Modified double-stranded RNA reagents

Abstract

One aspect of the present invention relates to double-stranded RNA (dsRNA) reagents capable of inhibiting target gene expression. The sense strand of the dsRNA reagent includes at least one thermally unstable nucleotide, and at least one thermally unstable nucleotide that appears at a relative site to the seed region (position 2-8) of the antisense strand; and the antisense strand of the dsRNA reagent includes at least two modified nucleotides that provide a spatial volume less than or equal to the spatial volume of 2'-OMe modification for the nucleotide, wherein the modified nucleotides are separated by 11 nucleotide lengths. Other aspects of the present invention relate to pharmaceutical compositions including these dsRNA reagents suitable for therapeutic use, and methods for inhibiting target gene expression by administering these dsRNA reagents, for example, for treating various disease conditions.

Description

[0001] This application is a divisional application of Chinese invention patent application No. 201580056832.8, whose application date is August 14, 2015 and whose invention name is “Modified double-stranded RNA reagent”.

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 093,919, filed December 18, 2014, U.S. Provisional Application No. 62 / 083,744, filed November 24, 2014, and U.S. Provisional Application No. 62 / 039,507, filed August 20, 2014, all of which are incorporated herein by reference in their entirety. Field of the Invention

[0003] The present invention relates to RNAi duplex agents having specific motifs that are beneficial for inhibiting target gene expression, as well as RNAi compositions suitable for therapeutic use. In addition, the present invention provides methods for inhibiting target gene expression by administering these RNAi duplex agents, for example, for treating various diseases. background

[0004] RNA interference, or "RNAi," is a term originally coined by Fire and colleagues to describe the observation that double-stranded RNAi (dsRNA) can block gene expression (Fire et al. (1998) Nature 391, 806-811; Elbashir et al. (2001) Genes Dev. 15, 188-200). Short dsRNAs direct gene-specific post-transcriptional silencing in many organisms, including vertebrates, and have provided a new tool for studying gene function. RNAi is mediated by the RNA-induced silencing complex (RISC), a sequence-specific multicomponent nuclease that destroys messenger RNA homologous to the silencing trigger. RISC is known to contain a short RNA (approximately 22 nucleotides) derived from the double-stranded RNA trigger, but the protein components of this activity remain unknown.

[0005] Drug development based on RNA interference (RNAi) requires double-stranded RNA (dsRNA) molecules with good gene silencing properties. The initial step in RNAi is the activation of the RNA-induced silencing complex (RISC), which requires degradation of the sense strand of the dsRNA duplex. The sense strand is known to be the first RISC substrate to be cleaved by Argonaute 2 in the middle of the duplex region. After the 5'-end and 3'-end fragments of the sense strand are removed from the nuclease Ago2, RISC is immediately activated by the antisense strand (Rand et al. (2005) Cell 123, 621).

[0006] It is believed that when sense strand cleavage is inhibited, endonucleolytic cleavage of the target mRNA is impaired (Leuschner et al. (2006) EMBO Rep., 7, 314; Lander et al. (2005) Cell 123, 621; Schwarz et al. (2004) Curr. Biol. 14, 787). Leuschner et al. demonstrated that incorporation of 2'-O-Me ribose into the Ago2 cleavage site in the sense strand inhibited RNAi in HeLa cells (Leuschner et al. (2006) EMBO Rep., 7, 314). Similar effects were observed with phosphorothioate modification, indicating that effective RNAi in mammals also requires sense strand cleavage.

[0007] Morrissey et al. used siRNA duplexes that included 2'-F modified residues at the Ago2 cleavage site in addition to other sites and modifications, and achieved comparable silencing compared to unmodified siRNA (Morrissey et al. (2005) Hepatology 41, 1349). However, Morrissey's modifications were not motif-specific, e.g., as long as pyrimidine residues were present, one modification included 2'-F modifications on all pyrimidines on both the sense and antisense strands without any selectivity; and therefore, based on these teachings, it is uncertain whether specific motif modifications at the cleavage site of the sense strand have any substantial effect on gene silencing activity.

[0008] Muhonen et al. used siRNA duplexes containing two 2'-F modified residues at the Ago2 cleavage site on either the sense or antisense strand and found that this was tolerated (Muhonen et al. (2007) Chemistry & Biodiversity 4, 858-873). However, Muhonen's modifications were also sequence-specific, e.g., for each specific strand, Muhonen only modified all pyrimidines or all purines without any selectivity.

[0009] Choung et al. used siRNA duplexes containing alternative modifications, using different combinations of 2'-OMe or 2'-F, 2'-OMe, and phosphorothioate modifications to stabilize siRNA in serum as Sur10058 (Choung et al. (2006) Biochemical and Biophysical Research Communications 342, 919-927). Choung suggested that in order to increase the stability of siRNA, the residues at the cleavage site of the antisense strand should not be modified with 2'-OMe.

