Modified double-stranded RNA agent

Abstract

To provide a double-stranded RNA agent for improving effectiveness of gene silencing of siRNA gene therapy.SOLUTION: A double-stranded RNA agent expressed by formula (I) includes a sense strand and an antisense strand, in which each strand has 14 to 40 nucleotides. (B1 to B3, B1' to B4': nucleotide including modification such as 2'-O alkyl and 2'-substituted alkoxy. C1: thermally destabilized nucleotide at a site on the opposite side of a seed region (position 2 to position 8) of the antisense strand. T1, T1' to T3': nucleotide including modification. n1, n3, q1: 4 to 15 nucleotide length. n5, q3, q7: 1 to 6 nucleotide length. q2, q6: 1 to 3 nucleotide length. q5: 0 to 10 nucleotide length. n2, n4, q4: 0 to 3 nucleotide 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, each of which is hereby incorporated by reference in its entirety.

[0002] The present invention relates to RNAi duplex agents containing specific motifs advantageous for inhibiting the expression of target genes, and RNAi compositions suitable for therapeutic use. In addition, the present invention provides methods for inhibiting the expression of target genes by administering these RNAi duplex agents, for example, for the treatment of various diseases.

Background Art

[0003] RNA interference or "RNAi" is a term first coined by Fire and colleagues to explain 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, and provide a new tool for studying gene function. RNAi is mediated by the RNA-induced silencing complex (RISC), a sequence-specific multi-component nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs (about 22 nucleotides) derived from double-stranded RNA triggers, but the protein components of this activity are still unknown.

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

[0005] It was thought that cleavage of the sense strand inhibits endonucleolytic cleavage of the 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 cleavage of the sense strand is required for efficient RNAi in mammals.

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

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

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

Summary of the Invention

Problems to be Solved by the Invention

[0009] Therefore, there is currently a need for an iRNA duplex agent to improve the effectiveness of gene silencing in siRNA gene therapy. The present invention relates to this need.

Means for Solving the Problems

[0010] The present invention provides an effective nucleotide or chemical motif of a dsRNA agent, optionally conjugated with at least one ligand, which is advantageous for inhibiting the expression of a target gene, and an RNAi composition suitable for therapeutic use.

[0011] In one aspect, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting the expression of a target gene. The dsRNA agent includes a sense strand and an antisense strand, and each strand has 14 to 40 nucleotides. The dsRNA agent is represented by formula (I):

Chemical Formula

[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'-O-N-methylacetamide (2'-O-NMA) modification.

[0013] C1 is a thermally labile nucleotide at a site opposite to the seed region of the antisense strand (i.e., positions 2 to 8 at the 5'-end of the antisense strand). For example, C1 is at a position of the sense strand that pairs with the nucleotides at positions 2 to 8 at the 5'-end of the antisense strand. In one example, C1 is at position 15 at the 5'-end of the sense strand. The C1 nucleotide may have a depurination modification; a mismatch with the opposing nucleotide of the double strand; and a sugar modification, such as a 2'-deoxy modification or an acyclic nucleotide, such as unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA), having a thermally labile modification. In one embodiment, C1 is (i) a mismatch with the opposing nucleotide of the antisense strand; (ii) a depurination modification selected from the group consisting of:

Chemical formula

Chemical formula

Chemical Structure

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

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

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

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

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

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

[0020] Alternatively, 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 are 1. In another example, n 4 is 0, and q 2 and q 6 are 1, and have modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 1 to 5 (counting from the 5' end of the sense strand) of the sense strand, as well as modifications of two phosphorothioate nucleotide internucleotide linkages at positions 1 and 2 (counting from the 5' end of the antisense strand) and 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 at the 5' end of the sense strand, and n 4 is 1. In one embodiment, C1 is at position 15 at the 5' end of the sense strand.

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

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

[0027] In an exemplary embodiment, T3' starts from position 2 at the 5' end of the antisense strand, and T1' starts from position 14 at the 5' end of the antisense strand. In one example, T3' starts from position 2 at the 5' end of the antisense strand, and q 6 is equal to 1, and T1' starts from position 14 at 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 in length (i.e., the T1' and T3' nucleotides are not counted).

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

[0030] In one embodiment, T3' is at position 2 at the 5' end of the antisense strand. In one example, T3' is at position 2 at the 5' end of the antisense strand, and q 6is equal to 1, and the modification is at the 2'-position, or is a non-ribose, acyclic, or backbone position providing a steric bulk less than or equal to that of the 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 in length, T1 is at position 11 of the 5'-end of the sense strand, and 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 of the 5'-end of the sense strand, and n 2 is 1.

[0032] In one embodiment, T2' starts at position 6 of the 5'-end of the antisense strand. In one example, T2' is at positions 6-10 of the 5'-end of the antisense strand, and q 4 is 1.

[0033] In an exemplary embodiment, when the sense strand is 19-22 nucleotides in length, T1 is, for example, at the cleavage site of the sense strand at position 11 of the 5'-end of the sense strand, 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 modification of T1' is at the 2'-position of the ribose sugar, or is a non-ribose, acyclic, or backbone position providing a steric bulk smaller than that of the 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 modification of T3' is at the 2'-position, or is a non-ribose, acyclic, or backbone position providing a steric bulk less than or equal to that of the 2'-OMe ribose.

[0034] In one embodiment, T2' starts at position 8 of the 5'-end of the antisense strand. In one example, T2' starts at position 8 of the 5'-end of the antisense strand, and q 4 is 2.

[0035] In one embodiment, T2’ starts at the 9th position of the 5’ end of the antisense strand. In one example, T2’ starts at the 9th position of the 5’ end of the antisense strand, and q 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; there are modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5’ end of the sense strand) of the sense strand, as well as modifications of the internucleotide linkages of two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand and modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 18 to 23.

[0037] In one embodiment, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, and q 1 is 9, T1’ is 2’-F, and q 2 is 1, B2’ is 2’-OMe or 2’-F, and q 3 is 4, T2’ is 2’-F, and q 4 is 1, B3’ is 2’-OMe or 2’-F, and q 5 is 6, T3’ is 2’-F, and q 6 is 1, B4’ is 2’-OMe, and q 7is 1; modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 1 to 5 (counting from the 5' end of the sense strand) of the sense strand, as well as modifications of two phosphorothioate nucleotide internucleotide linkages at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 18 to 23.

[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, q 6 is 1, B4’ is 2’-OMe, q 7 is 1; having modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 1 to 5 (counting from the 5’ end of the sense strand) of the sense strand, as well as modifications of two phosphorothioate nucleotide internucleotide linkages at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand and modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 18 to 23.

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

[0041] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 6, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 7, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, q 7 is 1; modification of the internucleotide linkage between two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5’ end of the sense strand) of the sense strand, and modification of the internucleotide linkage between two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand and modification of the internucleotide linkage between two phosphorothioate nucleotides within the range of positions 18 to 23.

[0042] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 1, B3’ is 2’-OMe or 2’-F, q 5 is 6, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, q 7 is 1.

[0043] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 1, B3’ is 2’-OMe or 2’-F, q 5 is 6, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, q 7 is 1; has modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 1 to 5 (counting from the 5’ end of the sense strand) of the sense strand, as well as modifications of two phosphorothioate nucleotide internucleotide linkages at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand and modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 18 to 23.

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

[0045] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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, having at least two additional TT at the 3'-end of the antisense strand; modification of two phosphorothioate internucleotide linkages within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, and modification of two phosphorothioate internucleotide linkages at positions 1 and 2 (counting from the 5'-end of the antisense strand) and modification of two phosphorothioate internucleotide linkages within the range of 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; having modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, and modifications of two phosphorothioate nucleotide internucleotide linkages at positions 1 and 2 (counting from the 5'-end of the antisense strand) and within the range of 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, q 6 is 1, B4’ is 2’-F, q 7 is 1.

[0049] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, q 7 is 1; having modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 1 to 5 (counting from the 5’ end of the sense strand) of the sense strand, as well as modifications of two phosphorothioate nucleotide internucleotide linkages at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand and modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 18 to 23.

[0050] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, 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; having modifications of two phosphorothioate internucleotide linkages within the range of positions 1 to 5 (counting from the 5’ end of the sense strand) of the sense strand, and modifications of two phosphorothioate internucleotide linkages at positions 1 and 2 (counting from the 5’ end of the antisense strand) and within the range of positions 18 to 23 of the antisense strand.

[0052] This dsRNA agent may contain a phosphorus-containing group at the 5’ end of the sense strand or the antisense strand. The 5’-terminal phosphorus-containing group is 5’-terminal phosphate (5’-P), 5’-terminal phosphorothioate (5’-PS), 5’-terminal phosphorodithioate (5’-PS2), 5’-terminal vinylphosphonate (5’-VP), 5’-terminal methylphosphonate (MePhos), or 5’-deoxy-5’-C-malonyl(

Chemical formula

[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 contains 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 contains 5'-P.

[0061] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, q 7 is 1. The dsRNA agent also contains 5’-VP. 5’-VP can be 5’-E-VP, 5’-Z-VP, or a combination thereof.

[0062] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, q 7 is 1. The dsRNA agent also contains 5’-PS2.

[0063] In one embodiment, B1 is 2’-OMe or 2’-F, n 1is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, q 7 is 1. The dsRNA agent also contains 5’-deoxy-5’-C-malonyl.

[0064] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, q 7is 1; it has modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of the internucleotide linkages of two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand and modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 18 to 23. The dsRNA agent also contains 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; it has modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of the internucleotide linkages of two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand and modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 18 to 23. The dsRNA agent also contains 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; having modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of two phosphorothioate nucleotide internucleotide linkages at positions 1 and 2 (counting from the 5'-end of the antisense strand) and within the range of positions 18 to 23 of the antisense strand. The dsRNA agent also includes 5'-VP. The 5'-VP can be 5'-E-VP, 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; it has modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of two phosphorothioate nucleotide internucleotide linkages at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand and modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 18 to 23. The dsRNA agent also contains 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; it has modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of two phosphorothioate nucleotide internucleotide linkages at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand and modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 18 to 23. The dsRNA agent also contains 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, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, q 4 is 0, B3’ is 2’-OMe or 2’-F, q 5 is 7, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, q 7 is 1. The dsRNA agent also includes 5’-VP. 5’-VP can be 5’-E-VP, 5’-Z-VP, or a combination thereof.

[0072] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, q 4 is 0, B3’ is 2’-OMe or 2’-F, q 5 is 7, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, q 7 is 1. The dsRNA agent also includes 5’-PS2.

[0073] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, 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 contains 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; it has modifications of two phosphorothioate internucleotide linkages within the range of positions 1 to 5 (counting from the 5’ end of the sense strand) of the sense strand, as well as modifications of two phosphorothioate internucleotide linkages at positions 1 and 2 (counting from the 5’ end of the antisense strand) and within the range of positions 18 to 23 of the antisense strand. The dsRNA agent also contains 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; has modifications of two phosphorothioate internucleotide linkages within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of two phosphorothioate internucleotide linkages at positions 1 and 2 (counting from the 5'-end of the antisense strand) and within the range of positions 18 to 23 of the antisense strand. The dsRNA agent also includes 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; it has modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of two phosphorothioate nucleotide internucleotide linkages at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand and modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 18 to 23. The dsRNA agent also contains 5'-VP. 5'-VP can be 5'-E-VP, 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; it has modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of two phosphorothioate nucleotide internucleotide linkages at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand and modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 18 to 23. The dsRNA agent also contains 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; it has modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5’ end of the sense strand) of the sense strand, as well as modifications of the internucleotide linkages of two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5’ end of the antisense strand) and within the range of positions 18 to 23 of the antisense strand. The dsRNA agent also contains 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 contains 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 contains 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 contains 5'-VP. The 5'-VP can be 5'-E-VP, 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 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 5'-deoxy-5'-C-malonyl.

[0084] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, q 7 is 1; having modifications of two phosphorothioate internucleotide linkages within the range of positions 1 to 5 (counting from the 5’ end of the sense strand) of the sense strand, and modifications of two phosphorothioate internucleotide linkages at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand and within the range of positions 18 to 23. The dsRNA agent also contains 5’-P.

[0085] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, q 7is 1; it has modifications of the phosphorothioate internucleotide linkages between two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of the phosphorothioate internucleotide linkages at positions 1 and 2 (counting from the 5'-end of the antisense strand) and within the range of positions 18 to 23 of the antisense strand. The dsRNA agent also contains 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; it has modifications of the phosphorothioate internucleotide linkages between two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of the phosphorothioate internucleotide linkages at positions 1 and 2 (counting from the 5'-end of the antisense strand) and within the range of positions 18 to 23 of the antisense strand. The dsRNA agent also contains 5'-VP. 5'-VP can be 5'-E-VP, 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; it has modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of the internucleotide linkages of two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand and modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 18 to 23. The dsRNA agent also contains 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; modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 1 to 5 (counting from the 5' end of the sense strand) of the sense strand, and modifications of two phosphorothioate nucleotide internucleotide linkages at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 18 to 23. The dsRNA agent also contains 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 contains 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, q 5 is 7, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, q 7 is 1. The dsRNA agent also includes 5’-PS.

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

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

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

[0094] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, q 4 is 0, B3’ is 2’-OMe or 2’-F, q 5 is 7, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, q 7is 1; it has modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of two phosphorothioate nucleotide internucleotide linkages at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand and modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 18 to 23. The dsRNA agent also contains 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; it has modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of two phosphorothioate nucleotide internucleotide linkages at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand and modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 18 to 23. The dsRNA agent also contains 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; it has modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5’ end of the sense strand) of the sense strand, as well as modifications of the internucleotide linkages of two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5’ end of the antisense strand) and within the range of positions 18 to 23 of the antisense strand. The dsRNA agent also includes 5’-VP. The 5’-VP can be 5’-E-VP, 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; it has modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of the internucleotide linkages of two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand and modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 18 to 23. The dsRNA agent also contains 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; it has modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of the internucleotide linkages of two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand and modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 18 to 23. The dsRNA agent also contains 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 contain modifications as described herein.

[0100] In one embodiment, the sense strand and the antisense strand of the dsRNA agent are each independently acyclic nucleotides, LNA, HNA, CeNA, 2'-methoxyethyl, 2'O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-O-N-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F modified.

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

[0102] In one embodiment, the dsRNA agent of formula (I) further comprises a 3' and / or 5' overhang that is 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, the dsRNA agent of the present invention does not contain any 2'-F modifications.

[0104] In one embodiment, the sense strand and / or the antisense strand of the dsRNA agent comprises one or more blocks of phosphorothioate or methylphosphonate internucleotide linkages. In one example, the sense strand comprises one block of two phosphorothioate or methylphosphonate internucleotide linkages. In one example, the antisense strand comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages. For example, the two blocks of phosphorothioate or methylphosphonate internucleotide linkages are separated by 16 to 18 phosphodiester nucleotide linkages.

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

[0106] In one embodiment, the nucleotide at position 1 at 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 the target RNA for hybridizing to the target RNA and inhibiting 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 the target RNA.

[0108] In one aspect, the present invention relates to a dsRNA agent as defined herein that can inhibit the expression of a target gene. The dsRNA agent includes a sense strand and an antisense strand, and each strand has 14 to 40 nucleotides. The sense strand includes at least one thermolabile nucleotide, and at least one of the thermolabile nucleotides is present at or near a site opposite to the seed region of the antisense strand (i.e., positions 2 to 8 at the 5'-end of the antisense strand). Each of the embodiments and aspects described herein related to the dsRNA represented by formula (I) can also be applied to dsRNAs containing thermolabile nucleotides.

[0109] The thermolabile nucleotide can be present, for example, between positions 14 and 17 at the 5'-end of the sense strand when the sense strand is 21 nucleotides in length. The antisense strand includes at least two modified nucleic acids that are smaller than the highly sterically demanding 2'-OMe modification. Preferably, the two modified nucleic acids that are smaller than the highly sterically demanding 2'-OMe are separated by 11 nucleotides in length. For example, the two modified nucleic acids are at positions 2 and 14 at the 5'-end of the antisense strand.

[0110] In one embodiment, the dsRNA agent further includes at least one ASGPR ligand. For example, the ASGPR ligand is a divalent or trivalent branched linker, such as:

Chemical formula

[0111] For example, the dsRNA agent described in this specification may include: (i) a phosphorus-containing group at the 5' end of the sense strand or the antisense strand; (ii) modification of the internucleotide linkages between two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5' end of the sense strand) of the sense strand, and modification of the internucleotide linkages between two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5' end of the antisense strand) and within the range of positions 18 to 23 of the antisense strand; and (iii) a ligand at the 5' or 3' end of the sense strand or the antisense strand, for example, an ASGPR ligand (such as one or more GalNAc derivatives). For example, this ligand may 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; it has modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of the internucleotide linkages of two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand and modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 18 to 23. The dsRNA agent also includes 5'-P and a target ligand. In one embodiment, 5'-P is at the 5'-end of the antisense strand and the target 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; it has modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of the internucleotide linkages of two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand and modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 18 to 23. The dsRNA agent also includes 5'-PS and a target ligand. In one embodiment, 5'-PS is at the 5'-end of the antisense strand and the target 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; it has modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of the internucleotide linkages of two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5'-end of the antisense strand) and within the range of 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 target ligand. In one embodiment, the 5'-VP is at the 5'-end of the antisense strand and the target 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, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, q 7 is 1; the modification of two phosphorothioate nucleotide internucleotide linkages within the range of positions 1 to 5 (counting from the 5’ end of the sense strand) of the sense strand, and the modification of two phosphorothioate nucleotide internucleotide linkages at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand and the modification of two phosphorothioate nucleotide internucleotide linkages within the range of positions 18 to 23. The dsRNA agent also includes 5’-PS2 and a target ligand. In one embodiment, 5’-PS2 is at the 5’ end of the antisense strand and the target ligand is at the 3’ end of the sense strand.

