I. The term "alternating motif" or "alternating pattern" as used herein refers to a motif having one or more modifications, each modification occurring at alternating nucleotides of a strand. The alternating nucleotides can refer to one at every other nucleotide, one at every third nucleotide, or similar patterns. For example, where A, B, and C each represent one type of modification to a nucleotide, the alternating motif can be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AAABBBAAABBB...", or "ABCABCABCABC...", etc.

[0177] The types of modifications included in the alternating motifs can be the same or different, for example, if A, B, C, D each represent one type of modification of a nucleotide, the alternation pattern, i.e. the modification of every other nucleotide, can be the same, but the sense or antisense strand can each choose from several possibilities of modifications within the alternating motif, e.g. "ABABAB...", "ACACAC...", "BDBDBD..." or "CDCDCD...".

[0178] In one embodiment, the dsRNA agent of the present invention comprises a modification pattern of an alternating motif in the sense strand that is shifted relative to the modification pattern of the alternating motif in the antisense strand. This shift can be such that the modification group of the nucleotide of the sense strand corresponds to a different modification group of the nucleotide of the antisense strand, or vice versa. For example, when the sense strand base pairs with the antisense strand in a dsRNA duplex, the alternating motif in the sense strand can begin with "ABABAB" from 5' to 3' of the sense strand, and the alternating motif in the antisense strand can begin with "BABABA" from 3' to 5' of the antisense strand in the duplex region. As another example, the alternating motif in the sense strand can begin with "AABBAABB" from 5' to 3' of the sense strand, and the alternating motif in the antisense strand can begin with "BBAABBAA" from 3' to 5' of the antisense strand in the duplex region, so that there is a complete or partial shift in the modification pattern between the sense strand and the antisense strand.

[0179] The dsRNA agent of the present invention can further comprise at least one phosphorothioate or methylphosphonate internucleotide bond.The phosphorothioate or methylphosphonate internucleotide bond modification can be present at any nucleotide in any position in sense strand and / or antisense strand, or both strands.For example, the internucleotide bond modification can be present at any nucleotide in sense strand and antisense strand; each internucleotide bond modification can be present in an alternating pattern in sense strand or antisense strand; or the sense strand or antisense strand comprises both internucleotide bond modifications in an alternating pattern.The alternating pattern of the internucleotide bond modification in sense strand can be the same or different from that of antisense strand, and the alternating pattern of the internucleotide bond modification in sense strand can have a shift with respect to the alternating pattern of the internucleotide bond modification in antisense strand.

[0180] In one embodiment, the dsRNA agent comprises a phosphorothioate or methylphosphonate internucleotide bond modification in the overhang region. For example, the overhang region comprises two nucleotides with a phosphorothioate or methylphosphonate internucleotide bond between the two nucleotides. The internucleotide bond modification can also be formed to link the overhang nucleotide to the terminal base-pairing nucleotide in the duplex region. For example, at least two, three, four, or all of the overhang nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide bonds, and optionally, there can be an additional phosphorothioate or methylphosphonate internucleotide bond that links the overhang nucleotide to the next base-pairing nucleotide. For example, there can be at least two phosphothioate internucleotide bonds between the terminal three nucleotides, two of which are overhang nucleotides, and the third nucleotide is the next base-pairing nucleotide of the overhang nucleotide. Preferably, these terminal three nucleotides can be at the 3' end of the antisense strand.

[0181] In one embodiment, a sense strand of a dsRNA agent contains 1 to 10 blocks of 2 to 10 phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is present at any position in the oligonucleotide sequence, and said sense strand is paired with an antisense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand containing either phosphorothioate, methylphosphonate, or phosphate linkages.

[0182] In one embodiment, the antisense strand of a dsRNA agent comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is present at any position in the oligonucleotide sequence, and said antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand containing either phosphorothioate, methylphosphonate, or phosphate linkages.

[0183] In one embodiment, the antisense strand of a dsRNA agent contains two blocks of three phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is present at any position in the oligonucleotide sequence, and said antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand containing either phosphorothioate, methylphosphonate, or phosphate linkages.

[0184] In one embodiment, the antisense strand of a dsRNA agent contains two blocks of four phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is present at any position in the oligonucleotide sequence, and said antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand containing either phosphorothioate, methylphosphonate, or phosphate linkages.

[0185] In one embodiment, the antisense strand of a dsRNA agent contains two blocks of five phosphorothioate or methylphosphonate internucleotide linkages separated by one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is present at any position in the oligonucleotide sequence, and said antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand containing either phosphorothioate, methylphosphonate, or phosphate linkages.

[0186] In one embodiment, the antisense strand of a dsRNA agent contains two blocks of six phosphorothioate or methylphosphonate internucleotide linkages separated by one, two, three, four, five, six, seven, eight, nine, or ten phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is present at any position in the oligonucleotide sequence, and said antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand containing either phosphorothioate, methylphosphonate, or phosphate linkages.

[0187] In one embodiment, the antisense strand of a dsRNA agent contains two blocks of seven phosphorothioate or methylphosphonate internucleotide linkages separated by one, two, three, four, five, six, seven, or eight phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is present at any position in the oligonucleotide sequence, and said antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand containing either phosphorothioate, methylphosphonate, or phosphate linkages.

[0188] In one embodiment, the antisense strand of a dsRNA agent contains two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by one, two, three, four, five, or six phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is present at any position in the oligonucleotide sequence, and said antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand containing either phosphorothioate, methylphosphonate, or phosphate linkages.

[0189] In one embodiment, the antisense strand of a dsRNA agent contains two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by one, two, three, or four phosphate internucleotide linkages, where one of the phosphorothioate or methylphosphonate internucleotide linkages is present at any position in the oligonucleotide sequence, and said antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand containing either phosphorothioate, methylphosphonate, or phosphate linkages.

[0190] In one embodiment, the dsRNA agent of the invention further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within the range of 1-10 terminal positions of the sense strand and / or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkages at one or both termini of the sense strand and / or antisense strand.

[0191] In one embodiment, the dsRNA agent of the invention further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within 1-10 internal regions of each duplex of the sense strand and / or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides can be linked by phosphorothioate, methylphosphonate internucleotide linkages in the duplex region from positions 8 to 16, counting from the 5' end of the sense strand; the dsRNA agent can optionally further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within 1-10 terminal positions.

[0192] In one embodiment, a dsRNA agent of the invention comprises 1-5 phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and 1-5 phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the sense strand, and further comprises 1-5 phosphorothioate or methylphosphonate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand and 1-5 phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the antisense strand.

[0193] In one embodiment, a dsRNA agent of the invention further comprises one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 5' end) and one phosphorothioate or methylphosphonate internucleotide linkage modification within positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the antisense strand.

[0194] In one embodiment, a dsRNA agent of the invention further comprises two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand.

[0195] In one embodiment, a dsRNA agent of the invention further comprises two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand.

[0196] In one embodiment, a dsRNA agent of the invention further comprises two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand.

[0197] In one embodiment, a dsRNA agent of the invention further comprises one phosphorothioate internucleotide linkage modification within the range of positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18-23 of the antisense strand.

[0198] In one embodiment, a dsRNA agent of the invention further comprises one phosphorothioate internucleotide linkage modification within the range of positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the antisense strand.

[0199] In one embodiment, the dsRNA agent of the invention further comprises one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand.

[0200] In one embodiment, a dsRNA agent of the invention further comprises two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand.

[0201] In one embodiment, a dsRNA agent of the invention further comprises two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand.

[0202] In one embodiment, a dsRNA agent of the invention further comprises two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand.

[0203] In one embodiment, a dsRNA agent of the invention further comprises two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand.

[0204] In one embodiment, the dsRNA of the invention further comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand.

[0205] In one embodiment, a dsRNA agent of the invention further comprises one phosphorothioate internucleotide linkage modification at position 1 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 of the antisense strand.

[0206] In one embodiment, a dsRNA agent of the invention further comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand.

[0207] In one embodiment, a dsRNA agent of the invention further comprises one phosphorothioate internucleotide linkage modification at position 1 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 of the antisense strand.

[0208] In one embodiment, a dsRNA agent of the invention further comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 22 and 23 of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand.

[0209] In one embodiment, a dsRNA agent of the invention further comprises one phosphorothioate internucleotide linkage modification at position 1 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 of the antisense strand.

[0210] In one embodiment, the dsRNA agent comprises a mismatch with the target, a mismatch within the duplex, or a combination thereof. The mismatch can occur in the overhang region, or in the duplex region. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., for the free energy of binding or dissociation of a particular pairing, the most straightforward approach is to evaluate base pairs on an individual base pair basis, but affinity or similar analysis can also be used). In terms of promoting dissociation, A:U is preferred over G:C; G:U is preferred over G:C; 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 pairings (A:T, A:U, G:C); base pairs containing universal bases are preferred over canonical pairings.

[0211] In one embodiment, the dsRNA agent of the invention includes at least one of the first one, two, three, four, or five base pairs in the duplex region from the 5' end of the antisense strand, which can be independently selected from the group of A:U, G:U, I:C, and a mismatch pairing, e.g., a non-canonical or non-canonical pairing, or a pairing that includes a universal base, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0212] In one embodiment, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U and dT.Alternatively, at least one of the first one, two or three base pairs in the double-stranded region from the 5' end of the antisense strand is an AU base pair.For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0213] The present inventors have found that the introduction of 4'- and / or 5'-modified nucleotides at the 3'-end of a dinucleotide phosphodiester (PO), phosphorothioate (PS), and / or phosphorodithioate (PS2) bond at any position in a single- or double-stranded oligonucleotide can have a steric influence on the internucleotide bond, thus protecting or stabilizing the internucleotide bond from nucleases.

[0214] In one embodiment, 5' modified nucleoside is introduced at the 3' end of dinucleotide at any position of single-stranded or double-stranded siRNA.For example, 5' alkylated nucleoside can be introduced at the 3' end of dinucleotide at any position of single-stranded or double-stranded siRNA.The alkyl group at the 5' position of ribose sugar can be racemic or chirally pure R or S isomer.An exemplary 5' alkylated nucleoside is 5' methyl nucleoside.5' methyl can be racemic or chirally pure R or S isomer.

[0215] In one embodiment, 4' modified nucleosides are introduced into the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA. For example, 4' alkylated nucleosides can be introduced into the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA. The alkyl group at the 4' position of ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4' alkylated nucleoside is 4' methyl nucleoside. 4' methyl can be racemic or chirally pure R or S isomer. Alternatively, 4'-O-alkylated nucleosides can be introduced into the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA. The 4'-O-alkyl of ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4'-O-alkylated nucleoside is 4'-O-methyl nucleoside. The 4'-O-methyl can be racemic or chirally pure R or S isomer.

[0216] In one embodiment, 5' alkylated nucleoside is introduced into any position of the sense or antisense strand of dsRNA, and such modification maintains or improves the efficacy of dsRNA. 5' alkyl can be racemic or chirally pure R or S isomer. Exemplary 5' alkylated nucleoside is 5'-methyl nucleoside. 5'-methyl can be racemic or chirally pure R or S isomer.

[0217] In one embodiment, 4' alkylated nucleoside is introduced into any position of the sense or antisense strand of dsRNA, and such modification maintains or improves the efficacy of dsRNA.4' alkyl can be racemic or chirally pure R or S isomer.Exemplary 4' alkylated nucleoside is 4'-methyl nucleoside.4'-methyl can be racemic or chirally pure R or S isomer.

[0218] In one embodiment, 4'-O-alkylated nucleoside is introduced into any position of the sense or antisense strand of dsRNA, and such modification maintains or improves the efficacy of dsRNA. 5' alkyl can be racemic or chirally pure R or S isomer. Exemplary 4'-O-alkylated nucleoside is 4'-O-methyl nucleoside. 4'-O-methyl can be racemic or chirally pure R or S isomer.

[0219] In one embodiment, the sense strand sequence of a dsRNA agent is represented by formula (Is): [ka] During the ceremony: B1, B2, and B3 each independently represent 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; C1 is a heat-destabilizing nucleotide (e.g., acyclic nucleotide, e.g., UNA or GNA, mismatch, abasic, or DNA) located opposite the antisense seed region (i.e., positions 2-8 of the 5' end of the antisense strand); T1 represents a nucleotide that contains a non-ribose, acyclic, or chemical modification at the 2' position or an equivalent position in the backbone that provides the nucleotide with less steric bulk than a 2'-OMe modification; for example, T1 is selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl; n 1 or n 3 are independently 4 to 15 nucleotides in length; n 5 is 1-6 nucleotides in length; n 4 is 1 to 3 nucleotides in length; 4 is 0, and n 2 is 0 to 3 nucleotides in length.

[0220] In one embodiment, the sense strand sequence of a dsRNA agent having a length of 19, 20, 21, or 22 nucleotides is represented by formula (Is): [ka] During the ceremony: B1, B2, and B3 each independently represent 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; C1 is a heat-destabilizing nucleotide (e.g., acyclic nucleotide, e.g., UNA or GNA, mismatch, abasic, or DNA) located opposite the antisense seed region (i.e., positions 2-8 of the 5' end of the antisense strand); T1 represents a nucleotide containing a chemical modification selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl; n 1 or n3 are independently 4 to 15 nucleotides in length; n 5 is 1-6 nucleotides in length; n 4 is 1 to 3 nucleotides in length; 4 is 0, and n 2 is 0 to 3 nucleotides in length.

[0221] In one embodiment, the dsRNA agent of formula (Is) further comprises a 3' and / or 5' overhang from 1 to 10 nucleotides in length. In one example, the dsRNA agent of formula (Is) comprises a 5' overhang.

[0222] In one embodiment, C1 comprises one heat destabilizing nucleotide at the 14th, 15th, 16th or 17th position of the 5' end of the sense strand. For example, C1 is a non-cyclic nucleotide (e.g., UNA or GNA), a mismatch, an abasic nucleotide, or DNA. In one particular example, C1 is GNA.

[0223] In one embodiment, T1 comprises DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl at position 11 of the 5' end of the sense strand.

[0224] In one embodiment, a dsRNA agent of the invention comprises a sense strand (Is), where C1 is an acyclic nucleotide (e.g., UNA or GNA), a mismatch, an abasic, or DNA; and T1 comprises DNA, RNA, LNA, 2'-F, or 2'-F-5'-methyl at position 11 of the 5' end of the sense strand.

[0225] In one embodiment, the antisense strand sequence of a dsRNA agent is represented by formula (Ia): [ka] During the ceremony: B1', B2', B3', and B4' each independently represent 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; T1', T2', and T3' each independently represent a nucleotide that contains a non-ribose, acyclic, or chemical modification at the 2' position or an equivalent position in the backbone that provides the nucleotide with less steric bulk than a 2'-OMe modification; for example, T1', T2', and T3' each independently are selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl; q 1 are independently 4 to 15 nucleotides in length; q 3 or q 7 are independently 1 to 6 nucleotides in length; q 2 or q 6 are independently 1 to 3 nucleotides in length; q 4 are independently 0 to 3 nucleotides in length: q 5 are independently 0 to 10 nucleotides in length.

[0226] In one embodiment, the antisense strand sequence of the dsRNA agent having a length of 19, 20, 21, 22, 23, 24, or 25 nucleotides is represented by Formula (Ia): [ka] During the ceremony: B1', B2', B3', and B4' each independently represent 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; T1', T2', and T3' each independently represent a nucleotide containing a chemical modification selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl; q 1are independently 4 to 15 nucleotides in length; q 3 or q 7 are independently 1 to 6 nucleotides in length; q 2 or q 6 are independently 1 to 3 nucleotides in length; q 4 are independently 0 to 3 nucleotides in length: q 5 are independently 0 to 10 nucleotides in length.

[0227] In one embodiment, the dsRNA of formula (Ia) further comprises a 3' and / or 5' overhang of 1 to 10 nucleotides in length. In one example, the dsRNA of formula (Ia) comprises a 3' overhang.

[0228] In one embodiment, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting expression of a target gene, the dsRNA agent comprising a sense strand and an antisense strand, each strand having 14-40 nucleotides: [ka] During the ceremony: B1, B2, B3, B1', B2', B3', and B4' each independently represent 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; C1 is an acyclic nucleotide (e.g., UNA or GNA); T1, T1', T2', and T3' each independently represent a nucleotide containing a chemical modification selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl; n 1 , n 3 , or q 1 are independently 4 to 15 nucleotides in length; n 5 , q 3 , or q 7are independently 1 to 6 nucleotides in length; n 4 , q 2 , or q 6 are independently 1 to 3 nucleotides in length; or 4 is 0; n 2 or q 4 are independently 0 to 3 nucleotides in length: q 5 are independently 0 to 10 nucleotides in length; and The dsRNA agent has 3' and / or 5' overhangs of 1-10 nucleotides in length on the antisense and / or sense strands.

[0229] In one embodiment, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting expression of a target gene, the dsRNA agent comprising a sense strand and an antisense strand, each strand having 14-40 nucleotides: [ka] During the ceremony: B1, B2, B3, B1', B2', B3', and B4' each independently represent 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; C1 is an acyclic nucleotide (e.g., UNA or GNA); T1, T1', T2', and T3' each independently represent a nucleotide containing a chemical modification selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl; n 1 , n 3 , or q 1 are independently 4 to 15 nucleotides in length; n 5 , q 3 , or q 7 are independently 1 to 6 nucleotides in length; n 4 , q 2 , or q6 are independently 1 to 3 nucleotides in length; or 4 is 0; n 2 or q 4 are independently 0 to 3 nucleotides in length: q 5 are independently 0 to 10 nucleotides in length; and The dsRNA agent has a 2 nucleotide long 3' overhang at the 3' end of the antisense strand.