[0010] Therefore, there is a continuing need for iRNA duplex reagents for improving the gene silencing efficacy of siRNA gene therapy. The present invention addresses this need. SUMMARY OF THE INVENTION

[0011] The present invention provides effective nucleotide or chemical motifs for dsRNA agents, optionally conjugated to at least one ligand, that facilitate inhibition of target gene expression, as well as RNAi compositions suitable for therapeutic use.

[0012] In one aspect, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting target gene expression. The dsRNA agent includes a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The dsRNA agent is represented by formula (I):

[0013]

[0014] In formula (I), B1, B2, B3, B1', B2', B3', and B4' are each independently a nucleotide containing a modification selected from the group consisting of 2'-O-alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halo, ENA, and BNA / LNA. In one embodiment, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe modification. In one embodiment, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe or 2'-F modification. In one embodiment, at least one B1, B2, B3, B1', B2', B3', and B4' contains a 2'-ON-methylacetamido (2'-O-NMA) modification.

[0015] C1 is a thermally unstable nucleotide located at a site opposite to the seed region of the antisense strand (i.e., position 2-8 of the 5'-end of the antisense strand). For example, C1 is located at the position of the sense strand that is paired with the nucleotides at positions 2-8 of the 5'-end of the antisense strand. In one example, C1 is located at position 15 from the 5'-end of the sense strand. C1 nucleotides carry thermally unstable modifications that may include abscisic modifications; mismatches with the nucleotides relative to the duplex; and sugar modifications, such as 2'-deoxy modifications or acyclic nucleotides (e.g., unlocked nucleic acids (UNA) or glycerol nucleic acids (GNA)). In one embodiment, C1 has a thermally unstable modification selected from the group consisting of: i) mismatches with the nucleotides relative to the antisense strand; ii) abscisic modifications selected from the group consisting of:

[0016] and iii) sugar modifications selected from the group consisting of:

[0017] and Wherein, B is a modified or unmodified nucleobase, R 1 and R 2 are independently H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. In one embodiment, the thermally labile modification in C1 is a mismatch selected from the group consisting of G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, and U:T; and optionally, at least one nucleobase in the mismatch pair is a 2'-deoxynucleobase. In one example, the thermally labile modification in C1 is GNA or

[0018] T1, T1', T2', and T3' each independently represent a nucleotide comprising a modification that provides a steric volume to the nucleotide that is less than or equal to the steric volume of the 2'-OMe modification. Steric volume refers to the sum of the steric effects of the modifications. Methods for determining the steric effects of modifications of nucleotides are known to those skilled in the art. The modification may be at the 2' position of the ribose of the nucleotide, or a modification of a non-ribonucleotide, an acyclic nucleotide, or a backbone of the nucleotide that is similar to or equal to the 2' position of the ribose, and provides a steric volume to the nucleotide that is less than or equal to the steric volume 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.

[0019] n 1 、n 3 , and q 1 The length of is independently 4 to 15 nucleotides.

[0020] n 5 ,q 3 , and q 7 are independently 1-6 nucleotides in length.

[0021] n 4 ,q 2 , and q 6 The length of n is independently 1-3 nucleotides; alternatively, n 4 It is 0.

[0022] q 5are independently 0-10 nucleotides in length.

[0023] n 2 and q 4 are independently 0-3 nucleotides in length.

[0024] Alternatively, n 4 The length is 0-3 nucleotides.

[0025] In one embodiment, n 4 Can be 0. In one example, n 4 is 0, and q 2 and q 6 is 1. In another example, n 4 is 0, and q 2 and q 6 is 1, having two phosphorothioate internucleotide linkage modifications in positions 1-5 (counting from the 5'-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the sense strand, and two phosphorothioate internucleotide linkage modifications in positions 18-23 (counting from the 5'-end of the antisense strand) of the antisense strand.

[0026] In one embodiment, n 4 ,q 2 , and q 6 Each is 1.

[0027] In one embodiment, n 2 、n 4 ,q 2 ,q 4 , and q 6 Each is 1.

[0028] In one embodiment, C1 is at position 14-17 of the 5'-end of the sense strand, when the sense strand is 19-22 nucleotides in length, and n 4 is 1. In one embodiment, C1 is at position 15 of the 5'-end of the sense strand.

[0029] In one embodiment, T3' begins at position 2 from the 5' end of the antisense strand. In one example, T3' is at position 2 from the 5' end of the antisense strand, and q 6 Equal to 1.

[0030] In one embodiment, T1' begins at position 14 from the 5' end of the antisense strand. In one example, T1' is at position 14 from the 5' end of the antisense strand, and q 2 Equal to 1.