[0116] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, q 7is 1; it has modifications of the phosphorothioate internucleotide linkages between two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of the phosphorothioate internucleotide linkages between two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5'-end of the antisense strand) and within the range of positions 18 to 23 of the antisense strand. The dsRNA agent also contains 5'-deoxy-5'-C-malonyl and a target ligand. In one embodiment, 5'-deoxy-5'-C-malonyl is at the 5'-end of the antisense strand and the target 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; it has modifications of the phosphorothioate internucleotide linkages between two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of the phosphorothioate internucleotide linkages between two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5'-end of the antisense strand) and within the range of positions 18 to 23 of the antisense strand. The dsRNA agent also contains 5'-P and a target ligand. In one embodiment, 5'-P is at the 5'-end of the antisense strand and the target 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; it has modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of the internucleotide linkages of two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5'-end of the antisense strand) and within the range of positions 18 to 23 of the antisense strand. The dsRNA agent also includes 5'-PS and a target ligand. In one embodiment, 5'-PS is at the 5'-end of the antisense strand and the target 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, q 5 is 7, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, q 7 is 1; modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 1 to 5 (counting from the 5’ end of the sense strand) of the sense strand, and modifications of two phosphorothioate nucleotide internucleotide linkages at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand and modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 18 to 23. The dsRNA agent also includes 5’-VP (e.g., 5’-E-VP, 5’-Z-VP, or combinations thereof) and a target ligand. In one embodiment, the 5’-VP is at the 5’ end of the antisense strand and the target ligand is at the 3’ end of the sense strand.

[0120] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, q 4 is 0, B3’ is 2’-OMe or 2’-F, q 5 is 7, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, q 7is 1; has modifications of the phosphorothioate internucleotide linkages between two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of the phosphorothioate internucleotide linkages at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand and modifications of the phosphorothioate internucleotide linkages within the range of positions 18 to 23. The dsRNA agent also includes 5'-PS2 and a target ligand. In one embodiment, 5'-PS2 is at the 5'-end of the antisense strand and the target ligand is at the 3'-end of the sense strand.

[0121] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, 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; has modifications of the phosphorothioate internucleotide linkages between two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of the phosphorothioate internucleotide linkages at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand and modifications of the phosphorothioate internucleotide linkages within the range of positions 18 to 23. The dsRNA agent also includes 5'-deoxy-5'-C-malonyl and a target ligand. In one embodiment, 5'-deoxy-5'-C-malonyl is at the 5'-end of the antisense strand and the target 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; it has modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of the internucleotide linkages of two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand and modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 18 to 23. The dsRNA agent also includes 5'-P and a target ligand. In one embodiment, 5'-P is at the 5'-end of the antisense strand and the target 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, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, q 7 is 1; modifications of internucleotide linkages of two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5’ end of the sense strand) of the sense strand, and modifications of internucleotide linkages of two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5’ end of the antisense strand) and modifications of internucleotide linkages of two phosphorothioate nucleotides within the range of positions 18 to 23 of the antisense strand. The dsRNA agent also includes 5’-PS and a target ligand. In one embodiment, 5’-PS is at the 5’ end of the antisense strand and the target ligand is at the 3’ end of the sense strand.

[0124] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, q 7is 1; it has modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5' end of the sense strand) of the sense strand, as well as modifications of the internucleotide linkages of two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 18 to 23. The dsRNA agent also includes 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a target ligand. In one embodiment, the 5'-VP is at the 5' end of the antisense strand and the target 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; it has modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5' end of the sense strand) of the sense strand, as well as modifications of the internucleotide linkages of two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 18 to 23. The dsRNA agent also includes 5'-PS2 and a target ligand. In one embodiment, the 5'-PS2 is at the 5' end of the antisense strand and the target ligand is at the 3' end of the sense strand.

[0126] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, 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; it has modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of the internucleotide linkages of two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5'-end of the antisense strand) and within the range of positions 18 to 23 of the antisense strand. The dsRNA agent also includes 5'-deoxy-5'-C-malonyl and a target ligand. In one embodiment, 5'-deoxy-5'-C-malonyl is at the 5'-end of the antisense strand and the target 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, q 3 is 4, q 4 is 0, B3’ is 2’-OMe or 2’-F, q 5 is 7, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, q 7 is 1; Modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 1 to 5 (counting from the 5’ end of the sense strand) of the sense strand, as well as modifications of two phosphorothioate nucleotide internucleotide linkages at positions 1 and 2 (counting from the 5’ end of the antisense strand) and within the range of positions 18 to 23 of the antisense strand. The dsRNA agent also includes 5’-P and a target ligand. In one embodiment, 5’-P is at the 5’ end of the antisense strand and the target ligand is at the 3’ end of the sense strand.

[0128] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, q 4 is 0, B3’ is 2’-OMe or 2’-F, q 5 is 7, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, q 7is 1; has modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 1 to 5 (counting from the 5' end of the sense strand) of the sense strand, as well as modifications of two phosphorothioate nucleotide internucleotide linkages at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 18 to 23. The dsRNA agent also includes 5'-PS and a target ligand. In one embodiment, the 5'-PS is at the 5' end of the antisense strand and the target 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; has modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 1 to 5 (counting from the 5' end of the sense strand) of the sense strand, as well as modifications of two phosphorothioate nucleotide internucleotide linkages at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand and modifications of two phosphorothioate nucleotide internucleotide linkages within the range of positions 18 to 23. The dsRNA agent also includes 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a target ligand. In one embodiment, the 5'-VP is at the 5' end of the antisense strand and the target 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; it has modifications of the internucleotide linkages of two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5'-end of the sense strand) of the sense strand, as well as modifications of the internucleotide linkages of two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5'-end of the antisense strand) and within the range of positions 18 to 23 of the antisense strand. The dsRNA agent also includes 5'-PS2 and a target ligand. In one embodiment, 5'-PS2 is at the 5'-end of the antisense strand and the target ligand is at the 3'-end of the sense strand.

[0131] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, 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; modifications of internucleotide linkages of two phosphorothioate nucleotides within the range of positions 1 to 5 (counting from the 5’ end of the sense strand) of the sense strand, as well as modifications of internucleotide linkages of two phosphorothioate nucleotides at positions 1 and 2 (counting from the 5’ end of the antisense strand) and within the range of positions 18 to 23 of the antisense strand. The dsRNA agent also includes 5’-deoxy-5’-C-malonyl and a target ligand. In one embodiment, 5’-deoxy-5’-C-malonyl is at the 5’ end of the antisense strand and the target ligand is at the 3’ end of the sense strand.

[0132] In a specific embodiment, the dsRNA agent of the present invention is: (a) a sense strand comprising: (i) 21 nucleotides in length; (ii) an ASGPR ligand added to the 3’ end including three GalNAc derivatives added by a trivalent branched linker; and (iii) 2’-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 17, 19, and 21 (counting from the 5’ end) and 2’-OMe modifications at positions 2, 4, 6, 8, 12, 14 to 16, 18, and 20 of the sense strand; and (b) an antisense strand comprising: (i) 23 nucleotides in length; (ii) 2’-OMe modifications at positions 1, 3, 5, 9, 11 to 13, 15, 17, 19, 21, and 23 (counting from the 5’ end) and 2’F modifications at positions 2, 4, 6 to 8, 10, 14, 16, 18, 20, and 22; and (iii) It includes 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; This dsRNA agent has an antisense strand having two nucleotide overhangs 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 present invention is: (a) A sense strand that: (i) Is 21 nucleotides in length; (ii) Contains three GalNAc derivatives attached by a trivalent branched linker, an ASGPR ligand attached to the 3'-end; (iii) Has 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 that: (i) Is 23 nucleotides in length; (ii) Has 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) Includes an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5'-end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; This dsRNA agent contains an antisense strand having a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0134] In another specific embodiment, the dsRNA agent of the present invention is: (a) A sense strand comprising: (i) 21 nucleotides in length; (ii) An ASGPR ligand added to the 3'-end, comprising three GalNAc derivatives added 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 nucleotides 1 and 2 (counting from the 5'-end) and between nucleotides 2 and 3; And (b) An antisense strand comprising: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 7, 9, 11, 13, 15, 17, and 19 to 23 (counting from the 5'-end), and 2'-F modifications at positions 2, 4 to 6, 8, 10, 12, 14, 16, and 18; and (iii) An antisense strand having phosphorothioate internucleotide linkages between nucleotides 1 and 2 (counting from the 5'-end), between nucleotides 2 and 3, between nucleotides 21 and 22, and between nucleotides 22 and 23; This dsRNA agent has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0135] In another specific embodiment, the dsRNA agent of the present invention is: (a) A sense strand comprising: (i) 21 nucleotides in length; (ii) An ASGPR ligand added to the 3'-end, comprising three GalNAc derivatives added 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, comprising: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 5, 7, 9, 11, 13, 15, 17, 19, and 21 to 23 (counting from the 5'-end), and 2'-F modifications at positions 2 to 4, 6, 8, 10, 12, 14, 16, 18, and 20; and (iii) An antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5'-end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; This dsRNA agent has two nucleotide overhangs 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 present invention is: (a) A sense strand, comprising: (i) 21 nucleotides in length; (ii) An ASGPR ligand added to the 3'-end, comprising three GalNAc derivatives added 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 nucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; and (b) An antisense strand comprising: (i) 23 nucleotides in length; (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 nucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; This dsRNA agent has two nucleotide overhangs 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 present invention is: (a) A sense strand comprising: (i) 21 nucleotides in length; (ii) An ASGPR ligand added to the 3' end, comprising three GalNAc derivatives added 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, 14-21; and (iv) A sense strand having phosphorothioate nucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; and (b) An antisense strand comprising: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5-7, 9, 11-13, 15, 17-19, and 21-23 (counting from the 5' end), and 2'-F modifications at positions 2, 4, 8, 10, 14, 16, and 20; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; This dsRNA agent has two nucleotide overhangs 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 present invention is: (a) a sense strand comprising: (i) 21 nucleotides in length; (ii) an ASGPR ligand added to the 3' end, comprising three GalNAc derivatives added 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 comprising: (i) 25 nucleotides in length; (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) It includes an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5'-end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; This dsRNA agent has a 4-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 present invention is: (a) A sense strand that: (i) Is 21 nucleotides in length; (ii) Contains an ASGPR ligand added to the 3'-end, which includes 3 GalNAc derivatives added by a trivalent branched linker; (iii) Has 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 that: (i) Is 23 nucleotides in length; (ii) Has 2'-OMe modifications at positions 1, 3 to 5, 7, 8, 10 to 13, 15, and 17 to 23 (counting from the 5'-end), and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16; and (iii) Includes an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5'-end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; This dsRNA agent has a 2-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 present invention is: (a) A sense strand comprising: (i) 21 nucleotides in length; (ii) An ASGPR ligand added to the 3'-end, comprising three GalNAc derivatives added by a trivalent branched linker; (iii) 2'-OMe modifications at positions 1-6, 8, and 12-21, and 2'-F modifications at positions 7 and 9-11; and (iv) A sense strand having phosphorothioate internucleotide linkages between nucleotides 1 and 2 (counting from the 5'-end) and between nucleotides 2 and 3; And (b) An antisense strand comprising: (i) 23 nucleotides in length; (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 nucleotides 1 and 2 (counting from the 5'-end), between nucleotides 2 and 3, between nucleotides 21 and 22, and between nucleotides 22 and 23; This dsRNA agent has two nucleotide overhangs at the 3'-end of the antisense strand and blunt ends at the 5'-end of the antisense strand. [[ID=2�]]

[0141] In another specific embodiment, the dsRNA agent of the present invention is: (a) A sense strand comprising: (i) 19 nucleotides in length; (ii) An ASGPR ligand added to the 3'-end, comprising three GalNAc derivatives added by a trivalent branched linker; (iii) 2'-OMe modifications at positions 1-4, 6, and 10-19, and 2'-F modifications at positions 5 and 7-9; and (iv) A sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; and (b) An antisense strand comprising: (i) 21 nucleotides in length; (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) having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21; This dsRNA agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0142] In one embodiment, the dsRNA agent described herein further comprises a thermolabile 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 includes a sense strand and an antisense strand, and each strand has 14 to 40 nucleotides. The sense strand includes at least one thermolabile nucleotide, and at least one of the thermolabile nucleotides is present at a site opposite to or in the vicinity of the seed region of the antisense strand (i.e., positions 2 to 8 at the 5'-end of the antisense strand). For example, when the sense strand is 21 nucleotides in length, the thermolabile nucleotide is present between positions 14 and 17 at the 5'-end of the sense strand. The antisense strand includes two modified nucleic acids that are smaller than the sterically demanding 2'-OMe modification and are separated by 11 nucleotides. For example, the two modified nucleic acids are 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 includes 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 at the 5'-end of the sense strand.

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

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

[0147] 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.

[0148] The present invention further relates to a dsRNA agent as defined herein for use in inhibiting the expression of a target gene in a subject. The subject can be any animal, preferably a mammal, more preferably a 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 includes a sense strand and an antisense strand, and each strand has 14 to 40 nucleotides. The sense strand includes an endonuclease-sensitive modified nucleotide (e.g., DNA, RNA, or 2'-F) near the cleavage site of the sense strand. For example, the endonuclease-sensitive modified nucleotide is at the 11th position at the 5' end of the sense strand. The endonuclease sensitivity modification present near the cleavage site can affect the sensitivity of the cleavage site. For example, a heat destabilizing modification near the cleavage site can provide endonuclease sensitivity to that cleavage site. The antisense strand includes two modified nucleic acids smaller than the highly sterically demanding 2'-OMe modification separated by 11 nucleotides. For example, the two modified nucleic acids are at the 2nd and 14th positions at the 5' end of the antisense strand.

[0150] In another aspect, the present invention further provides a method for delivering the dsRNA agent of the present invention to a specific target of a subject by subcutaneous administration or intravenous administration. The present invention further provides the dsRNA agent of the present invention for use in a method for delivering the dsRNA agent to a specific target of a subject by subcutaneous administration or intravenous administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0151]

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Mode for Carrying Out the Invention

[0152] The inventors have found that when the 2nd and 14th nucleotides at the 5'-end of the antisense strand have 2'-OMe modifications, the gene silencing activity of the dsRNA agent is suppressed. By introducing chemical modifications with a smaller steric bulk than the 2'-OMe modification at specific positions of the antisense strand and / or the sense strand into non-ribose, acyclic, or the 2'-position or equivalent position in the backbone, the dsRNA agent was able to regain its gene silencing activity. The inventors also concluded that when thermally labile nucleotides are introduced into the sense strand at a site opposite to the seed region (i.e., positions 2 to 8 at the 5'-end of the antisense strand) of the antisense strand, the gene silencing activity is improved.

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

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

[0155] The sense strand and the antisense strand form 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 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotide pairs in length.

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

[0157] In one embodiment, the nucleotides in the overhang region of the dsRNA agent of the present invention are each independently, but not limited to, 2'-sugar modifications, such as 2-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyladenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof, and may be modified nucleotides or unmodified nucleotides. For example, TT can be used as 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 target gene sequence or other sequences.

[0158] The 5' or 3' overhangs in 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 contains two nucleotides with phosphorothioate in between, and these two nucleotides may be the same nucleotide or different nucleotides. In one embodiment, the overhang is present at the 3' end of the sense strand, antisense strand, or both strands. In one embodiment, this 3' overhang is present in the antisense strand. In one embodiment, this 3' overhang is present in the sense strand.

[0159] The dsRNA agent of the present invention may contain only one overhang that can enhance the interference activity of dsRNA without affecting the overall stability. For example, the single-stranded overhang is located at the 3' end of the sense strand or the 3' end of the antisense strand. The dsRNA may have a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand), or vice versa. 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 and the 3' end overhang of the antisense strand prefer the guide strand added to 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, the dsRNA agent of the present invention may also have two blunt ends at both ends of the dsRNA duplex.

[0161] In one embodiment, the dsRNA agent of the present invention is a 19-nucleotide-long blunt-ended double-stranded (double ended bluntmer), and the sense strand contains at least one thermally destabilizing nucleotide, and this at least one thermally destabilizing nucleotide is present at or near the site opposite to the seed region of the antisense strand (i.e., positions 2 to 8 at the 5' end of the antisense strand). For example, the thermally destabilizing nucleotide is present between positions 14 and 17 at the 5' end of the sense strand. The antisense strand contains at least two modified nucleic acids smaller than the highly sterically demanding 2'-OMe; preferably, these two modified nucleic acids smaller than the highly sterically demanding 2'-OMe are at positions 2 and 14 at the 5' end of the antisense strand.

[0162] In one embodiment, the dsRNA agent of the present invention is a blunt-ended double-strand of 20 nucleotide lengths. The sense strand contains at least one thermally labile nucleotide, and this at least one thermally labile nucleotide is present at a site opposite to or in the vicinity of the seed region of the antisense strand (i.e., positions 2 to 8 at the 5'-end of the antisense strand). For example, the thermally labile nucleotide is present between positions 14 and 17 at the 5'-end of the sense strand. The antisense strand contains at least two modified nucleic acids that are smaller than 2'-OMe with high steric requirements; preferably, these two modified nucleic acids that are smaller than 2'-OMe with high steric requirements are at positions 2 and 14 at the 5'-end of the antisense strand.