[0230] In one embodiment, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting expression of a target gene, the dsRNA agent comprising a sense strand and an antisense strand, each strand having 15-30 nucleotides: [ka] During the ceremony: B1, B2, B3, B1', B2', B3', and B4' each independently represent a nucleotide containing a 2'-OMe modification; C1 is the acyclic nucleotide GNA; T1, T1', T2', and T3' are each independently DNA or RNA; n 1 , n 3 , or q 1 are independently 4 to 15 nucleotides in length; n 5 , q 3 , or q 7 are independently 1 to 6 nucleotides in length; n 4 , q 2 , or q 6 are independently 1 to 3 nucleotides in length; or 4 is 0; n 2 or q 4 are independently 0 to 3 nucleotides in length: q 5 are independently 0 to 10 nucleotides in length; and The dsRNA agent has a 3' overhang at the antisense 3' end that is 1-6 nucleotides long.

[0231] In one embodiment, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting expression of a target gene, the dsRNA agent comprising a sense strand and an antisense strand, each strand having 19-23 nucleotides: [ka] During the ceremony: B1, B2, B3, B1', B2', B3', and B4' each independently represent a nucleotide containing a 2'-OMe modification; C1 is the acyclic nucleotide GNA; T1, T1', T2', and T3' are independently DNA or RNA; n 1 , n 3 , q 1 , or q 3 are independently 4 to 15 nucleotides in length; n 5 , q 3 , or q 7 are independently 1 to 6 nucleotides in length; n 4 , q 2 , or q 6 are independently 1 to 3 nucleotides in length; or 4 is 0; n 2 , q 4 , or q 5 are independently 0 to 3 nucleotides in length: q 5 are independently 0 to 10 nucleotides in length; and The dsRNA agent has a 2 nucleotide long 3' overhang at the antisense 3' end.

[0232] In one embodiment, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting expression of a target gene, the dsRNA agent comprising a sense strand and an antisense strand, each strand having 14-40 nucleotides: [ka] During the ceremony: B1, B2, B3, B1', B2', B3', and B4' each independently represent 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; C1 is an acyclic nucleotide (e.g., UNA or GNA); T1, T1', T2', and T3' each independently represent a nucleotide containing a chemical modification selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl; n 1 , n 3 , or q 1 are independently 4 to 15 nucleotides in length; n 5 , q 3 , or q 7 are independently 1 to 6 nucleotides in length; n 4 , q 2 , or q 6 are independently 1 to 3 nucleotides in length; or 4 is 0; n 2 or q 4 are independently 0 to 3 nucleotides in length: q 5 are independently 0 to 10 nucleotides in length; and The dsRNA agent has a 5' overhang at the sense 5' end that is 1-10 nucleotides long.

[0233] In one embodiment, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting expression of a target gene, the dsRNA agent comprising a sense strand and an antisense strand, each strand having 14-40 nucleotides: [ka] During the ceremony: B1, B2, B3, B1', B2', B3', and B4' each independently represent 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; C1 is an acyclic nucleotide (e.g., UNA or GNA); T1, T1', T2', and T3' each independently represent a nucleotide containing a chemical modification selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl; n 1 , n 3 , or q 1 are independently 4 to 15 nucleotides in length; n 5 , q 3 , or q 7 are independently 1 to 6 nucleotides in length; n 4 , q 2 , or q 6 are independently 1 to 3 nucleotides in length; or 4 is 0; n 2 or q 4 are independently 0 to 3 nucleotides in length: q 5 are independently 0 to 10 nucleotides in length; and The dsRNA agent has a 5' overhang at the sense 5' end that is 1-6 nucleotides long.

[0234] In one embodiment, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting expression of a target gene, the dsRNA agent comprising a sense strand and an antisense strand, each strand having 14-40 nucleotides: [ka] During the ceremony: B1, B2, B3, B1', B2', B3', and B4' each independently represent 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; C1 is an acyclic nucleotide (e.g., UNA or GNA); T1, T1', T2', and T3' each independently represent a nucleotide containing a chemical modification selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl; n 1 , n 3 , or q 1 are independently 4 to 15 nucleotides in length; n 5 , q 3 , or q 7 are independently 1 to 6 nucleotides in length; n 4 , q 2 , or q 6 are independently 1 to 3 nucleotides in length; or 4 is 0; n 2 or q 4 are independently 0 to 3 nucleotides in length: q 5 are independently 0 to 10 nucleotides in length; and The dsRNA agent has a 5' overhang 1-10 nucleotides long on the 5' end of the sense strand and a 3' overhang 1-10 nucleotides long on the 5' end of the antisense strand.

[0235] thermal destabilization modification A dsRNA agent can be optimized for RNA interference by introducing a thermostabilizing modification in the sense strand at a site opposite the seed region of the antisense strand (i.e., positions 2-8 of the 5' end of the antisense strand) to enhance the dissociation or melting propensity of the dsRNA duplex (reducing the free energy of binding of the duplex). This modification can enhance the dissociation or melting propensity of the duplex at the seed region of the antisense strand.

[0236] Thermally destabilizing modifications can include abasic modifications; mismatches with the opposing nucleotide in the opposing strand; and sugar modifications, such as 2'-deoxy modifications or acyclic nucleotides, such as unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA).

[0237] Exemplary abasic modifications are as follows: [ka]

[0238] Exemplary sugar modifications are as follows: [ka]

[0239] The term "non-cyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar in which, for example, there is no bond between the ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') and / or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4'), alone or in combination, is absent from the nucleotide. In some embodiments, an acyclic nucleotide is: [ka] where B is a modified or unmodified nucleobase and R 1 and R 2 are independently H, halogen, OR3 , or alkyl; R 3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. The term "UNA" refers to unlocked acyclic nucleic acids in which all bonds of the sugar are removed to form an unlocked "sugar" residue. In one example, UNA includes monomers in which the C1'-C4' bond (i.e., the carbon-oxygen-carbon covalent bond between the C1' and C4' carbons) is removed. In another example, the C2'-C3' bond of the sugar (i.e., the carbon-carbon covalent bond between the C2' and C3' carbons) is removed (see Mikhailov et.al., Tetrahedron Letters, 26 (17):2059 (1985); and Fluiter et al., Mol. Biosyst., 10:1039 (2009), each of which is incorporated by reference in its entirety). Acyclic derivatives provide greater flexibility of the backbone without affecting Watson-Crick base pairing. The acyclic nucleotides can be linked by 2'-5' or 3'-5' linkages.

[0240] The term "GNA" refers to glycol nucleic acid, which is a polymer similar to DNA or RNA, but differs in the composition of its "backbone" as it consists of repeating glycerol units linked by phosphodiester bonds: [ka]

[0241] The heat destabilizing modification can be a mismatch (i.e., non-complementary base pair) between the heat destabilizing nucleotide and the opposite nucleotide in the opposite strand in the dsRNA duplex. Exemplary mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or combinations thereof. Other mismatch base pairings known in the art are also suitable for the present invention. Mismatches can occur between nucleotides that are naturally occurring or modified nucleotides, i.e., mismatch base pairings can occur between nucleobases from each nucleotide that are independent of the modification in the ribose sugar of the nucleotide. In certain embodiments, the dsRNA agent comprises at least one nucleobase that is a 2'-deoxynucleobase in mismatch pairing; for example, the 2'-deoxynucleobase is in the sense strand.

[0242] Further examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications are described in detail in WO 2011 / 133876, the entire contents of which are incorporated herein by reference.

[0243] Thermodestabilizing modifications can include universal bases that have reduced or lost the ability to form hydrogen bonds with the opposing base, and phosphate modifications.

[0244] Nucleobase modifications that reduce or completely abolish the ability to form hydrogen bonds with bases in the opposing strand were evaluated for destabilizing the central region of the dsRNA agent duplex as described in WO 2010 / 0011895, the entire contents of which are incorporated herein by reference. Exemplary nucleobase modifications are as follows: [ka]

[0245] Exemplary phosphate modifications known to reduce the thermal stability of dsRNA duplexes compared to natural phosphodiester linkages are: [ka]

[0246] In one embodiment, the dsRNA agent of the present invention can include 2'-5' linkages (with 2'-H, 2'-OH, and 2'-OMe, and with P=O or P=S). For example, modifications of the 2'-5' linkage can be used to increase nuclease resistance or to inhibit binding of the sense strand to the antisense strand, or at the 5' end of the sense strand to inhibit activation of the sense strand by RISC.

[0247] In another embodiment, the dsRNA agent of the present invention can include L sugars (e.g., L-ribose, L-arabinose, including 2'-H, 2'-OH, and 2'-OMe).For example, these L sugar modifications can be used to increase nuclease resistance or inhibit the binding of sense strand to antisense strand, or can be used at the 5' end of sense strand to inhibit the activation of sense strand by RISC.

[0248] In one embodiment, the dsRNA agent is a multimer comprising at least two duplexes represented by formula (I), said duplexes being linked by a linker.This linker can be cleavable or non-cleavable.Optionally, said multimer further comprises a ligand.Each of the dsRNA agents can target the same gene or two different genes; or each of the dsRNA agents can target the same gene at two different target sites.

[0249] In some embodiments, the dsRNA agent is a multimer that comprises three, four, five, six or more double strands represented by formula (I), and the double strands are linked by a linker.The linker can be cleavable or non-cleavable.Optionally, the multimer further comprises a ligand.Each of the dsRNA agents can target the same gene or two different genes; or each of the dsRNA agents can target the same gene at two different target sites.

[0250] In one embodiment, two dsRNA agents of formula (I) are linked to each other at their 5' ends, and one or both of their 3' ends are optionally conjugated with a ligand. Each of the dsRNAs may target the same gene or two different genes; or each of the dsRNAs may target the same gene at two different target sites.

[0251] Various publications describe multimeric siRNA, and all such siRNAs can be used with the dsRNA of the present invention.Such publications include WO2007 / 091269, US Patent No. 7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520, the full disclosure of which is incorporated herein by reference.

[0252] A dsRNA agent that includes one or more sugar moieties conjugated to a dsRNA agent can optimize one or more properties of the dsRNA agent. In many cases, the sugar moiety is attached to a modified subunit of the dsRNA agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, e.g., a non-carbohydrate carrier (preferably cyclic), to which a sugar ligand is attached. A ribonucleotide subunit in which the ribose sugar of the subunit is replaced in this manner is referred to herein as a ribose-replacement modified subunit (RRMS). The cyclic carrier can be a carbocyclic ring system, i.e., a ring system in which all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., a ring system in which one or more ring atoms can be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier can be a single ring system or can include two or more rings, e.g., fused rings. The cyclic carrier can be a fully saturated ring system or can include one or more double bonds.

[0253] The ligand can be attached to the polynucleotide by a carrier. The carrier comprises (i) at least one "backbone attachment point", preferably two "backbone attachment points", and (ii) at least one "tethering attachment point". As used herein, "backbone attachment point" refers to a functional group, e.g., a hydroxyl group, or generally a bond that is available and suitable for incorporation of the carrier into the backbone of a ribonucleic acid, e.g., a phosphate backbone, or, e.g., a sulfur-containing modified phosphate backbone. A "tethering attachment point" (TAP) refers, in some embodiments, to a constituent ring atom, e.g., a carbon atom or a heteroatom (different from the atom providing the backbone attachment point), of the cyclic carrier that connects the selected moiety. The selected moiety can be, for example, a carbohydrate, e.g., a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, and a polysaccharide. Optionally, the selected moiety is connected to the cyclic carrier by an intervening tether. Thus, cyclic carriers often contain functional groups, such as amino groups, or generally allow for attachment suitable for incorporation or tethering of another chemical entity, such as a ligand, to the constituent ring.

[0254] In one embodiment, the dsRNA agent of the invention is conjugated to the ligand via a carrier, which can be a cyclic or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is selected from a serinol backbone or a diethanolamine backbone.

[0255] The double-stranded RNA (dsRNA) of the present invention may be optionally conjugated with one or more ligands. The ligand can be attached to the sense strand, the antisense strand, or both strands at the 3' end, the 5' end, or both ends. For example, the ligand can be conjugated to the sense strand, particularly the 3' end of the sense strand.

[0256] In one embodiment, the dsRNA agent of the invention is 5' phosphorylated or contains a phosphoryl analog at the 5' terminus. 5' phosphate modifications include modifications compatible with RISC-mediated gene silencing. Suitable modifications include: 5'-monophosphate ((HO) 2 (O)PO-5'; 5'-diphosphate ((HO) 2 (O)POP(HO)(O)-O-5'); 5'-triphosphate ((HO) 2 (O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO) 2 (S)PO-5'; 5'-monodithiophosphates (phosphorodithioates; (HO)(HS)(S)PO-5'), 5'-phosphorothiolates ((HO)2(O)PS-5'); oxygen / sulfur substituted monophosphates, diphosphates, and triphosphates (e.g., 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-phosphoramidates ((HO) 2 (O)P-NH-5', (HO)(NH 2 )(O)PO-5'), 5'-alkyl phosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)-O-5'-, 5'-alkenyl phosphonates (i.e., vinyl, substituted vinyl), (OH) 2(O)P-5'-CH2-), 5'-alkyl ether phosphonates (R=alkyl ether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5'-). In one example, the modifications can be placed in the antisense strand of the dsRNA agent.

[0257] Ligand A wide variety of substances can be attached to the oligonucleotides of the invention. Preferred moieties are ligands, attached directly or indirectly through an intervening tether, preferably covalently.

[0258] In a preferred embodiment, the ligand alters the distribution, targeting, or life span of the molecule into which it is incorporated. In a preferred embodiment, the ligand enhances the affinity for a selected target, e.g., a molecule, a cell or cell type, a compartment, a receptor, e.g., a cell or organ compartment, a tissue, an organ, or a region of the body, for example, compared to a species in which such a ligand is not present. A ligand that enhances the affinity for a selected target is also called a targeting ligand.

[0259] Some ligands may have endosomolytic properties. Endosomolytic ligands promote the lysis of endosomes and / or the transport of the composition of the present invention or its components from endosomes to the cytoplasm of cells. Endosomolytic ligands may be polyanionic peptides or peptidomimetics that exhibit pH-dependent membrane activity and fusogenicity. In one embodiment, endosomolytic ligands are assumed to adopt their active conformation at endosomal pH. An "active" conformation is a conformation in which the endosomolytic ligand promotes the lysis of endosomes and / or the transport of the composition of the present invention or its components from endosomes to the cytoplasm of cells. Exemplary endosomolytic ligands include GALA peptides (Subbarao et al., Biochemistry, 1987, 26:2964-2972, the entire contents of which are incorporated herein by reference), EALA peptides (Vogel et al., J. Am. Chem. Soc., 1996, 118:1581-1586, the entire contents of which are incorporated herein by reference), and derivatives thereof (Turk et al., Biochem. Biophys. Acta, 2002, 1559:56-68, the entire contents of which are incorporated herein by reference). In one embodiment, the endosomolytic component may contain chemical groups (e.g., amino acids) that undergo a change in charge or protonation in response to a change in pH. The endosomolytic component may be linear or branched.

[0260] The ligands can improve the transport, hybridization, and specificity properties, and can also improve the nuclease resistance of the resulting natural or modified oligoribonucleotides, or polymeric molecules comprising any combination of the monomers and / or natural or modified ribonucleotides described herein.

[0261] Ligands may generally include therapeutic modifiers, e.g., to enhance uptake; diagnostic compounds or reporter groups, e.g., to monitor distribution; cross-linking agents; and moieties that confer nuclease resistance. Common examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptidomimetics.

[0262] Ligands can include naturally occurring substances, such as proteins (e.g., human serum albumin (HSA), low density lipoprotein (LDL), high density lipoprotein (HDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids, oligonucleotides (e.g., aptamers). Examples of polyamino acids that include polyamino acids include polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic anhydride copolymer, poly (L-lactide-co-glycolide) copolymer, divinyl ether maleic anhydride copolymer, N- (2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly (2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of polyamine, or alpha helix peptide.

[0263] The ligand can also include a targeting group, such as a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid, or protein, such as an antibody, that binds to a specific cell type, such as a kidney cell. The targeting group can be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acid, polyvalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, biotin, RGD peptide, RGD peptide mimic, or aptamer. Table 2 shows some examples of targeting ligands and their associated receptors.

[0264] Other examples of ligands include dyes, intercalating agents (e.g., acridines), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases or chelating agents (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG] 2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0265] The ligand may be a protein, e.g., a glycoprotein, or a peptide, e.g., a molecule with specific affinity for a co-ligand, or an antibody, e.g., an antibody that binds to a particular cell type, such as a cancer cell, an endothelial cell, or a bone cell. Ligands may also include hormones and hormone receptors. Ligands may also include non-peptide species, e.g., lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, multivalent fucose, or aptamers. Ligands may be, for example, lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

[0266] The ligand can be an agent, e.g., a drug, that can increase cellular uptake of the iRNA agent, e.g., by disrupting the cytoskeleton of a cell, e.g., by disrupting the microtubules, microfilaments, and / or intermediate filaments of the cell. The drug can be, e.g., taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0267] Ligands can increase cellular uptake of oligonucleotides, for example, by activating an inflammatory response. Exemplary ligands that can have such an effect include tumor necrosis factor alpha (TNF-α), interleukin-1β, or gamma interferon.

[0268] In one embodiment, the ligand is a lipid or lipid-based molecule. Such lipid or lipid-based molecule preferably binds to serum protein, for example, human serum albumin (HSA). HSA-binding ligand allows the conjugate to distribute to target tissue, for example, non-renal target tissue of the body. For example, the target tissue can be the liver, including liver parenchymal cells. Other molecules that can bind to HSA can also be used as ligand. For example, naproxen or aspirin can be used. Lipid or lipid-based ligand can (a) increase the resistance of the conjugate to degradation, (b) increase the targeting or transport to target cell or cell membrane, and / or (c) can be used to regulate the binding to serum protein, for example, HSA.

[0269] Lipid-based ligand can be used to regulate, for example, control, the binding of conjugate to target tissue.For example, lipid or lipid-based ligand that binds more strongly to HSA is less likely to target kidney, and therefore is less likely to be removed from the body.Lipid or lipid-based ligand that binds less strongly to HSA can be used to make conjugate target kidney.