[0031] In an exemplary embodiment, T3' starts at position 2 from the 5' end of the antisense strand, and T1' starts at position 14 from the 5' end of the antisense strand. In an example, T3' starts at position 2 from the 5' end of the antisense strand, and q 6 = 1, and T1' starts at position 14 from the 5' end of the antisense strand, and q 2 Equal to 1.

[0032] In one embodiment, T1' and T3' are separated by a length of 11 nucleotides (ie, the T1' and T3' nucleotides are not counted).

[0033] In one embodiment, T1' is at position 14 from the 5' end of the antisense strand. In one example, T1' is at position 14 from the 5' end of the antisense strand, and q 2 =1, and the modification is at the 2' position, or at a non-ribose, acyclic, or backbone position that provides less space than 2'-OMe ribose.

[0034] In one embodiment, T3' is at position 2 from the 5' end of the antisense strand. In one example, T3' is at position 2 from the 5' end of the antisense strand, and q 6 =1, and the modification is at the 2' position, or at a non-ribose, acyclic, or backbone position that provides a steric bulk less than or equal to that of the 2'-OMe ribose.

[0035] In one embodiment, T1 is at the cleavage site of the sense strand. In one example, T1 is at position 11 from the 5' end of the sense strand when the sense strand is 19-22 nucleotides long and n 2 is 1. In an exemplary embodiment, T1 is at the cleavage site of the sense strand at position 11 from the 5' end of the sense strand, when the length of the sense strand is 19-22 nucleotides, and n 2 It's 1 o'clock.

[0036] In one embodiment, T2' begins at position 6 from the 5' end of the antisense strand. In one example, T2' is at position 6-10 from the 5' end of the antisense strand, and q 4 It is 1.

[0037] In an exemplary embodiment, T1 is at the cleavage site of the sense strand, for example, at position 11 from the 5' end of the sense strand, when the sense strand is 19-22 nucleotides in length, and n 2 is 1; T1' is at position 14 from the 5' end of the antisense strand, and q 2=1, and the modification to T1' is at the 2' position of ribose, or at a non-ribose, acyclic, or backbone position that provides less steric bulk than 2'-OMe ribose; T2' is at positions 6-10 from the 5' end of the antisense strand, and q 4 is 1; and T3' is at position 2 from the 5' end of the antisense strand, and q 6 =1, and the modification to T3' is at the 2' position, or at a non-ribose, acyclic, or backbone position that provides a steric bulk less than or equal to that of the 2'-OMe ribose.

[0038] In one embodiment, T2' starts at position 8 from the 5' end of the antisense strand. In one example, T2' starts at position 8 from the 5' end of the antisense strand, and q 4 It's 2.

[0039] In one embodiment, T2' begins at position 9 from the 5' end of the antisense strand. In one example, T2' is at position 9 from the 5' end of the antisense strand, and q 4 It is 1.

[0040] In one embodiment, 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, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications in positions 1-5 (counting from the 5'-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the sense strand, and two phosphorothioate internucleotide linkage modifications in positions 18-23 (counting from the 5'-end of the antisense strand).

[0041] In one embodiment, 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, q6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications in positions 1-5 (counting from the 5'-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the sense strand, and two phosphorothioate internucleotide linkage modifications in positions 18-23 (counting from the 5'-end of the antisense strand).

[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 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 It 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, 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, and q 7is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0044] 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, and q 7 It is 1.

[0045] 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0046] 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, and q 7 It is 1.

[0047] 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, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and a phosphorothioate internucleotide linkage modification in positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0048] 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 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, and q 7 is 1; optionally having at least 2 additional TTs at the 3'-end of the antisense strand.

[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 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, and q 7 is 1; optionally has at least 2 additional TT at the 3'-end of the antisense strand; has two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[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 3 It is 4, 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, and q 7 It is 1.

[0051] 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 It 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, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications in positions 1-5 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 (counting from the 5' end) of the antisense strand.

[0052] 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, and q 7 It is 1.

[0053] In one embodiment, B1 is 2'-OMe or 2'-F,n 1 is 8, T1 is 2'F, n2 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, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications in positions 1-5 (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the sense strand, and two phosphorothioate internucleotide linkage modifications in positions 18-23 (counting from the 5' end of the antisense strand).

[0054] 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 It 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, and q 7 It is 1.

[0055] 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, q3 It 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, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications in positions 1-5 (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the sense strand, and two phosphorothioate internucleotide linkage modifications in positions 18-23 (counting from the 5' end of the antisense strand).