[0163] In one embodiment, the dsRNA agent of the present invention is a blunt-ended double-strand of 21 nucleotide lengths. The sense strand contains at least one thermally labile nucleotide, and this at least one thermally labile nucleotide is present at a site opposite to or in the vicinity of the seed region of the antisense strand (i.e., positions 2 to 8 at the 5'-end of the antisense strand). For example, the thermally labile nucleotide is present between positions 14 and 17 at the 5'-end of the sense strand. The antisense strand contains at least two modified nucleic acids that are smaller than 2'-OMe with high steric requirements; preferably, these two modified nucleic acids that are smaller than 2'-OMe with high steric requirements are at positions 2 and 14 at the 5'-end of the antisense strand.

[0164] In one embodiment, the dsRNA agent of the present invention includes a sense strand of 21 nucleotides (nt) and an antisense strand of 23 nucleotides (nt). The sense strand includes at least one thermally labile nucleotide, and this at least one thermally labile nucleotide is present at a site opposite to or in the vicinity of the seed region of the antisense strand (i.e., positions 2 to 8 at the 5'-end of the antisense strand). For example, when the sense strand is 21 nucleotides in length, the thermally labile nucleotide is present between positions 14 and 17 at the 5'-end of the sense strand. The antisense strand includes at least two modified nucleic acids that are smaller than the highly sterically demanding 2'-OMe; preferably, these two modified nucleic acids that are smaller than the highly sterically demanding 2'-OMe are at positions 2 and 14 at the 5'-end of the antisense strand. One end of the dsRNA is blunt, while the other end includes a 2-nucleotide overhang. Preferably, the 2-nucleotide overhang is at the 3'-end of the antisense strand. Optionally, the dsRNA further includes a ligand (preferably a receptor ligand, i.e., an ASGPR ligand).

[0165] In one embodiment, the dsRNA agent of the present invention includes a sense strand and an antisense strand: The sense strand is 25 to 30 nucleotide residues in length, starting from the nucleotide at the 5'-end (position 1), and positions 1 to 23 of the sense strand include at least 8 ribonucleotides; The antisense strand is 36 to 66 nucleotide residues in length, starting from the nucleotide at the 3'-end, and at least 8 ribonucleotides are at positions where they pair with positions 1 to 23 of the sense strand to form a double strand; At least the nucleotides at the 3'-end of the antisense strand do not pair with the sense strand, and a maximum of 6 consecutive nucleotides at the 3'-end do not pair with the sense strand, thereby forming a 3'-single-stranded overhang of 1 to 6 nucleotides; The 5'-end of the antisense strand includes 10 to 30 consecutive nucleotides that do not pair with the sense strand, thereby forming a single-stranded 5'-overhang of 10 to 30 nucleotides; At least the nucleotides at the 5'-end and 3'-end of the sense strand base pair with the nucleotides of the antisense strand when the sense strand and the antisense strand are aligned to obtain maximum complementarity, thereby forming a substantial double-stranded region between the sense strand and the antisense strand; And when the double-stranded nucleic acid is introduced into mammalian cells, the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the length of the antisense strand so that the expression of the target gene is reduced; And the sense strand includes at least one thermally labile nucleotide, and this at least one thermally labile nucleotide is present at or near a site on the opposite side of the seed region of the antisense strand (i.e., positions 2 to 8 at the 5'-end of the antisense strand). For example, the thermally labile nucleotide is present between positions 14 and 17 at the 5'-end of the sense strand. The antisense strand includes at least two modified nucleic acids that are smaller than the highly sterically demanding 2'-OMe; Preferably, these two modified nucleic acids that are smaller than the highly sterically demanding 2'-OMe are at positions 2 and 14 at the 5'-end of the antisense strand.

[0166] In one embodiment, the dsRNA agent of the present invention comprises a sense strand and an antisense strand. The dsRNA agent comprises a sense strand having a nucleotide length of at least 25 and at most 29 nucleotides, and an antisense strand having a nucleotide length of at most 30 nucleotides. The sense strand contains a modified nucleotide that is easily enzymatically degraded at the 11th position at the 5' end. The antisense strand contains two modified nucleic acids that are smaller than the highly sterically demanding 2'-OMe. These two modified nucleic acids are at the 2nd and 14th positions at the 5' end of the antisense strand; the 3' end of the sense strand and the 5' end of the antisense strand form a blunt end, the antisense strand is 1 to 4 nucleotides longer than the sense strand at its 3' end, the double-stranded region is at least 25 nucleotides long, and when the dsRNA agent is introduced into mammalian cells, the expression of the target gene is reduced. The antisense strand is sufficiently complementary to the target mRNA along at least 19 nucleotides of the length of the antisense strand, and the Dicer cleavage of the dsRNA preferentially occurs in the siRNA containing the 3' end of the antisense strand, thereby reducing the expression of the target gene in mammals. Optionally, the dsRNA agent further comprises a ligand.

[0167] In one embodiment, the sense strand contains a modified nucleotide that is easily enzymatically degraded at the 11th position at the 5' end. The antisense strand contains two modified nucleic acids that are smaller than the highly sterically demanding 2'-OMe. These two modified nucleic acids are at the 2nd and 14th positions at the 5' end of the antisense strand.

[0168] In one embodiment, the antisense strand contains two modified nucleic acids that are smaller than the highly sterically demanding 2'-OMe. These two modified nucleic acids are at the 2nd and 14th positions at the 5' end of the antisense strand.

[0169] In one embodiment, each nucleotide in the sense strand and the antisense strand of the dsRNA agent may be modified. Each nucleotide may be modified with the same or different modifications, which may include one or more alterations of one or both of the non-bridging phosphate oxygen and / or one or more of the bridging phosphate oxygens; alterations of the ribose sugar moiety, e.g., alteration of the 2'-hydroxyl of the ribose sugar; substantial substitution of the phosphate moiety with a "dephospho" linker; modification or substitution of the natural base; and substitution or modification of the ribose-phosphate backbone.

[0170] Because nucleic acids are polymers of subunits, many modifications, e.g., of the base, the phosphate moiety, or the non-bridging O of the phosphate moiety, are present at repeated positions within the nucleic acid. In some cases, the modification is present at all desired positions within the nucleic acid, but in many cases it is not. As an example, the modification may be present only at the 3' or 5' terminal positions, or only in the terminal region, e.g., the position of the terminal nucleotide or only the last 2, 3, 4, 5, or 10 nucleotides of the strand. The modification may be present in double-stranded regions, single-stranded regions, or both. The modification may be present only in the double-stranded region of the RNA, or only in the single-stranded region of the RNA. For example, phosphorothioate modification at the non-bridging oxygen position may be present only at one or both termini, or only in the terminal region, e.g., the position of the terminal nucleotide or only the last 2, 3, 4, 5, or 10 nucleotides of the strand, or alternatively in the double-stranded and single-stranded regions, particularly at the termini. The 5' terminus or both termini may be phosphorylated.

[0171] For example, it may be possible to increase stability, include specific bases in the overhang, or include modified nucleotides or nucleotide surrogates in single-stranded overhangs, such as 5' or 3' overhangs, or both overhangs. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases of the 3' or 5' overhang may be modified, for example, with the modifications described herein. Modifications can include, for example, the use of modifications known in the art for modification at the 2' position of the ribose sugar, such as the use of deoxyribonucleotides instead of ribonucleotides of the nucleobase, 2'-deoxy-2'-fluoro (2'-F), or 2'-O-methyl modification, and modifications of the phosphate group, such as phosphorothioate modification. The overhang need not be homologous to the target sequence.

[0172] In one embodiment, each residue of the sense strand and the 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. The sense strand and the antisense strand can include two or more modifications. In one embodiment, each residue of the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

[0173] Typically, at least two different modifications are present in the sense strand and the antisense strand. These two modifications can be 2'-deoxy, 2'-O-methyl or 2'-fluoro, acyclic nucleic acids, or others.

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

[0175] In one embodiment, each residue of the sense strand and the antisense strand is independently modified with a 2'-O-methyl nucleotide, 2'-deoxynucleotide, 2'-deoxyfluoronucleotide, 2'-O-N-methylacetamide (2'-O-NMA) nucleotide, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotide, 2'-O-aminopropyl (2'-O-AP) nucleotide, or 2'-ara-F nucleotide.

[0176] In one embodiment, the dsRNA agent of the present invention, as shown in Formula I, includes modifications in an alternating pattern, particularly in the B1, B2, B3, B1', B2', B3', B4' regions. As used herein, the terms "alternating motif" or "alternating pattern" refer to a motif having one or more modifications where each modification is present in alternating nucleotides of one strand. Alternating nucleotides can refer to every other nucleotide, 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 can be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AAABBBAAABBB...", or "ABCABCABCABC...", etc.

[0177] The types of modifications included in the alternating motif may be the same or different. For example, if A, B, C, D each represent one type of modification to a nucleotide, the alternating pattern, i.e., the modification of every other nucleotide, may be the same, but the sense strand or the antisense strand can each be selected from several possibilities of internal modifications of the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...", etc.

[0178] In one embodiment, the dsRNA agent of the present invention includes a modification pattern of alternating motifs in the sense strand that is shifted relative to the modification pattern of alternating motifs in the antisense strand. This shift may be a shift such that the modifying groups of the nucleotides of the sense strand correspond to different modifying groups of the nucleotides of the antisense strand, or vice versa. For example, when the sense strand forms a base pair with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand may start with "ABABAB" from the 5' to 3' of the sense strand, and the alternating motif in the antisense strand may start with "BABABA" from the 3' to 5' of the antisense strand within the duplex region. As another example, the alternating motif in the sense strand may start with "AABBAABB" from the 5' to 3' of the sense strand, and the alternating motif in the antisense strand may start with "BBAABBAA" from the 3' to 5' of the antisense strand within the duplex region, so that there is a complete or partial shift in the modification pattern between the sense strand and the antisense strand.

[0179] The dsRNA agent of the present invention may further include at least one phosphorothioate or methylphosphonate internucleotide linkage. The modification of the phosphorothioate or methylphosphonate internucleotide linkage may be present in any nucleotide at any position in the sense strand and / or antisense strand, or both strands. For example, the modification of the internucleotide linkage may be present in any nucleotide of the sense strand and antisense strand; the modification of each internucleotide linkage may be present in an alternating pattern in the sense strand or antisense strand; or the sense strand or antisense strand may include the modification of both internucleotide linkages in an alternating pattern. The alternating pattern of the modification of the internucleotide linkage in the sense strand may be the same as or different from that in the antisense strand, and the alternating pattern of the modification of the internucleotide linkage in the sense strand may have a shift relative to the alternating pattern of the modification of the internucleotide linkage in the antisense strand.

[0180] In one embodiment, the dsRNA agent comprises a modification of the internucleotide linkage of phosphorothioate or methylphosphonate in the overhang region. For example, the overhang region comprises two nucleotides having an internucleotide linkage of phosphorothioate or methylphosphonate between two nucleotides. The modification of the internucleotide linkage can also be formed to bind the overhang nucleotide to the terminal base-pairing nucleotide within the double-stranded region. For example, at least two, three, four, or all of the overhang nucleotides can be linked by an internucleotide linkage of phosphorothioate or methylphosphonate, and optionally, there may be an additional internucleotide linkage of phosphorothioate or methylphosphonate that binds the overhang nucleotide to its next base-pairing nucleotide. For example, there may be at least two phosphorothioate internucleotide linkages between the three terminal nucleotides, two of the three terminal nucleotides are overhang nucleotides, and the third nucleotide is the next base-pairing nucleotide of the overhang nucleotide. Preferably, these three terminal nucleotides can be present at the 3' end of the antisense strand.

[0181] In one embodiment, the sense strand of the dsRNA agent comprises 1 to 10 blocks of 2 to 10 internucleotide linkages of phosphorothioate or methylphosphonate separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, १३, १४, १५, or 16 phosphate internucleotide linkages, one of the internucleotide linkages of phosphorothioate or methylphosphonate is present at any position of the oligonucleotide sequence, and the sense strand pairs with an antisense strand comprising any combination of internucleotide linkages of phosphorothioate, methylphosphonate, and phosphate, or an antisense strand comprising any of the linkages of phosphorothioate, methylphosphonate, or phosphate.

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

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

[0184] In one embodiment, the antisense strand of the dsRNA agent comprises two blocks of five 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, with one of the phosphorothioate or methylphosphonate internucleotide linkages present at any position in the oligonucleotide sequence, and the antisense strand pairs with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising any of phosphorothioate, methylphosphonate, or phosphate linkages.

[0185] In one embodiment, the antisense strand of the dsRNA agent comprises two blocks of five phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate internucleotide linkages, with one of the phosphorothioate or methylphosphonate internucleotide linkages present at any position in the oligonucleotide sequence, and the antisense strand pairs with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising any of phosphorothioate, methylphosphonate, or phosphate linkages.

[0186] In one embodiment, the antisense strand of the 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, with one of the phosphorothioate or methylphosphonate internucleotide linkages being present at any position in the oligonucleotide sequence, and the antisense strand pairs with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising any of the phosphorothioate, methylphosphonate, or phosphate linkages.

[0187] In one embodiment, the antisense strand of the dsRNA agent comprises two blocks of seven phosphorothioate or methylphosphonate internucleotide linkages separated by one, two, three, four, five, six, seven, or eight phosphate internucleotide linkages, with one of the phosphorothioate or methylphosphonate internucleotide linkages being present at any position in the oligonucleotide sequence, and the antisense strand pairs with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising any of the phosphorothioate, methylphosphonate, or phosphate linkages.

[0188] In one embodiment, the antisense strand of the dsRNA agent comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by one, two, three, four, five, or six phosphate internucleotide linkages, with one of the phosphorothioate or methylphosphonate internucleotide linkages being present at any position in the oligonucleotide sequence, and the antisense strand pairs with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising any of the phosphorothioate, methylphosphonate, or phosphate linkages.

[0189] In one embodiment, the antisense strand of the dsRNA agent comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by internucleotide linkages of one, two, three, or four phosphates, with one of the phosphorothioate or methylphosphonate internucleotide linkages present at any position in the oligonucleotide sequence, and the antisense strand pairs with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising any of phosphorothioate, methylphosphonate, or phosphate linkages.

[0190] In one embodiment, the dsRNA agent of the present invention further comprises a modification of one or more phosphorothioate or methylphosphonate internucleotide linkages within the range of 1 to 10 terminal positions of the sense strand and / or the antisense strand. For example, at least two, three, four, five, six, seven, eight, nine, or ten nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkages at one or both ends of the sense strand and / or the antisense strand.

[0191] In one embodiment, the dsRNA agent of the present invention further comprises a modification of one or more phosphorothioate or methylphosphonate internucleotide linkages within the range of 1 to 10 internal regions of each double strand of the sense strand and / or the antisense strand. For example, at least two, three, four, five, six, seven, eight, nine, or ten nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkages in the double-stranded region at positions 8 to 16 counted from the 5' end of the sense strand; the dsRNA agent can optionally further comprise a modification of one or more phosphorothioate or methylphosphonate internucleotide linkages within the range of 1 to 10 terminal positions.

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

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

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

[0195] In one embodiment, the dsRNA agent of the present invention further comprises modifications of two phosphorothioate nucleotide linkages within the range of positions 1 to 5 (counting from the 5'-end) of the sense strand and modifications of two phosphorothioate nucleotide linkages within the range of positions 18 to 23, as well as modifications of one phosphorothioate nucleotide linkage at positions 1 and 2 (counting from the 5'-end) of the antisense strand and modifications of two phosphorothioate nucleotide linkages within the range of positions 18 to 23.

[0196] In one embodiment, the dsRNA agent of the present invention further comprises modifications of two phosphorothioate nucleotide linkages within the range of positions 1 to 5 (counting from the 5'-end) of the sense strand and modifications of two phosphorothioate nucleotide linkages within the range of positions 18 to 23, as well as modifications of one phosphorothioate nucleotide linkage at positions 1 and 2 (counting from the 5'-end) of the antisense strand and modifications of one phosphorothioate nucleotide linkage within the range of positions 18 to 23.

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

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

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

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

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

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

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

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

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

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

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

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

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

[0210] In one embodiment, the dsRNA agent comprises a mismatch with the target, a mismatch within the double strand, or a combination thereof. The mismatch can occur in the overhang region or the double strand region. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., with respect to the free energy of binding or dissociation of a particular pairing, and the simplest approach is to evaluate base pairs on an individual base pair basis, although related or similar analyses can also be used). With respect to promoting dissociation, A:U is preferred over G:C; G:U is preferred over G:C; I:C is preferred over G:C (where I = inosine). Mismatches, e.g., non-canonical or non-standard pairings (described elsewhere in this specification), are preferred over canonical pairings (A:T, A:U, G:C); base pairs containing universal bases are preferred over canonical pairings.

[0211] In one embodiment, the dsRNA agent of the present invention comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the double strand 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-standard pairing, or a pairing containing a universal base, for promoting dissociation of the antisense strand at the 5' end of the double strand.

[0212] In one embodiment, the nucleotide at position 1 within the double strand region from the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pairs within the double strand region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair within the double strand region from the 5' end of the antisense strand is an AU base pair.

