Modified double-stranded RNA agents

Abstract

To provide double-stranded RNA (dsRNA) agent to inhibit expression of a target gene.SOLUTION: A double-stranded RNA agent comprises a sense strand sequence and an antisense strand sequence complementary to at least one portion of a mRNA corresponding to a target gene, and the sense strand is represented by the formula (Is) in the figure, where: each of B1-3 is 2'-OMe; C1 is a thermally destabilizing nucleotide, selected from among a nucleotide that forms a mismatch pair with the opposing nucleotide in the antisense strand, a nucleotide having an abasic modification, and a nucleotide having a sugar modification, and placed at a site opposite to the seed region of the antisense strand; T1 is a nucleotide comprising a chemical modification selected from among DNA, RNA, LNA, 2'-F and 2'-F-5'-methyl; n2 is 1 to 3; n1 or n3 is independently 4 to 15 nucleotides in length; n5 is 1 to 6 nucleotides in length; and n4 is independently 0 to 3 nucleotides in length.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 RNAi duplex agents that contain specific motifs that are advantageous for inhibiting the expression of target genes, and RNAi compositions that are suitable for therapeutic use.In addition, the present invention provides the method for 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 first coined by Fire and colleagues to describe the observation that double-stranded RNAi (dsRNA) can block gene expression (Non-Patent Document 1). Short dsRNAs induce gene-specific post-transcriptional silencing in a variety of organisms, including vertebrates, providing a new tool for studying gene function. RNAi is mediated by the RNA-induced silencing complex (RISC), a sequence-specific multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs (approximately 22 nucleotides) derived from the double-stranded RNA trigger, but the protein components of this activity remain unknown.

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

[0005] It was thought 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 inhibits 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 used siRNA duplexes that contain 2'-F modified residues at Ago2 cleavage site among other sites and modifications, and have obtained suitable silencing compared with unmodified siRNA (Non-Patent Document 5).However, Morrissey's modification is not motif specific, for example, if one modification exists in both sense and antisense strands, it includes 2'-F modification in all pyrimidine residues without any preference;Therefore, based on these teachings, it is unclear whether specific motif modification 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 either the sense or antisense strand and found that this siRNA duplex was tolerated (Non-Patent Document 6). However, Muhonen's modification is also sequence-specific; for example, for each specific strand, Muhonen's modification simply modifies all pyrimidines or all purines without any selectivity.

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

[0009] Therefore, there is currently a need for iRNA duplex agents 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 a nucleotide containing a modification selected from the group consisting of 2'-O-alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halo, ENA, and BNA / LNA. In one embodiment, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe modification. In one embodiment, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe modification or a 2'-F modification. In one embodiment, at least one of B1, B2, B3, B1', B2', B3', and B4' contains a 2'-ON-methylacetamide (2'-O-NMA) modification.

[0013] C1 is a thermolabile nucleotide located at 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, C1 is located in the sense strand at a position that pairs with nucleotides 2-8 of the 5' end of the antisense strand. In one example, C1 is located at position 15 of the 5' end of the sense strand. The C1 nucleotide has a thermolabile modification that can include an abasic modification; a mismatch with the opposing nucleotide of the duplex; and a sugar modification, such as a 2'-deoxy modification, or an acyclic nucleotide, such as an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA). In one embodiment, C1 (i) is a mismatch with the opposing nucleotide of the antisense strand; or (ii) an abasic modification selected from the group consisting of: [ka] and (iii) a sugar modification selected from the group consisting of: [ka] wherein B is a modified or unmodified nucleobase and R 1 and R 2are independently H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. In one embodiment, the thermolabilizing modification of 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 thermolabilizing modification in C1 is GNA or [ka] is.

[0014] T1, T1', T2', and T3' each independently represent a nucleotide containing a modification that provides the nucleotide with steric bulk equal to or less than that of a 2'-OMe modification. Steric bulk refers to the total steric effect of the modification. Methods for determining the steric effect of a nucleotide modification 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 non-ribose nucleotide, an acyclic nucleotide, or a similar or equivalent modification at the 2' position of the ribose sugar, and provides the nucleotide with steric bulk equal to or less than that of a 2'-OMe modification. For example, T1, T1', T2', and T3' are each independently selected from DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl. In one embodiment, T1 is DNA. In one embodiment, T1' is DNA, RNA, or LNA. In one embodiment, T2' is DNA or RNA. In one embodiment, T3' is DNA or RNA.

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

[0016] n 5 , q 3 , and q 7are 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, 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 of the antisense strand.

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

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

[0024] In one embodiment, when the sense strand is 19 to 22 nucleotides in length, C1 is at positions 14 to 17 of the 5' end of the sense strand, and n 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. In one example, T3' begins at position 2 of the 5' end of the antisense strand, and q 6 is equal to 1, T1' starts from position 14 of the 5' end of the antisense strand, and q 2 is equal to 1.

[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. In one example, T3' is at position 2 of the 5' end of the antisense strand, and 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, n 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 at 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, and 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, when the sense strand is 19-22 nucleotides in length, and n 2 is 1; T1' is at position 14 of the 5' end of the antisense strand, and 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, and 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; 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 of the antisense strand.

[0037] In one embodiment, n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; 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 of the antisense strand.

[0038] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0039] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q5 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 of the antisense strand.

[0040] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 6, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 7, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0041] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 6, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 7, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and 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 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0043] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 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; 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 of the antisense strand.

[0044] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 5, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; optionally with at least two additional TTs at the 3' end of the antisense strand.

[0045] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 5, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; optionally has at least two additional TTs at the 3' end of the antisense strand; 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), 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0047] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; 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 of the antisense strand.

[0048] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; 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 of the antisense strand.

[0050] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1.

[0051] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; 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 of the antisense strand.

[0052] The dsRNA agent can include a phosphorus-containing group at the 5'-end of either the sense or antisense strand. The 5'-terminal phosphorus-containing group can be 5'-terminal phosphate (5'-P), 5'-terminal phosphorothioate (5'-PS), 5'-terminal phosphorodithioate (5'-PS2), 5'-terminal vinylphosphonate (5'-VP), 5'-terminal methylphosphonate (MePhos), or 5'-deoxy5'-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 includes a phosphorus-containing group at the 5'-end of the sense strand.In one embodiment, the dsRNA agent includes a phosphorus-containing group at the 5'-end of the antisense strand.

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

[0058] In one embodiment, the dsRNA agent comprises a 5'-PS2. In one embodiment, the dsRNA agent comprises a 5'deoxy5'-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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The dsRNA agent also includes a 5'-PS.

[0060] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The dsRNA agent also includes a 5'-P.

[0061] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The dsRNA agent also includes a 5'-PS2.

[0063] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-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'-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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; 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'-PS2.

[0068] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; 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 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The dsRNA agent also includes a 5'-PS.

[0071] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The dsRNA agent also includes a 5'-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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The dsRNA agent also includes a 5'-PS2.

[0073] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; 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'-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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; 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'-PS2.

[0078] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 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 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNA agent also includes a 5'-P.

[0080] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNA agent also includes a 5'-PS.

[0081] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0082] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNA agent also includes a 5'-PS2.

[0083] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNA agent also includes a 5'-deoxy-5'-C-malonyl.

[0084] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; 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'-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, and n 2 is 3, B2 is 2'-OMe, and n3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; 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'-PS2.

[0088] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; 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 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNA agent also includes a 5'-P.

[0090] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 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 includes a 5'-PS.

[0091] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0092] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNA agent also includes a 5'-PS2.

[0093] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNA agent also includes a 5'-deoxy-5'-C-malonyl.

[0094] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 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'-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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; 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'-PS2.

[0098] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; 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 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 the nucleotides present in the dsRNA agent comprise 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, the sense and antisense strands of the dsRNA agent each contain 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 to 10 nucleotides in length. In one example, the dsRNA agent of Formula (I) comprises a 3' overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand. In another example, the dsRNA agent has a 5' overhang at the 5' end of the sense strand.

[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 a 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 to 18 phosphate internucleotide linkages.

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

[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 to 40 nucleotides. The sense strand comprises at least one thermolabile nucleotide, at least one of which is located opposite or near the seed region of the antisense strand (i.e., positions 2 to 8 of the 5' end of the antisense strand). Each of the embodiments and aspects described herein relating to a dsRNA represented by Formula (I) can also be applied to a dsRNA comprising a thermolabile nucleotide.

[0109] For example, if the sense strand is 21 nucleotides long, the thermally destabilizing nucleotide may be located between positions 14 and 17 at the 5' end of the sense strand. The antisense strand comprises at least two modified nucleic acids that are smaller than sterically demanding 2'-OMe modifications. Preferably, the two modified nucleic acids that are smaller than sterically demanding 2'-OMe are spaced 11 nucleotides apart. For example, the two modified nucleic acids are located 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 can comprise: (i) a phosphorus-containing group at the 5'-end of the sense strand or antisense strand; (ii) two phosphorothioate internucleotide bond modifications within the 1st to 5th positions (counting from the 5'-end of the sense strand) of the sense strand, and two phosphorothioate internucleotide bond modifications at the 1st and 2nd positions (counting from the 5'-end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide bond modifications within the 18th to 23rd positions; and (iii) a ligand at the 5'-end or 3'-end of the sense strand or antisense strand, for example, an ASGPR ligand (for example, one or more GalNAc derivatives).For example, this ligand can be present 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; 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 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 to 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 to 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; 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 of the antisense strand. The dsRNA agent also includes a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0116] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; 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 of the antisense strand. The dsRNA agent also includes 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0117] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; 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 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 to 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 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 to 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 to 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; 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 of the antisense strand. The dsRNA agent also includes a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0121] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; 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 of the antisense strand. The dsRNA agent also includes 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0122] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; 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 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4is 2, B3' is 2'-OMe or 2'-F, 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 (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 to 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 to 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; 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 of the antisense strand. The dsRNA agent also includes a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0126] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; 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 of the antisense strand. The dsRNA agent also includes 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0127] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-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 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 to 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 to 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; 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 of the antisense strand. The dsRNA agent also includes a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0131] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 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; 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 of the antisense strand. The dsRNA agent also includes 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0132] In one particular embodiment, a dsRNA agent of the invention: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3' end, 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 specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) a 3′-terminally attached ASGPR ligand containing 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 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.