[0056] The dsRNA agent can include a phosphorus-containing group at the 5'-end of the sense strand or antisense strand. The 5'-terminal phosphorus-containing group can be a 5'-terminal phosphate (5'-P), a 5'-terminal phosphorothioate (5'-PS), a 5'-terminal phosphorodithioate (5'-PS2), a 5'-terminal vinylphosphonate (5'-VP), a 5'-terminal methylphosphonate (MePhos), or a 5'-deoxy-5'-C-malonyl monophosphate. When the 5'-terminal phosphorus-containing group is a 5'-terminal vinyl phosphonate (5'-Vp), the 5'-Vp may be a 5'-E-VP isomer (ie, trans-vinyl phosphate, ), 5'-Z-VP isomer (i.e., cis-vinyl phosphate, ), or a mixture thereof.

[0057] In one embodiment, the dsRNA agent comprises a phosphorus-containing group at the 5'-end of the sense strand. In one embodiment, the dsRNA agent comprises a phosphorus-containing group at the 5'-end of the antisense strand.

[0058] In one embodiment, the dsRNA agent comprises a 5'-P. In one embodiment, the dsRNA agent comprises a 5'-P on the antisense strand.

[0059] In one embodiment, the dsRNA agent comprises a 5'-PS. In one embodiment, the dsRNA agent comprises a 5'-PS on the antisense strand.

[0060] In one embodiment, the dsRNA agent comprises a 5'-VP. In one embodiment, the dsRNA agent comprises a 5'-VP on the antisense strand. In one embodiment, the dsRNA agent comprises a 5'-E-VP on the antisense strand. In one embodiment, the dsRNA agent comprises a 5'-Z-VP on the antisense strand.

[0061] In one embodiment, the dsRNA agent comprises a 5'-PS2. In one embodiment, the dsRNA agent comprises a 5'-PS2 on the antisense strand.

[0062] In one embodiment, the dsRNA agent comprises a 5'-PS2. In one embodiment, the dsRNA agent comprises a 5'-deoxy-5'-C-malonyl on the antisense strand.

[0063] 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, and q 7 1. The dsRNA reagent also includes a 5'-PS.

[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, and q 7 is 1. The dsRNA reagent also includes a 5'-P

[0065] In one embodiment, B1 is 2'-OMe or 2'-F,n 1 is 8, T1 is 2'F, n2 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, and q 7 1. The dsRNA agent further comprises a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0066] 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, and q 7 1. The dsRNA reagent also includes a 5'-PS2.

[0067] 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 4is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 1. The dsRNA reagent also includes a 5'-deoxy-5'-C-malonyl.

[0068] 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The dsRNA agent further comprises a 5'-P.

[0069] 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, and q 7is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The dsRNA agent further comprises a 5'-PS.

[0070] 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The dsRNA agent further comprises a 5'-VP. The 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0071] 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, q6 is 1, B4' is 2'-OMe, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The dsRNA agent further comprises a 5'-PS2.

[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, 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The dsRNA agent further comprises a 5'-deoxy-5'-C-malonyl.

[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 2 is 1, B2' is 2'-OMe or 2'-F, q 3 It 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'-OMe, and q 7 is 1. The dsRNA reagent also includes a 5'-P

[0074] 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 It 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, and q 7 1. The dsRNA reagent also includes a 5'-PS.

[0075] 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 It 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, and q 7 1. The dsRNA agent further comprises a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0076] 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 4is 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 It 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, and q 7 1. The dsRNA reagent also includes a 5'-PS2.

[0077] 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 It 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, and q 7 1. The dsRNA reagent also includes a 5'-deoxy-5'-C-malonyl.

[0078] 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 It 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, and q 7is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 (counted from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 (counted from the 5' end) of the antisense strand. The dsRNA agent further comprises a 5'-P.

[0079] 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 It 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 (counted from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 (counted from the 5' end) of the antisense strand. The dsRNA agent further comprises a 5'-PS.

[0080] 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 It 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, and q 71; having two phosphorothioate internucleotide linkage modifications in positions 1-5 (counted from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 (counted from the 5' end) of the antisense strand. The dsRNA agent further comprises a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0081] 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 It 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 (counted from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 (counted from the 5' end) of the antisense strand. The dsRNA agent further comprises a 5'-PS2.

[0082] 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 It 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, and q 7is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 (counted from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 (counted from the 5' end) of the antisense strand. The dsRNA agent further comprises a 5'-deoxy-5'-C-malonyl.

[0083] 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, and q 7 is 1. The dsRNA reagent also includes a 5'-P

[0084] 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, and q 7 1. The dsRNA reagent also includes a 5'-PS.

[0085] 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, 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, and q 7 1. The dsRNA agent further comprises a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0086] 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, and q 7 1. The dsRNA reagent also includes a 5'-PS2.

[0087] 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 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, and q 7 1. The dsRNA reagent also includes a 5'-deoxy-5'-C-malonyl.

[0088] 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5'-end of the antisense strand). The dsRNA agent further comprises a 5'-P.