[0213] The inventors have found that the introduction of 4'-modifications and / or 5'-modified nucleotides to the 3'-end of phosphodiester (PO), phosphorothioate (PS), and / or phosphorodithioate (PS2) linkages of dinucleotides at any position of single-stranded or double-stranded oligonucleotides can have a steric effect on the internucleotide linkages, and thus protect or stabilize the internucleotide linkages from nucleases.

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

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

[0216] In one embodiment, a 5'-alkylated nucleoside is introduced at any position of the sense or antisense strand of the dsRNA, and such a modification maintains or improves the potency of the dsRNA. The 5'-alkyl can be a racemate or a chirally pure R or S isomer. Exemplary 5'-alkylated nucleosides are 5'-methyl nucleosides. 5'-Methyl can be a racemate or a chirally pure R or S isomer.

[0217] In one embodiment, a 4'-alkylated nucleoside is introduced at any position of the sense or antisense strand of the dsRNA, and such a modification maintains or improves the potency of the dsRNA. The 4'-alkyl can be a racemate or a chirally pure R or S isomer. Exemplary 4'-alkylated nucleosides are 4'-methyl nucleosides. 4'-Methyl can be a racemate or a chirally pure R or S isomer.

[0218] In one embodiment, a 4'-O-alkylated nucleoside is introduced at any position of the sense or antisense strand of the dsRNA, and such a modification maintains or improves the potency of the dsRNA. The 5'-alkyl can be a racemate or a chirally pure R or S isomer. Exemplary 4'-O-alkylated nucleosides are 4'-O-methyl nucleosides. 4'-O-Methyl can be a racemate or a chirally pure R or S isomer.

[0219] In one embodiment, the sense strand sequence of the dsRNA agent is represented by formula (Is):

Chemical formula

[0220] In one embodiment, the sense strand sequence having a length of 19, 20, 21, or 22 nucleotides of the dsRNA agent is represented by the formula (Is):

Chemical formula

[0221] In one embodiment, the dsRNA agent of formula (Is) further comprises a 3' and / or 5' overhang that is 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 thermally labile nucleotide at position 14, 15, 16, or 17 at the 5' end of the sense strand. For example, C1 is an acyclic nucleotide (e.g., UNA or GNA), a mismatch, an abasic site, 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 at the 5' end of the sense strand.

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

[0225] In one embodiment, the antisense strand sequence of the dsRNA agent is represented by formula (Ia):

Chemical formula

[0226] In one embodiment, the antisense strand sequence having a nucleotide length of 19, 20, 21, 22, 23, 24, or 25 of the dsRNA agent is represented by formula (Ia):

Chemical formula

[0227] In one embodiment, the dsRNA of formula (Ia) further comprises 3' and / or 5' overhangs that are 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 the expression of a target gene. This dsRNA agent comprises a sense strand and an antisense strand, and each strand has 14 to 40 nucleotides:

Chemical formula

[0229] In one embodiment, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting the expression of a target gene. This dsRNA agent comprises a sense strand and an antisense strand, and each strand has 14 to 40 nucleotides:

Chemical Formula

[0230] In one embodiment, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting the expression of a target gene. This dsRNA agent includes a sense strand and an antisense strand, and each strand has 15 to 30 nucleotides:

Chemical formula

[0231] In one embodiment, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting the expression of a target gene. This dsRNA agent comprises a sense strand and an antisense strand, and each strand has 19 to 23 nucleotides:

Chemical formula

[0232] In one embodiment, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting the expression of a target gene. This dsRNA agent includes a sense strand and an antisense strand, and each strand has 14 to 40 nucleotides: [Chemical formula] Wherein: 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 is independently 4 to 15 nucleotides in length; n 5 , q 3 , or q 7 is independently 1 to 6 nucleotides in length; n 4 , q 2 , or q 6 is independently 1 to 3 nucleotides in length; alternatively, n 4 is 0; n 2 or q 4 is independently 0 to 3 nucleotides in length: q 5 is independently 0 to 10 nucleotides in length; and this dsRNA agent has a 5’ overhang of 1 to 10 nucleotides in length at the 5’ end of the sense strand.

[0233] In one embodiment, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting the expression of a target gene. The dsRNA agent includes a sense strand and an antisense strand, and each strand has 14 to 40 nucleotides:

Chemical formula

[0234] In one embodiment, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting the expression of a target gene. This dsRNA agent includes a sense strand and an antisense strand, and each strand has 14 to 40 nucleotides:

Chemical formula

[0235] Thermal destabilizing modification The dsRNA agent can optimize RNA interference by introducing a thermolabile modification to the sense strand at a site on the opposite side of the seed region of the antisense strand (i.e., positions 2 to 8 at the 5'-end of the antisense strand), thereby enhancing the dissociation or melting properties of the dsRNA duplex (decreasing the free energy of duplex binding). This modification can enhance the dissociation or melting properties of the duplex in the seed region of the antisense strand.

[0236] Thermolabile 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 (UNA) or glycerol nucleic acids (GNA).

[0237] Exemplary abasic modifications are as follows:

Chemical formula

[0238] Exemplary sugar modifications are as follows:

Chemical formula

[0239] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar in which there is no bond at all between carbons of ribose (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4'), and / or at least one of the carbons or oxygens of ribose (e.g., C1', C2', C3', C4', or O4') is absent from the nucleotide, either alone or in combination. In some embodiments, the acyclic nucleotide is as follows,

Chemical formula

[0240] The term "GNA" refers to glycol nucleic acid, a polymer similar to DNA or RNA but having a different "backbone" composition, consisting of repeating glycerol units linked by phosphodiester bonds:

Chemical formula

[0241] The heat destabilizing modification can be a mismatch (i.e., a non-complementary base pair) between a heat destabilizing nucleotide and the opposing nucleotide in the opposing strand within 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. The mismatch can occur between nucleotides that are either naturally occurring nucleotides or modified nucleotides, i.e., the mismatch base pairing can occur between the nucleobases from each nucleotide independent of the modification in the ribose sugar of the nucleotide. In certain embodiments, the dsRNA agent includes at least one nucleobase that is a 2'-deoxy nucleobase in the mismatch pairing; for example, the 2'-deoxy nucleobase 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 International Publication No. WO 2011 / 133876, the entire contents of which are incorporated herein by reference.

[0243] The heat destabilizing modification can include universal bases and phosphate modifications that have a reduced or lost ability to form hydrogen bonds with opposing bases.

[0244] Nucleobase modifications with a reduced or completely lost ability to form hydrogen bonds with bases in the opposing strand were evaluated for destabilization of the central region of a 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:

Chemical formula

[0245] Exemplary phosphate modifications known to reduce the thermal stability of a dsRNA duplex compared to the native phosphodiester bond are as follows: [Chem.]

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

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

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

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

[0251] Various publications describe multimeric siRNAs, and all such siRNAs can be used together with the dsRNAs of the present invention. Such publications include International Publication No. WO 2007 / 091269, U.S. Patent No. 7,858,769, International Publication No. WO 2010 / 141511, WO 2007 / 117686, WO 2009 / 014887, and WO 2011 / 031520, the entire disclosures of which are incorporated herein by reference.

[0252] A dsRNA agent comprising conjugation to one or more carbohydrate moieties can optimize one or more properties of the dsRNA agent. In many cases, the carbohydrate moiety attaches to a modified subunit of the dsRNA agent. For example, the ribose sugar of one or more ribonucleotide subunits of the dsRNA agent can be replaced with another moiety, such as a non-carbohydrate carrier (preferably cyclic) to which a carbohydrate ligand attaches. A ribonucleotide subunit in which the ribose sugar of the subunit is thus replaced is herein referred to as a ribose substitution modified subunit (RRMS). The cyclic carrier can be a carbocyclic system, i.e., a ring system in which all ring atoms are carbon atoms, or a heterocyclic system, i.e., a ring system in which one or more ring atoms can be heteroatoms, such as nitrogen, oxygen, sulfur. The cyclic carrier can be monocyclic or can include two or more rings, such as fused rings. The cyclic carrier can be a completely saturated ring system or can include one or more double bonds.

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

[0254] In one embodiment, the dsRNA agent of the present invention is conjugated to a ligand via a carrier, and this carrier can be a cyclic group or an 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, 5'-end, or both ends. For example, the ligand can be conjugated to the sense strand, particularly to the 3'-end of the sense strand.

[0256] In one embodiment, the dsRNA agent of the present invention is 5'-phosphorylated or contains a phosphoryl analog at the 5'-end. The 5'-phosphate modification includes modifications compatible with RISC-mediated gene silencing. Suitable modifications include: 5'-monophosphate ((HO)2(O)P-O-5'); 5'-diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-guanosine cap (methylated or non-methylated at the 7-position) (7m-G-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)P-O-5'); 5'-dithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), 5'-phosphorothiolate ((HO)2(O)P-S-5'); oxygen / sulfur-substituted monophosphate, diphosphate, and triphosphate (e.g., 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-phosphoramidate ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'), 5'-alkylphosphonate (R = alkyl = methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)-O-5'-), 5'-alkenylphosphonate (i.e., vinyl, substituted vinyl), (OH)2(O)P-5'-CH2-), 5'-alkyl ether phosphonate (R = alkyl ether = methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5'-), and any further combination thereof. In one example, the modification can be placed on the antisense strand of the dsRNA agent.

[0257] Ligand A wide variety of substances can bind to the oligonucleotide of the present invention. Preferred moieties are ligands that are bound directly or indirectly, preferably covalently, either directly or via an intervening tether.

[0258] In preferred embodiments, the ligand alters the distribution, targeting, or lifetime of the molecule into which the ligand is incorporated. In preferred embodiments, the ligand enhances the affinity for a selected target, such as a molecule, cell or cell type, compartment, receptor, such as a compartment of a cell or organ, tissue, organ, or region of the body, compared to a species in which such ligand is absent. A ligand that enhances the affinity for a selected target is also referred to as a targeting ligand.

[0259] Some ligands may have endosomolytic properties. An endosomolytic ligand promotes the lysis of endosomes and / or the transport of the compositions of the invention or components thereof from the endosome to the cytoplasm of the cell. The endosomolytic ligand may be a polyanionic peptide or peptidomimetic that exhibits pH-dependent membrane activity and fusogenicity. In one embodiment, the endosomolytic ligand is presumed to adopt its active conformational structure at the pH of the endosome. The "active" conformational structure is the conformational structure in which the endosomolytic ligand promotes the lysis of endosomes and / or the transport of the compositions of the invention or components thereof from the endosome to the cytoplasm of the cell. Exemplary endosomolytic ligands include the GALA peptide (Subbarao et al., Biochemistry, 1987, 26:2964 - 2972, the entire contents of which are incorporated herein by reference), the EALA peptide (Vogel et al., J. Am. Chem. Soc., 1996, 118:1581 - 1586, the entire contents of which are incorporated herein by reference), and derivatives thereof (Turk et al., Biochem. Biophys. Acta, 2002, 1559:56 - 68, the entire contents of which are incorporated herein by reference). In one embodiment, the endosomolytic component may include 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 ligand can improve the properties of transport, hybridization, and specificity, and can also improve the nuclease resistance of the resulting natural or modified oligoribonucleotides, or polymeric molecules comprising any combination of monomers and / or natural or modified ribonucleotides described herein.

[0261] The ligand can generally include, for example, therapeutic modifiers for enhancing uptake; diagnostic compounds or reporter groups for monitoring distribution, for example; crosslinking agents; and moieties conferring nuclease resistance. General examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptidomimetics.

[0262] Examples of ligands include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low density lipoprotein (LDL), high density lipoprotein (HDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or lipids. The ligand may be a recombinant molecule or a synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid, an oligonucleotide (e.g., an aptamer). Examples of polyamino acids containing 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 polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudo-peptide-polyamine, peptide-mimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of polyamine, or alpha helix peptide.

[0263] As ligands, there may be mentioned targeting groups, for example, cell targeting agents or tissue targeting agents, such as lectins, glycoproteins, lipids, or proteins, such as antibodies, that bind to specific cell types, such as kidney cells. Targeting groups may be thyrotropin, melanotropin, lectins, glycoproteins, surfactant protein A, mucin glycans, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamic acid salts, polyaspartic acid salts, lipids, cholesterol, steroids, bile acids, folates, vitamin B12, biotin, RGD peptides, RGD peptide mimetics, or aptamers. Table 2 shows some examples of target ligands and their related receptors.

[0264] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinking agents (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, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl) lithocholic acid, O3-(oleoyl) cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphates, aminos, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyaminos, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption promoters (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole cluster, acridine-imidazole conjugate, Eu3+ complex of tetraazamacrocycle), dinitrophenyl, HRP, or AP.

[0265] A ligand can be a molecule having specific affinity for a protein, such as a glycoprotein, or a peptide, such as a co-ligand, or an antibody that binds to a specific cell type such as a cancer cell, an endothelial cell, or an osteocyte. Hormones and hormone receptors can also be mentioned as ligands. Non-peptide species such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, or aptamers can also be mentioned as ligands. A ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

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

[0267] A ligand can increase the uptake of an oligonucleotide into a cell, for example, by activating an inflammatory response. Exemplary ligands that can have such an effect include tumor necrosis factor α (TNF-α), interleukin-1β, or gamma interferon.

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

[0269] Lipid-based ligands can be used to modulate, e.g., control, the binding of the conjugate to the target tissue. For example, lipids or lipid-based ligands that bind more strongly to HSA are less likely to target the kidney and, thus, less likely to be removed from the body. Lipids or lipid-based ligands that do not bind as strongly to HSA can be used such that the conjugate targets the kidney.

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

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

[0272] In another aspect, the ligand is a moiety that is taken up by target cells, e.g., proliferating cells, such as a vitamin. These are particularly useful, for example, in treating disorders characterized by unwanted cell proliferation, such as malignant or non-malignant types, e.g., cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include vitamins 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 aspect, the ligand is a cell permeant, preferably a helical cell permeant. Preferably, the agent is amphiphilic. Exemplary agents are peptides, such as tat or antennapedia. When the agent is a peptide, modifications can be made including peptidomimetics, reverse isomers, non-peptide or pseudo-peptide bonds, and the use of D-amino acids. Preferably, this helical cell permeant is preferably an alpha-helical agent having a lipophilic phase and a lipophobic phase.

[0274] The ligand may be a peptide or a peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule that can fold into a defined three-dimensional structure similar to that of a natural peptide. The peptide portion or peptidomimetic portion may be about 5 to 50 amino acids in length, for example, 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 amphiphilic peptide, or a hydrophobic peptide (e.g., composed mainly of Tyr, Trp, or Phe). The peptide portion may be a dendrimer peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide portion may contain a hydrophobic membrane transport sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having 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 portion may be a "delivery" peptide, which can transport large polar molecules, including peptides, oligonucleotides, and proteins, across the cell membrane. For example, sequences derived from the HIV Tat protein (GRKKRRQRRRPPQ) and the Drosophila antennapedia protein (RQIKIWFQNRRMKWKK) have been shown to function as delivery peptides. The peptide or peptidomimetic, for example, a peptide identified from a phage display library or a one-bead one-compound (OBOC) combinatorial library, may be encoded by a random sequence of DNA (Lam et al., Nature, 354:82-84, 1991, the entire content of which is incorporated herein by reference). Preferably, the peptide or peptidomimetic linked to the iRNA agent via incorporated monomer units is a cell-targeting peptide, for example, an arginine-glycine-aspartic acid (RGD)-peptide or an RGD mimetic. The peptide portion can range from about 5 amino acids in length to about 40 amino acids in length.The peptide moiety may have structural changes, for example, to enhance stability or direct structural properties. Any of the following structural changes can be utilized. The RGD peptide moiety 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 content of which is incorporated herein by reference). The RGD peptide can promote the 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 content of which is incorporated herein by reference). Preferably, the RGD peptide promotes the targeting of iRNA agents to the kidney. The RGD peptide can be linear or cyclic and can be modified, for example, glycosylated or methylated, to promote targeting to a specific tissue. For example, a glycosylated RGD peptide is α. v It is possible to deliver an iRNA agent to tumor cells expressing v α.β3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001, the entire content of which is incorporated herein by reference). Peptides that target markers abundant in proliferating cells can be used. For example, RGD-containing peptides and RGD-containing peptide mimetics can target cancer cells, particularly cells presenting integrin. 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, for example, the oncogenes described herein.

[0275] "Cell-penetrating peptides" can penetrate cells, such as microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. Peptides that penetrate microbial cells can be, for example, α-helical linear peptides (such as LL-37 or seropin P1), disulfide bond-containing peptides (such as α-defensin, β-defensin, or bactericidin), or peptides containing only one or two dominant amino acids (such as PR-39 or indolicidin). The cell-penetrating peptide may also include a nuclear localization signal (NLS). For example, the cell-penetrating peptide may be a bipartite amphipathic peptide derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen, such as MPG (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003, the entire content of which is incorporated herein by reference).

[0276] In one embodiment, the targeting peptide may be an amphiphilic α-helical peptide. Exemplary amphiphilic α-helical peptides include, but are not limited to, cecropin, lycotoxin, paradoxin, bactenecin, CPF, bombinin-like peptide (BLP), cathelicidin, ceratotoxin, S. clava peptide, HFIAP, magainin, brevinin-2, dermaseptin, melittin, pleurocidin, H2A peptide, Xenopus peptide, esculentinis-1, and caerin. Preferably, a number of factors are considered to maintain the integrity of helix stability. For example, the maximum number of helix-stabilizing residues (e.g., leu, ala, or lys) is utilized, and the minimum number of helix-destabilizing residues (e.g., proline, or cyclic monomer units) is utilized. Capping residues are also considered (e.g., Gly is an exemplary N-capping residue and / or C-terminal amidation can be used to achieve additional hydrogen bonds to stabilize the helix). It can be stabilized by the formation of salt bridges between residues with opposite charges separated by i±3 positions, or i±4 positions. Cationic residues, such as lysine, arginine, homo-arginine, ornithine, or histidine, can form salt bridges with anionic residues glutamic acid or aspartic acid.