[0134] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) a 3′-terminally attached ASGPR ligand containing three GalNAc derivatives attached by a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, 10, and 12 to 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; The dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0135] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) a 3′-terminally attached ASGPR ligand containing 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; The dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0136] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) a 3′-terminally attached ASGPR ligand containing 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; The dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0137] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) a 3′-terminally attached ASGPR ligand containing 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; The dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0138] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) a 3′-terminally attached ASGPR ligand containing 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; The dsRNA agent has a four nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0139] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) a 3′-terminally attached ASGPR ligand containing 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; The dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0140] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) a 3′-terminally attached ASGPR ligand containing 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; The dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0141] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 19 nucleotides long; (ii) a 3′-terminally attached ASGPR ligand containing three GalNAc derivatives attached by a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 4, 6, and 10 to 19, and 2'-F modifications at positions 5 and 7 to 9; and (iv) 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; The dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[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 the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The sense strand comprises at least one thermolabile nucleotide, which is located at or near the opposite site of the seed region of the antisense strand (i.e., positions 2 to 8 at the 5' end of the antisense strand). For example, if the sense strand is 21 nucleotides long, the thermolabile nucleotide is located between positions 14 and 17 at the 5' end of the sense strand. The antisense strand comprises two modified nucleic acids separated by 11 nucleotides and less sterically demanding than 2'-OMe modifications. For example, the two modified nucleic acids are located at positions 2 and 14 at 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 less than or equal to that of a 2'-OMe modification, and the third modified nucleotide is located at positions 6-10 of the 5' end of the antisense strand. For example, the third modified nucleotide is located at position 10 of the 5' end of the antisense strand.

[0146] Embodiments of thermally destabilized nucleotides are similar to the various embodiments described above for C1 in formula (I). Embodiments of modified nucleic acids that are less sterically demanding than 2'-OMe modifications are similar to the various embodiments described above for T1', T2', and T3' in formula (I). The embodiments describing length, overhangs, additional modifications, and ligand attachment for dsRNA agents of formula I above are appropriate here.

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

[0148] The present invention also relates to the dsRNA agent defined herein, which is used to inhibit the expression of target gene in subject.This 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 the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The sense strand comprises 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 located at position 11 of the 5' end of the sense strand. The endonuclease-sensitive modification present near the cleavage site can affect the sensitivity of the cleavage site. For example, a thermolabile modification near the cleavage site can provide endonuclease sensitivity to that cleavage site. The antisense strand comprises two modified nucleic acids that are less than sterically demanding 2'-OMe modifications separated by 11 nucleotides. For example, the two modified nucleic acids are located 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 the dsRNA agent to a specific target in a subject by subcutaneous or intravenous administration. [Brief explanation 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. [Figure 2] FIG. 2 is a chart showing the effect of position on the in vitro efficacy of heat-destabilizing GNA modifications spanning positions 16-18 of the sense strand, evaluated at concentrations of 10 nM and 0.1 nM. [Figure 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, evaluated 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, evaluated at concentrations of 10 nM and 0.1 nM. [Figure 5] FIG. 5 is a graph showing mTTR silencing in mice after a single SC dose of 2.5 mg / kg. [Figure 6] Figure 6 is a chart showing the dose response of non-F siRNAs AD-61398 and AD-64273 to the parental 2PS (AD-43527) and 6PS (AD-57727): single SC administration, protein levels measured 96 hours after administration. [Figure 7] FIG. 7 is a chart showing the reduction of mTTR protein in plasma after 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 the enhanced activity with Stability Enhanced Conjugate Chemistry (SEC-C), where liver was assessed for activity (mRNA) 7 days after administration. [Figure 13] FIG. 13 shows a chart demonstrating the approximately 4-fold improvement in activity for the new motifs (motifs 1 and 2) compared to the parent compound. [Figure 14] FIG. 14 shows a chart demonstrating 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 following a single SC administration of hAAV 1×10 11 GC / mouse, 3 mg / kg. [Figure 16] Figure 16 illustrates a schematic of Ago2-loaded siRNA and the modified phosphate, 5'-vinylphosphonate (5'-VP), which mimics the stable phosphate. The 5'-phosphonate is added by the cytoplasmic Clp1 kinase and serves as a key anchor for Ago2 loading. [Figure 17]Figure 17 shows a chart illustrating 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 and 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 illustrating how subtle changes in the 5' end of the antisense strand can significantly 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 5'P-dependent sequences (F9 activity measured on day 3 after a single SC dose of 3 mg / kg). The chart on the right shows approximately 3-fold improved potency with dN(PS)2 relative to the parent, as well as VP (LDL activity measured for ApoB on day 3 after a single SC dose of 10 mg / kg). [Figure 21] Figures 21A and 21B show SAR analyses comparing the in vitro and in vivo activity of ApoB siRNA containing a 5'-OH modification (at the 5' end of the antisense strand) and ApoB siRNA containing a 5'-E-VP modification. Figure 21A shows results in in vitro transfected mouse hepatocytes. Figure 21B shows LDL levels 3 days after a single administration (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 mouse primary hepatocytes. [Figure 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. [Figure 25] Figure 25 shows the enzymatic stability results of 5'-OH, 5'-C-malonyl, and 5'-phosphate siRNAs incubated with rat liver tritosomes. The siRNA target sequences are shown in Table 10. Data were normalized to untreated controls. [Figure 26] Figure 26 shows the results of RISC addition (5' modification of the antisense strand) of 5'-OH, 5'-C-malonyl, and 5'-phosphate siRNAs, as determined by immunoprecipitation of Ago2 from mouse primary stem cells and RT-PCR amplification of the Ago2-added single strands. Endogenous miR122 levels were determined as a control. The siRNA target sequences are shown in Table 10. [Figure 27] Figure 27 is a graph showing in vitro knockdown of TTR using siRNA modified with one (S)-GNA nucleotide. TTR mRNA levels were 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 in vitro knockdown of TTR using siRNA modified with one (S)-GNA base pair. TTR mRNA levels were 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 28B shows a mix and match duplex in which the sense and antisense strands containing one (S)-GNA nucleotide are paired as a 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 2.5 mg / kg siRNA. Before or at the indicated times after administration, animals were bled, and serum samples were measured using a sandwich ELISA assay utilizing an HRP-conjugated antibody and 3,3',5,5'-tetramethylbenzidine for reading at 450 nm. All samples were measured in duplicate, and each data point represents the average of mice within each cohort (n=3). [Figure 30] Figure 30 is a graph showing in vivo quantification of TTR mRNA levels. Animals received a single dose of 2.5 mg / kg siRNA. RNA extraction was performed on whole liver homogenates at the indicated times post-administration. TTR mRNA was assessed by RT-qPCR using the ΔΔCt method with GAPDH as the control transcript, as described above, and normalized to PBS-treated animals. Dark bars represent results at day 21; white bars represent results at day 7. DETAILED DESCRIPTION OF THE INVENTION

[0152] The present inventors have found that 2'-OMe modifications at nucleotide positions 2 and 14 of the 5' end of the antisense strand suppress the gene silencing activity of dsRNA agents. By introducing non-ribose, acyclic, or backbone-specific chemical modifications at the 2' or equivalent positions in the backbone that are less sterically bulky than 2'-OMe modifications at specific positions in the antisense and / or sense strands, the dsRNA agents were able to regain gene silencing activity. The present inventors have also determined that introducing a heat-labile nucleotide into the sense strand at a site opposite the antisense strand's seed region (i.e., positions 2-8 at the 5' end of the antisense strand) improves gene silencing activity.

[0153] The sense strand and antisense strand of dsRNA agent can be completely modified.Optionally, dsRNA agent is conjugated with, for example, asialoglycoprotein receptor (ASGPR) ligand on sense strand.The obtained 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 be in the range of 12 to 40 nucleotides in length. For example, each strand can be in the range of 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 to 40 nucleotide pairs in length. For example, the double-stranded region can be 14 to 40 nucleotide pairs in length, 17 to 30 nucleotide pairs in length, 25 to 35 nucleotide pairs in length, 27 to 35 nucleotide pairs in length, 17 to 23 nucleotide pairs in length, 17 to 21 nucleotide pairs in length, 17 to 19 nucleotide pairs in length, 19 to 25 nucleotide pairs in length, 19 to 23 nucleotide pairs in length, 19 to 21 nucleotide pairs in length, 21 to 25 nucleotide pairs in length, or 21 to 23 nucleotide pairs in length. In another example, the double-stranded region is selected from the group consisting of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotide pairs in length.

[0156] In one embodiment, a dsRNA agent of the invention includes one or more overhang regions and / or capping groups at the 3' end, 5' end, or both ends of the strand. The overhangs 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 overhangs 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 overhangs can form mismatches with the target mRNA, or they can be complementary to the targeted gene sequence or other sequences. 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 modified nucleotides, 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 a phosphorothioate between them, and these two nucleotides may be the same or different. In one embodiment, the overhang is present at the 3' end of the sense strand, the 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 can have 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 can have a blunt end located at the 5'-end of the antisense strand (or the 3'-end of the sense strand), or vice versa.Generally, the antisense strand of dsRNA has a nucleotide overhang at the 3'-end, and the 5'-end is blunt.Without being bound by theory, the asymmetric blunt end at the 5'-end of the antisense strand and the 3'-end overhang of the antisense strand favors the guide strand added in the RISC process.For example, one 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 at either end of the dsRNA duplex.

[0161] In one embodiment, a dsRNA agent of the invention is a 19-nucleotide double-ended bluntmer, and the sense strand contains at least one thermolabile nucleotide, which is located opposite or near 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 located between positions 14-17 of the 5' end of the sense strand. The antisense strand contains at least two modified nucleic acids that are smaller than sterically demanding 2'-OMe; preferably, the two modified nucleic acids that are 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, a dsRNA agent of the invention is a 20-nucleotide blunt-ended duplex, wherein the sense strand contains at least one thermolabile nucleotide, the at least one thermolabile nucleotide being located opposite or near 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 located between positions 14-17 of the 5' end of the sense strand. The antisense strand contains at least two modified nucleic acids that are smaller than sterically demanding 2'-OMe; preferably, the two modified nucleic acids that are 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, a dsRNA agent of the invention is a 21-nucleotide blunt-ended duplex, wherein the sense strand contains at least one thermolabile nucleotide, the at least one thermolabile nucleotide being located opposite or near 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 located between positions 14-17 of the 5' end of the sense strand. The antisense strand contains at least two modified nucleic acids that are smaller than sterically demanding 2'-OMe; preferably, the two modified nucleic acids that are 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, a dsRNA agent of the invention comprises a 21-nucleotide (nt) sense strand and a 23-nucleotide (nt) antisense strand, wherein the sense strand comprises at least one thermolabile nucleotide, the at least one thermolabile nucleotide being located 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, if the sense strand is 21 nucleotides long, the thermolabile 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, and one end of the dsRNA is blunt, while the other end comprises a two-nucleotide overhang. Preferably, the two-nucleotide overhang is located 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 present invention comprises a sense strand and an antisense strand: the sense strand is 25 to 30 nucleotide residues long, starting from the 5'-terminal nucleotide (position 1), and positions 1 to 23 of the sense strand comprise at least 8 ribonucleotides; the antisense strand is 36 to 66 nucleotide residues long, starting from the 3'-terminal nucleotide, and at least 8 ribonucleotides are positioned to pair with positions 1 to 23 of the sense strand to form a duplex; at least the 3'-terminal nucleotide of the antisense strand is not paired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are not paired with the sense strand, thereby forming a 3' single-stranded overhang of 1 to 6 nucleotides; the 5'-end of the antisense strand comprises 10 to 30 consecutive nucleotides that are not paired with the sense strand, thereby forming a duplex of 10 to 6 nucleotides. A 30-nucleotide single-stranded 5' overhang is formed; at least the 5'- and 3'-terminal nucleotides of the sense strand base-pair with nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantial duplex region between the sense and antisense strands; and the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand so 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 thermolabile nucleotide, which is located opposite or near 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 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, the 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, the sense strand comprising a modified nucleotide at position 11 of its 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 of 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 being at least 25 nucleotides in length; the antisense strand is sufficiently complementary to a target mRNA along at least 19 nucleotides of its length 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 comprising the 3' end of the antisense strand, thereby reducing expression of the target gene in a mammal. Optionally, the dsRNA agent further comprises a ligand.