[0270] In a preferred embodiment, the lipid-based ligand binds to HSA.Preferably, the lipid-based ligand binds to HSA with sufficient affinity so that the conjugate is preferably distributed to non-renal tissues.However, preferably, this affinity is not so strong that HSA-ligand binding is irreversible.

[0271] In another preferred embodiment, the lipid-based ligand binds weakly or not at all to HSA, such that the conjugate is preferably distributed to the kidney. Other moieties that target renal cells can be used in place of or in addition to the lipid-based ligand.

[0272] In another embodiment, the ligand is a moiety, e.g., a vitamin, that is taken up by target cells, e.g., proliferating cells. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., malignant or non-malignant, e.g., cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include vitamin B, e.g., folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by cancer cells. Also included are HAS, low density lipoprotein (LDL), and high density lipoprotein (HDL).

[0273] In another embodiment, the ligand is a cell-penetrating agent, preferably a helical cell-penetrating agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide, such as tat or antennapedia. When the agent is a peptide, it can be modified, including peptidyl mimics, inverted isomers, non-peptide or pseudo-peptide bonds, and the use of D-amino acids. Preferably, the helical cell-penetrating agent is an alpha-helical agent, preferably having a lipophilic phase and a lipophobic phase.

[0274] The ligand may be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule that can fold into a defined three-dimensional structure similar to a natural peptide. The peptidic or peptidomimetic moiety may be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. The peptide or peptidomimetic may be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptidic moiety may be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptidic moiety may include a hydrophobic membrane transport sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP. RFGF analogs containing hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP) may also be targeting moieties. The peptide moiety may be a "delivery" peptide, which can transport large polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK) have been shown to function as delivery peptides. Peptides or peptidomimetics, such as peptides identified from phage display libraries or one bead one compound (OBOC) combinatorial libraries, may be encoded by random sequences of DNA (Lam et al., Nature, 354:82-84, 1991, the entire contents of which are incorporated herein by reference). Preferably, the peptide or peptidomimetic linked to the iRNA agent via an incorporated monomer unit is a cell targeting peptide, such as an arginine-glycine-aspartic acid (RGD)-peptide or RGD mimic. The peptide moiety may range from about 5 amino acids in length to about 40 amino acids in length.The peptide moiety may have a structural change, for example, to enhance stability or direct conformational properties. Any of the following structural changes may be utilized: The RGD peptide moiety may be used to target tumor cells, such as endothelial tumor cells or breast cancer tumor cells (Zitzmann et al., Cancer Res., 62:5139-43, 2002, the entire contents of which are incorporated herein by reference). The RGD peptide may facilitate targeting of the iRNA agent to tumors of various other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001, the entire contents of which are incorporated herein by reference). Preferably, the RGD peptide facilitates targeting of the iRNA agent to the kidney. The RGD peptide may be linear or cyclic, and may be modified, for example, glycosylated or methylated, to facilitate targeting to a particular tissue. For example, a glycosylated RGD peptide may be alpha. v β 3 iRNA agents can be delivered to tumor cells expressing integrins (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001, the entire contents of which are incorporated herein by reference). Peptides that target markers abundant in proliferating cells can be used. For example, RGD-containing peptides and RGD-containing peptidomimetics can target cancer cells, particularly cells that present integrins. Thus, RGD peptides, cyclic peptides containing RGD, RGD peptides containing D-amino acids, and synthetic RGD mimetics can be used. In addition to RGD, other moieties that target integrin ligands can also be used. In general, such ligands can be used to control proliferating cells and angiogenesis. Preferred conjugates of this type of ligand target PECAM-1, VEGF, or other oncogenes, such as those described herein.

[0275] A "cell-penetrating peptide" can penetrate a cell, for example, a microbial cell, for example, a bacterial cell or a fungal cell, or a mammalian cell, for example, a human cell. A peptide that penetrates a microbial cell can be, for example, an α-helical linear peptide (for example, LL-37 or Seropin P1), a disulfide bond-containing peptide (for example, an α-defensin, a β-defensin, or a bactenecin), or a peptide that contains only one or two predominant amino acids (for example, PR-39 or indolicidin). A cell-penetrating peptide can also contain a nuclear localization signal (NLS). For example, a cell-penetrating peptide can be a bipartite amphipathic peptide, for example, MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003, the entire contents of which are incorporated herein by reference).

[0276] In one embodiment, the targeting peptide may be an amphipathic α-helical peptide. Exemplary amphipathic α-helical peptides include, but are not limited to, cecropin, lycotoxin, paradaxin, buforin, CPF, bombinin-like peptide (BLP), cathelicidin, ceratotoxin, S. clava peptide, hagfish intestinal antimicrobial peptide (HFIAP), magainin, brevinin-2, dermaseptin, melittin, pleurocidin, HFIAP, and the like. 2A peptide, Xenopus peptide, esculentinis-1, and caerin. Preferably, a number of factors are considered to maintain the integrity of helix stability. For example, a maximum number of helix-stabilizing residues (e.g., leu, ala, or lys) are utilized, and a minimum number of helix-destabilizing residues (e.g., proline, or cyclic monomer units are utilized. Capping residues are also considered (e.g., Gly is an exemplary N-capping residue, and / or C-terminal amidation can be used to provide additional hydrogen bonds to stabilize the helix). Stabilization can be achieved by the formation of salt bridges between oppositely charged residues spaced i±3 or i±4 positions apart. Cationic residues, such as lysine, arginine, homo-arginine, ornithine, or histidine, can form salt bridges with anionic residues glutamic acid or aspartic acid.

[0277] Peptide and peptidomimetic ligands include ligands having natural or modified peptides, such as D- or L-peptides; alpha, beta, or gamma peptides; N-methyl peptides; azapeptides; peptides having one or more amide bonds replaced by one or more urea, thiourea, carbamate, or sulfonylurea bonds, i.e., peptidyl bonds; or cyclic peptides.

[0278] The targeting ligand may be any ligand that can target a specific receptor. Examples include folate, GalNAc, galactose, mannose, mannose-6P, glycoclusters, such as GalNAc clusters, mannose clusters, galactose clusters, or aptamers. A cluster is a combination of two or more sugar units. Targeting ligands also include integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL ligands, and HDL ligands. The ligand may be based on a nucleic acid, such as an aptamer. The aptamer may be unmodified or may have any combination of modifications disclosed herein.

[0279] Endosomal release agents include imidazoles, poly- or oligoimidazoles, PEI, peptides, fusogenic peptides, polycarboxylates, polycations, masked oligo- or polycations or anions, acetals, polyacetals, ketals / polyketyals, orthoesters, polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges.

[0280] PK modulator refers to a pharmacokinetic modulator. PK modulators include lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkyl glycerides, diacyl glycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, etc. Oligonucleotides containing multiple phosphorothioate linkages are also known to bind to serum proteins, and therefore short oligonucleotides, such as oligonucleotides of about 5 bases, 10 bases, 15 bases, or 20 bases, containing multiple phosphorothioate linkages in the backbone, are also applicable to the present invention as ligands (e.g., as PK-modulating ligands).

[0281] In addition, aptamers that bind to serum components (eg, serum proteins) are also applicable to the present invention as PK-regulating ligands.

[0282] Other ligand conjugates applicable to the present invention are described in U.S. patent application Ser. Nos. 10 / 916,185, filed Aug. 10, 2004; 10 / 946,873, filed Sep. 21, 2004; 10 / 833,934, filed Aug. 3, 2007; 11 / 115,989, filed Apr. 27, 2005, and 11 / 944,227, filed Nov. 21, 2007, the entire disclosures of which are incorporated herein by reference.

[0283] When there are two or more ligands, the ligands may all have the same properties, or all have different properties, or some ligands may have the same properties while other ligands have different properties. For example, the ligands may have targeting properties, endosomal activity, or PK modulating properties. In a preferred embodiment, all ligands have different properties.

[0284] The ligand can be attached to the oligonucleotide at various positions, e.g., the 3' end, the 5' end, and / or internal positions. In a preferred embodiment, the ligand is attached to the oligonucleotide via an intervening tether, e.g., a carrier as described herein. The ligand or tether ligand may be present on the monomer when the monomer is incorporated into a growing chain. In some embodiments, the ligand can be incorporated by attachment to a "precursor" monomer after the monomer has been incorporated into a growing chain. For example, an amino-terminated tether (i.e., no ligand is attached), e.g., TAP-(CH 2 ) n NH 2 can be incorporated into a growing oligonucleotide chain. In a subsequent operation, i.e., after the precursor monomer is incorporated into the chain, a ligand bearing an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can then be attached to the precursor monomer by binding of the electrophilic group of the ligand with the terminal nucleophilic group of the tether of the precursor monomer.

[0285] In another example, monomers bearing chemical groups suitable for participating in click chemistry reactions can be incorporated into tethers / linkers, for example, azide or alkyne terminated tethers / linkers. Subsequent operations, i.e., after the precursor monomers are incorporated into the chain, ligands bearing complementary chemical groups, for example, alkyne or azide, can be attached to the precursor monomers by linking the alkyne and azide together.

[0286] In the case of double-stranded oligonucleotides, the ligand can be attached to one or both strands.In some embodiments, the double-stranded iRNA agent contains a ligand conjugated to the sense strand.In other embodiments, the double-stranded iRNA agent contains a ligand conjugated to the antisense strand.

[0287] In some embodiments, the ligand can be conjugated to the nucleobase, sugar moiety, or internucleoside linkage of the nucleic acid molecule. Conjugation to the purine nucleobase or its derivatives can occur at any position, including endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-position of the purine nucleobase is attached to the conjugate moiety. Conjugation to the pyrimidine nucleobase or its derivatives can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of the pyrimidine nucleobase can be replaced with a conjugate moiety. Conjugation to the sugar moiety of the nucleoside can occur at any carbon atom. Examples of carbon atoms of the sugar moiety that can be attached to the conjugate moiety include the 2', 3', and 5' carbon atoms. The 1' position can also be attached to a conjugate moiety, for example, an abasic residue. The internucleoside linkage can also carry a conjugate moiety. In the case of phosphorus-containing linkages (such as phosphodiesters, phosphorothioates, phosphorodithioates, and phosphoramidates), the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom that is attached to the phosphorus atom. In the case of internucleoside linkages that contain amines or amides (e.g., PNA), the conjugate moiety can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

[0288] While any suitable ligand in the field of RNA interference can be used, such ligands are typically carbohydrates, such as monosaccharides (eg, GalNAc), disaccharides, trisaccharides, tetrasaccharides, polysaccharides.

[0289] Linkers for conjugating the ligand to the nucleic acid include those described above. For example, the ligand may be one or more GalNAc (N-acetylglucosamine) derivatives attached by a monovalent, divalent or trivalent branched linker.

[0290] In one embodiment, the dsRNA of the invention is conjugated to bivalent and trivalent branched linkers comprising the structure shown in any of the following formulas (IV) to (VII): [ka] During the ceremony, q 2A , q 2B , q 3A , q 3B , q4 A , q 4B , q 5A , q 5B , and q 5C represents, independently for each occurrence, 0 to 20, and the repeating units may be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 5A , T 5B , T 5C are independent of each other and are either absent, CO, NH, O, S, OC(O), NHC(O), CH 2 , C.H. 2 NH, or CH 2 Represents O; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C represents independently for each occurrence, absent, alkylene, or substituted alkylene; one or more methylenes are O, S, S(O), SO 2 , N(R N), C(R')=C(R''), C≡C, or C(O); R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C are independently determined for each occurrence: NH, O, S, CH 2 , C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocyclyl; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B , and L 5C represents a ligand; i.e., each occurrence independently represents a monosaccharide (e.g., GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, or a polysaccharide; and R a is H or an amino acid side chain.

[0291] Trivalent conjugated GalNAc derivatives, such as those of formula (VII), are particularly useful for use with RNAi agents to inhibit expression of target genes: [ka] In the formula, L 5A , L 5B , and L 5C represents a monosaccharide, e.g., a GalNAc derivative.

[0292] Suitable divalent and trivalent branched linker group conjugated GalNAc derivatives include, but are not limited to, the following compounds: [ka] [ka]

[0293] definition As used herein, "dsRNA", "siRNA" and "iRNA agent" are used interchangeably with the agent that can mediate the silencing of target RNA, for example, mRNA, for example, the transcript of a gene that codes for a protein.For convenience, such mRNA is also referred to herein as the mRNA that is silenced.Such a gene is also referred to as a target gene.Generally, the RNA that is silenced is an endogenous gene or a pathogenic gene.In addition, RNA other than mRNA, for example, tRNA and viral RNA can also be targeted.

[0294] As used herein, the phrase "mediate RNAi" refers to the ability to sequence-specifically silence a target RNA. Without wishing to be bound by theory, it is believed that silencing uses the RNAi machinery or process and a guide RNA, e.g., a 21-23 nucleotide siRNA agent.

[0295] As used herein, "specifically hybridizable" and "complementary" are terms used to indicate sufficient complementarity such that stable specific binding occurs between the compound of the present invention and target RNA molecule.Specific binding requires sufficient complementarity to avoid specific binding of oligomeric compounds to non-target sequences under conditions where specific binding is desired, i.e., under physiological conditions in the case of assay or treatment, or under the conditions where the assay is performed in the case of in vitro assay.Non-target sequences typically differ by at least 5 nucleotides.

[0296] In one embodiment, the dsRNA agent of the present invention is "sufficiently complementary" to the target RNA, e.g., the target mRNA, so that the dsRNA agent silences the production of the protein encoded by the target mRNA. In another embodiment, the dsRNA agent of the present invention is "exactly complementary" to the target RNA, e.g., the target RNA and the dsRNA duplex agent anneal to form a hybrid consisting of only Watson-Crick base pairs in the exactly complementary region. The "sufficiently complementary" target RNA can include an internal region (e.g., at least 10 nucleotides) that is exactly complementary to the target RNA. Furthermore, in some embodiments, the dsRNA agent of the present invention specifically discriminates between one nucleotide difference. In this case, the dsRNA agent mediates RNAi only when exact complementarity is found in the region (e.g., within 7 nucleotides) where one nucleotide difference exists.

[0297] The term "oligonucleotide" as used herein refers to a nucleic acid molecule (RNA or DNA) of, for example, less than 100, less than 200, less than 300, or less than 400 nucleotides.

[0298] The term "BNA" refers to bridged nucleic acid, often referred to as restricted or sequestered RNA. BNAs are nucleic acids that contain "fixed" C3'-endosugar puckering (C 3 The bridges may include 5, 6, or even 7 membered bridge structures with a 2'-endo sugar puckering. The bridges are typically included at the 2', 4' positions of the ribose to give 2',4'-BNA nucleotides (e.g., LNA or ENA). Examples of BNA nucleotides include the following nucleosides: [ka]

[0299] The term "LNA" refers to locked nucleic acid, often referred to as restricted or sequestered RNA. LNA is a modified RNA nucleotide. The ribose moiety of an LNA nucleotide is modified with an additional bridge (e.g., a methylene or ethylene bridge) that connects the 2' hydroxyl to the 4' carbon of the same ribose sugar. For example, this bridge can "lock" the ribose into the following 3'-endo (North) conformation: [ka]

[0300] The term "ENA" refers to ethylene-bridged nucleic acid, often referred to as restricted or sequestered RNA.

[0301] As used herein, "cleavage site" refers to a backbone bond in a target gene or sense strand that is cleaved by the RISC mechanism by utilizing an iRNA agent. And the target cleavage site region includes at least one or at least two nucleotides on either side of the cleavage site. In the case of a sense strand, the cleavage site is the backbone bond of the sense strand that is cleaved when the sense strand itself is the target to be cleaved by the RNAi mechanism. The cleavage site can be determined using methods known in the art, for example, the 5'-RACE assay detailed in Soutschek et al., Nature (2004) 432,173-178, the entire contents of which are incorporated herein by reference. As is well understood in the art, the cleavage site region of a conical double stranded RNAi agent, which includes two strands 21 nucleotides long (these strands form a double-stranded region of 19 consecutive base pairs with a 2-nucleotide single-stranded overhang at the 3' end), corresponds to positions 9-12 of the 5' end of the sense strand.

[0302] The term "halo" refers to any radical of fluorine, chlorine, bromine, or iodine. The term "alkyl" refers to saturated and unsaturated non-aromatic hydrocarbon chains, which may be straight or branched, containing the indicated number of carbon atoms, optionally containing N, O, or S, including, but not limited to, propyl, allyl, or propargyl. For example, C 1 ~C 10 indicates that the group may have 1 to 10 (inclusive) carbon atoms in it. The term "alkoxy" refers to an -O-alkyl radical. The term "alkylene" refers to a divalent alkyl (i.e., -R-). The term "alkylenedioxo" refers to a divalent species of the structure -ORO-, where R represents an alkylene. The term "aminoalkyl" refers to an alkyl substituted with an amino. The term "mercapto" refers to an -SH radical. The term "thioalkoxy" refers to an S-alkyl radical.

[0303] The term "aryl" refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system in which 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. Examples of aryl groups include phenyl and naphthyl. The term "arylalkyl" or "aralkyl" refers to an alkyl substituted with an aryl. The term "arylalkoxy" refers to an alkoxy substituted with an aryl.

[0304] The term "cycloalkyl" as used herein includes saturated and partially saturated cyclic hydrocarbon groups having 3 to 12 carbons, e.g., 3 to 8 carbons, and e.g., 3 to 6 carbons, which cycloalkyl groups may be further optionally substituted. Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0305] The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms in the monocyclic, 1-6 heteroatoms in the bicyclic, or 1-9 heteroatoms in the tricyclic, the heteroatoms being selected from O, N, or S (e.g., 1-3, 1-6, or 1-9 carbon atoms and N, O, or S heteroatoms in the monocyclic, bicyclic, or tricyclic, respectively), and 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. Examples of heteroaryl groups include pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolinyl, and thiazolyl. The term "heteroarylalkyl" or "heteroaralkyl" refers to an alkyl substituted with a heteroaryl. The term "heteroarylalkoxy" refers to an alkoxy substituted with a heteroaryl.