[0277] Peptide ligands and peptidomimetic ligands include natural peptides or modified peptides, such as D-peptides or L-peptides; α-peptides, β-peptides, or γ-peptides; N-methyl peptides; azapeptides; peptides having one or more amide bonds, i.e., peptide bonds, substituted with one or more urea bonds, thiourea bonds, carbamic acid bonds, or sulfonylurea bonds; or ligands having cyclic peptides.

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

[0279] Examples of endosome release agents include imidazole, poly or oligoimidazole, PEI, peptides, fusogenic peptides, polycarboxylates, polycations, masked oligo or polycations or anions, acetals, polyacetals, ketals / polyketials, orthoesters, polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges.

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

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

[0282] Other ligand conjugates applicable to the present invention are described in U.S. Patent Application No. 10 / 916,185, filed Aug. 10, 2004; U.S. Patent Application No. 10 / 946,873, filed Sep. 21, 2004; U.S. Patent Application No. 10 / 833,934, filed Aug. 3, 2007; U.S. Patent Application No. 11 / 115,989, filed Apr. 27, 2005, and U.S. Patent Application No. 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 have different properties, or some ligands may have the same properties while other ligands have different properties. For example, the ligands may have targeting properties, endosomal activity, or PK modulating properties. In a preferred embodiment, all ligands have different properties.

[0284] The ligand can bind to the oligonucleotide at various positions, such as the 3'-end, 5'-end, and / or internal positions. In a preferred embodiment, the ligand is bound to the oligonucleotide via an intervening tether, such as a carrier described herein. When the monomer is incorporated into the growing chain, the ligand or tethered ligand may be present in this monomer. In some embodiments, the ligand can be incorporated by binding to the "precursor" monomer after the "precursor" monomer has been incorporated into the growing chain. For example, a tether with an amino terminus (i.e., no ligand is attached), such as TAP-(CH2) n NH2, can be incorporated into the growing oligonucleotide chain. Subsequent operations, i.e., after the precursor monomer has been incorporated into the chain, a ligand having an electrophilic group, such as a pentafluorophenyl ester or aldehyde group, can then be bound to the precursor monomer by the binding of the electrophilic group of the ligand to the terminal nucleophilic group of the tether of the precursor monomer.

[0285] In another example, a monomer having a chemical group suitable for participating in a click chemical reaction can be incorporated into a tether / linker with an azide or alkyne terminus, for example. Subsequent operations, i.e., after the precursor monomer has been incorporated into the chain, a ligand having a complementary chemical group, such as an alkyne or azide, can be bound to the precursor monomer by binding both the alkyne and azide together.

[0286] In the case of a double-stranded oligonucleotide, the ligand can bind 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 conjugate to the nucleobase, sugar moiety, or internucleoside linkage of a nucleic acid molecule. Conjugation to a purine nucleobase or a derivative thereof can occur at any position including atoms within the ring and atoms outside the ring. In some embodiments, the 2-, 6-, 7-, or 8-position of the purine nucleobase is attached to the conjugate moiety. Also, conjugation to a pyrimidine nucleobase or a derivative thereof can occur at any position. In some embodiments, the 2-, 5-, and 6-positions of the pyrimidine nucleobase can be substituted with the conjugate moiety. Conjugation to the sugar moiety of a nucleoside can occur at any carbon atom. Examples of carbon atoms of the sugar moiety that can be attached to the conjugate moiety include the 2’, 3’, and 5’ carbon atoms. The 1’ position can also be attached to a conjugate moiety, such as an abasic residue. The internucleoside linkage can also hold the conjugate moiety. In the case of a phosphorus-containing linkage (such as a phosphodiester, phosphorothioate, phosphorodithiotate, and phosphoramidate, etc.), the conjugate moiety can be directly attached to the phosphorus atom or attached to an O, N, or S atom attached to the phosphorus atom. In the case of an internucleoside linkage containing an amine or amide (such as PNA), the conjugate moiety can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

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

[0289] Linkers for conjugating the ligand to the nucleic acid include the linkers 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 present invention conjugates to a divalent and trivalent branched linker containing a structure represented by any of the following formulas (IV) to (VII):

Chemical formula

Chem.

[0291] The trivalent conjugate GalNAc derivatives, such as those of formula (VII), are particularly useful when used with an RNAi agent for inhibiting the expression of a target gene:

Chem.

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

Chem.

Chem.

[0293] Definition As used herein, the terms "dsRNA", "siRNA", and "iRNA agent" are used interchangeably with an agent capable of mediating the silencing of a target RNA, e.g., an mRNA, e.g., a transcript of a gene encoding a protein. For convenience, such an mRNA is also referred to herein as the mRNA to be silenced. Such a gene is also referred to as the target gene. Generally, the RNA to be silenced is an endogenous gene or a pathogenic gene. In addition, RNAs other than mRNA, e.g., 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, silencing is thought to use the RNAi machinery or process and a guide RNA, e.g., an siRNA agent of 21-23 nucleotides.

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

[0296] In one embodiment, the dsRNA agent of the present invention is "sufficiently complementary" to a target RNA, such as a target mRNA, such 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 "perfectly 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 type base pairs in the perfectly complementary region. A "sufficiently complementary" target RNA may include an internal region (e.g., at least 10 nucleotides) that is perfectly complementary to the target RNA. Further, in some embodiments, the dsRNA agent of the present invention specifically discriminates a difference of one nucleotide. In this case, the dsRNA agent mediates RNAi only when perfect complementarity is found in a region with a difference of one nucleotide (e.g., within 7 nucleotides).

[0297] As used herein, the term "oligonucleotide" 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 and is often referred to as restricted or isolated RNA. BNA may include 5-, 6-, and even 7-membered bridged structures having "fixed" C3'-endo sugar puckering. This bridge typically results in 2',4'-BNA nucleotides (e.g., LNA or ENA), including at the 2' and 4' positions of ribose. Examples of BNA nucleotides include the following nucleosides:

Chemical formula

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

Chemical formula

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

[0301] As used herein, "cleavage site" means the backbone linkage in the target gene or sense strand that is cleaved by the RISC mechanism by utilizing an iRNA agent. And the target cleavage site region includes at least one or at least two nucleotides on both sides of the cleavage site. In the case of the sense strand, the cleavage site is the backbone linkage of the sense strand that is to be cleaved if the sense strand itself is the target to be cleaved by the RNAi mechanism. The cleavage site can be determined by methods known in the art, for example, the 5'-RACE assay detailed in Soutschek et al., Nature (2004) 432, 173 - 178, the entire content of which is incorporated herein by reference. As is well understood in the art, the cleavage site region of a conical double stranded RNAi agent containing two strands 21 nucleotides in length (these strands form a double-stranded region of 19 consecutive base pairs with a single-stranded overhang of 2 nucleotides at the 3'-end) corresponds to positions 9 to 12 at 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 a saturated and unsaturated non-aromatic hydrocarbon chain which may be straight or branched, and which may optionally contain carbon atoms with an index number and may optionally insert N, O, or S (including, but not limited to, propyl, allyl, or propargyl). For example, C1~C 10 indicates that the group may have 1 to 10 (inclusive) carbon atoms therein. 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 -O-R-O-, where R represents an alkylene. The term "aminoalkyl" refers to an alkyl substituted with an amino. The term "mercapto" refers to an -SH radical. The term "thioalkoxy" refers to an S-alkyl radical.

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

[0304] As used herein, the term "cycloalkyl" includes saturated and partially saturated cyclic hydrocarbon groups having 3 to 12 carbons, such as 3 to 8 carbons, and, for example, 3 to 6 carbons, and the cycloalkyl group may further be optionally substituted. Examples of 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 1 to 3 heteroatoms in the monocyclic case, 1 to 6 heteroatoms in the bicyclic case, or 1 to 9 heteroatoms in the tricyclic case, said heteroatoms being selected from O, N, or S (e.g., 1 to 3, 1 to 6, or 1 to 9 heteroatoms of carbon atoms and N, O, or S respectively in the monocyclic, bicyclic, or tricyclic cases), and 0, 1, 2, 3, or 4 atoms of each ring may be substituted with substituents. Examples of heteroaryl groups include pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolinyl, and thiazolyl, among others. The term "heteroarylalkyl" or "heteroaralkyl" refers to an alkyl substituted with heteroaryl. The term "heteroarylalkoxy" refers to an alkoxy substituted with 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 1 to 3 heteroatoms in the monocyclic case, 1 to 6 heteroatoms in the bicyclic case, or 1 to 9 heteroatoms in the tricyclic case, said heteroatoms being selected from O, N, or S (e.g., 1 to 3, 1 to 6, or 1 to 9 heteroatoms of carbon atoms and N, O, or S respectively in the monocyclic, bicyclic, or tricyclic cases), and 0, 1, 2, or 3 atoms of each ring may be substituted with substituents. Examples of heterocyclyl groups include trizolyl, tetrazolyl, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, and tetrahydrofuranyl, among others.

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

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

[0309] The term "substituted" refers to the replacement of one or more hydrogen radicals in a given structure with the radical of a specific substituent, which specific substituents include, but are 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 to be understood that the substituents may be further substituted.

[0310] Cleavable linking group A cleavable linking group is a linking group that is sufficiently stable extracellularly but is cleaved upon entry into the target cell to release the two moieties that the linker holds together. In a preferred embodiment of the dsRNA agent according to the present invention, the cleavable linking group cleaves at least 10-fold faster, preferably at least 100-fold faster, intracellularly or under a first reference condition (e.g., a condition that can be selected to mimic or achieve intracellular conditions) than in the blood of the subject or under a second reference condition (e.g., a condition that can be selected to mimic or achieve conditions present in blood or serum).

[0311] Cleavable linking groups are susceptible to the effects of cleaving agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleaving agents are present at more widespread, or higher levels or activities, intracellularly rather than in serum or blood. Examples of such degrading agents include redox agents that can cleave a redox-cleavable linking group by reduction, such as oxidases or reductases or reducing agents, such as mercaptans, present intracellularly, which are selected for, or have substrate specificity for, a particular substrate; esterases; endosomes or agents that create an acidic environment, such as a pH of 5 or less; general acids, peptidases (which may be substrate specific), and enzymes that can hydrolyze or degrade an acid-cleavable linking group by functioning as phosphatases.

[0312] Cleavable linking groups, such as disulfide bonds, are susceptible to the effects of pH. The pH of human serum is 7.4, but the average intracellular pH is slightly lower, in the range of about 7.1 - 7.3. Endosomes have a more acidic pH in the range of 5.5 - 6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers are cleaved at a preferred pH, thereby releasing a cationic lipid from a ligand into the cell or within a desired compartment of the cell, having a cleavable linking group.

[0313] Linkers may contain cleavable linking groups that are cleavable by specific enzymes. The type of cleavable linking group incorporated into a linker can vary depending on the target cell. For example, a ligand targeting the liver can be linked to a cationic lipid via a linker containing an ester group. Hepatocytes are rich in esterases, and thus, the linker is cleaved more efficiently in hepatocytes than in cell types that are not rich in esterases. Other cell types rich in esterases include cells of the lung, renal cortex, and testis.

[0314] When targeting cell types rich in peptidases, such as hepatocytes and synoviocytes, a linker containing a peptide bond can be used.

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

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

[0317] Phosphate-based cleavable linking group Phosphate-based cleavable linkers can be used in the dsRNA agents according to the present invention and are cleaved by agents that decompose or hydrolyze phosphate groups. As an example of an agent that cleaves phosphate groups in cells, intracellular enzymes such as phosphatases can be mentioned. Examples of phosphate-based linkers include -O-P(O)(ORk)-O-, -O-P(S)(ORk)-O-, -O-P(S)(SRk)-O-, -S-P(O)(ORk)-O-, -O-P(O)(ORk)-S-, -S-P(O)(ORk)-S-, -O-P(S)(ORk)-S-, -S-P(S)(ORk)-O-, -O-P(O)(Rk)-O-, -O-P(S)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(S)(Rk)-O-, -S-P(O)(Rk)-S-, -O-P(S)(Rk)-S-. Preferred embodiments are -O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S-P(O)(OH)-O-, -O-P(O)(OH)-S-, -S-P(O)(OH)-S-, -O-P(S)(OH)-S-, -S-P(S)(OH)-O-, -O-P(O)(H)-O-, -O-P(S)(H)-O-, -S-P(O)(H)-O-, -S-P(S)(H)-O-, -S-P(O)(H)-S-, -O-P(S)(H)-S-. A preferred embodiment is -O-P(O)(OH)-O-. These candidates can be evaluated using methods similar to the above methods.

[0318] Acid-cleavable linker An acid-cleavable linking group can be used in the dsRNA agent according to the present invention and is a linking group that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linking group is cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0, or less), or by an agent that can function as a general acid, such as an enzyme agent. In cells, certain acidic organelles with a low pH, such as endosomes and lysosomes, can provide a cleavage environment for the acid-cleavable linking group. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and amino acid esters. The acid-cleavable group can have the general formula: -C=NN-, C(O)O, or -OC(O). A preferred embodiment is when the carbon bonded 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 a method similar to the above method.

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

[0320] Peptide-based cleavage linking group Peptide-based cleavable linkers can be used in the 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 that result in oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable linkers do not contain an amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids that results in peptides and proteins. Peptide-based cleavage groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids that result in peptides and proteins and do not include the entire amide functional group. Peptide-based cleavable linkers generally have the general formula: -NHCHR A C(O)NHCHR B C(O)-, where R A and R B are the R groups of two adjacent amino acids. These candidates can be evaluated using a method similar to the above method. As used herein, "carbohydrate" refers to the carbohydrate itself formed from one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic) with an oxygen atom, nitrogen atom, or sulfur atom bonded to each carbon atom; or a compound having as part of it a carbohydrate moiety formed from one or more monosaccharide units each having at least six carbon atoms (which may be linear, branched, or cyclic) with an oxygen atom, nitrogen atom, or sulfur atom bonded to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4 to 9 monosaccharide units), as well as polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Specific monosaccharides include C5 and the above (preferably C5-C8) sugars; disaccharides and trisaccharides include sugars having two or three monosaccharide units (preferably C5-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 further relates to the use of a dsRNA agent as defined herein for use in inhibiting the expression of a target gene in a subject. The subject can be any animal, such as a mammal, such as a mouse, rat, sheep, cow, dog, cat, or human.

[0323] In one embodiment, the dsRNA agent of the present invention is administered by dissolving it in a buffer.

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

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

[0326] The siRNA preparation can be formulated in combination with another agent, such as another therapeutic agent, or an agent that stabilizes the siRNA, such as a protein that forms a complex with the siRNA to form an iRNP. Other agents include chelating agents, such as EDTA (e.g., for removing divalent cations, such as Mg 2+ ), salts, and RNase inhibitors (e.g., broad - specificity RNase inhibitors, such as RNasin).

[0327] In one embodiment, the siRNA preparation includes another siRNA compound, such as a second siRNA capable of mediating RNAi against a second gene or the same gene. Other preparations may include at least 3, 5, 10, 20, 50, or 100 or more different siRNA species. Such siRNAs can mediate RNAi against a similar number of different genes.

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

[0329] Exemplary formulations that can be used for the administration of the dsRNA agents according to the present 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, these formulations, compositions, and methods can be practiced with other siRNA compounds, such as modified siRNAs, and it will be understood that such practice is within the scope of the invention. siRNA compounds, such as double-stranded siRNA compounds, or ssiRNA compounds, (e.g., precursors, such as larger siRNA compounds that can be processed into ssiRNA compounds, or siRNA compounds, such as DNA encoding double-stranded siRNA compounds, or ssiRNA compounds, or their precursors) preparations can be formulated into membrane molecular assemblies, such as liposomes or micelles, for delivery. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids consisting of at least one bilayer, e.g., one bilayer or multiple bilayers. Liposomes include unilamellar vesicles and multilamellar vesicles having a membrane formed from lipophilic materials and an aqueous interior. The aqueous portion contains the siRNA composition. The lipophilic material separates the aqueous interior from the aqueous exterior, which typically does not contain the siRNA composition but may in some cases. Liposomes are useful for the transfer and delivery of the active ingredient to the site of action. Since the liposome membrane is structurally similar to the biological membrane, when the liposome contacts the tissue, the liposome bilayer fuses with the bilayer of the cell membrane. As the integration of the liposome and the cell progresses, the internal aqueous component containing the siRNA is delivered into the cell, where the siRNA can specifically bind to the target RNA and mediate RNAi. In some cases, the liposomes are specifically targeted, e.g., to direct the siRNA to a particular cell type.

[0331] Liposomes containing siRNA can be prepared in various ways. In one example, the lipid component of the liposome is dissolved in a detergent, whereby micelles are formed from the lipid component. For example, the lipid component can be an amphiphilic cationic lipid or a lipid conjugate. The detergent can have a high critical micelle concentration and can be nonionic. Exemplary detergents include cholate, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. The siRNA preparation is then added to the micelles containing the lipid component. The cationic groups on the lipid interact with the siRNA and condense around the siRNA to form liposomes. After condensation, the detergent is removed, for example, by dialysis, to obtain a liposome preparation of siRNA.