[0167] In one embodiment, the sense strand contains an enzymatically susceptible modified nucleotide at the 5' end at position 11. The antisense strand contains two modified nucleic acids that are 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.

[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 strand and antisense strand 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 linking phosphate oxygens; changes to components of the ribose sugar, such as the 2' hydroxyl of the ribose sugar; heavy substitution of the phosphate moiety with a "dephospho" linker; modifications or substitutions of natural bases; and substitutions or modifications of the ribose-phosphate backbone.

[0170] Because nucleic acids are polymers of subunits, many modifications, such as modifications of bases, phosphate moieties, or non-linked Os in phosphate moieties, occur at repeated positions within nucleic acids. In some cases, modifications occur at all desired positions within a nucleic acid, but often not. For example, modifications may occur only at the 3' or 5' terminal positions, or only in terminal regions, such as terminal nucleotide positions or the last two, three, four, five, or ten nucleotides of a chain. Modifications may occur in double-stranded regions, single-stranded regions, or both. Modifications may occur only in double-stranded regions of an RNA or only in single-stranded regions of an RNA. For example, phosphorothioate modifications at non-linked oxygen positions may occur only at one or both ends, or only in terminal regions, such as terminal nucleotide positions or the last two, three, four, five, or ten nucleotides of a chain, or in both double-stranded and single-stranded regions, especially at the ends. The 5' 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 the modifications described herein. Modifications may include, for example, the use of modifications at the 2' position of the ribose sugar using modifications known in the art, e.g., the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F), or 2'-O-methyl modifications rather than ribosugars in the nucleobase, 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, and 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 a 2'-O-methyl nucleotide, a 2'-deoxy nucleotide, a 2'-deoxyfluoro nucleotide, a 2'-ON-methylacetamide (2'-O-NMA) nucleotide, a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotide, a 2'-O-aminopropyl (2'-O-AP) nucleotide, or a 2'-ara-F nucleotide.

[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', and B4' regions, as shown in Formula I. As used herein, the term "alternating motif" or "alternating pattern" refers to a motif having one or more modifications, each modification occurring at alternating nucleotides in a strand. The alternating nucleotides may refer to one at every other nucleotide, one at every third nucleotide, or a similar pattern. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif may be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AAABBBAAABBB...", or "ABCABCABCABC...", etc.

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

[0178] In one embodiment, the dsRNA agent of the present invention comprises an alternating motif modification pattern in the sense strand that is shifted relative to the alternating motif modification pattern in the antisense strand. This shift can be such that the modified groups of the nucleotides in the sense strand correspond to different modified groups of the nucleotides in 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 within 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 within the duplex region, resulting in 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 modification of phosphorothioate or methylphosphonate internucleotide bond can be present at any nucleotide in sense strand and / or antisense strand, or at any position of both strands.For example, the modification of internucleotide bond can be present at any nucleotide in sense strand and antisense strand; the modification of each internucleotide bond can be present in an alternating pattern in sense strand or antisense strand; or the sense strand or antisense strand comprises the modification of both internucleotide bonds in an alternating pattern.The alternating pattern of the modification of internucleotide bond in sense strand can be the same or different from that of antisense strand, and the alternating pattern of the modification of internucleotide bond in sense strand can have a shift with respect to the alternating pattern of the modification of internucleotide bond in antisense strand.

[0180] In one embodiment, the dsRNA agent comprises phosphorothioate or methylphosphonate internucleotide bond modification in the overhang region.For example, the overhang region comprises two nucleotides with phosphorothioate or methylphosphonate internucleotide bond between the two nucleotides.Internucleotide bond modification can also be formed to allow the overhang nucleotide to be bound to the terminal base-pairing nucleotide in the double-stranded region.For example, at least two, three, four, or all of the overhang nucleotides can be bound by phosphorothioate or methylphosphonate internucleotide bond, and optionally, there can be an additional phosphorothioate or methylphosphonate internucleotide bond that connects 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 overhanging nucleotides, and the third nucleotide is the next base-pairing nucleotide of the overhanging nucleotide.Preferably, these terminal three nucleotides can be at the 3' end of the antisense strand.

[0181] In one embodiment, the 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 the 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 the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate linkages.

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

[0184] In one embodiment, the antisense strand of a dsRNA agent comprises two blocks of four phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is present at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising 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 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is present at any position in the oligonucleotide sequence, and the 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 comprises 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 wherein the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising 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 the 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 the 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, one of the phosphorothioate or methylphosphonate internucleotide linkages being present at any position in the oligonucleotide sequence, and the 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, a dsRNA agent of the invention further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within the 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, a dsRNA agent of the invention further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within an internal region of 1 to 10 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 of 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 an internal region of positions 1 to 10 of the terminal positions.

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

[0198] 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 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.

[0199] 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) 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 mismatches with the target, mismatches within the duplex, or a combination thereof. Mismatches can occur in overhang regions or duplex regions. Base pairs can be ranked based on their tendency to promote dissociation or melting (for example, based on the free energy of binding or dissociation of a particular pairing; the simplest 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 more preferred than G:C; G:U is more preferred than G:C; I:C is more preferred than G:C (I=inosine). Mismatches, such as non-canonical pairings or non-canonical pairings (described elsewhere herein), are more preferred than canonical pairings (A:T, A:U, G:C); base pairs containing universal bases are more preferred than canonical pairings.

[0211] In one embodiment, the dsRNA agent of the invention comprises 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 including 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 sterically influence 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 in single-stranded or double-stranded siRNA.For example, 5' alkylated nucleoside can be introduced at the 3' end of dinucleotide at any position in single-stranded or double-stranded siRNA.The alkyl group at the 5' position of ribose sugar can be racemic or chiral pure R or S isomer.An exemplary 5' alkylated nucleoside is 5' methyl nucleoside.5' methyl can be racemic or chiral pure R or S isomer.

[0215] In one embodiment, 4'-modified nucleosides are introduced at the 3'-end of dinucleotides at any position in single-stranded or double-stranded siRNA.For example, 4'-alkylated nucleosides can be introduced at the 3'-end of dinucleotides at any position in single-stranded or double-stranded siRNA.The alkyl group at the 4'-position of ribose sugar can be racemic or chirally pure R or S isomer.An exemplary 4'-alkylated nucleoside is 4'-methyl nucleoside.4'-methyl can be racemic or chirally pure R or S isomer.Alternatively, 4'-O-alkylated nucleosides can be introduced at the 3'-end of dinucleotides at any position in single-stranded or double-stranded siRNA.The 4'-O-alkyl 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 strand or antisense strand of dsRNA, and this modification maintains or improves the efficacy of dsRNA.5' alkyl 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.

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

[0218] In one embodiment, 4'-O-alkylated nucleoside is introduced into any position of the sense strand or antisense strand of dsRNA, and this modification maintains or improves the efficacy of dsRNA.5' alkyl can be racemic or chiral pure R or S isomer.Exemplary 4'-O-alkylated nucleoside is 4'-O-methyl nucleoside.4'-O-methyl can be racemic or chiral 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-labile nucleotide (e.g., an acyclic nucleotide, e.g., UNA or GNA, a mismatch, an abasic, or DNA) located opposite the antisense seed region (i.e., positions 2 to 8 of the 5' end of the antisense strand); T1 represents a nucleotide containing 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 to 6 nucleotides in length; n 4 is 1 to 3 nucleotides in length; alternatively, n 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-labile nucleotide (e.g., an acyclic nucleotide, e.g., UNA or GNA, a mismatch, an abasic, or DNA) located opposite the antisense seed region (i.e., positions 2 to 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 to 6 nucleotides in length; n 4 is 1 to 3 nucleotides in length; alternatively, n 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 an acyclic 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), wherein 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 the 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 includes a non-ribose, acyclic, or chemical modification at the 2'-position or 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 a 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 to 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 to 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 to 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 1-6 nucleotide long 3' overhang at the antisense 3' end.

[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 to 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 to 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 to 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 in length on the 5' end of the sense strand and a 3' overhang 1-10 nucleotides in length 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 into the sense strand at a site opposite the antisense strand's seed region (i.e., positions 2-8 at the 5' end of the antisense strand) to enhance the dissociation or melting properties of the dsRNA duplex (reducing the free energy of duplex binding). This modification can enhance the dissociation or melting properties of the duplex at the antisense strand's seed region.

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

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

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

[0239] The term "acyclic 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] wherein B is a modified or unmodified nucleobase and R 1 and R 2are independently H, halogen, OR3, or alkyl; R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. The term "UNA" refers to an unlocked acyclic nucleic acid in which all bonds of the sugar have been removed to form an unlocked "sugar" residue. In one example, a UNA also includes a monomer in which the C1'-C4' bond (i.e., the carbon-oxygen-carbon covalent bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond of the sugar (i.e., the carbon-carbon covalent bond between the C2' and C3' carbons) has been removed (see Mikhailov et al., Tetrahedron Letters, 26 (17):2059 (1985); and Fluiter et al., Mol. Biosyst., 10:1039 (2009), the entire contents of each of which are incorporated herein by reference). Acyclic derivatives offer greater backbone flexibility without affecting Watson-Crick base pairing. Acyclic nucleotides can be linked by 2'-5' or 3'-5' linkages.

[0240] The term "GNA" refers to glycol nucleic acid, a polymer similar to DNA or RNA, but differing 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 nucleotides or modified nucleotides, that is, mismatch base pairing can occur between the nucleobases from each nucleotide regardless 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 eliminated the ability to form hydrogen bonds with opposing bases, and phosphate modifications.

[0244] Nucleobase modifications that reduce or completely eliminate 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 International Publication No. 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 a 2'-5' linkage (with 2'-H, 2'-OH, and 2'-OMe, and with P=O or P=S). For example, 2'-5' linkage modifications can be used to increase nuclease resistance or to inhibit binding of the sense strand to the antisense strand, or can be used 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 comprise L-sugar (such as L-ribose, 2'-H, 2'-OH, and L-arabinose, including 2'-OMe).For example, these L-sugar modifications can be used to increase nuclease resistance, or to inhibit the binding of sense strand to antisense strand, or can be used at the 5' end of sense strand to 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), wherein the duplexes are linked by a linker. This linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each dsRNA agent may target the same gene or two different genes; or each dsRNA agent may target the same gene at two different target sites.