[0306] The term "heterocyclyl" refers to a non-aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms in the monocyclic, 1-6 heteroatoms in the bicyclic, or 1-9 heteroatoms in the tricyclic, the heteroatoms being selected from O, N, or S (e.g., 1-3, 1-6, or 1-9 carbon atoms and N, O, or S heteroatoms in the monocyclic, bicyclic, or tricyclic, respectively), and 0, 1, 2, or 3 atoms of each ring may be optionally substituted by a substituent. Examples of heterocyclyl groups include trizolyl, tetrazolyl, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, and tetrahydrofuranyl.

[0307] The term "oxo" refers to an oxygen atom which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.

[0308] The term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted by substituents.

[0309] The term "substituted" refers to the replacement of one or more hydrogen radicals in a given structure with the radical of a specified substituent, including, but not limited to, halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, and aliphatic. It is understood that the substituents may be further substituted.

[0310] Cleavable linking group Cleavable linking group is a linking group that is stable enough outside of cells, but when it enters target cells, it is cut off and releases the two parts that linker holds together.In the preferred embodiment of the dsRNA agent according to the present invention, cleavable linking group is cut at least 10 times faster in target cells or under first reference condition (for example, it can be selected to mimic or realize the conditions in cells) than in the blood of subject or under second reference condition (for example, it can be selected to mimic or realize the conditions present in blood or serum), preferably at least 100 times faster.

[0311] Cleavable linking groups are susceptible to the presence of cleaving agents, such as pH, redox potential, or degradable molecules.In general, cleaving agents are more prevalent or present at higher levels or activity in cells than in serum or blood.Examples of such degrading agents include oxidizing or reducing enzymes or reducing agents present in cells, which can degrade redox cleavable linking groups by reduction, such as redox agents that are selective for specific substrates or have no substrate specificity, including mercaptans; esterases; endosomes or agents that can create an acidic environment, such as a pH of 5 or less; enzymes that can hydrolyze or degrade acidic cleavable linking groups by acting as general acids, peptidases (which may be substrate specific), and phosphatases.

[0312] Cleavable linking groups, such as disulfide bonds, are sensitive to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly 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 about 5.0. Some linkers have cleavable linking groups that are cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand into the cell or into a desired compartment of the cell.

[0313] Linker can contain a cleavable linking group that can be cleaved by a specific enzyme.The type of cleavable linking group incorporated in linker can vary depending on the cell to be targeted.For example, a ligand that targets the liver can be linked to a cationic lipid through a linker that contains an ester group.Hepatocytes are rich in esterases, and therefore linkers are more efficiently cleaved in hepatocytes than in cell types that are not rich in esterases.Other cell types that are rich in esterases include lung, renal cortex, and testicular cells.

[0314] When targeting cell types that are rich in peptidases, such as hepatocytes and synovial cells, linkers containing peptide bonds can be used.

[0315] In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degrading agent (or condition) to cleave the candidate linking group. It is also desirable to test the candidate cleavable linking group for its ability to resist cleavage in blood or when in contact with other non-target tissues. Thus, the relative ease of cleavage between a first condition and a second condition can be determined, the first condition being selected to indicate cleavage in target cells, and the second condition being selected to indicate cleavage in other tissues or body fluids, such as blood or serum. Evaluation can be performed in a cell-free system, cells, cell cultures, organ or tissue cultures, or in whole animals. It may be useful to perform initial evaluations in a cell-free or cultured condition and confirm by further evaluation in whole animals. In a preferred embodiment, a useful candidate compound cleaves at least 2-fold, 4-fold, 10-fold, or 100-fold faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0316] Redox-cleavable linking groups One class of cleavable linking group is the redox cleavable linking group that can be used in the dsRNA agent according to the present invention and is cleaved upon reduction or oxidation. One example of a reductively cleavable linking group is a disulfide linking group (-SS-). To determine whether a candidate cleavable linking group is a suitable "reductively cleavable linking group" or is suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, the methods described herein can be checked. For example, the candidate can be evaluated by incubating with dithiothreitol (DTT) or other reducing agents using a reagent known in the art that mimics the cleavage rate that can be observed in cells, for example, target cells. The candidate can also be evaluated under conditions selected to mimic blood or serum conditions. In a preferred embodiment, the candidate compound is cleaved at most 10% in blood. In preferred embodiments, useful candidate compounds are degraded at least 2, 4, 10, or 100 times faster inside a cell (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic intracellular medium and compared to conditions selected to mimic extracellular medium.

[0317] Phosphate-based cleavable linkers Phosphate-based cleavable linkers can be used in dsRNA agents according to the invention and are cleaved by agents that degrade or hydrolyze phosphate groups. One example of an agent that cleaves phosphate groups within a cell is an intracellular enzyme, such as a phosphatase. Examples of phosphate-based linking groups include, -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. Preferred embodiments 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-. Preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0318] Acid cleavable linking group Acidic cleavable linking groups can be used in the dsRNA agent according to the present invention, and are linking groups that are cleaved under acidic conditions. In a preferred embodiment, acidic cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0 or less) or by agents that can function as general acids, such as enzymatic agents. In cells, certain organelles with low pH, such as endosomes and lysosomes, can provide a cleavage environment for acidic cleavable linking groups. Examples of acidic cleavable linking groups include, but are not limited to, hydrazones, esters, and amino acid esters. Acidic cleavable groups can have the general formula: -C=NN-, C(O)O, or -OC(O). A preferred embodiment is when the carbon attached to the enzyme of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group, such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0319] Ester Linking Group Ester-based cleavable linking group can be used in the dsRNA agent according to the present invention, and is cleaved by intracellular enzymes, such as esterase and amidase.Examples of ester-based cleavable linking group include, but are not limited to, the esters of alkylene, alkenylene, and alkynylene groups.Ester-based cleavable linking group has the general formula: -C(O)O- or -OC(O)-.These candidates can be evaluated using methods similar to those described above.

[0320] Peptide-Based Cleavable Linkers Peptide-based cleavable linkers can be used in the dsRNA agent according to the present invention and are cleaved by intracellular enzymes, such as peptidases and proteases. Peptide-based cleavable linkers are peptide bonds formed between amino acids resulting in oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable linkers 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 resulting in peptides and proteins. Peptide-based cleavage groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids resulting in peptides and proteins, and do not include the entire amide functionality. Peptide-based cleavable linkers can be represented by the general formula: -NHCHR A C(O)NHCHR B C(O)-, R A and R B are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above. As used herein, "carbohydrate" refers to a carbohydrate itself formed from one or more monosaccharide units (which may be linear, branched, or cyclic) having at least six carbon atoms, each of which has an oxygen, nitrogen, or sulfur atom attached to it; or to a compound having as part of it a carbohydrate moiety formed from one or more monosaccharide units, each of which has at least six carbon atoms, each of which has an oxygen, nitrogen, or sulfur atom attached to it. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4-9 monosaccharide units), and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide gums. Specific monosaccharides include C 5 and the above (preferably C 5 ~C 8 ) sugars; disaccharides and trisaccharides include sugars containing two or three monosaccharide units (preferably C 5 ~C 8 ) is an example of a sugar having the formula:

[0321] The present invention further relates to the use of a dsRNA agent as defined herein for inhibiting expression of a target gene. In one embodiment, the present invention further relates to the use of a dsRNA agent for inhibiting expression of a target gene in vitro.

[0322] The present invention further relates to the use of the dsRNA agent defined herein for inhibiting the expression of target gene in a subject.The subject can be any animal, for example, mammal, for example, mouse, rat, sheep, cow, dog, cat or human.

[0323] In one embodiment, a dsRNA agent of the invention is administered in a buffer solution.

[0324] In one embodiment, the siRNA compound described herein can be formulated for administration to a subject.The siRNA composition that is formulated can be in various states.In some examples, the composition is at least partially crystalline, uniformly crystalline, and / or anhydrous (e.g., less than 80%, less than 50%, less than 30%, less than 20%, or less than 10% water).In another example, the siRNA is dissolved in an aqueous phase, for example, a solution that contains water.

[0325] Aqueous phase and crystalline composition can be, for example, included in delivery vehicle, for example, liposome (especially for aqueous phase) or particle (e.g., microparticles, which may be suitable for crystalline composition).Generally, siRNA composition is formulated to be compatible with intended administration method described herein.For example, in certain embodiments, the composition is prepared by at least one of the following methods: spray drying, freeze drying, vacuum drying, evaporation, fluidized bed drying, or combination of these techniques; or ultrasonication, freeze drying, condensation, and other self-assembly using lipid.

[0326] The siRNA preparation can be formulated in combination with another agent, e.g., another therapeutic agent, or an agent that stabilizes the siRNA, e.g., a protein that complexes with the siRNA to form an iRNP. Still other agents include chelating agents, e.g., EDTA (e.g., chelating agents that bind divalent cations, e.g., Mg 2+ for removing ribosomal RNA), salts, and RNAse inhibitors (eg, broad specificity RNAse inhibitors such as RNAsin).

[0327] In one embodiment, siRNA preparation comprises another siRNA compound, for example, a second siRNA that can mediate RNAi for a second gene or the same gene.Yet other preparations can comprise at least 3, 5, 10, 20, 50, or 100 or more different siRNA species.Such siRNA can mediate RNAi for a similar number of different genes.

[0328] In one embodiment, the siRNA preparation comprises at least a second therapeutic agent (e.g., an agent other than RNA or DNA).For example, the siRNA composition for treating viral disease, e.g., HIV, comprises a known antiviral agent (e.g., a protease inhibitor or a reverse transcriptase inhibitor).In another example, the siRNA composition for treating cancer can further comprise a chemotherapeutic agent.

[0329] Exemplary formulations that can be used to administer a dsRNA agent according to the invention are described below.

[0330] Liposomes. For ease of explanation, the formulations, compositions, and methods in this section are described primarily with respect to unmodified siRNA compounds. However, it will be understood that these formulations, compositions, and methods can be practiced with other siRNA compounds, e.g., modified siRNAs, and such practice is within the scope of the present invention. siRNA compounds, e.g., double-stranded siRNA compounds, or ssiRNA compounds, (e.g., precursors, e.g., larger siRNA compounds that can be processed into ssiRNA compounds, or siRNA compounds, e.g., DNA encoding double-stranded siRNA compounds, or ssiRNA compounds, or precursors thereof) preparations can be formulated into membrane molecular assemblies, e.g., liposomes or micelles, for delivery. The term "liposomes" as used herein refers to vesicles made of amphiphilic lipids that are composed of at least one bilayer, e.g., one bilayer or multiple bilayers. Liposomes include unilamellar and multilamellar vesicles with a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the siRNA composition. The lipophilic material separates the aqueous interior from the aqueous exterior, which typically does not contain the siRNA composition, but may in some cases do. Liposomes are useful for transporting and delivering active ingredients to the site of action.Liposome membrane is structurally similar to biological membrane, so when liposome contacts tissue, liposome bilayer fuses with cell membrane bilayer.When liposome and cell are integrated, the aqueous content inside, including siRNA, is delivered into the cell, where siRNA can specifically bind to target RNA and mediate RNAi.In some cases, liposomes are specifically targeted, for example, directing siRNA to a specific cell type.

[0331] Liposomes containing siRNA can be prepared in various ways. In one example, the lipid components of liposomes are dissolved in detergent, which allows micelles to form with the lipid components. For example, the lipid components can be amphipathic cationic lipids or lipid conjugates. The detergent can have a high critical micelle concentration and can be non-ionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. Then, the siRNA preparation is added to the micelles containing the lipid components. The cationic groups on the lipids interact with the siRNA and condense around the siRNA to form liposomes. After condensation, the detergent is removed, for example, by dialysis, to obtain a liposome preparation of siRNA.

[0332] If necessary, carrier compounds that aid in condensation can be added during the condensation reaction, for example, by controlled addition.For example, carrier compounds can be polymers other than nucleic acids (for example, spermine or spermidine).pH can also be adjusted to be suitable for condensation.

[0333] Further details of methods for forming stable polynucleotide delivery vehicles that include polynucleotide / cationic lipid complexes as a structural component of the delivery vehicle are described in WO 96 / 37194. The formation of liposomes has been described in detail in the following publications: Felgner, PL et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. Nos. 4,897,355; 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75:4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. 728:339, 1983; and Fukunaga, et al. Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw and extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986, the entire contents of which are incorporated herein by reference). When consistently small (50-200 nm) and relatively uniform aggregates are desired, microfluidization can be used (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984, the entire contents of which are incorporated herein by reference). Such methods are readily adapted for packaging siRNA agent preparations into liposomes.

[0334] pH-sensitive or negatively charged liposomes do not form complexes with nucleic acid molecules, but instead entrap nucleic acid molecules. Both nucleic acid molecules and lipids are similarly charged, so repulsion occurs instead of complex formation. However, some nucleic acid molecules are entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes are used to deliver DNA encoding thymidine kinase gene to cell monolayers in culture. The expression of exogenous genes is detected in target cells (Zhou et al., Journal of Controlled Release, 19, (1992) 269-274, the entire contents of which are incorporated herein by reference).

[0335] One important type of liposome composition comprises phospholipids other than naturally occurring phosphatidylcholine. Neutral liposome composition can be formed from, for example, dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome composition is generally formed from dimyristoyl phosphatidylglycerol, and anionic fusogenic liposomes are mainly formed from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0336] Other examples of methods for introducing liposomes into cells in vitro include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94 / 00569; WO 93 / 24640; WO 91 / 16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

[0337] In one embodiment, cationic liposome is used.Cationic liposome has the advantage that it can fuse with cell membrane.Non-cationic liposome cannot fuse with plasma membrane efficiently, but it can be taken up by macrophage in vivo, so it can be used to deliver siRNA to macrophage.

[0338] Additional advantages of liposomes include: liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a variety of water-soluble and lipid-soluble drugs; liposomes can protect siRNA incorporated within the internal compartment from metabolism and degradation (Rosoff, in "Pharmaceutical Dosage Forms," ​​Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p.245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size, and aqueous volume of the liposomes.

[0339] The positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), can be used to form small liposomes that naturally interact with nucleic acids to form lipid-nucleic acid complexes that fuse with the negatively charged lipids of the plasma membrane of tissue culture cells, resulting in delivery of siRNA (see, e.g., Felgner, PL et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987, the entire contents of which are incorporated herein by reference, and U.S. Pat. No. 4,897,355, which describes DOTMA and its use with DNA).

[0340] A DOTMA analog, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP), can be used in combination with phospholipids to form vesicles that complex with DNA. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids to living tissue culture cells, which contain positively charged DOTMA liposomes that naturally interact with negatively charged polynucleotides to form complexes. When sufficiently positively charged liposomes are used, the net charge of the resulting complex is also positive. The positively charged complexes thus prepared naturally attach to negatively charged cell surfaces, fuse to the plasma membrane, and effectively deliver functional nucleic acids to, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Indiana), differs from DOTMA in that the oleoyl moieties are attached by ester rather than ether bonds.

[0341] Other reported cationic lipid compounds include, for example, those conjugated to one of two types of lipids and conjugated to various moieties, including compounds such as carboxyspermine containing 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (Transfectam™, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide ("DPPES") (see, for example, U.S. Pat. No. 5,171,678).

[0342] Another cationic lipid conjugate comprises lipid derivatives with cholesterol ("DC-Chol") formulated in liposomes in combination with DOPE (see Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine formed by conjugating polylysine to DOPE has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991, the entire contents of which are incorporated herein by reference). In certain cell lines, such liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and achieve more efficient transfection than DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland).Other cationic lipids suitable for delivery of oligonucleotides are described in WO 98 / 39359 and WO 96 / 37194.

[0343] Liposome formulations are particularly suitable for topical administration, and liposomes have several advantages over other formulations.These advantages include the reduced side effects associated with the high systemic absorption of administered drugs, the increased accumulation of administered drugs in desired targets, and the ability to administer siRNA to the skin.In some implementations, liposomes are also used to deliver siRNA to epithelial cells and to promote the penetration of siRNA into skin tissues, such as skin.For example, liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been demonstrated (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, RJ and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, RM and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y and See Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

[0344] Non-ionic liposomal systems have also been tested to determine their utility in delivering drugs to the skin, especially in systems containing non-ionic surfactants and cholesterol.Non-ionic liposomal formulations containing Novasome I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver drugs to the dermis of mouse skin.Such formulations containing siRNA are useful for treating skin disorders.

[0345] Liposomes containing siRNA can be made highly deformable. Such deformability can allow liposomes to enter pores smaller than the average radius of the liposome. For example, transfersomes are one type of deformable liposome. Transfersomes can be made by adding surface edge activators, usually surfactants, to standard liposome compositions. Transfersomes containing siRNA can be delivered, for example, by subcutaneous injection to deliver siRNA to keratinocytes in the skin. To pass through intact mammalian skin, lipid vesicles must pass through a series of micropores, each with a diameter of less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the properties of lipids, such transfersomes can be self-optimizing (e.g., adapting to the shape of skin pores), self-repairing, and often can reach the target without fragmentation, and can often be self-loading.

[0346] Other formulations applicable to the present invention are described in U.S. Provisional Patent Application Nos. 61 / 018,616, filed January 2, 2008; 61 / 018,611, filed January 2, 2008; 61 / 039,748, filed March 26, 2008; 61 / 047,087, filed April 22, 2008; and 61 / 051,528, filed May 8, 2008. International Application PCT / US2007 / 080331, filed October 3, 2007, also describes formulations applicable to the present invention.