[0332] Optionally, a carrier compound that aids condensation can be added during the condensation reaction, for example, by controlled addition. For example, the carrier compound can be a polymer other than nucleic acid (e.g., 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 a structural component of the delivery vehicle are described in WO 96 / 37194. Liposome formation may also include one or more aspects of the exemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No. 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 728:339, 1983; and Fukunaga, et al. Endocrinol. 115:757, 1984, the entire contents of which are incorporated herein by reference. Techniques commonly used to prepare lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thawing and extrusion (see, for example, Mayer, et al. Biochim. Biophys. Acta 858:161, 1986, the entire contents of which are incorporated herein by reference). Microfluidization can be used if consistently small (50-200 nm) relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984, the entire contents of which are incorporated herein by reference). Such methods are readily adaptable for packaging siRNA formulations into liposomes.

[0334] pH-sensitive or negatively charged liposomes do not form a complex with nucleic acid molecules, but rather incorporate them. Since both the nucleic acid molecules and the lipids are similarly charged, repulsion occurs rather than complex formation. Nevertheless, some nucleic acid molecules are incorporated into the aqueous interior of these liposomes. pH-sensitive liposomes are used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the foreign gene was detected in the 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 mainly 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] Examples of other methods for introducing liposomes into cells in vitro include U.S. Patent Nos. 5,283,185; 5,171,678; International Publication Nos. 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 liposomes are used. Cationic liposomes have the advantage of being able to fuse with cell membranes. Non-cationic liposomes cannot efficiently fuse with the plasma membrane, but can be taken up by macrophages in vivo and thus can be used to deliver siRNA to macrophages.

[0338] Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate various water-soluble and lipid-soluble drugs; liposomes can protect the 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 charge on the lipid surface, vesicle size, and the aqueous volume of the liposomes.

[0339] Small liposomes can be formed using the positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), which interacts spontaneously with nucleic acids to form lipid-nucleic acid complexes that can fuse with the negatively charged lipids of the cell membranes of tissue culture cells and as a result deliver siRNA (see, for example, Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987, which is hereby incorporated by reference in its entirety, and U.S. Patent No. 4,897,355, which describes the use of DOTMA and its complexes with DNA).

[0340] A DOTMA analog, 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP), can be used in combination with a phospholipid to form vesicles that form complexes with DNA. Lipofectin (trademark; 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 interact spontaneously 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 adhere spontaneously to negatively charged cell surfaces, fuse with the plasma membrane, and deliver functional nucleic acids effectively, for example, to tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonio)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Indiana), differs from DOTMA in that the oleoyl moieties are joined by esters rather than ether linkages.

[0341] Other reported cationic lipid compounds include those conjugated to one of two types of lipids, for example, to various moieties including carboxyspermine conjugated to a compound such as 5-carboxyspermidine dioctaoleylamide ("DOGS") (Transfectam (trademark), Promega, Madison, Wis.,) and dipalmitoylphosphatidylethanolamine 5-carboxyspermidine-amide ("DPPES") (see, for example, U.S. Patent No. 5,171,678).

[0342] Another cationic lipid conjugate contains a lipid derivative by cholesterol (``DC-Chol'') formulated to be included within liposomes in combination with DOPE (see Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylisine 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 conjugate cationic lipids are said to exhibit lower toxicity than DOTMA-containing compositions and achieve more efficient transfection. 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 International Publication Nos. 98 / 39359 and 96 / 37194.

[0343] Liposomal formulations are particularly suitable for topical administration, and liposomes have several advantages over other formulations. Such advantages include a reduction in side effects associated with high systemic absorption of the administered drug, an increase in the accumulation of the administered drug at the desired target, and the ability to administer siRNA to the skin. In some embodiments, liposomes are also used to deliver siRNA to epithelial cells and to promote the penetration of siRNA into skin tissue, such as the skin. For example, liposomes can be applied topically. Topical delivery of drugs formulated as liposomes has been demonstrated (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R.J. 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 Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987, the entire contents of which are incorporated herein by reference).

[0344] Nonionic liposomal systems have also been tested to determine their utility in the delivery of drugs to the skin, particularly in systems containing nonionic surfactants and cholesterol. Nonionic liposomal formulations containing Novasome I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) have been 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 may enable the liposomes to enter pores smaller than the average radius of the liposomes. For example, transferosomes are one type of deformable liposome. Transferosomes can be prepared by adding a surface activating factor, usually a surfactant, to a standard liposome composition. Transferosomes containing siRNA can be delivered, for example, by subcutaneous injection, to deliver the siRNA to keratinocytes in the skin. To pass through intact mammalian skin, lipid vesicles must pass through a series of micropores each having a diameter of less than 50 nm under the influence of an appropriate transdermal gradient. In addition, due to the properties of the lipids, such transferosomes can be self-optimizing (e.g., adapting to the shape of the pores in the skin), self-healing, and in many cases can reach the target without fragmentation and can often be self-loading.

[0346] Other formulations applicable to the present invention are described in U.S. Provisional Patent Application No. 61 / 018,616, filed on January 2, 2008; U.S. Provisional Patent Application No. 61 / 018,611, filed on January 2, 2008; U.S. Provisional Patent Application No. 61 / 039,748, filed on March 26, 2008; U.S. Provisional Patent Application No. 61 / 047,087, filed on April 22, 2008; and U.S. Provisional Patent Application No. 61 / 051,528, filed on May 8, 2008. The formulations applicable to the present invention are also described in the international application PCT / US2007 / 080331 pamphlet, filed on October 3, 2007.

[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 practiced with other siRNA compounds, such as modified siRNA compounds, and that such practice is within the scope of the present invention. Surfactants have found wide use 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 include surfactants. In one embodiment, the siRNA is formulated as an emulsion comprising a surfactant. The most common way to classify and rank the properties of a number of different types of both natural and synthetic surfactants is by use of the hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group serves as the most useful means for classifying the different surfactants used in formulations (Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker, Inc., New York, NY, 1988, p. 285).

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

[0349] When surfactant molecules have a negative charge when dissolved or dispersed in water, these surfactant molecules are classified as anionic. Examples of anionic surfactants include carboxylates such as soaps, acyl lactates, acylamides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates, and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are alkyl sulfates and soaps.

[0350] When surfactant molecules have a positive charge when dissolved or dispersed in water, these surfactant molecules are classified as cationic. Examples of cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most used members of this class.

[0351] When surfactant molecules have the ability to have either a positive or negative charge, these surfactant molecules are classified as amphoteric. Examples of amphoteric surfactants include acrylic acid derivatives, substituted alkyl amides, N-alkyl betaines, and phosphatides.

[0352] The use of surfactants in pharmaceutical products, formulations, and emulsions is reviewed (Rieger, in “Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

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

[0354] A mixed micelle formulation suitable for delivery through a transdermal membrane is an aqueous solution of an siRNA composition and an alkali metal C8-C 22It can be prepared by mixing an alkyl sulfate and a micelle-forming compound. Exemplary micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleate, monolaurate, rucic acid oil, evening primrose oil, menthol, trihydroxyoxocholanyl glycine and its pharmaceutically acceptable salts, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ether and its analogs, polydecanol alkyl ether and its analogs, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle-forming compound can be added simultaneously with or after the alkali metal alkyl sulfate. Mixed micelles are formed by mixing substantially any kind of components, but vigorous mixing is carried out to provide smaller-sized micelles.

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

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

[0357] To deliver the micelle formulation as a spray, the formulation can be placed in an aerosol dispenser and the dispenser can be filled with a propellant. The propellant under pressure is in liquid form within the dispenser. The component ratios are adjusted such that the aqueous phase and the propellant phase become one, i.e., only one phase exists. If two phases are present, it is necessary to shake the dispenser, for example, before dispensing a portion of the contents by means of a metering valve. The dispensed amount of the pharmaceutical is sprayed in a fine mist from the metering valve.

[0358] Examples of 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 experiments. For absorption from the oral cavity, it is often desirable to increase the dosage of administration by injection or via the gastrointestinal tract, for example, at least two or three times.

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

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

[0362] The pharmaceutical composition can be specially formulated to be administered in solid or liquid form, including but not limited to the following forms of administration: (1) oral administration, such as aqueous or non-aqueous solutions or suspensions, tablets, e.g., buccal, sublingual, and enteric-coated tablets, boluses, powders, granules, and pastes for application to the tongue; (2) parenteral administration, such as subcutaneous injection, intramuscular injection, intravenous injection, or epidural injection, as a sterile solution or suspension, or as a sustained-release formulation; (3) topical application, such as creams, ointments, or controlled-release patches or sprays applied to the skin; (4) vaginal or rectal administration, such as pessaries, creams, and foams; (5) sublingual administration; (6) intraocular administration; (7) transdermal administration; or (8) intranasal administration. Delivery using subcutaneous or infusion methods can be particularly advantageous.

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

[0364] As used herein, the term "pharmaceutically acceptable" is used to refer to compounds, materials, compositions, and / or dosage forms that are within the scope of sound medical judgment and are suitable for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic reaction, or other problems or complications, at a reasonable benefit / risk ratio.

[0365] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in the transport or delivery of the 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., lubricants, talc, magnesium, calcium stearate, zinc stearate, or steric acid), or a solvent for encapsulating the material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious 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 carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium state, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as polyethylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffering solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) fillers, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; and (22) other non-toxic compatible substances utilized in pharmaceutical formulations.

[0366] This formulation can be conveniently provided in unit dosage form and can be prepared by any method well known in the pharmaceutical art. The amount of the active ingredient that can be combined with the carrier material to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration. The amount of the active ingredient that can be combined with the carrier material to produce a single dosage form is generally the amount of the compound that will achieve a therapeutic effect. Generally, out of 100%, this amount ranges from about 0.1% to about 99% of the active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0367] In certain embodiments, excipients selected from the group consisting of cyclodextrin, cellulose, liposomes, micelle-forming agents such as bile acids, and polymeric carriers such as polyesters and polyanhydrides; and the compounds of the present invention are included in the formulations of the present invention. In certain embodiments, the above-described formulations enable oral administration of the compounds of the present invention.

[0368] The iRNA agent preparation can be formulated in combination with another agent, such as another therapeutic agent, or an agent that stabilizes the iRNA, such as a protein that forms a complex with the iRNA to form an iRNP. Still other agents include chelating agents such as EDTA (for example, for removing divalent cations such as Mg 2+ ), salts, and RNase inhibitors (such as nonspecific RNase inhibitors such as RNasin).

[0369] The method for preparing these formulations or compositions includes the step of combining the compounds of the present invention with a carrier and optionally one or more auxiliary components. Generally, this composition is prepared by uniformly and intimately binding the compounds of the present invention with a liquid carrier, a finely divided solid carrier, or both, and then shaping the product as necessary.

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

[0371] The compounds according to the invention can be formulated for administration by any convenient method used for drugs for humans or animals, depending on their similarity to other pharmaceuticals.

[0372] The term "treatment" is to include prophylaxis, therapy, and cure. The patients to be treated are any animals in need of treatment, including primates, particularly humans, and other mammals such as horses, cows, pigs, and sheep; as well as common poultry and pets.

[0373] Double-stranded RNAi agents are produced in cells in vivo, for example, by an exogenous DNA template delivered to the cells. For example, the DNA template can be inserted into a vector and used as a gene therapy vector. The gene therapy vector can be delivered to a subject, for example, by intravenous injection, topical administration (U.S. Patent 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). A pharmaceutical preparation of the gene therapy vector can contain the gene therapy vector in an acceptable diluent, or can contain a sustained-release matrix in which the gene delivery vehicle is embedded. For example, the DNA template can contain two transcription units, one transcription unit producing a transcript containing the upper strand of the dsRNA agent and the other transcription unit producing a transcript containing the lower strand of the dsRNA agent. When the template is transcribed, a dsRNA agent is produced and processed into fragments of the siRNA agent that mediate gene silencing.

[0374] Delivery routes The dsRNA agents as defined herein or pharmaceutical compositions containing the dsRNA agents as defined herein can be delivered to a subject using different delivery routes. Compositions containing 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 a pharmaceutical composition suitable for administration. Such compositions typically include one or more iRNAs and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any solvent, dispersion medium, coating, antibacterial agent, antifungal agent, isotonic agent, absorption delaying agent, and the like that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The use of any conventional media or agent is contemplated in the composition, except where it is incompatible with the active compound. Auxiliary 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 on the area to be treated. Administration can be local (including ocular, vaginal, rectal, nasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intratracheal or intracerebroventricular administration.

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

[0378] Dosage In one aspect, the present invention addresses a method of administering a dsRNA agent, such as an siRNA agent, to a subject (e.g., a human subject). In another aspect, the present invention relates to a dsRNA agent as defined herein for use in inhibiting the expression of a target gene in a subject. This method or medical use involves administering a unit dose of a dsRNA agent, such as an siRNA agent, such as 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., exogenous or pathogen target RNA), and optionally (c) includes at least one 3' overhang of 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 is less than 200 nmol of RNA agent (e.g., about 4.4×10 16 copies) per kg of body weight, or less than 1500 nmol, less than 750 nmol, less than 300 nmol, less than 150 nmol, less than 75 nmol, less than 15 nmol, less than 7.5 nmol, less than 1.5 nmol, less than 0.75 nmol, less than 0.15 nmol, less than 0.075 nmol, less than 0.015 nmol, less than 0.0075 nmol, less than 0.0015 nmol, less than 0.00075 nmol, or less than 0.00015 nmol of RNA agent per kg of body weight.

[0379] The specified amount can be an amount effective to treat or prevent a disease or disorder, such as a disease or injury associated with the target RNA. The unit dose can be administered, for example, by injection (e.g., intravenous, subcutaneous, or intramuscular), inhalation, or topical application. In some embodiments, the dose can be less than 10 mg, less than 5 mg, less than 2 mg, less than 1 mg, or less than 0.1 mg per kg of body weight.

[0380] In some embodiments, the unit dose is administered less than once a day, such as less than once every two days, less than once every four days, less than once every eight days, or less than once every thirty days. In another embodiment, the unit dose is not administered at regular intervals (e.g., not at a periodic frequency). For example, the unit dose can be administered once.

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

[0382] In one embodiment, a subject is administered a dsRNA agent, such as 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, such as an siRNA agent, or a precursor thereof) in a loading amount and one or more maintenance amounts. The one or more maintenance amounts can be the same as the loading amount or less than the loading amount, e.g., half of the loading amount. Maintenance therapy can include treating the subject using one or more doses in the range of 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. The maintenance amount can be administered, for example, no more than once every two days, no more than once every five days, no more than once every ten days, or no more than once every thirty days. Further, the treatment method can continue for a certain period, which can vary depending on the nature of the specific disease, its severity, and the overall condition of the patient. In certain embodiments, the dose can be administered no more than once a day, e.g., once every 24 hours, 36 hours, 48 hours, or more, e.g., once every five days or eight days. After treatment, the patient can be monitored for changes in the patient's condition and alleviation of the symptoms of the disease state. The dose of the compound can be increased if the patient does not respond significantly to the current dose level, or decreased if alleviation of the symptoms of the disease state is observed, if the disease state is alleviated, or if undesired side effects are observed.

[0383] The effective amount can be administered in a single or multiple administrations, as needed or when considered appropriate under specific circumstances. If it is desired to facilitate repeated or frequent infusions, the implantation of a delivery device, such as a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracapsular, or intra-articular), or reservoir can be proposed.

[0384] In one embodiment, the composition comprises a plurality of dsRNA agent species. In another embodiment, the dsRNA agent species have sequences that do not overlap or adjoin another species with respect to the native target sequence. In another embodiment, the plurality of dsRNA agent species are specific for different native target genes. In another embodiment, the dsRNA agent is allele-specific.

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

[0386] In one embodiment, administration of the dsRNA agent, e.g., siRNA agent, composition is parenteral, e.g., intravenous (e.g., as a bolus or as a continuous infusion), intradermal, intraperitoneal, intramuscular, subarachnoid, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, nasal, urethral, or intraocular. Administration can be performed by the subject or by another human, e.g., a healthcare provider. Pharmaceutical treatment can be performed at a measured dose or with a dispenser that delivers a measured dose. The delivery method selected is 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 the dsRNA agents of the invention used in the methods described above.

[0389] Method for inhibiting the expression of a target gene Embodiments of the present invention also relate to a method of inhibiting the expression of a target gene. This method includes the step of administering an amount of a dsRNA agent in any of the foregoing embodiments sufficient to inhibit the expression of the target gene. The present invention further relates to the use of a dsRNA agent as defined herein for inhibiting the expression of a target gene in a target cell. In a preferred embodiment, the present invention further relates to the use of a dsRNA agent for inhibiting the expression of a target gene in a target cell in vitro.

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

[0391] In certain embodiments, the present invention relates to a dsRNA agent of the present invention used in the above method.

[0392] The present invention is further illustrated by the following examples, which should not be construed as further limitations. The disclosure of all references, pending patent applications, and published patents mentioned in this application are hereby 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 in RPMI (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) in a 5% CO2 atmosphere at 37°C until almost confluent, and then detached from the plate by trypsin treatment. Transfection was performed by adding 14.8 μl of Opti-MEM and 0.2 μl of Lipofectamine RNAiMax (Invitrogen, Carlsbad CA, cat#13778-150) to each well containing 5 μl of siRNA duplex in a 96-well plate and incubating at room temperature for 15 minutes. 80 μl of antibiotic-free complete growth medium containing approximately 2×104 Hep3B cells was added to the siRNA mixture. The cells were incubated for 24 hours or 120 hours, and then the RNA was purified. 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 at the maximum dose of a final duplex concentration of 10 nM.