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

[0251] A variety of publications have described multimeric siRNA, and all of these siRNAs can be used with the dsRNA of the present invention.Such publications include International Publication No. WO2007 / 091269, United States Patent No. 7858769, International Publication No. WO2010 / 141511, United States Patent No. 2007 / 117686, United States Patent No. 2009 / 014887 and United States Patent No. 2011 / 031520, the entire disclosure of which is incorporated herein by reference.

[0252] A dsRNA agent containing one or more sugar moieties conjugated to the dsRNA agent can optimize one or more properties of the dsRNA agent. Often, 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, such as a non-carbohydrate carrier (preferably cyclic), to which a sugar ligand is attached. A ribonucleotide subunit in which the ribose sugar of the subunit has been 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, such as nitrogen, oxygen, or sulfur. The cyclic carrier can be a single ring system or can contain two or more rings, e.g., fused rings. The cyclic carrier can be a fully saturated ring system or can contain one or more double bonds.

[0253] The ligand can be attached to the polynucleotide via a carrier. The carrier comprises (i) at least one "backbone attachment point," preferably two "backbone attachment points," and (ii) at least one "tether attachment point." As used herein, "backbone attachment point" refers to a functional group, e.g., a hydroxyl group, or generally to a bond 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. In some embodiments, a "tether attachment point" (TAP) refers to a ring atom, e.g., a carbon atom or heteroatom (different from the atom providing the backbone attachment point), of the cyclic carrier to which the selected moiety is attached. The selected moiety can be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or 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, a dsRNA agent of the invention is conjugated to a 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 optionally be 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. The 5' phosphate modification includes a modification that is 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'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P O-5'), 5'-phosphorothiolate ((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)(NH2)(O)PO-5'), 5'-alkylphosphonates (R = alkyl = methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)-O-5'-, 5'-alkenylphosphonates (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 on the antisense strand of a 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 preferred embodiments, the ligand alters the distribution, targeting, or lifetime of the molecule into which it is incorporated. In preferred embodiments, the ligand increases 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, compared to, for example, a species in which such ligand is not present. Ligands that increase affinity for a selected target are also referred to as targeting ligands.

[0259] Some ligands may have endosomolytic properties. Endosomolytic ligands promote the lysis of endosomes and / or the transport of the compositions of the present invention or their 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, the endosomolytic ligand is presumed to adopt its 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 compositions of the present invention or their components from endosomes to the cytoplasm of cells. Exemplary endosomolytic ligands include GALA peptide (Subbarao et al., Biochemistry, 1987, 26:2964-2972, incorporated herein by reference in its entirety), EALA peptide (Vogel et al., J. Am. Chem. Soc., 1996, 118:1581-1586, incorporated herein by reference in its entirety), and derivatives thereof (Turk et al., Biochem. Biophys. Acta, 2002, 1559:56-68, incorporated herein by reference in its entirety). In one embodiment, the endosomolytic component may contain a chemical group (e.g., an amino acid) that undergoes 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 polymer molecules comprising any combination of the monomers and / or natural or modified ribonucleotides described herein.

[0261] Ligands generally may 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, or oligonucleotides (e.g., aptamers). Examples of 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, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helical peptides.

[0263] The ligand can also include a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid, or protein, e.g., 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 peptidomimetic, or aptamer. Table 2 lists some examples of targeting ligands and their associated receptors.

[0264] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases or chelating agents (e.g., EDTA), lipophilic molecules, such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-( oleoyl), cholenoic 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, Eu complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0265] Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules with specific affinity for a co-ligand, or antibodies, e.g., antibodies that bind to specific cell types, such as cancer cells, endothelial cells, or bone cells. Ligands can also include hormones and hormone receptors. Ligands can 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 can be, for example, lipopolysaccharides, activators of p38 MAP kinase, or activators of NF-κB.

[0266] The ligand can be a substance, e.g., a drug, that can increase cellular uptake of an iRNA agent, e.g., by disrupting the cytoskeleton of a cell, e.g., by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments. 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 conjugate to be distributed to target tissue, for example, non-renal target tissue of the body.For example, 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 conjugate to degradation, (b) increase targeting or transport to target cell or cell membrane, and / or (c) 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, the 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 body.The 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 preferably distributes to non-renal tissues. However, preferably, the 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, so that the conjugate preferably distributes to the kidney. Other moieties that target kidney cells can be used instead of or in addition to the lipid-based ligand.

[0272] In another embodiment, the ligand is a moiety, such as 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 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. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules that can fold into a defined three-dimensional structure similar to a natural peptide. The peptide or peptidomimetic moiety may be about 5 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. 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 peptide moiety may be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide moiety may contain a hydrophobic membrane transport sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP. An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP) may also be a targeting moiety. The peptide moiety can be a "delivery" peptide, which can transport large polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, the sequence (GRKKRRQRRRPPQ) from the HIV Tat protein and the sequence (RQIKIWFQNRRMKWKK) from the Drosophila Antennapedia protein 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, can also 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 mimetic. The peptide moiety can range from about 5 amino acids in length to about 40 amino acids in length.The peptide moiety can have a structural change, for example, to enhance stability or direct conformational properties. Any of the following structural changes can be utilized: RGD peptide moieties can 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). RGD peptides can promote targeting of iRNA agents to tumors in 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, RGD peptides promote targeting of iRNA agents to the kidney. RGD peptides can be linear or cyclic and can be modified, e.g., glycosylated or methylated, to promote targeting to specific tissues. For example, glycosylated RGD peptides can be α. v iRNA agents can be delivered to tumor cells expressing β3 (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 display 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. Generally, 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 cells, such as microbial cells, e.g., bacterial or fungal cells, or mammalian cells, e.g., human cells. Peptides that penetrate microbial cells can be, for example, α-helical linear peptides (e.g., LL-37 or seropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two predominant amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain a nuclear localization signal (NLS). For example, a cell-penetrating peptide can be a bipartite amphipathic peptide, e.g., 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, H2A peptide, Xenopus peptide, esculentinis-1, and caerin. Preferably, multiple factors are considered to maintain the integrity of helix stability. For example, utilize a maximum number of helix-stabilizing residues (e.g., leu, ala, or lys) and a minimum number of helix-destabilizing residues (e.g., proline, or cyclic monomer units). 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 at positions i±3, or i±4. Cationic residues, such as lysine, arginine, homo-arginine, ornithine, or histidine, can form salt bridges with the 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, i.e., peptidic bonds, replaced by one or more urea, thiourea, carbamate, or sulfonylurea bonds; or cyclic peptides.

[0278] The targeting ligand may be any ligand capable of targeting 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 glycoconjugates. 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, for example, 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 charge, dendrimers with masked or unmasked cationic or anionic charge.

[0280] PK modulators refer to pharmacokinetic modulators. PK modulators include lipophilics, 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, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, etc. Oligonucleotides containing multiple phosphorothioate linkages are also known to bind to serum proteins. Therefore, short oligonucleotides containing multiple phosphorothioate linkages in the backbone, for example, oligonucleotides of about 5, 10, 15, or 20 bases, are also applicable as ligands (e.g., as PK-modulating ligands) in the present invention.

[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 two or more ligands are present, the ligands may all have the same properties, all may have different properties, or some may have the same properties while others have different properties. For example, the ligands may have targeting properties, endosomal activity, or PK modulating properties. In a preferred embodiment, all of the ligands have different properties.

[0284] The ligand can be attached to the oligonucleotide at various positions, e.g., the 3'-terminus, the 5'-terminus, and / or an internal position. In preferred embodiments, the ligand is attached to the oligonucleotide via an intervening tether, e.g., a carrier described herein. The ligand or tethered 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 "precursor" monomer has been incorporated into a growing chain. For example, an amino-terminated tether (i.e., no ligand attached), e.g., TAP-(CH2) n Monomers bearing an NH can be incorporated into a growing oligonucleotide chain. Subsequent to this, 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 coupling 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 to this, i.e., after the precursor monomers are incorporated into the chain, a ligand bearing a complementary chemical group, e.g., an alkyne or azide, can be attached to the precursor monomer 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 a nucleic acid molecule. Conjugation to a purine nucleobase or its derivative can occur at any position, including endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-position of a purine nucleobase is bound to a conjugate moiety. Conjugation to a pyrimidine nucleobase or its derivative can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of a pyrimidine nucleobase can be substituted with a conjugate moiety. Conjugation to a sugar moiety of a nucleoside can occur at any carbon atom. Examples of carbon atoms of the sugar moiety that can be bound to a conjugate moiety include the 2', 3', and 5' carbon atoms. The 1' position can also be bound 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 (e.g., phosphodiester, phosphorothioate, phosphorodithioate, and phosphoramidate), the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom attached to the phosphorus atom. In the case of internucleoside linkages containing 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 that conjugate the ligand to the nucleic acid include those described above. For example, the ligand can 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)-(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 represents, independently for each occurrence, absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C represents independently at each occurrence absent, alkylene, or substituted alkylene, and one or more methylenes are O, S, S(O), SO, N(R N ), C(R')=C(R''), C≡C, or C(O); R 2A , R2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C are independently present in each occurrence: NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocyclyl; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B , and L 5C represents a ligand; i.e., each occurrence independently represents a monosaccharide (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or 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 in conjunction with RNAi agents to inhibit expression of target genes: [ka] In the formula, L 5A , L 5B , and L 5C represents a monosaccharide, for example, a GalNAc derivative.

[0292] Suitable bivalent 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 to mean the agent that can mediate the silencing of target RNA, for example, mRNA, for example, the transcript of the gene that codes for protein.For convenience, this mRNA is also referred to herein as the mRNA that is silenced.This gene is also referred to as target gene.Generally, the RNA that is silenced is endogenous gene or pathogenic gene.In addition, RNA other than mRNA, for example, tRNA and viral RNA can also be targeted.

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

[0295] As used herein, "specifically hybridizable" and "complementary" refer to sufficient complementarity to form stable and specific binding between the compound of the present invention and the target RNA molecule.Specific binding requires sufficient complementarity to avoid the specific binding of the oligomeric compound to non-target sequences under conditions where specific binding is desired, i.e., under physiological conditions in the case of assays or treatments, or under the conditions in which the assay is performed in the case of in vitro assays.The non-target sequences typically differ by at least five nucleotides.

[0296] In one embodiment, the dsRNA agent of the present invention is "sufficiently complementary" to the target RNA, for example, 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, for example, the target RNA and the dsRNA duplex agent anneal to form a hybrid consisting of only Watson-Crick base pairs in the exact complementary region.The "sufficiently complementary" target RNA can include an internal region (for example, 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 where one nucleotide difference exists (for example, within 7 nucleotides).