[0089] 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, and q 7is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5'-end of the antisense strand). The dsRNA agent further comprises a 5'-PS.

[0090] 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The dsRNA agent further comprises a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[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, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6is 1, B4' is 2'-F, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5'-end of the antisense strand). The dsRNA agent further comprises a 5'-PS2.

[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, 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5'-end of the antisense strand). The dsRNA agent further comprises a 5'-deoxy-5'-C-malonyl.

[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 It is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6is 1, B4' is 2'-F, and q 7 is 1. The dsRNA reagent also includes a 5'-P

[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 It 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, and q 7 1. The dsRNA reagent also includes a 5'-PS.

[0095] 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 It 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, and q 7 1. The dsRNA agent further comprises a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0096] 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 5is 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 It 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, and q 7 1. The dsRNA reagent also includes a 5'-PS2.

[0097] 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 It 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, and q 7 1. The dsRNA reagent also includes a 5'-deoxy-5'-C-malonyl.

[0098] 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 It 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, and q 7is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The dsRNA agent further comprises a 5'-P.

[0099] 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 It 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The dsRNA agent further comprises a 5'-PS.

[0100] 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 It 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, and q 7is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The dsRNA agent further comprises a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0101] 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 It 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The dsRNA agent further comprises a 5'-PS2.

[0102] 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 It 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, and q7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The dsRNA agent further comprises a 5'-deoxy-5'-C-malonyl.

[0103] In one embodiment, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of the dsRNA agents of the invention are modified. For example, when 50% of the dsRNA agent is modified, 50% of all nucleotides present in the dsRNA agent contain a modification as described herein.

[0104] In one embodiment, each sense strand and antisense strand of the dsRNA agent is independently modified with acyclic nucleotides, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-O-N-methylacetamido (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.

[0105] In one embodiment, each sense and antisense strand of the dsRNA agent contains at least two different modifications.

[0106] In one embodiment, the dsRNA agent of formula (I) further comprises one or more 3' and / or 5' overhangs of 1-10 nucleotides in length. In one example, the dsRNA agent of formula (I) comprises a 3' overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand. In another example, the dsRNA agent has a 5' overhang at the 5'-end of the sense strand.

[0107] In one embodiment, the dsRNA agents of the invention do not contain any 2'-F modifications.

[0108] In one embodiment, the sense strand and / or antisense strand of the dsRNA reagent comprises one or more blocks of phosphorothioate or methylphosphonate internucleotide linkages. In one example, the sense strand comprises a 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, two blocks of phosphorothioate or methylphosphonate internucleotide linkages are separated by 16-18 phosphate internucleotide linkages.

[0109] In one embodiment, each sense strand and antisense strand of the dsRNA agent has 15-30 nucleotides. In one example, the sense strand has 19-22 nucleotides, and the antisense strand has 19-25 nucleotides. In another example, the sense strand has 21 nucleotides, and the antisense strand has 23 nucleotides.

[0110] In one embodiment, the nucleotide at position 1 of the 5'-end of the antisense strand in the duplex is selected from the group consisting of A, dA, dU, U, and dT. In one embodiment, at least one of the first, second, and third base pairs from the 5'-end of the antisense strand is an AU base pair.

[0111] In one embodiment, the antisense strand of a dsRNA agent of the invention is 100% complementary to a target RNA, hybridizes thereto, and inhibits its expression by RNA interference. In another embodiment, the antisense strand of a dsRNA agent of the 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 a target RNA.

[0112] In one aspect, the present invention relates to a dsRNA reagent capable of inhibiting target gene expression as defined herein. The dsRNA reagent comprises a sense strand and an antisense strand, and each strand has 14 to 40 nucleotides. The sense strand contains at least one thermolabile nucleotide, wherein at least one of the thermolabile nucleotides occurs at a site relative to or near a site of the antisense strand's seed region (i.e., at the 5'-end position 2-8 of the antisense strand). Each embodiment and aspect described in this specification relates to a dsRNA represented by formula (I), and can also be applied to dsRNA containing thermolabile nucleotides.

[0113] The thermolabile nucleotide can occur, for example, between positions 14-17 of the 5'-end of the sense strand when the sense strand is 21 nucleotides long. The antisense strand contains at least two modified nucleic acids that are smaller than the sterically required 2'-OMe modification. Preferably, the two modified nucleic acids that are smaller than the sterically required 2'-OMe modification are separated by 11 nucleotides in length. For example, the two modified nucleic acids are at positions 2 and 14 of the 5' end of the antisense strand.

[0114] In one embodiment, the dsRNA agent further comprises at least one ASGPR ligand. For example, the ASGPR ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker, such as: In one example, the ASGPR ligand is attached to the 3' end of the sense strand.