[0347] Surfactants. For ease of explanation, the formulations, compositions, and methods in this section are described primarily with respect to unmodified siRNA compounds. However, it will be understood that these formulations, compositions, and methods can be implemented with other siRNA compounds, e.g., modified siRNA compounds, and such implementations are within the scope of the present invention. Surfactants have found wide application in formulations, e.g., emulsions (including microemulsions) and liposomes (above). siRNA (or precursors, e.g., larger dsiRNAs that can be processed into siRNAs, or DNAs that code for siRNAs or precursors) compositions can include surfactants. In one embodiment, siRNA is formulated as an emulsion that includes surfactants. The most common way to classify and rank the properties of the many different types of surfactants, both natural and synthetic, is by using hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group provides the most useful means for classifying different surfactants used in formulations (Rieger, in "Pharmaceutical Dosage Forms," ​​Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0348] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical products and are usable over a wide range of pH values. In general, their HLB values ​​range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters, such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers, are also included in this class. Polyoxyethylene surfactants are the most common members of the nonionic surfactant class.

[0349] If a surfactant molecule has a negative charge when dissolved or dispersed in water, the surfactant molecule is classified as anionic.Anionic surfactants include carboxylates such as soaps, acyl lactates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyltaurates and sulfosuccinates, and phosphates.The most important members of the anionic surfactant class are alkyl sulfates and soaps.

[0350] If a surfactant molecule has a positive charge when dissolved or dispersed in water, the surfactant molecule is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most used members of this class.

[0351] If a surfactant molecule is capable of carrying either a positive or negative charge, it is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phosphatides.

[0352] The use of surfactants in drug products, formulations, and emulsions has been reviewed (Rieger, in "Pharmaceutical Dosage Forms", Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0353] Micelles and other membranous formulations. For ease of explanation, micelles and other formulations, compositions, and methods in this section are described mainly with respect to unmodified siRNA compounds. However, it is understood that these micelles and other formulations, compositions, and methods can be implemented with other siRNA compounds, e.g., modified siRNA compounds, and such implementations are within the scope of the present invention. siRNA compounds, e.g., double-stranded siRNA compounds, or ssiRNA compounds, (e.g., precursors, e.g., larger siRNA compounds that can be processed into ssiRNA compounds, or siRNA compounds, e.g., DNAs encoding double-stranded siRNA compounds, or ssiRNA compounds, or precursors thereof) compositions can be provided as micellar formulations. A "micelle" is defined herein as a particular type of molecular assembly in which amphiphilic molecules are organized in a spherical structure such that all hydrophobic portions of the amphiphilic molecules face inward and hydrophilic portions contact the surrounding aqueous phase. If the environment is hydrophobic, it will exist in the opposite configuration.

[0354] Mixed micelle formulations suitable for transdermal delivery consist of an aqueous solution of the siRNA composition and an alkali metal C 8 ~C 22It can be prepared by mixing alkyl sulfate with micelle-forming compound.Exemplary micelle-forming compounds include lecithin, hyaluronic acid, hyaluronic acid pharmaceutically acceptable salt, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleate, monolaurate, borage oil, evening primrose oil, menthol, trihydroxyoxocholanylglycine and its pharmaceutically acceptable salt, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ether and its analogue, polidocanol alkyl ether and its analogue, chenodeoxycholate, deoxycholate, and mixtures thereof.Mixed micelles can be added simultaneously with or after the addition of alkali metal alkyl sulfate.Mixed micelles can be formed by mixing virtually any kind of components, but vigorous mixing is used to provide smaller size micelles.

[0355] In one method, a first micelle composition is prepared, which contains siRNA composition and at least alkali metal alkyl sulfate.Then, the first micelle composition is mixed with at least three kinds of micelle-forming compounds to form a mixed micelle composition.In another method, the micelle composition is prepared by mixing siRNA composition, alkali metal alkyl sulfate and at least one kind of micelle-forming compound, and then adding the remaining micelle-forming compounds and vigorously mixing.

[0356] Phenol and / or m-cresol can be added to the mixed micelle composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and / or m-cresol can be added together with the micelle forming components. An isotonicity agent, such as glycerin, can be added after the formation of the mixed micelle composition.

[0357] To deliver the micelle formulation as a spray, the formulation can be placed in an aerosol dispenser, which is filled with the propellant. The propellant under pressure is in liquid form in the dispenser. The ratio of components is adjusted so that the aqueous phase and the propellant phase are one, i.e., there is only one phase. If there are two phases, the dispenser must be shaken before dispensing a portion of the contents, for example, through a metering valve. The dispensed amount of the pharmaceutical product is sprayed in a fine mist from the metering valve.

[0358] Propellants can include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether, and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

[0359] The specific concentrations of the essential components can be determined by relatively simple experimentation. For absorption via the oral cavity, it is often desirable to increase the dosage administered by injection or via the gastrointestinal tract, for example by at least a factor of two or three.

[0360] Particles. For ease of explanation, the particles, formulations, compositions, and methods in this section are mainly described with respect to modified siRNA compounds. However, it is understood that these particles, formulations, compositions, and methods can be implemented with other siRNA compounds, such as unmodified siRNA compounds, and such implementation is within the scope of the present invention. In another embodiment, the siRNA compounds, such as double-stranded siRNA compounds, or ssiRNA compounds, (e.g., precursors, such as larger siRNA compounds that can be processed into ssiRNA compounds, or siRNA compounds, such as DNAs encoding double-stranded siRNA compounds, or ssiRNA compounds, or precursors thereof) preparations can be included in particles, such as microparticles. Microparticles can be produced by spray drying, but can also be produced by other methods, including freeze drying, evaporation, fluidized bed drying, vacuum drying, or a combination of these techniques.

[0361] Pharmaceutical Compositions The iRNA agent of the present invention can be formulated for pharmaceutical use.The present invention further relates to a pharmaceutical composition comprising the dsRNA agent defined herein.The pharmaceutically acceptable composition comprises a therapeutically effective amount of one or more dsRNA agents of any of the above-mentioned embodiments, either alone or formulated with one or more pharmaceutically acceptable carriers (additives), excipients, and / or diluents.

[0362] The pharmaceutical compositions can be specifically formulated to be administered in solid or liquid form, including administration adapted for: (1) oral administration, e.g., drenches (aqueous or non-aqueous solutions, or suspensions), tablets, e.g., tablets for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension, or sustained release formulation; (3) topical application, e.g., as a cream, ointment, or controlled release patch or spray applied to the skin; (4) vaginal or rectal administration, e.g., as a pessary, cream, foam; (5) sublingual administration; (6) intraocular administration; (7) transdermal administration; or (8) intranasal administration. Delivery using subcutaneous or instillation techniques can be particularly advantageous.

[0363] As used herein, the phrase "therapeutically effective amount" refers to an amount of a compound, material, or composition, including a compound of the invention, effective to obtain some desired therapeutic effect in at least a subpopulation of cells of an animal, at a reasonable benefit / risk ratio applicable to any treatment.

[0364] The phrase "pharmacologically acceptable" is utilized herein to refer to those compounds, materials, compositions, and / or dosage forms which, within the scope of sound medical judgment, are suitable for contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0365] The phrase "pharmacologically acceptable carrier" as used herein means a pharma- ceutically acceptable material, composition, or vehicle involved in carrying or transporting a compound of interest from one organ or part of the body to another, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium, calcium stearate, zinc stearate, or steric acid), or a solvent that encapsulates the material. Each carrier must be "acceptable" in that it is compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of materials that can serve as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium state; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as polyethylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar: (14 (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffers; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; and (22) other non-toxic compatible substances utilized in pharmaceutical formulations.

[0366] The formulations can be conveniently provided in unit dosage form and can be prepared by any method well known in the art of pharmacy. The amount of active ingredient that can be combined with carrier materials to produce a single dosage form varies depending on the recipient and the particular mode of administration. The amount of active ingredient that can be combined with carrier materials to produce a single dosage form is generally the amount of compound that provides a therapeutic effect. Generally, out of 100%, this amount ranges from about 0.1% to about 99% of the active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0367] In certain embodiments, the formulations of the invention include an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle-forming agents such as bile acids, and polymeric carriers such as polyesters and polyanhydrides; and the compounds of the invention. In certain embodiments, the formulations described above allow the compounds of the invention to be administered orally.

[0368] An iRNA agent preparation can be formulated in combination with another agent, e.g., another therapeutic agent, or an agent that stabilizes the iRNA, e.g., a protein that complexes with the iRNA to form an iRNP. Still other agents include chelating agents, e.g., EDTA (e.g., chelating agents that bind divalent cations, e.g., Mg 2+ for removing ribosomal RNA), salts, and RNAse inhibitors (eg, broad specificity RNAse inhibitors such as RNAsin).

[0369] The method of preparing these formulations or compositions includes the step of combining the compound of the present invention with the carrier and, optionally, one or more accessory ingredients.In general, the composition is prepared by uniformly and intimately combining the compound of the present invention with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

[0370] In some cases, it is desirable to delay the absorption of drugs by subcutaneous or intramuscular injection in order to prolong the effect of the drug. This can be achieved by using a liquid suspension of crystalline or amorphous material that is poorly soluble in water. The rate of absorption of the drug then depends on the dissolution rate, which may depend on the size of the crystals and the crystalline form. Alternatively, delayed absorption of parenterally administered drug forms can be achieved by dissolving or suspending the drug in an oil vehicle.

[0371] The compounds according to the invention may, by analogy with other pharmaceutical agents, be formulated for administration in any convenient way used in human or veterinary medicine.

[0372] The term "treatment" is intended to encompass prophylaxis, therapy, and cure. Patients receiving this treatment are any animal in need of treatment, including primates, particularly humans, and other mammals, such as horses, cows, pigs, and sheep; as well as common poultry and pets.

[0373] Double-stranded RNAi agents are produced in cells in vivo, for example, by exogenous DNA templates delivered to cells. For example, DNA templates can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to subjects, for example, by intravenous injection, local administration (US Pat. No. 5,328,470, the entire contents of which are incorporated herein by reference), or stereotactic injection (see, for example, Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057, the entire contents of which are incorporated herein by reference). Pharmaceutical preparations of gene therapy vectors can include gene therapy vectors in acceptable diluents, or can include slow release matrices in which gene delivery vehicles are embedded. For example, DNA templates can include two transcription units, one transcription unit producing transcripts that include the top strand of dsRNA agents, and another transcription unit producing transcripts that include the bottom strand of dsRNA agents. Once the template is transcribed, a dsRNA agent is produced which is processed into fragments of siRNA agents that mediate gene silencing.

[0374] Delivery route The dsRNA agent defined herein or the pharmaceutical composition comprising the dsRNA agent defined herein can be delivered to a subject using different delivery routes.The composition comprising the iRNA can be delivered to a subject by various routes.Exemplary routes include: intravenous, subcutaneous, topical, rectal, anal, vaginal, nasal, pulmonary, and ocular.

[0375] The iRNA molecules and / or dsRNA agents of the present invention can be included in pharmaceutical compositions suitable for administration. Such compositions typically include one or more iRNAs and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein is intended to include any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the composition is contemplated. Auxiliary active compounds may also be included in the composition.

[0376] The compositions of the present invention can be administered in various ways based on whether local or systemic treatment is desired and the area to be treated. Administration can be topical (including ophthalmic, vaginal, rectal, nasal, transdermal), oral, or parenteral. Parenteral administration includes infusion, subcutaneous, intraperitoneal or intramuscular injection, or intrabronchial or intraventricular administration.

[0377] The route and site of administration can be selected to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscle of interest would be a logical choice. Lung cells can be targeted by administering iRNA in a nebulized form. Vascular endothelial cells can be targeted by coating a balloon catheter with iRNA to mechanically introduce DNA.

[0378] dose In one aspect, the invention features a method of administering a dsRNA agent, e.g., a siRNA agent, to a subject (e.g., a human subject). In another aspect, the invention relates to a dsRNA agent, as defined herein, for use in inhibiting expression of a target gene in a subject. The method or medical use includes administering a unit dose of a dsRNA agent, e.g., a siRNA agent, e.g., a double-stranded siRNA agent, (a) where the double-stranded portion is 14-40 nucleotides in length, e.g., 21-23 nucleotides in length, (b) is complementary to a target RNA (e.g., a foreign or pathogen target RNA), and, optionally, (c) includes at least one 3' overhang 1-5 nucleotides in length. In one embodiment, the unit dose is less than 10 mg per kg of body weight, or less than 10 mg per kg of body weight, less than 5 mg, less than 2 mg, less than 1 mg, less than 0.5 mg, less than 0.1 mg, less than 0.05 mg, less than 0.01 mg, less than 0.005 mg, less than 0.001 mg, less than 0.0005 mg, less than 0.0001 mg, less than 0.00005 mg, or less than 0.00001 mg, and less than 200 nmol of RNA agent per kg of body weight (e.g., about 4.4×10 16 copies per kg of body weight), or less than 1500 nmol, less than 750 nmol, less than 300 nmol, less than 150 nmol, less than 75 nmol, less than 15 nmol, less than 7.5 nmol, less than 1.5 nmol, less than 0.75 nmol, less than 0.15 nmol, less than 0.075 nmol, less than 0.015 nmol, less than 0.0075 nmol, less than 0.0015 nmol, less than 0.00075 nmol, less than 0.00015 nmol.

[0379] The amount can be an amount that is effective for treating or preventing disease or disorder, for example, disease or injury associated with target RNA.The unit dose can be administered, for example, by injection (for example, intravenous, subcutaneous, or intramuscular), inhalation, or topical application.In some embodiments, the dose can be less than 10 mg, less than 5 mg, less than 2 mg, less than 1 mg, or less than 0.1 mg per kg of body weight.

[0380] In some embodiments, the unit dose is administered less than once a day, for example, less than once every 2 days, less than once every 4 days, less than once every 8 days, or less than once every 30 days. In another embodiment, the unit dose is not administered at a fixed frequency (e.g., not at a regular frequency). For example, the unit dose can be administered once.

[0381] In one embodiment, the effective amount is administered with other conventional therapeutic modalities.In one embodiment, the subject is a patient with viral infection, and the therapeutic modality is an antiviral agent other than dsRNA agent, for example, other than siRNA agent.In another embodiment, the subject is atherosclerosis, and an effective amount of dsRNA agent, for example, siRNA agent, is administered, for example, after surgical intervention, for example, in combination with angioplasty.

[0382] In one embodiment, the subject is administered an initial dose and one or more maintenance doses of a dsRNA agent, e.g., an siRNA agent, (e.g., a precursor, e.g., a larger dsRNA agent that can be processed into an siRNA agent, or a DNA encoding a dsRNA agent, e.g., an siRNA agent, or a precursor thereof). The one or more maintenance doses can be the same as the initial dose or less than the initial dose, e.g., half the initial dose. Maintenance therapy can include treating the subject with one or more doses ranging from 0.01 μg to 15 mg per kg of body weight per day, e.g., 10 mg, 1 mg, 0.1 mg, 0.01 mg, 0.001 mg, or 0.00001 mg per kg of body weight per day. Maintenance doses are administered, for example, no more than once every two days, no more than once every five days, no more than once every ten days, or no more than once every 30 days. Additionally, therapy can be continued for a period of time, which can vary depending on the nature of the particular disease, its severity, and the overall condition of the patient. In certain embodiments, dosage can be administered once a day or less, for example, once every 24 hours, 36 hours, 48 ​​hours, or more, for example, once every 5 or 8 days.After treatment, the patient can be monitored for changes in the patient's condition and relief of symptoms of the pathology.The dosage of the compound can be increased if the patient does not respond significantly to the current dosage level, or can be decreased if relief of symptoms of the disease state is observed, if the disease state is alleviated, or if undesirable side effects are observed.

[0383] An effective amount can be administered in a single dose or in two or more doses as needed or as deemed appropriate under the particular circumstances. Where it is desired to facilitate repeated or frequent infusions, the implantation of a delivery device, such as a pump, a semi-permanent stent (e.g., intravenous, intraperitoneal, intracapsular, or intraarticular), or a reservoir, can be suggested.

[0384] In one embodiment, the composition comprises multiple dsRNA agent species.In another embodiment, the dsRNA agent species has a sequence that is not overlapping or adjacent to another species with respect to natural target sequence.In another embodiment, the multiple dsRNA agent species are specific to different natural target genes.In another embodiment, the dsRNA agent is allele-specific.

[0385] The dsRNA agents of the invention described herein can be administered to mammals, particularly large animals, such as non-human primates or humans, in a variety of ways.

[0386] In one embodiment, administration of dsRNA agent, e.g., siRNA agent, composition is parenteral, e.g., intravenous (e.g., as a bolus or as a diffuse infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, nasal, urethral, ​​or intraocular.Administration can be performed by the subject or by another person, e.g., a medical provider.Medical treatment can be performed in a measured dose or with a dispenser that delivers a measured dose.Selected delivery modes are described in detail below.

[0387] The present invention provides methods, compositions, and kits for rectal administration or delivery of the dsRNA agents described herein.

[0388] In certain embodiments, the present invention relates to a dsRNA agent of the invention for use in the above methods.

[0389] Methods for inhibiting expression of a target gene The present invention also relates to a method for inhibiting the expression of target gene.The method comprises administering the dsRNA agent in any of the above-mentioned embodiments in an amount sufficient to inhibit the expression of target gene.The present invention further relates to the use of the dsRNA agent defined herein for inhibiting the expression of target gene in target cell.In a preferred embodiment, the present invention further relates to the use of the dsRNA agent for inhibiting the expression of target gene in target cell in vitro.

[0390] In another aspect, the invention relates to a method for modulating expression of a target gene in a cell, comprising the step of providing said cell with a dsRNA agent of the invention. In one embodiment, the target gene is selected from the group consisting of factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGFβ gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1 / 2 gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, hepcidin, activated protein C, cyclin D gene, VEGF gene, EGFR gene, The mutation is selected from the group consisting of a cyclin A gene, a cyclin E gene, a WNT-1 gene, a beta-catenin gene, a c-MET gene, a PKC gene, a NFKB gene, a STAT3 gene, a survivin gene, a Her2 / Neu gene, a topoisomerase I gene, a topoisomerase IIα gene, a mutation in the p73 gene, a mutation in the p21(WAF1 / CIP1) gene, a mutation in the p27(KIP1) gene, a mutation in the PPM1D gene, a mutation in the RAS gene, a mutation in the caveolin I gene, a mutation in the MIB I gene, a mutation in the MTAI gene, a mutation in the M68 gene, a mutation in a tumor suppressor gene, and a mutation in the p53 tumor suppressor gene.