[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 can contain 5-, 6-, or even 7-membered bridge structures with a "locked" C3'-endo sugar puckering. This bridge is typically included at the 2' and 4' positions of the ribose to yield 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, the term "cleavage site" refers to a backbone bond in a target gene or sense strand that is cleaved by the RISC mechanism using an iRNA agent. 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 the 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, such as 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 contains two 21-nucleotide-long strands (these strands form a double-stranded region of 19 consecutive base pairs with a two-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 (including, but not limited to, propyl, allyl, or propargyl) that may be straight or branched and contain the indicated number of carbon atoms, optionally containing N, O, or S. For example, C1-C 10 indicates that the group can 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 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 substituents. Examples of aryl groups include phenyl and naphthyl. The terms "arylalkyl" or "aralkyl" refer 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 further be 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- to 8-membered monocyclic, 8- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring system having one to three heteroatoms in the monocyclic ring, one to six heteroatoms in the bicyclic ring, or one to nine heteroatoms in the tricyclic ring, selected from O, N, or S (e.g., carbon atoms and one to three, one to six, or one to nine heteroatoms of N, O, or S in the monocyclic, bicyclic, or tricyclic ring, respectively), and zero, one, two, three, or four atoms in each ring may be optionally substituted. 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- to 8-membered monocyclic, 8- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring system having one to three heteroatoms in the monocyclic ring, one to six heteroatoms in the bicyclic ring, or one to nine heteroatoms in the tricyclic ring, selected from O, N, or S (e.g., carbon atoms and one to three, one to six, or one to nine heteroatoms of N, O, or S in the monocyclic, bicyclic, or tricyclic ring, respectively), and zero, one, two, or three atoms in each ring may be optionally substituted. 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 substituents can 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 cell, it is cut off and releases the two parts that linker holds together.In the preferred embodiment of the dsRNA agent of the present invention, cleavable linking group is cut off at least 10 times faster in target cell or under first reference condition (for example, it can be selected to mimic or realize the condition in cell) than in target blood or under second reference condition (for example, it can be selected to mimic or realize the condition that exists in blood or serum).

[0311] Cleavable linking groups are susceptible to cleaving agents, such as pH, redox potential, or the presence of degradable molecules.Generally, 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, for example, oxidizing enzymes or reductases or reducing agents present in cells, which can degrade redox-cleavable linking groups by reduction, such as mercaptans, which are selective for specific substrates or do not have substrate specificity; esterases; endosomes or agents that can create an acidic environment, for example, 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 linkers, such as disulfide bonds, are sensitive to pH. While the pH of human serum is 7.4, 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 linkers 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 comprise a cleavable linking group that can be cleaved by specific enzyme.The type of cleavable linking group incorporated into linker can vary depending on the cell to be targeted.For example, the ligand for targeting the liver can be linked to cationic lipid via a linker that comprises an ester group.Hepatocytes are rich in esterase, and therefore linker is more efficiently cleaved in hepatocytes than in cell types that are not rich in esterase.Other cell types that are rich in esterase include lung, renal cortex and testicular cells.

[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 tether can be evaluated by testing the ability of a degradation agent (or condition) to cleave the candidate tether. It may also be desirable to test the candidate cleavable tether for its ability to resist cleavage in blood or when in contact with other non-target tissues. Thus, the relative ease of cleavage can be determined between a first condition and a second condition, where the first condition is selected to indicate cleavage within target cells and the second condition is selected to indicate cleavage in other tissues or body fluids, such as blood or serum. Evaluations can be performed in cell-free systems, cells, cell cultures, organ or tissue cultures, or whole animals. It may be useful to perform initial evaluations in cell-free or culture conditions and confirm with further evaluations in whole animals. In preferred embodiments, useful candidate compounds cleave at least 2-fold, 4-fold, 10-fold, or 100-fold faster in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0316] Redox-cleavable linking groups One class of cleavable linkers that can be used in dsRNA agents according to the present invention are redox-cleavable linkers that are cleaved upon reduction or oxidation. An example of a reductively cleavable linker is a disulfide linker (-SS-). To determine whether a candidate cleavable linker is a suitable "reductively cleavable linker," or, for example, whether it is suitable for use with a particular iRNA moiety and a particular targeting agent, the methods described herein can be verified. For example, candidates can be evaluated by incubating with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate observed in cells, e.g., target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In a preferred embodiment, the candidate compound cleaves at most 10% in blood. In preferred embodiments, useful candidate compounds are degraded at least 2-fold, 4-fold, 10-fold, or 100-fold faster inside cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic intracellular 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. An 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-, and -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0318] acidic cleavable linking group Acidic cleavable linking groups can be used in the dsRNA agent of 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 (for example, about 6.0, 5.5, 5.0, or less), or by an agent that can function as a general acid, such as an enzymatic agent.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).In a preferred embodiment, 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-based linking group Ester-based cleavable linking group can be used in the dsRNA agent of 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 ester of alkylene group, alkenylene group and alkynylene group.Ester-based cleavable linking group has the general formula:-C(O)O- or -OC(O)-.These candidates can be evaluated by the method similar to the above method.

[0320] Peptide-based cleavable linkers Peptide-based cleavable linkers can be used in dsRNA agents 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 contain amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. Peptide bonds are a special type of amide bond formed between amino acids that produce peptides and proteins. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids that produce peptides and proteins, and do not include the entire amide functionality. Peptide-based cleavable linkers have the general formula: -NHCHR A C(O)NHCHR B C(O)- and 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 straight, branched, or cyclic) having at least six carbon atoms, each of which is bonded to an oxygen, nitrogen, or sulfur atom; or to a compound having a carbohydrate moiety formed from one or more monosaccharide units (which may be straight, branched, or cyclic), each of which has at least six carbon atoms, each of which is bonded to an oxygen, nitrogen, or sulfur atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4 to 9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide gums. Specific monosaccharides include C5 and above (preferably C5 to C8) sugars; disaccharides and trisaccharides include sugars having two or three monosaccharide units (preferably C5 to C8).

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

[0322] The present invention also relates to the use of dsRNA agent as defined herein, which is used to inhibit the expression of target gene in subject.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 subject.The siRNA composition that is formulated can be in various states.In some cases, this composition is at least partially crystalline, uniformly crystalline, and / or anhydrous (for example, less than 80%, less than 50%, less than 30%, less than 20%, or less than 10% water).In another example, siRNA is dissolved in an aqueous phase, for example, a solution that contains water.

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

[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 ribonucleotides), salts, and RNase inhibitors (for example, broad specificity RNase inhibitors such as RNAsin).

[0327] In one embodiment, the 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 diseases, such as 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 may 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 will be 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, such as modified siRNAs, and such implementations are within the scope of the present invention. siRNA compounds, such as 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, such as liposomes or micelles, for delivery. The term "liposome" as used herein refers to a vesicle composed of amphiphilic lipids, composed of at least one bilayer, e.g., one bilayer or multiple bilayers. Liposomes include unilamellar and multilamellar vesicles, whose membranes are 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 in some cases may. Liposome is useful for transporting and delivering active ingredients to the site of action.Because liposome membrane is structurally similar to biological membrane, when liposome contacts tissue, liposome bilayer fuses with cell membrane bilayer.When liposome and cell are integrated, the internal aqueous content containing siRNA is delivered into the cell, where siRNA can specifically bind to target RNA and mediate RNAi.In some cases, liposome is specifically targeted, for example, siRNA is directed to 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, thereby forming micelles 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 group on the lipid interacts with the siRNA and condenses 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 condensation can be added during the condensation reaction, for example, by controlled addition.For example, the carrier compounds can be polymers other than nucleic acids (for example, spermine or spermidine).The 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 structural components of the delivery vehicle are described in WO 96 / 37194. The formation of liposomes is 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; the entire contents of which are incorporated herein by reference. 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 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 easily adapted to package siRNA agent preparations into liposomes.

[0334] pH-sensitive or negatively charged liposomes entrap nucleic acid molecules rather than forming complexes with them. Because both nucleic acid molecules and lipids are similarly charged, repulsion occurs rather than complex formation. However, some nucleic acid molecules are entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding thymidine kinase genes to cultured cell monolayers. The expression of exogenous genes has been 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 contains phospholipids other than naturally occurring phosphatidylcholine. Neutral liposome compositions can be formed, for example, from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, and anionic fusogenic liposomes are primarily formed from dioleoylphosphatidylethanolamine (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; and 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 can fuse with negatively charged lipids in 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] The 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 delivering 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 adhere to negatively charged cell surfaces, fuse with 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 moiety is attached by an ester rather than an ether bond.

[0341] Other reported cationic lipid compounds include 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, e.g., 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 preparations are particularly suitable for topical administration, and liposomes have several advantages over other preparations.These advantages include the reduction of the side effects associated with the high systemic absorption of administered drugs, the increased accumulation of administered drugs in desired target, and the ability to administer siRNA to skin.In some implementations, liposomes are also used to deliver siRNA to epithelial cells and 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 (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, R M 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] Nonionic liposome systems have also been tested to determine their utility in delivering drugs to the skin, particularly in systems containing nonionic surfactants and cholesterol. Nonionic liposome 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 formed to be highly deformable. Such deformability can allow liposomes to enter pores smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposome. Transfersomes can be prepared by adding a surface edge activator, usually a surfactant, to a standard liposome composition. 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 an appropriate 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 their target without fragmentation, and are often self-loading.

[0346] Other formulations applicable to the present invention are described in U.S. Provisional Patent Applications 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 No. 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 will be 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, such as modified siRNA compounds, and such implementations are within the scope of the present invention. Surfactants have found wide application in formulations, such as emulsions (including microemulsions) and liposomes (described above). siRNA (or precursors, such as larger dsiRNAs that can be processed into siRNAs, or DNA encoding siRNAs or precursors) compositions can contain surfactants. In one embodiment, siRNA is formulated as an emulsion containing a surfactant. The most common way to classify and rank the properties of the many different types of surfactants, both natural and synthetic, is by using the hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group is the most useful tool 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. Their HLB values ​​generally range from 2 to approximately 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] When surfactant molecule has negative charge when dissolved or dispersed in water, this surfactant molecule is classified as anionic.Anionic surfactants include carboxylates such as soap, acyl lactate, acyl amide of amino acid, esters of sulfuric acid such as alkyl sulfate and ethoxylated alkyl sulfate, sulfonates such as alkyl benzene sulfonate, acyl isethionate, acyl taurate and sulfosuccinate, and phosphate.The most important members of anionic surfactant class are alkyl sulfate and soap.

[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 commonly used members of this class.

[0351] If a surfactant molecule has the ability to carry 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 will be described primarily with respect to unmodified siRNA compounds. However, it will be 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., DNA encoding double-stranded siRNA compounds, or ssiRNA compounds, or their precursors), compositions can be provided as micellar formulations. A "micelle" is defined herein as a specific type of molecular assembly in which amphiphilic molecules are organized into a spherical structure with all hydrophobic portions facing inward and hydrophilic portions in contact with the surrounding aqueous phase. In a hydrophobic environment, the opposite configuration exists.