[0115] For example, a dsRNA agent as defined herein can include i) a phosphorus-containing group at the 5'-end of the sense or antisense strand; ii) two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5'-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5'-end of the antisense strand); and iii) a ligand, such as an ASGPR ligand (e.g., one or more GalNAc derivatives), at the 5'-end or 3'-end of the sense or antisense strand. For example, the ligand can be 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, and q 7is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The dsRNA agent further comprises a 5'-P and a targeting ligand. In one embodiment, the 5'-P is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

[0117] 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The dsRNA agent further comprises a 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

[0118] 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, 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The dsRNA agent further comprises a 5'-Vp (e.g., a 5'-E-VP, 5'-Z-VP, or a combination thereof), and a targeting ligand. In one embodiment, the 5'-Vp is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

[0119] 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The dsRNA reagent further comprises a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

[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, n3 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The dsRNA agent further comprises a 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

[0121] 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 It 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, and q 7is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 (counted from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 (counted from the 5' end) of the antisense strand. The dsRNA agent further comprises a 5'-P and a targeting ligand. In one embodiment, the 5'-P is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

[0122] 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 It 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 (counted from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 (counted from the 5' end) of the antisense strand. The dsRNA agent further comprises a 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

[0123] 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 It is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 (counted from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 (counted from the 5' end) of the antisense strand. The dsRNA agent further comprises a 5'-Vp (e.g., a 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-Vp is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

[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 It 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 (counted from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 (counted from the 5' end) of the antisense strand. The dsRNA agent further comprises a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

[0125] 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 1is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 It 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 (counted from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 (counted from the 5' end) of the antisense strand. The dsRNA agent further comprises a 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

[0126] 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5'-end of the antisense strand). The dsRNA agent further comprises a 5'-P and a targeting ligand. In one embodiment, the 5'-P is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

[0127] 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, 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5'-end of the antisense strand). The dsRNA agent further comprises a 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

[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, 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, and q 7is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5'-end of the antisense strand). The dsRNA agent further comprises a 5'-VP (e.g., a 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

[0129] 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5'-end of the antisense strand). The dsRNA agent further comprises a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

[0130] 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, 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5'-end of the antisense strand). The dsRNA agent further comprises a 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

[0131] 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 It 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The dsRNA agent further comprises a 5'-P and a targeting ligand. In one embodiment, the 5'-P is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

[0132] 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, n3 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 It 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The dsRNA agent further comprises a 5'-Ps and a targeting ligand. In one embodiment, the 5'-PS is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

[0133] 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 It 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, and q 7is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The dsRNA agent further comprises a 5'-Vp (e.g., a 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

[0134] 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 It 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The dsRNA agent further comprises a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

[0135] 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, q3 It 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, and q 7 is 1; having two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The dsRNA agent further comprises a 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

[0136] In a specific embodiment, the dsRNA agent of the present invention comprises:

[0137] (a) a sense strand having:

[0138] (i) 21 nucleotides in length;

[0139] (ii) an ASGPR ligand attached to the 3' end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker; and

[0140] (iii) 2'-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 17, 19, and 21, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14 to 16, 18, and 20 (counting from the 5' end);

[0141] and

[0142] (b) an antisense strand having:

[0143] (i) 23 nucleotides in length;

[0144] (ii) 2'-OMe modifications at positions 1, 3, 5, 9, 11 to 13, 15, 17, 19, 21, and 23, and 2'F modifications at positions 2, 4, 6 to 8, 10, 14, 16, 18, 20, and 22 (counting from the 5' end); and

[0145] (iii) phosphorothioate internucleotide linkages between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end);

[0146] wherein the 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.

[0147] In another specific embodiment, the dsRNA agent of the present invention comprises:

[0148] (a) a sense strand having:

[0149] (i) 21 nucleotides in length;

[0150] (ii) an ASGPR ligand attached to the 3′ end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0151] (iii) 2'-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 15, 17, 19, and 21, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 (counting from the 5' end); and

[0152] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end);

[0153] and

[0154] (b) an antisense strand having:

[0155] (i) 23 nucleotides in length;

[0156] (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13, 15, 17, 19, and 21 to 23, and 2'F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5' end); and

[0157] (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end);

[0158] wherein the 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.

[0159] In another specific embodiment, the dsRNA agent of the present invention comprises:

[0160] (a) a sense strand having:

[0161] (i) 21 nucleotides in length;

[0162] (ii) an ASGPR ligand attached to the 3′ end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0163] (iii) 2'-OMe modifications at positions 1 to 6, 8, 10, and 12 to 21, 2'-F modifications at positions 7 and 9, and a deoxynucleotide (e.g., dT) at position 11 (counting from the 5' end); and

[0164] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end);

[0165] and

[0166] (b) an antisense strand having:

[0167] (i) 23 nucleotides in length;

[0168] (ii) 2'-OMe modifications at positions 1, 3, 7, 9, 11, 13, 15, 17, and 19 to 23, and 2'-F modifications at positions 2, 4 to 6, 8, 10, 12, 14, 16, and 18 (counting from the 5' end); and

[0169] (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end);

[0170] wherein the 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.