[0391] In certain embodiments, the present invention relates to a dsRNA agent of the invention for use in the above methods.

[0392] The present invention is further illustrated by the following examples, which should not be construed as further limiting. The disclosures of all references, pending patent applications, and published patents mentioned in this application are expressly incorporated herein by reference. EXAMPLES

[0393] Example 1: In vitro screening of siRNA duplexes Cell culture and transfection: Human Hep3B or rat H.II.4.E cells (ATCC, Manassas, VA) were grown to near confluence at 37°C in 5% CO2 atmosphere in RPMI (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) and then detached from the plate by trypsinization. Transfection was performed by adding 14.8 μl Opti-MEM and 0.2 μl Lipofectamine RNAiMax (Invitrogen, Carlsbad CA. cat#13778-150) per well to each well of a 96-well plate containing 5 μl of siRNA duplex and incubating for 15 min at room temperature. Approximately 2 × 104 Hep3B cells in 80 μl of complete growth medium without antibiotics were added to the siRNA mixture. Cells were incubated for 24 or 120 h before RNA purification. Single dose experiments were performed at final duplex concentrations of 10 nM and 0.1 nM, and dose-response experiments were performed using 8- and 4-fold serial dilutions with the highest dose being a final duplex concentration of 10 nM.

[0394] Isolation of total RNA using DYNABEADS mRNA isolation kit (Invitrogen, part #:610-12) Cells were harvested and lysed in 150 μl of lysis / binding buffer, then mixed for 5 minutes at 850 rpm using an Eppendorf Thermomixer (mixing speed was the same throughout). A mixture of 10 μl of magnetic beads and 80 μl of lysis / binding buffer was added to a round-bottom plate and mixed for 1 minute. The magnetic beads were captured using a magnetic stand, and the supernatant was removed without disturbing the beads. After removing the supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing the supernatant, the magnetic beads were washed twice with 150 μl of wash buffer A and mixed for 1 minute. The beads were captured again and the supernatant was removed. The beads were then washed with 150 μl of wash buffer B, the beads were captured, and the supernatant was removed. The beads were then washed with 150 μl of elution buffer, captured, and the supernatant was removed. The beads were then washed with 150 μl of elution buffer, captured, and the supernatant was removed. The beads were then washed with 150 μl of elution buffer, captured, and the supernatant was removed. The beads were then dried for 2 minutes. After drying, 50 μl of elution buffer was added and mixed for 5 minutes at 70° C. The beads were captured on a magnet for 5 minutes. 40 μl of the supernatant was removed and added to another 96-well plate.

[0395] cDNA synthesis using the ABI High Performance cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, Cat#4368813): Each reaction contained 1 μl of 10× buffer, 0.4 μl of 25× dTNP, 1 μl of random primers, 0.5 μl of reverse transcriptase, 0.5 μl of RNAse inhibitor, and 1.6 μl of H 2 A master mix containing O was added to 5 μl of total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler with the following steps: 25°C for 10 min, 37°C for 120 min, 85°C for 5 s, and a 4°C hold.

[0396] Real-time PCR 2 μl of cDNA was added to each well of a 384-well plate (Roche cat#04887301001) containing 0.5 μl of GAPDH TaqMan probe (Applied Biosystems Cat#4326317E (human) Cat#4308313 (rodent)), 0.5 μl of TTR TaqMan probe (Applied Biosystems cat#HS00174914_m1 (human) cat#Rn00562124_m1 (rat)), and 5 μl of Lightcycler 480 probe master mix (Roche Cat#04887301001). Real-time PCR was performed on a Roche LC 480 Real Time PCR machine (Roche). Unless otherwise stated, each duplex was tested in at least two separate transfections and each transfection was assayed in duplicate.

[0397] To calculate relative fold changes, real-time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955 or mock-transfected cells. IC 50 was calculated using a four-parameter fit model using XLFit and normalized to AD-1955 transfected or untreated cells for the same dose range or to its own lowest dose. 50 was calculated for each transfection and the combination, giving an IC 50 was fitted to the data from both transfections.

[0398] Gene silencing results of exemplary siRNA duplexes with various motif modifications of the present invention are shown in the table below.

[0399] Example 2. RNA synthesis and duplex annealing 1. Oligonucleotide synthesis: All oligonucleotides were synthesized on an AKTA oligopilot synthesizer or an ABI 394 synthesizer. Unless otherwise stated, oligonucleotides were synthesized using commercially available controlled pore glass solid supports (dT-CPG, 500 Å, Prime Synthesis) and RNA phosphoramidites with standard protecting groups, 5′-O-dimethoxytrityl N6-benzoyl-2'-t-butyldimethylsilyl-adenosine-3'-ON, N'-diisopropyl-2-cyanoethyl phosphoramidite, 5'-O-dimethoxytrityl-N4-acetyl-2'-t-butyldimethylsilyl-cytidine-3'-ON, N'-diisopropyl-2-cyanoethyl phosphoramidite, 5'-O-dimethoxytrityl-N2-isobutryl-2'-t-butyldimethylsilyl-guanosine-3'-ON, N'-diisopropyl-2-cyanoethyl phosphoramidite, and 5'-O-dimethoxytrityl-2'-t-butyldimethylsilyl-uridine-3'-ON, N'-diisopropyl-2-cyanoethyl phosphoramidite (Pierce Nucleic Acids Technologies) were used. 2'-F phosphoramidites, 5'-O-dimethoxytrityl-N4-acetyl-2'-fluoro-cytidine-3'-ON, N'-diisopropyl-2-cyanoethyl-phosphoramidite and 5'-O-dimethoxytrityl-2'-fluoro-uridine-3'-ON, N'-diisopropyl-2-cyanoethyl-phosphoramidite were purchased from Promega. All phosphoramidites were prepared using acetonitrile (CH) except for guanosine, which was used at a concentration of 0.2 M in 10% THF / ANC (v / v). 3 A concentration of 0.2 M in 2,6-lutidine / ACN (1:1 v / v) was used. A coupling / recycling time of 16 min was used. The activator was 5-ethylthiotetrazole (0.75 M, American International Chemicals), and for PO oxidation iodine / water / pyridine was used, and for PS oxidation PADS (2%) dissolved in 2,6-lutidine / ACN (1:1 v / v) was used.

[0400] The ligand-conjugated chains were synthesized using a solid support containing the corresponding ligand. For example, the introduction of a carbohydrate moiety / ligand (e.g., for GalNAc) at the 3' end of the sequence was achieved by initiating synthesis on the corresponding carbohydrate solid support. Similarly, the cholesterol moiety at the 3' end was introduced by initiating synthesis on a cholesterol support. In general, the ligand moiety was linked to trans-4-hydroxyprolinol via a tether selected from those described in the previous examples to obtain a hydroxyprolinol-ligand moiety. The hydroxyprolinol-ligand moiety was then attached to a solid support via a succinic acid linker or converted to a phosphoramidite by standard phosphitylation conditions to obtain the desired carbohydrate-conjugated building block. Fluorophore-labeled siRNA was synthesized on the corresponding phosphoramidite or solid support purchased from Biosearch Technologies. Oleyllithocholic acid (GalNAc) 3 The polymer support was made in-house with a loading of 38.6 μmol / gram. 3 The polymer support was also prepared in-house with a loading of 42.0 μmol / gram.

[0401] Conjugation of the selected ligand at the desired position, e.g., the 5' end of the sequence, was achieved by coupling the corresponding phosphoramidite to the growing chain under standard phosphoramidite coupling conditions unless otherwise stated. Anhydrous CH in the presence of 5-(ethylthio)-1H-tetrazole activator for solid-bound oligonucleotides. 3Long-term coupling of 0.1 M phosphoramidite solutions in CN was performed for 15 min. Oxidation of internucleotide phosphites to phosphates was performed using standard iodine water as reported in (1) or by treating the conjugated oligonucleotides with tert-butyl hydroperoxide / acetonitrile / water (10:87:3) with an oxidation wait time of 10 min. Phosphorothioates were introduced by oxidation of phosphites to phosphorothioates using sulfur transfer reagents such as DDTT (purchased from AM Chemicals), PADS, and / or Beaucage reagent. Cholesterol phosphoramidites were synthesized in-house and used at a concentration of 0.1 M in dichloromethane. Coupling time for cholesterol phosphoramidites was 16 min.

[0402] 2. Deprotection-I (Nucleobase Deprotection) After completion of the synthesis, the support was transferred to a 100 ml glass bottle (VWR). The oligonucleotides were cleaved from the support simultaneously with deprotection of the base and phosphate groups using a mixture of 80 mL of ethanol ammonia [ammonia:ethanol (3:1)] at 55 °C for 6.5 h. The bottle was briefly cooled on ice, and then the ethanol ammonia mixture was filtered into a new 250 mL bottle. The CPG was washed with 2 × 40 mL portions of ethanol / water (1:1 v / v). The volume of the mixture was then reduced to approximately 30 mL on a rotovap. The mixture was then frozen on dry ice and dried under vacuum in a SpeedVac.

[0403] 3. Deprotection-II (Removal of 2'TBDMS Group) The dry residue was resuspended in 26 ml of triethylamine, triethylamine trihydrofluoride (TEA.3HF), or pyridine-HF and DMSO (3:4:6) and heated at 60°C for 90 min to remove the tert-butyldimethylsilyl (TBDMS) group at the 2' position. The reaction was then quenched with 50 ml of 20 mM sodium acetate, the pH was adjusted to 6.5, and stored in the freezer until purification.

[0404] 4.Analysis Oligonucleotides are analyzed by high performance liquid chromatography (HPLC) prior to purification, with the choice of buffer and column dependent on the sequence and / or the nature of the conjugated ligand.

[0405] 5. HPLC Purification Ligand-conjugated oligonucleotides were purified by reverse-phase preparative HPLC. Unconjugated oligonucleotides were purified by anion-exchange HPLC on in-house packed TSK gel columns. The buffer was 10% CH 3 20 mM sodium phosphate (pH 8.5) and 10% CH in CN (buffer A) 3 CN, 20 mM sodium phosphate (pH 8.5) in 1 M NaBr (buffer B). Fractions containing full-length oligonucleotides were pooled, desalted, and lyophilized. The desalted oligonucleotides with an OD of approximately 0.15 were diluted with water to 150 μl and then pipetted into specialized vials for CGE and LC / MS analysis. Finally, the compounds were analyzed by LC-ESMS and CGE.

[0406] 6. siRNA Preparation For preparation of siRNA, equimolar amounts of sense and antisense strands were heated to 95° C. for 5 min in 1×PBS and slowly cooled to room temperature. The integrity of the duplex was confirmed by HPLC analysis.

[0407] Example 3: In vitro silencing activity using various chemical modifications to ANGPTL3 siRNA Cell culture and transfection Hep3B cells (ATCC, Manassas, VA) were cultured in RPMI (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) at 37 °C for 24 h at 37 °C in 5% CO 2Cells were grown at 37°C in a 5% CO atmosphere until near confluence and then detached from the plates by trypsinization. Transfection was performed by adding 14.8 μl Opti-MEM and 0.2 μl Lipofectamine RNAiMax (Invitrogen, Carlsbad CA. cat#13778-150) per well to each well of a 96-well plate containing 5 μl of siRNA duplex and incubating at room temperature for 15 min. Approximately 2 × 10 4 1000×1000 ribosomal RNA (1000×1000 ribosomal RNA) was added to the siRNA mixture. Cells were incubated for 24 or 120 hours before RNA purification. Unless otherwise stated, single dose experiments were performed at final duplex concentrations of 10 nM and 0.1 nM, and dose response experiments were performed at final duplex concentrations of 10 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, 0.005 nM, 0.001 nM, 0.0005 nM, 0.0001 nM, 0.00005 nM, and 0.00001 nM.

[0408] cDNA synthesis using the ABI High Performance cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, Cat#4368813) Each reaction contained 2 μl of 10× buffer, 0.8 μl of 25× dNTPs, 2 μl of random primers, 1 μl of reverse transcriptase, 1 μl of RNAse inhibitor, and 3.2 μl of H 2 A master mix containing O was added to 10 μl of total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, CA) with the following steps: 25°C for 10 min, 37°C for 120 min, 85°C for 5 s, and hold at 4°C.

[0409] Real-time PCR 2 μl of cDNA was added to each well of a 384-well 50-plate (Roche cat#04887301001) containing master mix (0.5 μl GAPDH TaqMan probe (Applied Biosystems Cat#4326317E), 0.5 μl ANGPTL TaqMan probe (Applied Biosystems cat#Hs00205581_m1), and 5 μl Lightcycler 480 probe master mix (Roche Cat#04887301001). Real-time PCR was performed on an ABI 7900HT Real-time PCR system (Applied Biosystems) using the ΔΔCt (RQ) assay. Unless otherwise stated in the summary table, each duplex was tested in two separate transfections and each transfection was assayed in duplicate.

[0410] To calculate relative fold changes, real-time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955 or mock-transfected cells. IC 50 was calculated using a four-parameter fit model using XLFit and normalized to cells transfected with AD-1955 or untreated cells for the same dose range or its own lowest dose. The AD-1955 sequence used as a negative control targets luciferase and has the following sequence: Sense strand: cuuAcGcuGAGuAcuucGAdTsdT; Antisense strand: UCGAAGuACUcAGCGuAAGdTsdT.

[0411] The above various embodiments can be combined to create other embodiments. All U.S. patents, published U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein are incorporated by reference in their entirety. Aspects of the embodiments can be modified if necessary to utilize concepts from various patents, patent applications, and published patent applications to create yet other embodiments.

[0412] These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the following claims to the specific embodiments disclosed in the specification and the appended claims, but should be construed to include all possible embodiments to the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by this disclosure.

[0413] Example 4: Chemical modification of siRNA and in vitro silencing of modified siRNA Sense strand design Ligand design and conjugation sites The sense strand was conjugated to a GalNAc ligand at the 3' position, similar to the parent compound.

[0414] Position 11 of the sense strand Position 11 of the sense strand at the putative cleavage site (opposite position 11 of the AS when the sense strand is 21 nucleotides long and the antisense strand is 23 nucleotides long) was modified with a nuclease-sensitive modification (e.g., DNA). Data from statistical analysis of a large number of different conjugates suggests the importance of this position.

[0415] Thermal destabilization of the sense 3' region (positions 16-18) This region was modified with a heat destabilizing modification, e.g., GNA or a mismatch to the opposite AS strand. Modifications at positions 16 or 17 appear to have the greatest impact. Figure 1 and Table 1 highlight this position / region and the impact of heat destabilization on in vitro efficacy. Efficient knockdown comparable to the parent template design was obtained with GNA or other heat destabilizing modifications, e.g., abasic (Y34) or a mismatch to the antisense strand. On the other hand, reduced silencing was generally observed with 2'-OMe modifications, or DNA modifications complementary to the opposite AS strand.

[0416] [Table 1]

[0417] [ka]

[0418] Figure 2 and Table 2 show the effect of the position of the heat destabilizing modified GNA in the 3' region (positions 16-18). The results show that GNA modifications at positions 16 and 17 showed good efficacy similar to the parent design, while the GNA at position 18 showed reduced activity.

[0419] [Table 2]

[0420] [Table 3]

[0421] Antisense strand design 2nd place in AS This position was identified by statistical analysis of the data set of the AS strand position affected by sterically demanding 2'-modifications, including 2'-OMe, and large conjugates. However, we found that some modifications, including DNA, possibly RNA, and other modifications that do not bring steric bulk to the 2' position, can be well tolerated in the context of non-F designs. Results from in vitro silencing studies are summarized in Figure 3 and Table 3, and suggest that DNA and RNA at the 2 position generally maintain the activity of non-F designs similar to the parent template designs, while 2'-OMe is generally not well tolerated and results in reduced activity.

[0422] 14th in AS This position was identified by statistical analysis of data sets of large conjugates and shifts in positions in the AS strand affected by sterically demanding 2'-modifications, including 2'-OMe. However, it was found that some modifications, including DNA, and possibly RNA, and other modifications that do not introduce steric bulk at the 2' position, can be well tolerated in the context of non-F designs. Results from in vitro silencing studies are summarized in Figure 4 and Table 4 and suggest that DNA and RNA at position 14 generally maintain activity of non-F designs similar to the parent template designs, while 2'-OMe is generally not well tolerated, resulting in reduced activity.

[0423] [Table 4]

[0424] [Table 5]

[0425] In vivo evaluation siRNA targeting mTTR Animals (n=3 / group) were administered a single dose of 2.5 mg / kg siRNA and FVII serum protein levels were measured pre-dose, 4, 7, 13, 22, 29, and 36 days after dosing. Figure 5 shows the concentration-time profile of FVII protein for two non-F siRNAs AD-61398 and 64273 compared to the parent compound AD-57727. Figure 6 shows the reduction of mTTR protein 96 hours after dosing for two non-F siRNAs at three different dose levels compared to the parent compound. Figure 7 shows the profile of the reduction of mTTR serum protein for a repeat-dosing regimen (1 mg / kg, QW) (total of 6 doses) up to 42 days.

[0426] Overall, these studies suggest that the non-F siRNAs AD-61398 and AD-642733 demonstrated similar in vivo efficacy and potency as the parental template designs.

[0427] TMPRSS6-targeting siRNA

[0428] [Table 6]

[0429] The results suggest that the in vivo efficacy of non-F designs varies depending on the exact location of the modification and the combination of the sense and AS strands. The in vitro data suggests that the non-F compounds had similar potency / efficacy as the parent compounds, although the non-F compound found to be the most active in vivo, AD-64604, was still significantly less effective than the parent AD-60940 (see Figure 8).

[0430] Further improvements of the non-F design were made and evaluated as summarized in Table 7. Figure 9 shows silencing of TMPRSS6 mRNA in the liver 7 days after a single SC dose of 3 mg / kg.