[0354] Mixed micelle formulations suitable for transdermal delivery are prepared by combining an aqueous solution of the siRNA composition with alkali metal ions (C8-C8). 22It can be prepared by mixing alkyl sulfate with a micelle-forming compound.Exemplary micelle-forming compounds include lecithin, hyaluronic acid, hyaluronic acid pharmaceutically acceptable salts, 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 salts, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ether and its analogs, polidocanol alkyl ether and its analogs, chenodeoxycholate, deoxycholate, and mixtures thereof.The micelle-forming compound can be added simultaneously with or after the addition of alkali metal alkyl sulfate.Mixed micelles can be formed by mixing virtually any type of components, but vigorously mixing is required to provide smaller micelles.

[0355] In one method, a first micelle composition is prepared, which contains siRNA composition and at least alkali metal alkyl sulfate.Then, this 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 mixed micelle composition is formed.

[0357] To deliver a micelle formulation as a spray, the formulation can be placed in an aerosol dispenser, which is filled with a propellant. The propellant under pressure is in liquid form within the dispenser. The ratio of components is adjusted so that the aqueous phase and the propellant phase are combined, i.e., only one phase is present. If two phases are present, the dispenser must be shaken before dispensing a portion of the contents, for example, via a metering valve. The dispensed amount of pharmaceutical agent is sprayed as 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, e.g., by at least two or three times.

[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, for example, unmodified siRNA compounds, and such implementations are within the scope of the present invention. In another embodiment, siRNA compounds, for example, double-stranded siRNA compounds, or ssiRNA compounds, (e.g., precursors, for example, larger siRNA compounds that can be processed into ssiRNA compounds, or siRNA compounds, for example, DNA encoding double-stranded siRNA compounds, or ssiRNA compounds, or their precursors) preparations can be contained in particles, for example, 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 Composition The iRNA agents of the present invention can be formulated for pharmaceutical use. The present invention further relates to pharmaceutical compositions comprising the dsRNA agents defined herein. Pharmaceutically acceptable compositions comprise a therapeutically effective amount of one or more dsRNA agents of any of the foregoing embodiments, alone or formulated with one or more pharmaceutically acceptable carriers (additives), excipients, and / or diluents.

[0362] Pharmaceutical compositions can be specially formulated for administration in solid or liquid form, including administration adapted for the following: (1) oral administration, e.g., drenches (aqueous or non-aqueous solutions, or suspensions), tablets, e.g., buccal, sublingual, and systemic tablets, boluses, powders, granules, and 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, or foam; (5) sublingual administration; (6) intraocular administration; (7) transdermal administration; or (8) intranasal administration. Delivery using subcutaneous or infusion 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 produce some desired therapeutic effect in at least a subpopulation of cells in an animal, at a reasonable benefit / risk ratio applicable to any treatment.

[0364] The phrase "pharmaceutically acceptable" is used 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 excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0365] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically 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 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 pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; and (7) lubricants, such as magnesium state. (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) (16) pyrogen-free water; (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 (24) other non-toxic, compatible substances utilized in pharmaceutical formulations.

[0366] The formulations can be conveniently presented in unit dosage form and prepared by any method well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the recipient and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of compound that provides a therapeutic effect. Generally, out of 100%, this amount will range 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 present 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 present invention. In certain embodiments, the formulations described above allow for oral administration of the compounds of the present invention.

[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 ribonucleotides), salts, and RNase inhibitors (for example, broad specificity RNase inhibitors such as RNAsin).

[0369] Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with a carrier, and, optionally, one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association a 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 from subcutaneous or intramuscular injections 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 drug absorption then depends on the dissolution rate, which may depend on the size and crystalline form of the crystals. 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 include any animal in need of treatment, including primates, particularly humans, and other mammals, such as horses, cattle, pigs, and sheep; and 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 by, for example, 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).The pharmaceutical preparation of gene therapy vectors can contain the gene therapy vector in an acceptable diluent, or can contain a slow-release matrix in which gene delivery vehicles are embedded.For example, the DNA template can contain two transcription units, one transcription unit producing transcripts containing the upper strand of dsRNA agent, and the other transcription unit producing transcripts containing the lower strand of dsRNA agent. When the template is transcribed, a dsRNA agent is produced and 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 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 contain one or more iRNAs and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, 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. Supplementary active compounds may also be included in the composition.

[0376] The compositions of the present invention can be administered in a variety of ways depending 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 target muscle 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 and mechanically introducing the DNA.

[0378] dose In one aspect, the invention features a method of administering a dsRNA agent, e.g., an 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., an siRNA agent, e.g., a double-stranded siRNA agent, wherein (a) the double-stranded portion is 14 to 40 nucleotides in length, e.g., 21 to 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 to 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 (e.g., about 4.4 x 10 16 copies), 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 specified amount can be the amount that is effective for treating or preventing disease or disorder, for example, the disease or injury associated with target RNA.The unit dose can be administered by, for example, injection (for example, intravenous, subcutaneous or intramuscular), inhalation administration or topical application.In some embodiments, the dose can be less than 10mg, less than 5mg, less than 2mg, less than 1mg or less than 0.1mg 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 two days, less than once every four days, less than once every eight days, or less than once every 30 days. In other embodiments, the unit dose is not administered at a fixed frequency (for example, not at a regular frequency). For example, the unit dose can be administered once.

[0381] In one embodiment, effective amount is administered with other conventional therapeutic modalities.In one embodiment, the subject is the patient with virus infection, and the therapeutic modality is the antiviral agent other than dsRNA agent, for example, other than siRNA agent.In another embodiment, the subject is atherosclerosis, and 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, a 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 of 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 10 days, or no more than once every 30 days. Furthermore, therapy can be continued for a period of time, which can vary depending on the nature of the particular disease, its severity, and the patient's overall condition. In certain embodiments, the dosage can be administered less than once a day, 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 the alleviation of symptoms of the disease state.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 the alleviation 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 intra-articular), or a reservoir, may be suggested.

[0384] In one embodiment, the composition comprises a plurality of 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 plurality of 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, the administration of dsRNA agent, for example, siRNA agent, composition is parenteral, for example, intravenous (for example, as bolus or 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 carried out by subject or by another person, for example, medical provider.Medical treatment can be carried out in measured dose or by a dispenser that delivers measured dose.Selected delivery mode will be 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 invention relates to a dsRNA agent of the invention for use in the above methods.

[0389] Methods for inhibiting expression of target genes The present invention also relates to a method for inhibiting the expression of target gene.This 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 also 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 also 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, the method comprising the step of providing the 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, PDGF beta 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 mutation in a cyclin A gene, a cyclin E gene, a WNT-1 gene, a beta-catenin gene, a c-MET gene, a PKC gene, an NFKB gene, a STAT3 gene, a survivin gene, a Her2 / Neu gene, a topoisomerase I gene, a topoisomerase IIα gene, a p73 gene mutation, a p21 (WAF1 / CIP1) gene mutation, a p27 (KIP1) gene mutation, a PPM1D gene mutation, a RAS gene mutation, a caveolin I gene mutation, a MIB I gene mutation, a MTAI gene mutation, a M68 gene mutation, a tumor suppressor gene mutation, and a p53 tumor suppressor gene mutation.

[0391] In certain embodiments, the 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. [Example]

[0393] Example 1: In vitro screening of siRNA duplexes Cell culture and transfection: Human Hep3B cells or rat H.II.4.E cells (ATCC, Manassas, VA) were grown to near confluence in RPMI (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) in a 5% CO2 atmosphere at 37°C and then detached from the plate by trypsinization. Transfection was performed by adding 14.8 μl of Opti-MEM and 0.2 μl of 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 minutes 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 hours 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-fold and 4-fold serial dilutions with the highest dose at a final duplex concentration of 10 nM.

[0394] Total RNA isolation 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 (the 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 again captured, 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 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): A master mix containing 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 HO was added to 5 μl of total RNA per reaction. 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 Two microliters of cDNA was added to a master mix 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) per well of a 384-well plate (Roche cat#04887301001). Real-time PCR was performed on a Roche LC 480 Real Time PCR machine (Roche). Unless otherwise noted, 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. 50 was calculated using a four-parameter fit model using XLFit and normalized to AD-1955 transfected or untreated cells over the same dose range or to its own lowest dose. 50 was calculated for each individual transfection and for the combination, resulting in one 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, such as 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 used at a concentration of 0.2 M in acetonitrile (CH3CN), except for guanosine, which was used at a concentration of 0.2 M in 10% THF / ACN (v / v). A 16-minute coupling / recycling time was used. The activating agent was 5-ethylthiotetrazole (0.75 M, American International Chemicals). iodine / water / pyridine was used for PO oxidation, and PADS (2%) dissolved in 2,6-lutidine / ACN (1:1 v / v) was used for PS oxidation.

[0400] Ligand-conjugated chains were synthesized using solid supports containing the corresponding ligands. 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, a cholesterol moiety at the 3' end was introduced by initiating synthesis on a cholesterol support. Generally, 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 using standard phosphitylation conditions to obtain the desired carbohydrate conjugate component. Fluorophore-labeled siRNA was synthesized using the corresponding phosphoramidite or solid support purchased from Biosearch Technologies. Oleyllithocholic acid (GalNAc)3 polymer supports were prepared in-house with a loading of 38.6 μmol / gram. Mannose (Man)3 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 noted. Long-term coupling to solid-bound oligonucleotides was performed using a 0.1 M phosphoramidite solution in anhydrous CH3CN in the presence of 5-(ethylthio)-1H-tetrazole activator for 15 min. Oxidation of internucleotide phosphites to phosphates was carried out using standard iodine water as reported in (1) or by treating the conjugated oligonucleotide with tert-butyl hydroperoxide / acetonitrile / water (10:87:3) with a 10-min oxidation wait time. Phosphorothioates were introduced by oxidation of phosphites to phosphorothioates using sulfur transfer reagents, e.g., DDTT (purchased from AM Chemicals), PADS, and / or Beaucage reagent. Cholesterol phosphoramidite was synthesized in-house and used at a concentration of 0.1 M in dichloromethane. The coupling time for cholesterol phosphoramidite was 16 min.

[0402] 2. Deprotection-I (Nucleobase Deprotection) After synthesis was complete, the support was transferred to a 100 mL glass bottle (VWR). The oligonucleotide was cleaved from the support simultaneously with deprotection of the base and phosphate groups using 80 mL of a mixture of ethanolic ammonia [ammonia:ethanol (3:1)] at 55 °C for 6.5 hours. The bottle was briefly cooled on ice, and the ethanolic ammonia mixture was then filtered into a new 250 mL bottle. The CPG was washed with two 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 the mixture was stored in a freezer until purification.

[0404] 4.Analysis Oligonucleotides are analyzed by high performance liquid chromatography (HPLC) before purification, with the choice of buffer and column dependent on the sequence and / or 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 an in-house packed TSK gel column. The buffers were 20 mM sodium phosphate (pH 8.5) in 10% CH3CN (Buffer A) and 20 mM sodium phosphate (pH 8.5) in 10% CH3CN, 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 minutes in 1x 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 grown to near confluence in RPMI (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) at 37°C in a 5% CO2 atmosphere and then detached from the plate by trypsinization. Transfection was performed by adding 14.8 μl of Opti-MEM and 0.2 μl of 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 minutes at room temperature. Approximately 2 × 10 4 80 μl of complete growth medium without antibiotics containing Hep3B cells was added to the siRNA mixture. Cells were incubated for 24 or 120 hours before RNA purification. Unless otherwise noted, 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) A master mix containing 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 HO was added to 10 μl of total RNA per reaction. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, CA) with the following steps: 25°C for 10 minutes, 37°C for 120 minutes, 85°C for 5 seconds, and a 4°C hold.