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

[0395] cDNA synthesis was performed using the ABI High Performance cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, Cat#4368813): For each reaction, a master mix containing 1 μl of 10× buffer, 0.4 μl of 25× dTNP, 1 μl of random primer, 0.5 μl of reverse transcriptase, 0.5 μl of RNase inhibitor, and 1.6 μl of H2O was added to 5 μl of total RNA. cDNA was prepared using a Bio-Rad C-1000 or S-1000 thermal cycler with steps of holding at 25 °C for 10 minutes, 37 °C for 120 minutes, 85 °C for 5 seconds, and then holding at 4 °C.

[0396] Real-time PCR 2 μl 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) in each 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 stated, each duplex was tested in at least two separate transfections, and each transfection was assayed in duplicate.

[0397] To calculate the relative fold change, real-time data was analyzed using the ΔΔCt method and normalized to assays performed on cells transfected with 10 nM AD-1955 or mock-transfected cells. IC 50 was calculated using a four-parameter fit model using XLFit and normalized to cells transfected with AD-1955 or untreated cells for the same dose range or its own minimum dose. IC 50 was calculated for individual transfections and combinations, and one IC 50 was fit to the data from both transfections.

[0398] The results of gene silencing of exemplary siRNA duplexes having various motif modifications of the present invention are shown in the following table.

[0399] Example 2. RNA Synthesis and Duplex Annealing 1. Oligonucleotide Synthesis: All oligonucleotides were synthesized using an AKTA oligopilot synthesizer or an ABI 394 synthesizer. Unless otherwise noted, oligonucleotide synthesis was performed using a commercially available glass solid support with controlled pores (dT-CPG, 500 Å, Prime Synthesis), and RNA phosphoramidites with standard protecting groups, 5'-O-dimethoxytrityl N6-benzoyl-2'-t-butyldimethylsilyl-adenosine-3'-O-N,N'-diisopropyl-2-cyanoethyl phosphoramidite, 5'-O-dimethoxytrityl-N4-acetyl-2'-t-butyldimethylsilyl-cytidine-3'-O-N,N'-diisopropyl-2-cyanoethyl phosphoramidite, 5'-O-dimethoxytrityl-N2-isobutryl-2'-t-butyldimethylsilyl-guanosine-3'-O-N,N'-diisopropyl-2-cyanoethyl phosphoramidite, and 5'-O-dimethoxytrityl-2'-t-butyldimethylsilyl-uridine-3'-O-N,N'-diisopropyl-2-cyanoethyl phosphoramidite (Pierce Nucleic Acids Technologies). 2'-F phosphoramidites, 5'-O-dimethoxytrityl-N4-acetyl-2'-fluoro-cytidine-3'-O-N,N'-diisopropyl-2-cyanoethyl-phosphoramidite and 5'-O-dimethoxytrityl-2'-fluoro-uridine-3'-O-N,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 / ANC (v / v). A coupling / recycling time of 16 minutes 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] The ligand conjugate chain was synthesized using a solid support containing the corresponding ligand. For example, the introduction of the carbohydrate moiety / ligand (e.g., for GalNAc) at the 3'-end of the sequence was achieved by starting the synthesis with the corresponding carbohydrate solid support. Similarly, the cholesterol moiety at the 3'-end was introduced by starting the synthesis with a cholesterol support. Generally, the ligand moiety was linked to trans-4-hydroxyproline via a tether selected from those described in the foregoing examples to obtain a hydroxyproline-ligand moiety. The hydroxyproline-ligand moiety was then either coupled to the solid support via a succinic acid linker or converted to a phosphoramidite under standard phosphitylation conditions to obtain the desired carbohydrate conjugate component. Fluorophore-labeled siRNA was synthesized with the corresponding phosphoramidite or solid support purchased from Biosearch Technologies. An oleyl lithocholic acid (GalNAc)3 polymer support was prepared in-house with a loading of 38.6 μmol / gram. A 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., at the 5' end of the array, was achieved by coupling the corresponding phosphoramidite to the growing chain under standard phosphoramidite coupling conditions, unless otherwise stated. A long coupling of 15 minutes with a 0.1 M phosphoramidite solution in anhydrous CH3CN in the presence of 5-(ethylthio)-1H-tetrazole activating substance to the solid-bound oligonucleotide was performed. Oxidation of internucleotide phosphites to phosphates was carried out using standard aqueous iodine as reported in (1), or by treatment of the conjugate oligonucleotide with tert-butyl hydroperoxide / acetonitrile / water (10:87:3) with a 10-minute oxidation waiting time. Phosphorothioates were introduced by oxidation of phosphites to phosphorothioates using a sulfur transfer reagent, 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 of cholesterol phosphoramidite was set to 16 minutes.

[0402] 2. Deprotection-I (Nucleobase Deprotection) After completion of the synthesis, the support was transferred to a 100 ml glass bottle (VWR). Using a mixture of 80 mL of ethanol ammonia [ammonia:ethanol (3:1)], the bases and phosphate groups were deprotected and the oligonucleotide was cleaved from the support at 55 °C for 6.5 hours. The bottle was cooled briefly on ice, then the ethanol ammonia mixture was filtered and placed in a new 250 mL bottle. The CPG was washed with 2 x 40 mL portions of ethanol / water (1:1 v / v). Then the volume of the mixture was reduced to about 30 mL on a rotary evaporator. Then this mixture was frozen on dry ice and dried under vacuum by speed vac.

[0403] 3. Deprotection-II (Removal of 2'-TBDMS Groups) 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 minutes 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 it was stored in a freezer until purification.

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

[0405] 5. HPLC Purification Oligonucleotides conjugated with ligands were purified by reverse phase preparative HPLC. Unconjugated oligonucleotides were purified by anion-exchange HPLC on a TSK gel column packed in-house. 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 special vials for CGE and LC / MS analysis. Finally, the compounds were analyzed by LC-ESMS and CGE.

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

[0407] Example 3: In Vitro Silencing Activity Using Various Chemical Modifications of ANGPTL3 siRNA Cell Culture and Transfection Hep3B cells (ATCC, Manassas, VA) were grown in RPMI (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) in a 5% CO2 atmosphere at 37°C until near confluence and then detached from the plate by trypsinization. 14.8 μl of Opti-MEM and 0.2 μl of Lipofectamine RNAiMax (Invitrogen, Carlsbad CA, cat# 13778-150) per well were added to each well containing 5 μl of siRNA duplex in a 96-well plate and transfection was performed by incubating at room temperature for 15 minutes. 80 μl of antibiotic-free complete growth medium containing approximately 2×10 4 Hep3B cells were added to the siRNA mixture. Cells were incubated for 24 hours or 120 hours and then RNA was purified. Unless otherwise stated, single-dose experiments were performed at final duplex concentrations of 10 nM and 0.1 nM, and dose-response experiments were performed at final duplex concentrations of 10 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, 0.005 nM, 0.001 nM, 0.0005 nM, 0.0001 nM, 0.00005 nM, and 0.00001 nM.

[0408] cDNA synthesis using the ABI High Performance cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, Cat# 4368813) For each reaction, a master mix containing 2 μl of 10× buffer, 0.8 μl of 25× dNTP, 2 μl of random primers, 1 μl of reverse transcriptase, 1 μl of RNase inhibitor, and 3.2 μl of H2O was added to 10 μl of total RNA. cDNA was prepared using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, CA) in the following steps: 10 minutes at 25°C, 120 minutes at 37°C, 5 seconds at 85°C, hold at 4°C.

[0409] Real-time PCR 2 μl of cDNA was added to the 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)) in each well of a 384-well 50 plate (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 the relative fold change, real-time data was analyzed using the ΔΔCt method and normalized to assays performed on cells transfected with 10 nM of AD-1955 or mock-transfected cells. IC 50 was calculated using a 4-parameter fit model using XLFit and normalized to cells transfected with AD-1955 or untreated cells over the same dose range or its own minimum dose. The AD-1955 sequence used as a negative control targets luciferase and has the following sequences: Sense strand: cuuAcGcuGAGuAcuucGAdTsdT; Antisense strand: UCGAAGuACUcAGCGuAAGdTsdT.

[0411] The various embodiments described above can be combined to create another embodiment. All U.S. patents, published U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to herein are hereby incorporated by reference in their entirety. If it is necessary to create still another embodiment using the concepts of the various patents, patent applications, and published patent applications, the aspects of the embodiments can be changed.

[0412] In view of the above detailed description, these and other modifications can be made to the embodiments. In general, in the following claims, the terms used should not be construed as limiting the following claims to the specific embodiments disclosed in this specification and the appended claims, but should be construed to include all possible embodiments within the full scope of equivalents given to such claims. Accordingly, the claims are not limited by the present disclosure.

[0413] Example 4: Chemical Modification of siRNA and In Vitro Silencing of Modified siRNA Design of Sense Strand Design of Ligand and Conjugation Site The sense strand was conjugated to the GalNAc ligand at the 3' position in the same manner as the parent compound.

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

[0415] Thermal Destabilization of the Sense 3' Region (Positions 16 - 18) This region was modified with a thermal destabilization modification, e.g., GNA or a mismatch to the opposite AS strand. Modification at position 16 or 17 appears to have the greatest effect. Figures 1 and 1 emphasize the effect of thermal destabilization on this position / region and in vitro efficacy. Effective knockdown corresponding to the parental template design was obtained with GNA or other thermal destabilization modifications, e.g., abasic (Y34) or a mismatch to the antisense strand. On the other hand, a decrease in silencing was generally observed with 2'-OMe modifications or DNA modifications complementary to the opposite AS strand.

[0416]

Table 1

[0417]

Chem.

[0418] Figure 2 and Table 2 showed the influence of the position of thermolabilized modified GNA in the 3’ region (positions 16 to 18). This result indicates that the GNA modifications at positions 16 and 17 showed excellent efficacy similar to the parental design, while the GNA at position 18 showed a decrease in activity.

[0419]

Table 2

[0420]

Table 3

[0421] Design of the antisense strand Position 2 of the AS This position was identified by the movement of the position in the AS strand affected by highly sterically demanding 2’-modifications including 2’-OMe and the statistical analysis of a large conjugate dataset. However, the inventors have found that some modifications including DNA and in some cases 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. The results from in vitro silencing studies are summarized in Figure 3 and Table 3, suggesting that DNA and RNA at position 2 generally maintain the activity of non-F designs similar to the parental template design, while 2’-OMe is generally not well tolerated and results in a decrease in activity.

[0422] Position 14 of the AS This position was identified by the shift of the position in the AS strand affected by the highly steric demanding 2'-modification including 2'-OMe and the statistical analysis of the dataset of large conjugates. However, it was found that several modifications including DNA and in some cases 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. The results from in vitro silencing studies are summarized in Figure 4 and Table 4, showing that DNA and RNA at position 14 generally maintain the activity of non-F designs similar to the design of the parental template, while 2'-OMe is generally not well tolerated and leads to a decrease in activity.

[0423] [Table 4]

[0424] [Table 5]

[0425] In vivo evaluation siRNA targeting mTTR Animals (n = 3 / group) were administered a single dose of 2.5 mg / kg of siRNA, and FVII serum protein levels were measured before dosing, 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 parental compound AD-57727. Figure 6 shows the decrease in mTTR protein 96 hours after dosing for two non-F siRNAs at three different dosing levels compared to the parental compound. Figure 7 shows the profile of the decrease in mTTR serum protein for a repeated-dose regimen (1 mg / kg, QW) up to 42 days (a total of 6 doses).

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

[0427] siRNA targeting TMPRSS6

[0428] [Table 6]

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

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

[0431] [Table 7]

[0432] As shown in Figure 9, the improvement yielded at least one non-F compound (AD-65105) with in vivo efficacy comparable to 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 the GalNAc ligand at the 3'-position using the same procedure as 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] Results in vitro As shown in Figure 10, all 10 sequences representing three targets, two motifs, namely, Motif 1 (modification of 6 phosphorothioate nucleotide internucleotide linkages in the sense and antisense strands; 4 2'-F modifications at positions 7 and 9-11 of the sense strand from the 5'-end of the sense strand, and 4 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 (modification of 6 phosphorothioate nucleotide internucleotide linkages in the sense and antisense strands; 4 2'-F modifications at positions 7 and 9-11 of the sense strand from the 5'-end of the sense strand, and 6 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 a statistically significant improvement in activity compared to the parent compound AD-57727.

[0436] Evaluation in vivo Target silencing of siRNA was evaluated by qPCR. The ability of the motifs targeting mTTR was evaluated. Animals (n = 3 / group) were administered 3 mg / kg of siRNA as a single dose, and liver levels were evaluated first before administration and then at 7 and 22 days later as shown in Figure 11.

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

[0438] Figure 13 shows an improvement in activity (about a four-fold improvement in activity) with the new motifs (Motifs 1 and 2) compared to the parent compound using data evaluated 7 days after administration. This data demonstrates the effect of the motifs on in vivo activity. The several-fold improvement is consistent across all sequences.

[0439] Figure 14 shows a significant improvement in duration across all three sequences, demonstrating that the new motifs result in an improvement in duration.

[0440] Figure 15 shows the results of ApoC3-GalNAc3 SAR in hAAV 1×10 11 GC / mice with a single subcutaneous administration of 3 mg / kg.

[0441] Example 5: VP and PS2 Modifications at the 5' End of the Antisense Strand Exemplary protocols for the synthesis of oligonucleotides containing 5'-vinylphosphonate (VP) and for the synthesis of oligonucleotides containing 2'-deoxythymidine linked by phosphorodithioate (PS2) bonds at the 5'-terminus of the oligonucleotide are described below. One of ordinary skill in the art will understand that similar oligonucleotides can be synthesized using these same or similar techniques. Other synthetic techniques known to those of ordinary skill in the art can also be used to synthesize and prepare these and similar oligonucleotides and modifications, such synthetic techniques including, but not limited to, Whittaket et al., “Stereoselective synthesis of highly functionalized P-stereogenic nucleosides via palladium-catalyzed P-C 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 the synthetic techniques disclosed in U.S. Patent Application Publication No. 2013 / 0084576, the entire contents of each of which are incorporated herein by reference.

[0442] Protocol for the Synthesis of Oligonucleotides Containing 5'-Vinylphosphonate Introduction of Pivaloyloxymethyl-(POM)-protected VP [Chemical formula] Coupling and oxidation: The coupling of amidites was carried out under standard synthetic conditions using 0.25 M 5-(ethylthio)-1H-tetrazole in acetonitrile for activation. A standard thiolation protocol using either 3-(dimethylaminomethylene)amino-3H-1,2,4-dithiazole-5-thione (DDTT) or phenylacetyl disulfide (PADS) was performed to convert the phosphite triesters to phosphorothioate linkages. Since the vinyl phosphonate component does not contain a DMT protecting group at the 5'-position, the final detritylation step was omitted.

[0443] Deprotection and cleavage: After synthesis, the vinyl phosphonate-containing oligonucleotide was deprotected in a 3:1 mixture of aqueous NH3 and EtOH with 40 volumes % of a 1 - 2.5% methylamine solution added at 60 °C for 5 h or 35 °C for 16 h.

[0444] Introduction of ethyl-protected VP

Chemical formula

[0445] Deprotection and cleavage: A solution of acetonitrile (ACN) and pyridine (Pyr) 50:1 (v / v) was prepared, and 3×10 -8A molecular sieve of cm (3 angstroms) was added and the mixture was dried as much as possible. To this mixture, 3.5 mL (5 g) of iodotrimethylsilane (TMSI) was added per 135 mL of the ACN / Pyr solution. This solution had to be freshly prepared and its maximum shelf life was 1 day. Next, a 0.5 M mercaptoethanol solution was prepared in 1:1 (v / v) acetonitrile-triethylamine and 3×10 -8 cm (3 angstroms) of molecular sieve was added. With the 5'-VP-containing oligonucleotide on the resin in the synthesis column, the TMSI solution was slowly added at about 5 - 10 CV and reacted for 15 minutes. This step was repeated twice for a total exposure time of about 45 minutes. Subsequently, the resin was extensively washed with ACN, then about 5 - 10 column volumes of the mercaptoethanol solution was passed through the column and reacted 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 phosphorodithioate bonds at the 5'-end of the oligonucleotide [Chemical formula] Coupling and oxidation: The phosphoramidite solution was prepared from commercially available dT-thiophosphoramidite (Glen Research) according to the manufacturer's protocol in dry acetonitrile at a concentration of 0.15 M. Coupling was carried out under standard synthetic 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 synthetic conditions.

[0447] Deprotection and cleavage: The solid support (on the column) was washed with 0.5 M piperidine in ACN and then (exposure time of 2 × 15 minutes), the resin was transferred to a suitable container and treated under standard conditions (e.g., 3:1 aqueous NH₃:EtOH solution, 5 hours at 60 °C or 16 hours at 35 °C), and the oligonucleotide was cleaved from the solid support and deprotected.

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

[0449] Figure 16 illustrates a schematic diagram of siRNA with Ago2 added. Generally, 5'-phosphate-functionalized siRNA (ESC chemistry) shows improved in vitro activity. For example, approximately 80% of the sequences tested showed improved intrinsic potency when transfected in vitro, and approximately 30% showed an IC 50 effect. However, in vitro, the 5'-phosphate rapidly disappears in the endo / lysosome compartment. A modified phosphate, 5'-vinylphosphonate (5'-VP), which mimics a stable phosphate, was also added to the 5'-end of the modified oligonucleotide and is shown in Figure 16. This phosphonate was first designed by Merck.