[0431] [Table 7]

[0432] As shown in Figure 9, improvements have resulted in at least one non-F compound (AD-65105) with in vivo efficacy comparable to that of the parent (AD-60940). This compound contains a sense strand with DNA at positions 6 and 11, and an antisense strand with RNA at position 2 and DNA at positions 10 and 14.

[0433] Motif design When designing the motif, the sense strand was conjugated to a GalNAc ligand at the 3' position using the same procedure used for the parent compound. Additional motifs were designed according to embodiments of the present invention. Representative sequences are listed in Table 8.

[0434] [Table 8]

[0435] In vitro results As shown in FIG. 10, all ten sequences representing three targets and two motifs, namely motif 1 (six phosphorothioate internucleotide linkage modifications in the sense and antisense strands; four 2'-F modifications at positions 7 and 9-11 of the sense strand from the 5' end of the sense strand, and four 2'-F modifications at positions 2, 6, 14, and 16 of the antisense strand from the 5' end of the antisense strand) and motif 2 (six phosphorothioate internucleotide linkage modifications in the sense and antisense strands; four 2'-F modifications at positions 7 and 9-11 of the sense strand from the 5' end of the sense strand, and six 2'-F modifications at positions 2, 6, 8-9, 14, and 16 of the antisense strand from the 5' end of the antisense strand), were found to have statistically significant improvements in activity compared to the parent compound AD-57727.

[0436] In vivo evaluation The target silencing of siRNA was evaluated by qPCR. The ability of the motif to target mTTR was evaluated. Animals (n=3 / group) were administered a single dose of 3mg / kg siRNA, and liver levels were evaluated before administration, and then after 7 and 22 days, as shown in Figure 11.

[0437] Figure 12 shows the enhancement of activity with stability-enhanced conjugate chemistry (SEC-C), and liver was assessed for activity (mRNA) 7 days after dosing. Animals were dosed with a single dose (SC) of 3 mg / kg. The data demonstrate the effect of the motif on in vivo activity.

[0438] Figure 13 shows the improved activity (approximately 4-fold improvement in activity) for the new motifs (motifs 1 and 2) compared to the parent compound with data evaluated after 7 days of dosing. This data demonstrates the influence of the motifs on in vivo activity. The several-fold improvement is consistent for all sequences.

[0439] FIG. 14 shows a significant improvement in duration for all three sequences, demonstrating that the new motif results in improved duration.

[0440] Figure 15 shows hAAV 1 × 10 11 GC / mouse, ApoC3-GalNAc3 SAR results following a single subcutaneous dose of 3 mg / kg.

[0441] Example 5: VP and PS at the 5' end of the antisense strand 2 qualification The synthesis of oligonucleotides containing 5'-vinylphosphonate (VP) and phosphorodithioate (PS) at the 5' end of oligonucleotides are described below. 2Exemplary protocols for the synthesis of oligonucleotides containing 2'-deoxythymidine linked by a 2'-deoxythymidine bond are described below. Those skilled in the art will appreciate that similar oligonucleotides can be synthesized using these same or similar techniques. Other synthetic techniques known to those of skill in the art can also be used to synthesize and prepare these and similar oligonucleotides and modifications, including, but not limited to, those disclosed in Whittaket et al., "Stereoselective synthesis of highly functionalized P-stereogenic nucleosides via palladium-catalyzed PC cross-coupling reactions," Tetrahedron Letters 49:6984-87 (2008); Zhao and Caruthers, "Synthesis and Preliminary Biochemical Studies with 5'-Deoxy-5'-methylidyne Phosphonate Linked Thymidine Oligonucleotides," Tetrahedron Letters 37(35):6239-42 (1996); and U.S. Patent Application Publication No. 2013 / 0084576, the entire contents of each of which are incorporated herein by reference.

[0442] Protocol for the synthesis of oligonucleotides containing 5'-vinylphosphonates Introduction of pivaloyloxymethyl-(POM)-protected VP [ka] Coupling and oxidation: Coupling of the amidites was carried out under standard synthesis conditions using 0.25M 5-(ethylthio)-1H-tetrazole in acetonitrile for activation. Standard thiolation protocols using either 3-(dimethylaminomethylene)amino-3H-1,2,4-dithiazole-5-thione (DDTT) or phenylacetyl disulfide (PADS) were performed to convert the phosphite triesters to phosphorothioate linkages. The final detritylation step was omitted because the vinyl phosphonate building blocks do not contain a DMT protecting group at the 5' position.

[0443] Deprotection and cleavage: After synthesis, vinylphosphonate-containing oligonucleotides were deprotected in aqueous NH 3 and EtOH in a 3:1 mixture.

[0444] Introduction of ethyl-protected VP [ka] Coupling and oxidation: Coupling of the amidites was carried out under standard synthesis conditions using 0.25M 5-(ethylthio)-1H-tetrazole in acetonitrile for activation. Standard thiolation protocols using either 3-(dimethylaminomethylene)amino-3H-1,2,4-dithiazole-5-thione (DDTT) or phenylacetyl disulfide (PADS) were performed to oxidize the phosphite triesters and induce phosphorothioate linkages. The final detritylation step was omitted because the vinyl phosphonate building blocks do not contain a DMT protecting group at the 5' position.

[0445] Deprotection and cleavage: Prepare a solution of acetonitrile (ACN) and pyridine (Pyr) 50:1 (v / v) and add 3 × 10 -8The mixture was dried as much as possible by adding 3 cm (3 Angstrom) molecular sieves. To this mixture, 3.5 mL (5 g) of iodotrimethylsilane (TMSI) was added for every 135 mL of ACN / Pyr solution. This solution must be freshly prepared and has a maximum shelf life of 1 day. Next, a solution of 0.5 M mercaptoethanol in 1:1 (v / v) acetonitrile-triethylamine was prepared and diluted with 3 x 10 -8 cm (3 Angstroms) of molecular sieves were added. While the 5'-VP-containing oligonucleotide was on the resin in the synthesis column, the TMSI solution was slowly added at about 5-10 CV and allowed to react for 15 min. This step was repeated twice for a total exposure time of about 45 min. The resin was then washed extensively with ACN, and then the column was flushed with about 5-10 column volumes of mercaptoethanol solution and allowed to react for 10 min. This step was repeated once for a total exposure time of 20 min. After another extensive wash with ACN, the support-bound oligonucleotide was deprotected and cleaved from the support using standard conditions.

[0446] Protocol for the synthesis of oligonucleotides containing 2'-deoxythymidine linked by a phosphorodithioate bond at the 5' end of the oligonucleotide [ka] Coupling and oxidation: A phosphoramidite solution was prepared from commercially available dT-thiophosphoramidite (Glen Research) according to the manufacturer's protocol at a concentration of 0.15 M in dry acetonitrile. Coupling was performed under standard synthesis conditions with 0.25 M 5-(ethylthio)-1H-tetrazole in acetonitrile for a total coupling time of 17 min. The capping step was omitted from this synthesis cycle. Oxidation (thiolation) was performed with 3-(dimethylaminomethylene)amino-3H-1,2,4-dithiazole-5-thione (DDTT) by extending the reagent delivery and reaction time to 3 x 10 min. The final detritylation step was performed using standard synthesis conditions.

[0447] Deprotection and cleavage: The solid support (on the column) was washed with 0.5 M piperidine in ACN (2 x 15 min exposure time) and the resin was then transferred to a suitable vessel and treated under standard conditions (e.g., 3:1 aqueous NH:EtOH solution at 60°C for 5 h or 35°C for 16 h) to cleave the oligonucleotides from the solid support and deprotect them.

[0448] The remaining steps of the oligonucleotide synthesis process are similar to those described in Example 2.

[0449] Figure 16 illustrates a schematic of Ago2-tagged siRNA. In general, 5'-phosphate functionalized siRNA (ESC chemistry) shows improved in vitro activity. For example, about 80% of the sequences tested showed improved intrinsic potency when transfected in vitro, and about 30% showed IC of about 10-fold. 50 It has been shown that 5'-phosphates are rapidly lost in the endo / lysome compartment in vitro. A modified phosphate that mimics a stable phosphate, 5'-vinylphosphonate (5'-VP), has also been added to the 5' end of a modified oligonucleotide, as shown in Figure 16. This phosphonate was originally designed by Merck.

[0450] One embodiment of the present invention relates to a 5' end modification (RISC addition) for improved potency, which provides a stable phosphomimetic and promotes endogenous phosphorylation.

[0451] Figure 17 shows a chart illustrating how the presence of 5'-VP generally improves in vitro activity, based on evaluation of four different ApoB sequences. LDL levels 7 days after a single SC dose of 3 mg / kg were analyzed for the four conjugates (with or without the 5'-VP modification). As can be seen from the chart, the ED 50A three-fold improvement in β-blockade is seen for certain ApoR sequences. In vivo benefit was confirmed for additional compounds / targets including ApoC3, Tmprssr6, and TTR. ApoB sequences are listed in Table 9.

[0452] [Table 9]

[0453] FIG. 18 shows the phosphorodithioate (PS ) complex, which promotes endogenous phosphorylation. 2 ) and methylphosphonate (MePhos). Modified siRNAs are generally not good substrates for Clp1 kinase, likely due to interference from the 2'OMe modification at the first nucleotide of the AS strand. However, the 2'-OMe modification, along with phosphorothioate linkages, is desirable for exonuclease protection. Replacement of the 2'-OMe modification, for example with 2'F, and modification of the PS linkage can promote exonuclease protection while maintaining metabolic stability.

[0454] FIG. 19 shows the structures of 2'-OMe-MePhos, 2'-OMe-PS, and dN(PS 2 ), and 2'F-PS. As shown in the chart, dn(PS 2 ) linkage and 2'F-PS showed improved in vitro activity relative to the parent (2'OMe-PS). 2 was stable in an in vitro tritosome assay, whereas 2'F-PS exhibited metabolic liability. Transfection of mouse primary hepatocytes at 10 nM and 0.1 nM (n=4) was performed with the two ApoB conjugates.

[0455] Figure 20 shows two charts that show how subtle changes in the antisense 5' end can dramatically improve in vivo efficacy. Figure 20A shows that a 2'F-PS at position 1 of the antisense strand can improve the activity of a 5'P-dependent sequence, and Figure 20B shows that a dN(PS) at position 1 of the antisense strand can improve the activity of a 5'P-dependent sequence, similar to a 5'-VP, relative to the parent. 2 These results show approximately three-fold improved efficacy.

[0456] Example 6: 5'-VP modification and evaluation of siRNA activity Synthesis of 5' vinylphosphonate phosphoramidites using pivaloxymethyl protecting groups: [ka] Reagents and reaction conditions for Scheme 1: (a) Dess-Martin periodinane, DCM, 0° C.; (b) NaH, tetra(pivaloyloxymethyl)bisphosphonate, THF, −78° C., followed by stirring at 0° C., 70% (E and Z isomers); (c) formic acid:water, 1:1, 24 h, E and Z isomers separated by silica column chromatography or by RP-HPLC (reverse phase HPLC); (d) 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphordiamidite, 5-(ethylthio)-1H-tetrazole, ACN, 6 h, room temperature, 65%.

[0457] Synthesis of Tetra(pivaloyloxymethyl)-bis-phosphonate (X) [ka] Tetramethyl methylene bisphosphonate (120 g, 0.51 mol), NaI (308 g, 2 mol), chloromethyl pivalate (387 g, 2.5 mol), and acetonitrile (400 ml) were mixed and refluxed overnight. Product formation was confirmed by TLC in 5% methanol in EtOAc. The reaction mixture was diluted with ether (1000 ml), washed with water (2 x 1000 ml), and then diluted with Na 2 SO 3The solid residue was washed with cold hexane and dried in vacuum to give 148 g (45%) of X as a pale yellow solid. 1 H NMR (500MHz, CDCl 3 ):δ 5.73-5.63(m,8H),2.65(t,2H),1.22(s,36H); 31 P NMR (500MHz, CDCl 3 ):δ 18.61.

[0458] Preparation of compound 2 [ka] To an ice-cold solution of compound 1 (3.0 g, 8 mmol) in 150 mL of anhydrous dichloromethane was added Dess-Martin periodinane (DMP) (1.4 equiv.; 4.7 g, 11.2 mmol). The reaction mixture was stirred at 0° C. for 1 h and then at room temperature for 3 h. TLC confirmed the formation of the product. The reaction mixture was then diluted with 10% Na 2 S 2 O 3 and saturated NaHCO 3 A 200 ml solution of (1:1) was added followed by 200 ml of ethyl acetate. The crude aldehyde was extracted with ethyl acetate and concentrated to dryness under reduced pressure. This crude aldehyde was used in the next step without any purification.

[0459] Yield = 2.87 gm (97%); about 70% purity by NMR; LC-MS: m / z 371.

[0460] Preparation of compound 3 [ka] A solution of tetrapolyoxometalate (POM)-bisphosphonate sodium salt was prepared by adding bisphosphonate (X) (12.6 gm, 20 mmol) in 14 ml of THF to a suspension of NaH (0.58 g, 24 mmol) in 20 mL of THF at -78°C and stirring for 15 minutes.

[0461] A solution of aldehyde 2 (2.86 g) in 40 mL of anhydrous THF was added dropwise to the POM-bisphosphonate sodium salt solution prepared above at -78 °C. The reaction mixture was stirred at -78 °C for 1 h, at 0 °C for the next 1 h, and at room temperature for another 1 h. TLC confirmed the formation of the product (EtOAc:Hexane 7:3). The crude reaction mixture was added to 300 ml of saturated ammonium chloride and extracted with 300 ml of ethyl acetate. The organic layer was washed with brine and dried over sodium sulfate. The solution was then concentrated under reduced pressure and the residue was purified by silica gel column chromatography (EtOAc in hexane = 20-100%) to give compound 3 (4.0 g) as a mixture of E / Z isomers (88 / 12) in 72% yield.

[0462] Preparation of compound 4 [ka] 200 mL HCOOH / H 2 A solution of 3 (4 g, 5.7 mmol) in 2H2O (1:1, v:v) was stirred at room temperature for 24 h. TLC revealed the product (MeOH:CH 2 CL 2 =5:95) was confirmed.

[0463] The solution was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (MeOH:CH 2 CL 2 = 7:93, v:v). The fractions were tested by RP-HPLC (C18 column, buffer A = 0.05% TFA in water, buffer B = 0.05% TFA in ACN; gradient 5-95% in 25 min) to confirm the purity of the two isomers (E and Z isomers): the E isomer eluted at 14.1 min and the Z isomer eluted at 14.9 min. The first fraction from the silica gel column chromatography contained the E and Z isomers, the remaining fractions were the E isomer. The fractions containing a mixture of E and Z isomers were purified by RP-HPLC. 2.3 g of 4-E isomer was obtained, a yield of 71%.

[0464] E Opposite sex: 1 H NMR(400MHz,アセトニトリル-d 3 ):δ 8.98(s,1H),7.30(d,J=8.1Hz,1H),6.80(ddd,J=23.7,17.2,5.0Hz,1H),6.02(ddd,J=21.6,17.1,1.7Hz,1H),5.77(d,J=3.2Hz ,1H),5.57(m,5H),4.32(m,1H),4.01(dd,J=7.0,5.4Hz,1H),3.82(dd,J=5.5,3.2Hz,1H),3.41(s,3H),1.14(d,J=1.5Hz,18H); 31 P NMR (162 MHz, アセトトトリル-d 3 ):δ 18.29.

[0465] Z heterosexual: 1 H NMR(500MHz,アセトトトリル-d 3 ):δ 9.50(s,1H),7.44(d,J=8.1Hz,1H),6.69(ddd,J=54.4,13.3,8.7Hz,1H),5.93(ddd,J=17.8,13.3,1.3Hz,1H),5.80(d,J=2.9Hz,1 H),5.69-5.58(m,5H),5.22(m,1H),4.01(dd,J=7.1,5.3Hz,1H),3.88(dd,J=5.3,2.9Hz,1H),3.49(s,3H),1.19(d,J=5.8Hz,18H); 31 P NMR (202 MHz, アセトトトリル-d 3 ):δ 18.75.

[0466] Modulation of Compound 5

change

[0467] E isomer: .20(m,1H),3.99(m,1H),3.92-3.57(m,4H),3.44(s,3H),2.73-2.64(m,2H),2.14(s,1H),1.24-1.14(m,30H); 31 P NMR (162MHz, acetonitrile-d 3 ):δ 151.79(d,J=71.3Hz),18.07(d,J=54.0Hz).

[0468] Z isomer: 1 H NMR (400MHz, acetonitrile-d 3 ):δ 9.02(s,1H),7.41(dd,J=8.1,1.6Hz,1H),6.62(dddd,J=53.7,13.1,9.7,7.0Hz,1H),5.97(dd,J=17.4,13.1Hz,1H),5.80(dd,J=7.0,3.5Hz,1H),5. 70-5.52(m,5H),5.41(m,1H),4.40-4.10(m,1H),4.06-3.98(m,1H),3.93 -3.56(m,4H),3.47(s,3H),2.68(m,2H),2.14(s,1H),1.33-1.11(m,30H); 31 P NMR (202MHz, acetonitrile-d 3 ):δ 150.81(d,J=141.4Hz),15.17.

[0469] Protocol for the synthesis of oligonucleotides containing 5'-vinylphosphonates The synthesis of vinylphosphonate monomers and 5'-VP modified oligonucleotides was carried out similarly to the procedures described in the literature (WO 2008 / 100447 to Chen et al.; Lima et al. "Single-Stranded siRNAs Activate RNAi in Animals," Cell 150:883-894 (2012); Prakash et al., "Identification of metabolically stable 5-phosphate analogs that support single-stranded siRNA activity," Nucleic Acids Research 43:2993-3011 (2015)), each of which is incorporated herein by reference in its entirety. Briefly, the 5'-phosphate is protected by ethyl ether, and the ethyl ether protected phosphate is then subjected to a two-step deprotection: (1) TMS-I on solid support under anhydrous conditions, and (2) standard oligonucleotide deprotection to obtain 5'-VP modified oligonucleotides. This process is also described in Example 5.