[0409] Real-time PCR Two microliters of cDNA was added to each well of a 384-well 50-plate (Roche cat#04887301001) containing a master mix containing 0.5 μl of GAPDH TaqMan probe (Applied Biosystems cat#4326317E), 0.5 μl of ANGPTL TaqMan probe (Applied Biosystems cat#Hs00205581_m1), and 5 μl of 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 noted 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. 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 various embodiments described above can be combined to create additional 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 as needed to utilize concepts from the various patents, patent applications, and published patent applications to create additional 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 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 range of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the present 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] Sense strand position 11 Position 11 of the sense strand at the predicted 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 many 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 thermolabile modification, e.g., GNA or a mismatch to the opposite AS strand. Modifications at positions 16 or 17 appeared to have the greatest impact. Figure 1 and Table 1 highlight this position / region and the impact of thermolabile modification on in vitro efficacy. Efficient knockdown comparable to the parental template design was obtained with GNA or other thermolabile modifications, e.g., an 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 thermolabile modified GNA in the 3' region (positions 16-18). The results show that GNA modifications at positions 16 and 17 showed excellent efficacy similar to the parent design, while GNA at position 18 showed reduced activity.

[0419] [Table 2]

[0420] [Table 3]

[0421] Antisense strand design 2nd in AS This position was identified by statistical analysis of data sets of large conjugates and shifts in the AS strand affected by sterically demanding 2'-modifications, including 2'-OMe. However, we 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, summarized in Figure 3 and Table 3, suggest that DNA and RNA at the 2' position generally maintain activity of non-F designs similar to the parent template design, while 2'-OMe is generally not well tolerated, resulting in reduced activity.

[0422] 14th in AS This position was identified by statistical analysis of data sets of large conjugates and shifts in the AS strand affected by sterically demanding 2'-modifications, including 2'-OMe. However, several modifications, including DNA and possibly RNA, and other modifications that do not introduce steric bulk at the 2' position, were found to be well tolerated in the context of non-F designs. Results from in vitro silencing studies, summarized in Figure 4 and Table 4, 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) received a single dose of 2.5 mg / kg siRNA, and FVII serum protein levels were measured pre-dose and 4, 7, 13, 22, 29, and 36 days after dosing. Figure 5 shows the concentration-time profiles of FVII protein for two non-F siRNAs AD-61398 and 64273 compared to the parent compound AD-57727. Figure 6 shows the reduction in 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 reduction in mTTR serum protein for a repeat-dose 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 exhibited in vivo efficacy and potency similar to the parental template designs.

[0427] TMPRSS6-targeting si RNA

[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 sense and AS strands. While the in vitro data suggest that the non-F compounds had similar potency / efficacy to their parent compounds, the non-F compound AD-64604, which was found to be the most active in vivo, 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 Figure 10, all 10 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 3 mg / kg siRNA, and liver levels were assessed before administration, and then 7 and 22 days later, as shown in Figure 11.

[0437] Figure 12 shows the enhanced activity with stability-enhanced conjugate chemistry (SEC-C), where liver was assessed for activity (mRNA) 7 days after administration. Animals were administered a single SC dose of 3 mg / kg. The data demonstrate the influence 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 using data evaluated 7 days after administration. This data demonstrates the influence of motifs on in vivo activity. The several-fold improvement is consistent across 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 the results of hAAV 1 × 10 11 GC / mouse, ApoC3-GalNAc3 SAR results after a single subcutaneous administration of 3 mg / kg are shown.

[0441] Example 5: VP and PS2 modifications at the 5' end of the antisense strand The following describes exemplary protocols for synthesizing oligonucleotides containing 5'-vinylphosphonate (VP) and 2'-deoxythymidine linked by phosphorodithioate (PS2) bonds at the 5' end of the oligonucleotide. Those skilled in the art will understand that similar oligonucleotides can be synthesized using the 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 5'-vinylphosphonate-containing oligonucleotides Introduction of pivaloyloxymethyl-(POM)-protected VP [ka] Coupling and oxidation: Amidite coupling was performed under standard synthesis conditions using 0.25 M 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 triester to a phosphorothioate linkage. Because the vinylphosphonate building block does not contain a DMT protecting group at the 5' position, the final detritylation step was omitted.

[0443] Deprotection and cleavage: After synthesis, vinylphosphonate-containing oligonucleotides were deprotected in a 3:1 mixture of aqueous NH3 and EtOH to which 40% by volume of 1–2.5% methylamine solution was added for 5 h at 60 °C or 16 h at 35 °C.

[0444] Introduction of ethyl-protected VP [ka] Coupling and oxidation: Amidite coupling was performed under standard synthesis conditions using 0.25 M 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 triester and introduce the phosphorothioate linkage. Because the vinylphosphonate building block does not contain a DMT protecting group at the 5' position, the final detritylation step was omitted.

[0445] Deprotection and cleavage: Prepare a solution of acetonitrile (ACN) and pyridine (Pyr) 50:1 (v / v) and add 3 x 10 -8The mixture was dried as much as possible by adding 3 cm (3 Å) 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 0.5 M solution of mercaptoethanol in 1:1 (v / v) acetonitrile-triethylamine was prepared and diluted with 3 × 10 -8 3 Å (1 / 3 cm) of molecular sieves were added. While the 5'-VP-containing oligonucleotide was still on the resin in the synthesis column, the TMSI solution was slowly added over approximately 5-10 CV and allowed to react for 15 minutes. This step was repeated twice for a total exposure time of approximately 45 minutes. The resin was then washed extensively with ACN, and then approximately 5-10 column volumes of mercaptoethanol solution were applied to the column and allowed to react for 10 minutes. This step was repeated once for a total exposure time of 20 minutes. 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) at a concentration of 0.15 M in dry acetonitrile according to the manufacturer's protocol. Coupling was carried out under standard synthesis conditions using 0.25 M 5-(ethylthio)-1H-tetrazole in acetonitrile for a total coupling time of 17 minutes. The capping step was omitted from this synthesis cycle. Oxidation (thiolation) was carried out using 3-(dimethylaminomethylene)amino-3H-1,2,4-dithiazole-5-thione (DDTT) by extending the reagent delivery and reaction time to 3 × 10 minutes. The final detritylation step was carried out 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 then the resin was transferred to a suitable container and treated under standard conditions (e.g., 3:1 aqueous NH:EtOH solution, 60 °C for 5 h or 35 °C for 16 h) to cleave the oligonucleotide from the solid support and deprotect it.

[0448] The remaining steps in 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) exhibits improved in vitro activity. For example, approximately 80% of the sequences tested showed improved intrinsic potency when transfected in vitro, and approximately 30% exhibited an IC of approximately 10-fold. 50 However, in vitro, the 5'-phosphate is rapidly lost in the endo / lysomal compartment. 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 and without the 5'-VP modification). As can be seen from the chart, the ED 50A three-fold improvement in activity is seen with certain ApoR sequences. In vivo benefit was confirmed with additional compounds / targets, including ApoC3, Tmprssr6, and TTR. ApoB sequences are listed in Table 9.

[0452] [Table 9]

[0453] Figure 18 shows different chemical modifications that can replace PS bonds, including phosphorodithioate (PS2) and methylphosphonate (MePhos), which promote endogenous phosphorylation. 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, 2'-OMe modifications, along with phosphorothioate bonds, are desirable for exonuclease protection. Substitution of 2'-OMe modifications, such as with 2'F, and modification of PS bonds can promote exonuclease protection while maintaining metabolic stability.

[0454] 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. As shown in the chart, the dn(PS2) conjugate and 2'F-PS showed improved in vitro activity relative to the parent (2'OMe-PS). Notably, dn(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 illustrating how subtle changes in the antisense 5' end can significantly improve in vivo efficacy: Figure 20A shows that 2'F-PS at position 1 of the antisense strand can improve the activity of 5'P-dependent sequences, and Figure 20B shows the approximately 3-fold improved potency of dN(PS)2 over the parent, similar to 5'-VP.

[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 hours, 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 hours, 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 × 1000 mL), dried over NaSO, and evaporated. The solid residue was washed with cold hexane and dried in vacuo to give 148 g (45%) of X as a pale yellow solid. 1 H NMR(500MHz,CDCI3):δ 5.73-5.63(m,8H),2.65(t,2H),1.22(s,36H); 31 P NMR (500 MHz, CDCl): 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 equivalents; 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 added to 200 mL of a solution of 10% NaSO and saturated NaHCO (1:1), followed by the addition of 200 mL of ethyl acetate. The crude aldehyde was extracted with ethyl acetate, dried, and concentrated under reduced pressure. This crude aldehyde was used in the next step without any purification.

[0459] Yield = 2.87 gm (97%); approx. 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 in 14 ml of THF by adding bisphosphonate (X) (12.6 gm, 20 mmol) 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, then at 0°C for another 1 h, and then at room temperature for an additional 1 h. TLC confirmed the formation of the product (7:3 EtOAc:hexane). 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 (20% to 100% EtOAc in hexane) 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] A solution of 3 (4 g, 5.7 mmol) in 200 mL of HCOOH / HO (1:1, v:v) was stirred at room temperature for 24 h. TLC confirmed the formation of the product (MeOH:CHCL = 5:95).

[0463] The solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (MeOH:CH2Cl2 = 7:93, v:v). Fractions were tested by RP-HPLC (C18 column, buffer A = 0.05% TFA in water, buffer B = 0.05% TFA in ACN; 25 min gradient 5-95%) 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, and the remaining fractions were the E isomer. The fraction containing the mixture of E and Z isomers was purified by RP-HPLC. 2.3 g of 4-E isomer was obtained, a 71% yield.

[0464] E isomer: 1 H NMR (400MHz, acetonitrile-d3):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, acetonitrile-d): δ 18.29.

[0465] Z isomer: 1 H NMR (500MHz, acetonitrile-d3):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); 31P NMR (202 MHz, acetonitrile-d): δ 18.75.

[0466] Preparation of Compound 5 [ka] To a solution of compound 4-E isomer (2.1 g, 3.62 mmol) and ethylthiotetrazole (0.46 g, 3.62 mmol) in ACN (40 mL) was added 2-cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite (1.311 g, 4.35 mmol). The mixture was stirred at room temperature for 2 h. TLC in hexane:EtOAc (2:8 in 0.15% TEA) confirmed product formation. The reaction mixture was filtered, concentrated, and loaded onto a silica column. The sample was eluted with 20% to 100% EtOAc in hexane containing TEA (0.15%) to give compound 5 (1.75 g, 62%) as a white foam.