[0171] In another specific embodiment, the dsRNA agent of the present invention comprises:

[0172] (a) a sense strand having:

[0173] (i) 21 nucleotides in length;

[0174] (ii) an ASGPR ligand attached to the 3′ end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0175] (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

[0176] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end);

[0177] and

[0178] (b) an antisense strand having:

[0179] (i) 23 nucleotides in length;

[0180] (ii) 2'-OMe modifications at positions 1, 5, 7, 9, 11, 13, 15, 17, 19, and 21 to 23, and 2'-F modifications at positions 2 to 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counting from the 5' end); and

[0181] (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end);

[0182] wherein the 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.

[0183] In another specific embodiment, the dsRNA agent of the present invention comprises:

[0184] (a) a sense strand having:

[0185] (i) 21 nucleotides in length;

[0186] (ii) an ASGPR ligand attached to the 3′ end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0187] (iii) 2'-OMe modifications at positions 1 to 9, and 12 to 21, and 2'-F modifications at positions 10 and 11; and

[0188] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end);

[0189] and

[0190] (b) an antisense strand having:

[0191] (i) 23 nucleotides in length;

[0192] (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13, 15, 17, 19, and 21 to 23, and 2'-F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5' end); and

[0193] (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end);

[0194] wherein the 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.

[0195] In another specific embodiment, the dsRNA agent of the present invention comprises:

[0196] (a) a sense strand having:

[0197] (i) 21 nucleotides in length;

[0198] (ii) an ASGPR ligand attached to the 3′ end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0199] (iii) 2'-F modifications at positions 1, 3, 5, 7, 9 to 11, and 13, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, and 14 to 21; and

[0200] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end);

[0201] and

[0202] (b) an antisense strand having:

[0203] (i) 23 nucleotides in length;

[0204] (ii) 2'-OMe modifications at positions 1, 3, 5 to 7, 9, 11 to 13, 15, 17 to 19, and 21 to 23, and 2'-F modifications at positions 2, 4, 8, 10, 14, 16, and 20 (counting from the 5' end); and

[0205] (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end);

[0206] wherein the 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.

[0207] In another specific embodiment, the dsRNA agent of the present invention comprises:

[0208] (a) a sense strand having:

[0209] (i) 21 nucleotides in length;

[0210] (ii) an ASGPR ligand attached to the 3′ end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0211] (iii) 2'-OMe modifications at positions 1, 2, 4, 6, 8, 12, 14, 15, 17, and 19 to 21, and 2'-F modifications at positions 3, 5, 7, 9 to 11, 13, 16, and 18; and

[0212] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end);

[0213] and

[0214] (b) an antisense strand having:

[0215] (i) 25 nucleotides in length;

[0216] (ii) 2'-OMe modifications at positions 1, 4, 6, 7, 9, 11 to 13, 15, 17, and 19 to 23, 2'-F modifications at positions 2, 3, 5, 8, 10, 14, 16, and 18, and deoxynucleotides (e.g., dT) at positions 24 and 25 (counting from the 5' end); and

[0217] (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end);

[0218] wherein the dsRNA agent has a four nucleotide overhang at the 3'-end of the antisense strand, and a blunt end at the 5'-end of the antisense strand.

[0219] In another specific embodiment, the dsRNA agent of the present invention comprises:

[0220] (a) a sense strand having:

[0221] (i) 21 nucleotides in length;

[0222] (ii) an ASGPR ligand attached to the 3′ end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0223] (iii) 2'-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2'-F modifications at positions 7, and 9 to 11; and

[0224] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end);

[0225] and

[0226] (b) an antisense strand having:

[0227] (i) 23 nucleotides in length;

[0228] (ii) 2'-OMe modifications at positions 1, 3 to 5, 7, 8, 10 to 13, 15, and 17 to 23, and 2'-F modifications at positions 2, 6, 9, 14, and 16 (counting from the 5' end); and

[0229] (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end);

[0230] wherein the 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.

[0231] In another specific embodiment, the dsRNA agent of the present invention comprises:

[0232] (a) a sense strand having:

[0233] (i) 21 nucleotides in length;

[0234] (ii) an ASGPR ligand attached to the 3′ end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0235] (iii) 2'-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2'-F modifications at positions 7, and 9 to 11; and

[0236] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end);

[0237] and

[0238] (b) an antisense strand having:

[0239] (i) 23 nucleotides in length;

[0240] (ii) 2'-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 23, and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end); and

[0241] (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end);

[0242] wherein the 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.