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

[0451] Figure 17 shows a chart indicating how the presence of 5'-VP generally improves in vitro activity based on the evaluation of four different ApoB sequences. The LDL levels 7 days after a single SC administration of 3 mg / kg were analyzed for four conjugates (with or without 5'-VP modification). As can be seen from the chart, ED 50A three-fold improvement is seen with specific ApoR sequences. In vivo advantages were 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 the PS bond, including phosphorodithioate (PS2) and methylphosphonate (MePhos), which promote endogenous phosphorylation. Modified siRNAs are generally not good substrates for Clp1 kinase, probably due to interference by the 2’OMe modification at the first nucleotide of the AS strand. However, the 2’-OMe modification, together with the phosphorothioate bond, is desirable for exonuclease protection. Substitution of the 2’-OMe modification, e.g., with 2’F, and modification of the PS bond 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) bond and 2’F-PS showed improved in vitro activity compared to the parent (2’OMe-PS). In particular, dn(PS)2 was stable in the in vitro tritosome assay, while 2’F-PS showed metabolic liability. Transfection of primary mouse hepatocytes at 10 nM and 0.1 nM (n = 4) was performed for two ApoB conjugates.

[0455] Figure 20 shows two charts demonstrating how minor changes at the antisense 5'-end can significantly improve in vivo efficacy. Figure 20A shows that the 2'-F-PS at position 1 of the antisense strand can improve the activity of the 5'-P-dependent sequence, and Figure 20B shows that, similar to 5'-VP, the potency is improved by approximately 3-fold with dN(PS)2 compared to the parent.

[0456] Example 6: Evaluation of 5'-VP Modification and siRNA Activity Synthesis of 5'-Vinylphosphonate Phosphoramidite Using the Pivaloxymethyl Protecting Group:

Chemical Structure

[0457] Synthesis of Tetra(pivaloyloxymethyl)-bis-phosphonate (X)

Chemical Structure

[0458] Preparation of Compound 2

Chemical formula

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

[0460] Preparation of Compound 3

Chemical formula

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

[0462] Preparation of Compound 4

Chemical Structure

[0463] This solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (MeOH:CH2CL2 = 7:93, v:v). The fractions were tested by RP-HPLC (C18 column, buffer A = in water, 0.05% TFA, buffer B = in ACN, 0.05% TFA; gradient of 5 - 95% for 25 minutes) to confirm the purity of the two isomers (E and Z isomers): The E isomer eluted at 14.1 minutes and the Z isomer eluted at 14.9 minutes. The first fraction from the silica gel column chromatography contained both 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, with a yield of 71%.

[0464] E isomer: 1 H NMR (400 MHz, acetonitrile-d3): δ 8.98 (s, 1H), 7.30 (d, J = 8.1 Hz, 1H), 6.80 (ddd, J = 23.7, 17.2, 5.0 Hz, 1H), 6.02 (ddd, J = 21.6, 17.1, 1.7 Hz, 1H), 5.77 (d, J = 3.2 Hz, 1H), 5.57 (m, 5H), 4.32 (m, 1H), 4.01 (dd, J = 7.0, 5.4 Hz, 1H), 3.82 (dd, J = 5.5, 3.2 Hz, 1H), 3.41 (s, 3H), 1.14 (d, J = 1.5 Hz, 18H); 31 P NMR (162 MHz, acetonitrile-d3): δ 18.29.

[0465] Z isomer: 1 H NMR (500 MHz, acetonitrile-d3): δ 9.50 (s, 1H), 7.44 (d, J = 8.1 Hz, 1H), 6.69 (ddd, J = 54.4, 13.3, 8.7 Hz, 1H), 5.93 (ddd, J = 17.8, 13.3, 1.3 Hz, 1H), 5.80 (d, J = 2.9 Hz, 1H), 5.69 - 5.58 (m, 5H), 5.22 (m, 1H), 4.01 (dd, J = 7.1, 5.3 Hz, 1H), 3.88 (dd, J = 5.3, 2.9 Hz, 1H), 3.49 (s, 3H), 1.19 (d, J = 5.8 Hz, 18H); 31³¹P NMR (202 MHz, acetonitrile-d3): δ 18.75.

[0466] Preparation of Compound 5 [Chemical formula] 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), 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphorodiamidite (1.311 g, 4.35 mmol) was added. The mixture was stirred at room temperature for 2 hours. The formation of the product was confirmed by TLC in hexane:EtOAc (2:8 in 0.15% TEA). The reaction mixture was filtered, concentrated, and loaded onto a silica column. The sample was eluted with 20% - 100% EtOAc in hexane containing TEA (0.15%) to give Compound 5 as a white foam (1.75 g, 62%).

[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 ³¹P NMR (162 MHz, acetonitrile-d3): δ 151.79 (d, J = 71.3 Hz), 18.07 (d, J = 54.0 Hz).

[0468] Z isomer: 11H NMR (400 MHz, acetonitrile-d3): δ 9.02 (s, 1H), 7.41 (dd, J = 8.1, 1.6 Hz, 1H), 6.62 (dddd, J = 53.7, 13.1, 9.7, 7.0 Hz, 1H), 5.97 (dd, J = 17.4, 13.1 Hz, 1H), 5.80 (dd, J = 7.0, 3.5 Hz, 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 31P NMR (202 MHz, acetonitrile-d3): δ 150.81 (d, J = 141.4 Hz), 15.17.

[0469] Protocol for the synthesis of oligonucleotides containing 5'-vinylphosphonate The synthesis of vinylphosphonate monomers and 5'-VP modified oligonucleotides was carried out in a similar manner to the procedures of the literature (International Publication No. WO 2008 / 100447 pamphlet granted to Chen et al., Lima et al. “Single-Stranded siRNAs Activate RNAi in Animals,” Cell 150:883-894 (2012); Prakash et al., “Identification of metabolically stable 5-phosphate analogs that support single-stranded siRNA activity,” Nucleic Acids Research 43:2993-3011 (2015), the entire contents of each of which are incorporated herein by reference). Briefly, the 5'-phosphate was protected by ethyl ether, and then the phosphate protected by this ethyl ether was 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 (E-) and (Z-) 5'-Vinylphosphonates on siRNA Activity Double-stranded small interfering RNAs (siRNAs) with 5'-phosphorylated antisense strands facilitate efficient loading into the RNA-induced silencing complex (RISC) and induce strong RNAi-mediated gene silencing. Thus, endogenous 5'-phosphorylation of synthetic siRNAs by Clp1 kinase is crucial for RISC loading and strand selection (Weitzer et al., “The human RNA kinase hClp1 is active on 3’ transfer RNA exons and short interfering RNAs,” Nature 447:222-226 (2007)). Phosphonate analogs with metabolic stability linkages have been used to improve gene silencing activity against antiviral agents as nucleoside modifications (International Publication No. WO 2008 / 100447 pamphlet granted to Chen et al.), corresponding unphosphorylated siRNAs, particularly 5'-end modifications of siRNAs for improving gene silencing activity against 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 phosphonate analogs in double-stranded siRNAs were evaluated both in vitro and in vivo.

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

[0473] [Table 10]

[0474] [Table 11]

[0475] The effects on the duplex siRNA activity of 5'-vinylphosphonate (VP) with E- and Z-geometries were compared. This result indicates that the in vivo efficacy of chemically modified siRNA can be improved with 5'-trans-(E-)VP, which sufficiently mimics natural phosphate, while 5'-cis-(Z-)VP did not show an improvement in efficacy, suggesting that the Z isomer does not sufficiently mimic natural phosphate.

[0476] Figures 21A and 21B show SAR analyses comparing the in vitro and in vivo activities of Ap...

Claims

1. A double-stranded RNA (dsRNA) agent capable of suppressing the expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the dsRNA agent has the formula (I): 【Chemical 1】 represented by In the formula: 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 a thermolabile nucleotide at a site opposite to the seed region (positions 2 to 8) of the antisense strand; T1, T1', T2', and T3' each independently represent a nucleotide containing a modification that provides a steric bulk to the nucleotide less than or equal to the steric bulk of the 2'-OMe modification; n １ 、 n ３ 、 and q １ are each independently 4 to 15 nucleotides in length; n ５ 、q ３ 、and q ７ are each independently 1 to 6 nucleotides in length; q ２ and q ６ are each independently 1 to 3 nucleotides in length; q ５ is independently 0 to 10 nucleotides in length; and n ２ 、 n ４ 、 and q ４ are each independently a double-stranded RNA (dsRNA) agent having a length of 0 to 3 nucleotides.

2. n ４ 、 q ２ 、 and q ６ The dsRNA agent according to claim 1, wherein n, q, and q are each 1.

3. n ２ 、n ４ 、q ２ 、q ４ 、and q ６ is 1 respectively, the dsRNA agent according to claim 1.

4. The dsRNA agent according to claim 1, wherein T1' and T3' are separated by 11 nucleotides.

5. The dsRNA agent according to claim 1, wherein C1 is at a site opposite to positions 5 to 8 at the 5'-end of the antisense strand.

6. The sense strand has a length of 19 to 22 nucleotides, and when n ４ is 1, C1 is at positions 14 to 17 at the 5'-end of the sense strand, and the dsRNA agent according to claim 5.

7. T1' is at the 14th position at the 5' end of the antisense strand, and q ２ The dsRNA agent according to claim 1, wherein q is 1.

8. T3' is at the 2nd position of the 5'-end of the antisense strand, and q ６ is 1, the dsRNA agent according to claim 1.

9. The dsRNA agent according to claim 1, wherein T1 is at the cleavage site of the sense strand.

10. The sense strand has a length of 19 to 22 nucleotides, and when n ２ is 1, T1 is at the 11th position at the 5' end of the sense strand, and the dsRNA agent according to claim 1.

11. T2' is at positions 6 to 10 at the 5'-end of the antisense strand, and q ４ is 1, the dsRNA agent according to claim 1.

12. The dsRNA agent according to claim 1, wherein B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe modification.

13. C1 is a mismatch with the opposing nucleotide of the antisense strand; 【Chemical 2】 a deoxyribose modification selected from the group consisting of: ; and [Chemical Formula 3] (wherein B is a modified or unmodified nucleobase, and R １ and R ２ are independently H, halogen, OR ３ , or alkyl; and R ３ is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), the dsRNA agent according to claim 1, having a heat destabilizing modification selected from the group consisting of a sugar modification selected from the group consisting of:

14. The dsRNA agent according to claim 13, wherein the thermolabile modification 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; and optionally, at least one nucleobase in the mismatch pair is a 2'-deoxy nucleobase.

15. 【Chemical 4】 The dsRNA agent according to claim 13, wherein the thermolabile modification is GNA or

16. The dsRNA agent according to claim 1, wherein T1, T1', T2', and T3' are each independently selected from DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl.

17. The dsRNA agent according to claim 1, wherein T1 is DNA.

18. The dsRNA agent according to claim 1, wherein T1' is DNA, RNA, or LNA.

19. The dsRNA agent according to claim 1, wherein T2' is DNA or RNA.

20. The dsRNA agent according to claim 1, wherein T3' is DNA or RNA.

21. The dsRNA agent according to claim 1, wherein the sense strand and the antisense strand are each independently acyclic nucleotides, LNA, HNA, CeNA, 2'-methoxyethyl, 2'O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-O-N-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F modified.

22. The dsRNA agent according to claim 1, wherein the sense strand and the antisense strand each contain at least two different modifications.

23. The dsRNA agent according to claim 1, which does not contain any 2'-F modification.

24. The dsRNA agent according to claim 1, wherein the sense strand and / or the antisense strand contain one or more blocks of phosphorothioate or methylphosphonate nucleotide linkages.

25. The dsRNA agent according to claim 24, wherein the sense strand contains one block of two phosphorothioate or methylphosphonate nucleotide linkages.

26. The dsRNA agent according to claim 24, wherein the antisense strand contains two blocks of two phosphorothioate or methylphosphonate nucleotide linkages separated by 16 to 18 phosphate nucleotide linkages.

27. The dsRNA agent according to claim 1, wherein the sense strand and the antisense strand each have 15 to 30 nucleotides.

28. The dsRNA agent according to claim 1, wherein the sense strand has 19 to 22 nucleotides and the antisense strand has 19 to 25 nucleotides.

29. The dsRNA agent according to claim 1, wherein the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides.

30. The dsRNA agent according to claim 1, having 3' and / or 5' overhangs of 1 to 10 nucleotides in length.

31. The dsRNA agent according to claim 1, having a 3' overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

32. The dsRNA agent according to claim 1, having a 5' overhang at the 5' end of the sense strand.

33. The dsRNA agent according to claim 1, wherein the nucleotide at the 1st position at the 5' end of the antisense strand of the double strand is selected from the group consisting of A, dA, dU, U, and dT.

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

35. The dsRNA agent according to claim 1, further comprising at least one ASGPR ligand.

36. The dsRNA agent according to claim 35, wherein the ASGPR ligand is added to the 3' end of the sense strand.

37. The dsRNA agent according to claim 35, wherein the ASGPR ligand is one or more GalNAc derivatives added by a divalent or trivalent branched linker.

38. The ASGPR ligand is: 【Chemical Formula 5】 The dsRNA agent according to claim 37.

39. A dsRNA agent capable of suppressing the expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 40 nucleotides: The sense strand contains at least one thermolabile nucleotide, and at least one of the thermolabile nucleotides is present at a site on the opposite side of the seed region (positions 2 to 8) of the antisense strand; and The antisense strand contains at least two modified nucleotides that provide a steric bulk below that of the 2'-OMe modification to the nucleotides, and the modified nucleotides are separated by 11 nucleotides in length.

40. The dsRNA agent according to claim 39, wherein the sense strand further comprises an endonuclease-sensitive modified nucleotide at the cleavage site of the sense strand.

41. The antisense strand further comprises a third modified nucleotide that provides a nucleotide with a steric bulk below that of the 2'-O-Me modification, and the third modified nucleotide is at positions 6 to 10 at the 5' end of the antisense strand. The dsRNA agent according to claim 39.

42. The thermolabile nucleotide is at positions 14 to 17 at the 5' end of the sense strand. The dsRNA agent according to claim 39.

43. The two modified nucleotides that provide a nucleotide with a steric bulk below that of the 2'-O-Me modification are at positions 2 and 14 at the 5' end of the antisense strand. The dsRNA agent according to claim 39.

44. The endonuclease-sensitive modified nucleotide is at position 11 at the 5' end of the sense strand. The dsRNA agent according to claim 40.

45. The third modified nucleotide that provides a nucleotide with a steric bulk below that of the 2'-O-Me modification is at position 10 at the 5' end of the antisense strand. The dsRNA agent according to claim 41.

46. The thermolabile nucleotide is a mismatch with the nucleotide opposite the antisense strand; a depurination modification selected from the group consisting of: [Chemical Formula 6] ; and a sugar modification selected from the group consisting of: 【Chemical Formula 7】 (wherein B is a modified or unmodified nucleobase, and R １ and R ２ are independently H, halogen, OR ３ , or alkyl; and R ３ is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), the dsRNA agent according to claim 39, comprising a modification selected from the group consisting of

47. The modified nucleotide that provides a nucleotide with a steric bulk below that of the 2'-O-Me modification includes a modification independently selected from DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl. The dsRNA agent according to claim 39.

48. The sense strand and the antisense strand 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'-O-N-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F. The dsRNA agent according to claim 39.

49. The sense strand and the antisense strand each contain at least two different modifications. The dsRNA agent according to claim 39.

50. The dsRNA agent according to claim 39 that does not contain any 2'-F modification.

51. The dsRNA agent according to claim 39, wherein the sense strand has 19 to 22 nucleotides and the antisense strand has 19 to 25 nucleotides.

52. The dsRNA agent according to claim 39, wherein the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides.

53. The dsRNA agent according to claim 39, having a 3' overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

54. The dsRNA agent according to claim 39, further comprising at least one ASGPR ligand.

55. The dsRNA agent according to claim 54, wherein the ASGPR ligand is one or more GalNAc derivatives added by a divalent or trivalent branched linker.

56. A pharmaceutical composition comprising the dsRNA agent according to any one of the preceding claims, alone or in combination with a pharmaceutically acceptable carrier or excipient.

57. A method for suppressing the expression of a target gene, comprising the step of administering the dsRNA agent according to any one of the preceding claims in an amount sufficient to suppress the expression of the target gene.

58. The method according to claim 57, wherein the dsRNA agent is administered by subcutaneous administration or intravenous administration.

59. A method for delivering a polynucleotide to a specific target of a subject by administering the dsRNA agent according to any one of the preceding claims.

60. The method according to claim 59, wherein the administering step is performed by an administration means including intramuscular administration, intratracheal administration, intrapleural administration, intraperitoneal administration, intraarterial administration, lymphatic administration, intravenous administration, subcutaneous administration, cerebrospinal administration, or a combination thereof.

61. A method for delivering a polynucleotide to a specific target of a subject, comprising: delivering the dsRNA agent according to any one of the preceding claims to the subject by subcutaneous administration such that the polynucleotide is delivered to the specific target of the subject.

62. The dsRNA agent according to claim 1, wherein formula (I) further comprises 5'-vinylphosphonate (VP).

63. The dsRNA agent according to claim 1, wherein formula (I) further comprises 2'-deoxythymidine linked by a phosphorodithioate (PS ２ ) bond at the 5' end of the antisense strand or the sense strand.

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