[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-d3): δ 151.79(d,J=71.3Hz),18.07(d,J=54.0Hz).

[0468] Z isomer: 1H NMR (400MHz, acetonitrile-d3):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 (202 MHz, acetonitrile-d): δ 150.81 (d, J = 141.4 Hz), 15.17.

[0469] Protocol for the synthesis of 5'-vinylphosphonate-containing oligonucleotides Vinyl phosphonate monomers and 5'-VP-modified oligonucleotides were synthesized similarly to literature procedures (Chen et al., International Publication No. 2008 / 100447; 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)), the entire contents of each of which are incorporated herein by reference. Briefly, the 5'-phosphate was protected with ethyl ether, and the ethyl ether-protected phosphate was then deprotected in two steps: (1) TMS-I on a solid support under anhydrous conditions, and (2) standard oligonucleotide deprotection to obtain the 5'-VP-modified oligonucleotide. 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 addition to the RNA-induced silencing complex (RISC) and induces robust RNAi-mediated gene silencing. Thus, endogenous 5'-phosphorylation of synthetic siRNA by Clp1 kinase is crucial for RISC addition 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 (Chen et al., International Publication No. 2008 / 100447) and to modify the 5' ends of siRNAs to improve the gene silencing activity of 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 siRNA 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] We compared the effects of 5'-vinylphosphonate (VP) with E- and Z-geometry on double-stranded siRNA activity. The results showed that the in vivo efficacy of chemically modified siRNA 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] Figures 21A and 21B show SAR analyses comparing the in vitro and in vivo activity of ApoB siRNA containing a 5'-OH modification (at the 5' end of the antisense strand) and ApoB siRNA containing a 5'-E-VP modification. Figure 21A shows the results in in vitro transfected mouse hepatocytes. Figure 21B shows the LDL levels 3 days after a single administration (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 on mTTR and F9 siRNA-GalNAc conjugates. The results were obtained from in vitro transfected mouse primary hepatocytes. As shown in the figure, 5'-E-VP-modified siRNA conjugates showed maintained or improved potency, while 5'-Z-VP-modified siRNA conjugates 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 exhibited improved gene silencing activity relative to the 5'-OH control, while 5'-Z-VP-modified siRNA conjugates exhibited activity similar to that of the 5'-OH control.

[0479] The results in these figures demonstrate 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-vinylphosphonate (5'-E-VP), which adequately mimics 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 an argon atmosphere under anhydrous conditions. 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), broadband signal (br), or multiplet (m). 31 P NMR spectra were recorded at 162 MHz in proton-isolated 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 consisted of 100 mM 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and 16.3 mM triethylamine (TEA) in HO, and buffer B was 100% methanol.

[0481] [ka] Reagents and conditions for Scheme 2: (a) benzyloxymethyl acetal chloride (BOM), DBU, DMF, 30 minutes, 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, the entire contents of each of which are incorporated herein by reference). (2009)); (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 atmosphere, i-PrOH / HO (10:1, v / v), 0.05 equivalents formic acid, 12 h, room temperature, 98% (Aleiwi et al., “A reliable Pd-mediated hydrogenolytic deprotection of BOM group of uridine ureido nitrogen,” Tetrahedron Lett. 53:3758-3762, the entire contents of which are incorporated herein by reference). (2012)); (e) NEt3-3HF, THF, THF, 48 hours, room temperature, 88%; (d) 2-cyanoethyl N,N-diisopropylphosphoramidite, DIEA, DCM, 18 hours, room temperature, 56%; (g) 1M aqueous piperidine, 24 hours, room temperature; then 30% aqueous ammonia / ethanol (3:1, v / v), 36 hours, room temperature, quantitative, Z + =Piperidinium.

[0482] N 3 Synthesis of 2-benzyloxymethyl-2'-O-methyl-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 5'-benzyloxymethyl-5'-deoxy-5'-iodo-2'-O-methyl-3'-O-tert-butyldimethylsilyluridine (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 min. Methanol (200 mL) was added to the reaction, and the mixture was stirred for an additional 15 min. The solvent was evaporated to dryness; the residue was dissolved in dichloromethane (DCM) and washed with a 5% solution of NaSO, followed by water. The organic layer was collected, dried over NaSO, filtered, and evaporated to dryness. The crude residue was purified by silica gel chromatography using 0–50% ethyl acetate (EtOAc) in hexane as the eluent to give 3 (11.2 g, 92%) as a white foam. 1 H NMR(400MHz,DMSO-d6):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(126MHz,DMSO-d6):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 IN2NaO6Si(M+Na) + The calculated value for is 625.1207; the measured 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, and 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. Refluxing was continued for 24 h. The reaction mixture was cooled to room temperature, and methanol (50 mL) was added to quench the reaction. The solvent and volatiles were evaporated in vacuo. The crude residue was purified by silica gel chromatography using 0–100% EtOAc in hexane as eluent to give compound 4 (9.2 g, 92%) as a colorless oil. 1 H NMR(400MHz,DMSO-d6):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=13.4...

Claims

1. A double-stranded RNA (dsRNA) agent capable of suppressing expression of a target gene, comprising a sense strand sequence and an antisense strand sequence, wherein the antisense strand sequence is at least 80% complementary to an mRNA corresponding to the target gene, and comprises an antisense strand sequence having a mismatch with the target mRNA at the first nucleotide from the 5' end of the antisense strand and the first 5 nucleotides within a terminal region at the 3' end of the antisense strand, the sense strand being 19-22 nucleotides in length and comprising at least one phosphorothioate bond, and having the formula (Is): 【Chemistry 1】 It is expressed as During the ceremony: B1 is selected from 2'-OMe and 2'-F modified nucleotides; B2 and B3 each consist exclusively of 2'-OMe modified nucleotides; C1 is glycol nucleic acid (GNA) or unlocked nucleic acid (UNA); each T1 represents a 2'-F modified nucleotide; n ２ is 3; n １ and n ３ are independently 4 to 15 nucleotides in length; n ５ is 1 to 6 nucleotides in length; and n ４ is 0 or 1 nucleotide in length, wherein one of the T1 nucleotides is: at position 11 from the 5' end of the sense strand; wherein the antisense strand is: (i) is 19-25 nucleotides in length and contains at least two different nucleotide modifications selected from the group consisting of 2'-deoxy, 2'-O-methyl, 2'-fluoro modifications, and acyclic nucleotides; and (ii) comprising two 2'-fluoro modified nucleotides at positions 2 and 14, counting from the 5' end of the antisense strand; Here, n ４ is 1, C1 is at one of positions 16-17 counting from the 5' end of the sense strand.

2. The dsRNA agent of claim 1, wherein the 2'-F modified nucleotides are at positions 7 and 9 from the 5' end of the sense strand.

3. The dsRNA agent of claim 2, wherein 2'-F modified nucleotides are only at positions 7 and 9-11 from the 5' end of the sense strand.

4. n ４ The dsRNA agent of claim 1, wherein

5. n ４ The dsRNA agent of claim 1, wherein

6. The dsRNA agent of claim 1, wherein the antisense strand comprises a third modified nucleotide at one or more of positions 6-10 at the 5' end of the antisense strand.

7. The dsRNA agent of claim 1, wherein the sense strand comprises one block of two phosphorothioate or methylphosphonate internucleotide linkages.

8. The dsRNA agent of claim 7, wherein the sense strand comprises two interphosphorothioate linkage modifications within positions 1-5 of the sense strand, counting from the 5' end of the sense strand.

9. 10. The dsRNA agent of claim 1, wherein the dsRNA agent is covalently linked to one or more ligands that enhance nuclease resistance.

10. The dsRNA agent of claim 1, having a 3' and / or 5' overhang from 1 to 10 nucleotides in length.

11. The dsRNA agent of claim 1, further comprising at least one ligand.

12. The dsRNA agent of claim 11, further comprising at least one ASGPR ligand attached to the 5' or 3' end of the sense strand.

13. The dsRNA agent of claim 11, further comprising at least one ASGPR ligand attached to the 3' end of the sense strand.

14. 14. The dsRNA agent of claim 12 or 13, wherein the ASGPR ligand is one or more GalNAc derivatives attached by a bivalent or trivalent branched linker.

15. The ASGPR ligand: 【Chemistry 2】 The dsRNA agent of claim 14, wherein

16. The dsRNA agent of claim 1, wherein each nucleotide of the antisense strand comprises a modification selected from the group consisting of 2'-deoxy, 2'-O-methyl, 2'-fluoro modified, UNA and GNA.

17. The dsRNA agent of claim 1, wherein each nucleotide of the antisense strand comprises a modification selected from the group consisting of a 2'-O-methyl and a 2'-fluoro modification.

18. 10. The dsRNA agent of claim 1, wherein one end of the dsRNA agent is blunt while the other end comprises a two nucleotide overhang.

19. The dsRNA agent of claim 18, wherein the overhang is at the 3' end of the antisense strand.

20. The dsRNA agent of claim 1, wherein both ends of the dsRNA agent are blunt and the sense and antisense strands each have 19-23 nucleotides.

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

22. The dsRNA agent of claim 1, wherein the first base pair from the 5' end of the antisense strand is an AU base pair.

23. The dsRNA agent of claim 1, wherein each B1 comprises a 2'-O methyl modification.

24. Formula (Is) is a phosphorodithioate (PS ２ 2. The dsRNA agent of claim 1, further comprising a 2'-deoxythymidine or a 5'-vinylphosphonate (VP) linked by a 5'-vinylphosphonate (VP) bond.

25. The dsRNA agent of claim 1, wherein the antisense strand sequence is at least 80% complementary to an mRNA corresponding to a target gene, has a mismatch with the target mRNA in an overhang region of the antisense strand, and 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 an AU base pair.

26. A pharmaceutical composition comprising the dsRNA agent of any one of claims 1-25 in combination with a pharma- ceutically acceptable carrier or excipient.

27. 26. A pharmaceutical composition for use in a method of inhibiting expression of a target gene, said method comprising the step of administering a dsRNA agent of any one of claims 1-25 in an amount sufficient to inhibit expression of said target gene, wherein said pharmaceutical composition comprises the dsRNA agent of any one of claims 1-25.

28. 28. The pharmaceutical composition of claim 27, wherein the dsRNA agent is administered by subcutaneous or intravenous administration.

29. 26. A pharmaceutical composition for use in a method of delivering a polynucleotide to a specific target in a subject by administering a dsRNA agent of any one of claims 1-25, wherein said pharmaceutical composition comprises the dsRNA agent of any one of claims 1-25.

30. 30. The pharmaceutical composition of claim 29, wherein the administering step is performed 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.

31. 26. A pharmaceutical composition for use in a method of delivering a polynucleotide to a specific target in a subject, said method comprising the step of delivering to said subject by subcutaneous administration a dsRNA agent of any one of claims 1-25, such that said polynucleotide is delivered to said specific target in said subject, wherein said pharmaceutical composition comprises a dsRNA agent of any one of claims 1-25.

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