[0470] Effect of metabolic stability of (E-) and (Z-) 5'-vinylphosphonates on siRNA activity Double-stranded small interfering RNA (siRNA) with a 5'-phosphorylated antisense strand promotes efficient loading into the RNA-induced silencing complex (RISC) to induce robust RNAi-mediated gene silencing. Thus, endogenous 5'-phosphorylation of synthetic siRNA by Clp1 kinase is crucial for RISC loading and strand selection (Weitzer et al., "The human RNA kinase hClp1 is active on 3' transfer RNA exons and short interfering RNAs," Nature 447:222-226 (2007)). Phosphate analogs with metabolically stable linkages have been used to modify nucleosides as antiviral agents (WO 2008 / 100447 to Chen et al.) and to modify the 5' end of siRNAs to improve gene silencing activity over corresponding non-phosphorylated siRNAs, particularly single-stranded siRNAs (Lima et al. "Single-Stranded siRNAs Activate RNAi in Animals," Cell 150:883-894 (2012);Prakash et al., "Identification of metabolically stable 5-phosphate analogs that support single-stranded siRNA activity," Nucleic Acids Research 43:2993-3011 (2015)).

[0471] In this example, the effects of phosphate analogs in double-stranded siRNAs were evaluated both in vitro and in vivo.

[0472] The siRNA sequences used in this example are shown in the table below.

[0473] [Table 10]

[0474] [Table 11]

[0475] The effects of 5'-vinylphosphonates (VPs) with E- and Z-geometry on double-stranded siRNA activity were compared. The results showed that the in vivo efficacy of chemically modified siRNAs could be improved with 5'-trans-(E-)VP, which adequately mimics the natural phosphate, whereas 5'-cis-(Z-)VP showed no improvement in efficacy, suggesting that the Z isomer does not adequately mimic the natural phosphate.

[0476] Figure 21A and Figure 21B show SAR analysis comparing the in vitro and in vivo activity of ApoB siRNA containing 5'-OH modification (at the 5' end of the antisense strand) and ApoB siRNA containing 5'-E-VP modification. Figure 21A shows the results in in vitro transfected mouse hepatocytes. Figure 21B shows the level of LDL 3 days after a single dose (SC administration). The results in Figure 21B demonstrate that ApoB siRNA modified with 5'-E-VP showed improved activity.

[0477] Figure 22 shows the results of a comparison of the in vitro efficacy of 5'-E-VP and 5'-Z-VP modifications for mTTR and F9 siRNA-GalNAc conjugates. The results were obtained from in vitro transfected mouse primary hepatocytes. As shown in the figure, siRNA conjugates modified with 5'-E-VP showed maintained or improved potency, while siRNA conjugates modified with 5'-Z-VP showed reduced potency.

[0478] Figure 23 shows the results of an in vivo comparison of 5'-E-VP and 5'-Z-VP modifications to F9 siRNA-GalNAc conjugates (single SC administration). The results demonstrate that 5'-E-VP modified siRNA conjugates showed improved gene silencing activity relative to the 5'-OH control, while 5'-Z-VP modified siRNA conjugates showed activity similar to that of the 5'-OH control.

[0479] The results in these figures show that 5'-phosphorylation of the antisense strand is desirable for efficient RNAi-mediated gene silencing. The efficacy of chemically modified siRNAs can be improved with 5'-trans-vinylphosphonates (5'-E-VP), which adequately mimic the natural phosphate.

[0480] Example 7: 5'-C-malonyl modification and evaluation of siRNA activity Synthesis of 5'-C-malonyl nucleotides and incorporation into the 5' end of siRNA General experimental conditions: All moisture-sensitive reactions were carried out under anhydrous conditions under an argon atmosphere. Flash chromatography was performed on a Teledyne ISCO (Lincoln, NE) Combi Flash system using prepacked ReadySep Teledyne ISCO silica gel columns. Electrospray ionization-high resolution mass spectrometry (ESI-HRMS) spectra were recorded on a Waters (milford, MA) Q-Tof API-US spectrometer using direct flow injection in the positive mode (capillary = 3000 kV, cone = 35, source temperature = 120 °C, and desolvation temperature = 350 °C). 1 H and 13 C NMR spectra were obtained at 400 MHz ( 1 H) and 126MHz ( 13C) were recorded at room temperature on a Varian spectrometer (Palo Alto, CA) and chemical shifts in ppm were relative to the residual solvent peak. Coupling constants were given in Hertz. Signal splitting patterns were expressed as singlet (s), doublet (d), triplet (t), quartet (q), broad signal (br), or multiplet (m). 31 P NMR spectra were recorded at 162 MHz in proton isolation mode, and chemical shifts were relative to external H3PO4 (80%). LC / ESI-MS was performed on an Agilent (Santa Clara, CA) 6130 single quadrupole LC / MS system using an XBridge C8 column (2.1 × 50 mm, 2.5 μm) at 60 °C. Buffer A was H 2 Buffer A consisted of 100 mM 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and 16.3 mM triethylamine (TEA) in O, with buffer B being 100% methanol.

[0481] [ka] Reagents and conditions for Scheme 2: (a) benzyloxymethyl acetal chloride (BOM), DBU, DMF, 30 min, 0° C., quantitative (Kurosu et al., “Synthetic studies towards the identification of novel capuramycin analogs with mycobactericidal activity,” Heterocycles 77:217-225 (2009); Kurosu et al., “Concise Synthesis of Capuramycin,” Org. Lett. 11:2393-2396 (2009) each of which is incorporated by reference in its entirety); (b) methyltriphenoxyphosphonium iodide, DMF, 15 min, room temperature, 92%; (c) sodium methoxide, dimethyl malonate, 1,2-DME, 24 h, reflux, 92%; (d) 10% Pd / C, H 2 Atmosphere, i-PrOH / H 2HO (10:1, v / v), 0.05 equivalents formic acid, 12 hours, room temperature, 98% (Aleiwi et al., “A reliable Pd-mediated hydrogenolytic deprotection of BOM group of uridine ureido nitrogen,” Tetrahedron Lett. 53:3758-3762 (2012) which is incorporated herein by reference in its entirety); (e) NEt 3 -3HF, THF, THF, 48 h, room temperature, 88%; (d) 2-cyanoethyl N,N-diisopropylphosphoramidite, DIEA, DCM, 18 h, room temperature, 56&; (g) 1M aqueous piperidine, 24 h, room temperature; then 30% aqueous ammonia / ethanol (3:1, v / v), 36 h, room temperature, quantitative, Z + =Piperidinium.

[0482] N 3 Synthesis of 2'-benzyloxymethyl-3'-O-tert-butyldimethylsilyluridine (2) 2'-O-Methyl-3'-O-tert-butyldimethylsilyluridine (1, 20 g, 53.7 mmol) was converted to 2 (26.5 g, quantitative) following a modification of a previously reported procedure.

[0483] N 3 Synthesis of 1-benzyloxymethyl-5'-deoxy-5'-iodo-2'-O-methyl-3'-O-tert-butyldimethylsilyl uridine (3) Compound 2 (10 g, 20.3 mmol) was dissolved in 100 mL of anhydrous DMF and 20 g (40.6 mmol) of methyltriphenoxyphosphonium iodide was added. The mixture was stirred at room temperature for 15 minutes. Methanol (200 mL) was added to the reaction and the mixture was stirred for an additional 15 minutes. The solvent was evaporated to dryness; the residue was dissolved in dichloromethane (DCM) and Na 2 S 2 O 3 The organic layer was collected and washed with a 5% solution of Na 2 SO 4The crude residue was purified by silica gel chromatography using 0-50% ethyl acetate (EtOAc) in hexanes as eluent to give 3 (11.2 g, 92%) as a white foam. 1 H NMR (400MHz, DMSO-d 6 ):δ 7.77(d,J=8.2Hz,1H),7.30(m,5H),5.90(d,J=5.2Hz,1H),5.85(d,J=8.2Hz,1H),5.33(d,J=13. 0Hz,1H),5.30(d,J=13.0Hz,1H),4.58(s,2H),4.23(t,J=4.5Hz,1H),4.07(t,J=5.1Hz,1H),3.87 (q,J=6.1Hz,1H),3.55(dd,J=10.6,6.3Hz,1H),3.39(dd,J=10.6,6.3Hz,1H),3.32(s,3H),0.89(s,9H),0.14(s,3H),0.12(s,3H). 13 C NMR (126 MHz, DMSO-d 6 ):δ 161.7,150.7,140.2,138.0,128.2,127.4,127.3,101.6,87.9,83.3,80.8,72.7,71.0,70.1,57.6,25.6,17.7,6.2,-4.7,-4.8.

[0484] HRMS-ESI:C 24 H 35 IN 2 NaO 6 Si(M+Na) + The calculated value for is 625.1207; the actual value is: 625.1205.

[0485] N 3 Synthesis of 5'-benzyloxymethyl-5'-deoxy-5'-C-(dimethylmalonyl)-2'-O-methyl-3'-O-tert-butyldimethylsilyluridine (4) Sodium methoxide (2 g, 33 mmol) was placed in a dry round-bottom flask, dimethyl malonate (12 mL, 100 mmol) and anhydrous 1,2-dimethoxyethylene (DME, 100 mL) were added, and the mixture was refluxed. Compound 3 (10 g, 16.5 mmol), after coevaporation twice with anhydrous acetonitrile, was dissolved in 70 mL of anhydrous DME and added to the refluxing solution of dimethyl malonate and sodium methoxide. Reflux was continued for 24 h. The reaction mixture was cooled to room temperature and quenched by the addition of methanol (50 mL). Solvent and volatiles were evaporated in vacuo. The crude residue was purified by silica gel chromatography using 0-100% EtOAc in hexanes as eluent to give compound 4 (9.2 g, 92%) as a colorless oil. 1 H NMR (400MHz, DMSO-d 6 ):δ 7.66(d,J=8.2Hz,1H),7.30(m,5H),5.80(d,J=8.2Hz,1H),5.76(d,J=4. 0Hz,1H),5.33(d,J=13.4Hz,1H),5.30(d,J=13.4Hz,1H),4.58(s,2H),4. 14(t,J=5.4Hz,1H),3.91(m,1H),3.76(m,1H),3.64(m,4H),3.60(s,3H) ,3.33(s,3H),2.37-2.09(m,2H),0.87(s,9H),0.09(s,3H),0.08(s,3H). 13 C NMR (126 MHz, DMSO-d 6 ):δ 169.1,168.8,161.9,150.6,140.4,138.0,128.1,127.4,127.3,101.3,88.6,88.5,8 1.3,80.9,73.1,71.0,70.0,59.7,57.5,52.5,48.0,31.5,25.6,17.7,-4.76,-5.06.

[0486] HRMS-ESI:C 29 H 42 N 2 NaO 10 Si(M+Na) +The calculated value for is 629.2506; the actual value is: 629.2508.

[0487] Synthesis of 5'-deoxy-5'-C-(dimethylmalonyl)-2'-O-methyl-3'-O-tert-butyldimethylsilyluridine (5) Compound 4 (8.7 g, 14.3 mmol) was dissolved in 660 mL of isopropanol / water (10:1, v / v) and 0.9 g of 10% Pd / C was added, followed by 27 mL (0.7 mmol) of formic acid. The air in the flask was removed under vacuum; the reaction flask was flushed with hydrogen and stirred at room temperature under standard pressure for 12 h. The reaction mixture was filtered through Celite and rinsed with ethanol. The filtrate was collected and evaporated to dryness. The crude re...

Claims

1. A double-stranded RNA (dsRNA) agent capable of inhibiting expression of a target gene, wherein the dsRNA agent comprises a sense strand and an antisense strand that is complementary to at least a portion of an mRNA corresponding to the target gene: the sense and antisense strands form a duplex region 17-23 nucleotide pairs in length; each of the sense and antisense strands is 19-25 nucleotides in length; each of the sense and antisense strands comprises at least two different nucleotide modifications, where each nucleotide modification is independently selected from the group consisting of acyclic nucleotides, LNA, HNA, CeNA, 2'-methoxyethyl, 2'O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-O-N-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), and 2'-ara-F; and The antisense strand comprises a 2'-F modification, and the 2'-F modification of the antisense strand is present and is represented by one of (A)-(C) below: of the antisense strand from the 5' end of the antisense strand, (A) positions 2, 6, 14, and 16; (B) positions 2, 6, 9, 14, and 16; or (C) positions 2, 6, 8-9, 14, and 16; exists only at the position dsRNA agents.

2. The dsRNA agent of claim 1, wherein each nucleotide in the sense strand and the antisense strand is a modified nucleotide.

3. The dsRNA agent of claim 1, wherein the antisense strand comprises phosphorothioate modifications within the first five nucleotides at the 5' end of the antisense strand.

4. 3. The dsRNA agent of claim 2, wherein each nucleotide comprises a modification selected from the group consisting of an acyclic nucleotide, a 2'-deoxy, a 2'-O-methyl or a 2'-fluoro modification.

5. The dsRNA agent of claim 2, wherein each nucleotide comprises a modification selected from the group consisting of a 2'-O-methyl and a 2'-fluoro modification.

6. 2. The dsRNA agent of claim 1, wherein the sense strand, the antisense strand, or both the antisense strand and the sense strand comprise one or more blocks of phosphorothioate or methylphosphonate internucleotide linkages.

7. The dsRNA agent of claim 6, wherein the sense strand comprises one block of two phosphorothioate or methylphosphonate internucleotide linkages.

8. 7. The dsRNA agent of claim 6, wherein the antisense strand comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 14, 15, 16, 17, or 18 phosphate internucleotide linkages.

9. The dsRNA agent of claim 6, wherein the antisense strand comprises two blocks of phosphorothioate or methylphosphonate internucleotide linkages separated by 16-18 phosphate internucleotide linkages.

10. The dsRNA agent of claim 1, wherein the sense strand has 19-22 nucleotides and the antisense strand has 19-25 nucleotides.

11. 2. The dsRNA agent of claim 1, wherein the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides.

12. 10. The dsRNA agent of claim 1, wherein the dsRNA agent has a 3' and / or a 5' overhang.

13. 2. The dsRNA agent of claim 1, wherein the dsRNA agent has a 3' overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

14. The dsRNA agent of claim 1, wherein the dsRNA agent has a 5' overhang at the 5' end of the sense strand.

15. The dsRNA agent of claim 1, wherein the nucleobase of the nucleotide at position 1 of the 5' end of the antisense strand in the duplex is selected from the group consisting of adenine, uracil, and thymine.

16. The dsRNA agent of claim 1, wherein at least one of the first, second, and third base pairs at the 5' end of the antisense strand is an AU base pair.

17. 10. The dsRNA agent of claim 1, wherein the dsRNA agent is conjugated to at least one ligand.

18. 20. The dsRNA agent of claim 17, wherein at least one ligand enhances nuclease resistance of the dsRNA agent.

19. 18. The dsRNA agent of claim 17, wherein at least one ligand is an ASGPR ligand.

20. 20. The dsRNA agent of claim 19, wherein an ASGPR ligand is attached to the 5' or 3' end of the sense strand.

21. 20. The dsRNA agent of claim 19, wherein an ASGPR ligand is attached to the 3' end of the sense strand.

22. 20. The dsRNA agent of claim 19, wherein the ASGPR ligand is one or more GalNAc derivatives attached by a bivalent or trivalent branched linker.

23. The ASGPR ligand is 【Chemistry 1】 23. The dsRNA agent of claim 22, wherein

24. The dsRNA agent of claim 1, wherein the dsRNA has a blunt end at the 5' end of the antisense strand.

25. 2. The dsRNA agent of claim 1, wherein the antisense strand comprises four 2'-F modifications and comprises four 2'-F modifications only at positions 2, 6, 14, and 16 of the antisense strand from the 5' end of the antisense strand.

26. 2. The dsRNA agent of claim 1, wherein the antisense strand comprises five 2'-F modifications and comprises five 2'-F modifications only at positions 2, 6, 9, 14, and 16 of the antisense strand from the 5' end of the antisense strand.

27. 2. The dsRNA agent of claim 1, wherein the antisense strand comprises six 2'-F modifications and comprises six 2'-F modifications only at positions 2, 6, 8-9, 14, and 16 of the antisense strand from the 5' end of the antisense strand.

28. 2. The dsRNA agent of claim 1, wherein at least one of the first 1, 2, 3, 4, or 5 base pairs in the duplex region from the 5' end of the antisense strand is independently selected from the group consisting of A:U, G:U, I:C, and mismatch pairing.

29. The dsRNA agent of claim 1, wherein the dsRNA agent further comprises a 5'-terminal phosphorus-containing group.

30. 30. The dsRNA agent of claim 29, wherein the 5'-terminal phosphorus-containing group is 5'-vinylphosphonate (VP).

31. The dsRNA agent may have a phosphorodithioate (PS ２ 2. The dsRNA agent of claim 1, comprising a 2'-deoxythymidine linked by a 2'-(N-terminal) bond.

32. The dsRNA agent of claim 1, wherein the first nucleotide at the 5' end of the antisense strand is modified with 2'-OMe.

33. The dsRNA agent of claim 1, having two blunt ends on both ends of the dsRNA agent.

34. A pharmaceutical composition comprising the dsRNA agent of any one of claims 1-33, alone or in combination with a pharma- ceutically acceptable carrier or excipient.

35. 34. Use of a dsRNA agent according to any one of claims 1 to 33 in the manufacture of a medicament for inhibiting expression of a target gene.

36. 36. The use of claim 35, wherein the dsRNA agent is administered by subcutaneous or intravenous administration.

37. 41. Use of a dsRNA agent according to any one of claims 1-33 in the manufacture of a medicament for delivering a polynucleotide to a specific target in a subject.

38. 38. The use of claim 37, wherein the dsRNA agent is administered by an administration means comprising intramuscular administration, intrabronchial administration, intrapleural administration, intraperitoneal administration, intraarterial administration, lymphatic administration, intravenous administration, subcutaneous administration, cerebrospinal administration, or a combination thereof.

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