[0243] In another specific embodiment, the dsRNA agent of the present invention comprises:

[0244] (a) a sense strand having:

[0245] (i) 19 nucleotides in length;

[0246] (ii) an ASGPR ligand attached to the 3′ end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0247] (iii) 2'-OMe modifications at positions 1 to 4, 6, and 10 to 19, and 2'-F modifications at positions 5, and 7 to 9; and

[0248] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end);

[0249] and

[0250] (b) an antisense strand having:

[0251] (i) 21 nucleotides in length;

[0252] (ii) 2'-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 21, and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end); and

[0253] (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counting from the 5' end);

[0254] wherein the 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.

[0255] In one embodiment, the dsRNA agent described herein further comprises a thermolabile modification at position 7 counting from the 5 '-end of the antisense form, at position 15 counting from the 5 '-end of the sense strand, at position 21 counting from the 5 '-end of the sense strand, or a combination thereof.

[0256] In one aspect, the present invention relates to a dsRNA reagent that can inhibit target gene expression.The dsRNA reagent includes a sense strand and an antisense strand, and each strand has 14 to 40 nucleotides.The sense strand contains at least one thermally unstable nucleotide, wherein at least one described thermally unstable nucleotide occurs at a site relative to the seed region of the antisense strand or at a site close to the antisense strand (i.e., at the position 2-8 of the 5'-end), for example, when the length of the sense strand is 21 nucleotides, the thermally unstable nucleotide occurs between the position 14-17 of the 5'-end of the sense strand.The antisense strand includes two modified nucleic acids that are less than the 2'-OMe modified space required to be separated by 11 nucleotide lengths.For example, these two modified nucleic acids are at positions 2 and 14 of the 5'-end of the antisense strand.

[0257] In one embodiment, the sense strand of the dsRNA agent further comprises a nucleotide susceptible to endonuclease modification at the cleavage site of the sense strand. In an example, the nucleotide susceptible to endonuclease modification is at position 11 from the 5' end of the sense strand.

[0258] In one embodiment, the antisense strand further comprises a third modified nucleotide that provides a steric volume for the nucleotide that is less than or equal to the steric volume of the 2'-OMe modification, and the third modified nucleotide is at positions 6-10 from the 5' end of the antisense strand. For example, the third modified nucleotide is at position 10 from the 5' end of the antisense strand.

[0259] The embodiments for the thermolabile nucleotides are analogous to the various embodiments described above for C1 in Formula (I). The embodiments for modified, less than sterically required 2'-OMe-modified nucleic acids are analogous to the various embodiments described above for T1', T2', and T3' in Formula (I). The embodiments describing the length, overhangs, additional modifications, and ligands of the dsRNA agent T bound to Formula I are suitable herein.

[0260] The present invention further relates to the use of a dsRNA agent as described herein for inhibiting target gene expression. In one embodiment, the present invention further relates to the use of a dsRNA agent for inhibiting target gene expression in vitro.

[0261] The present invention further relates to a dsRNA agent as described herein for inhibiting target gene expression in a subject. The subject can be any animal, preferably a mammal, more preferably a mouse, rat, sheep, cow, dog, cat, or human.

[0262] In one aspect, the present invention relates to a dsRNA reagent that can inhibit target gene expression. The dsRNA reagent includes a sense strand and an antisense strand, and each strand has 14 to 40 nucleotides. The sense strand includes a nucleotide (for example, DNA, RNA, or 2'-F) that is easily modified by an endonuclease near the cleavage site of the sense strand. For example, the nucleotide that is easily modified by an endonuclease is at position 11 from the 5' end of the sense strand. The modification that occurs in the endonuclease near the cleavage site can affect the sensitivity of the cleavage site. For example, the thermal instability modification near the cleavage site can provide the endonuclease sensitivity at the cleavage site. The antisense strand includes two modified nucleic acids that are less than the 2'-OMe modified space required to be separated by 11 nucleotide lengths. For example, these two modified nucleic acids are at position 2 and 14 at the 5' end of the antisense strand.

[0263] In another aspect, the present invention further provides a method for delivering a dsRNA of the present invention to a specific target in a subject by subcutaneous or intravenous administration. The present invention further provides a dsRNA agent of the present invention for use in a method for delivering the agent to a specific target in a subject by subcutaneous or intravenous administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0264] Figure 1A-1C is a graph showing the effect of different modifications at position 17 of the sense strand on in vitro efficacy evaluated at 10 nM and 0.1 nM concentrations: (A) siRNA targeting mTTR with a non-F sense strand paired with a parental AS strand; (B) siRNA targeting mTTR with a non-F sense strand paired with a non-F AS strand; (C) siRNA targeting ANG, ApoC3, and TTRSC with a non-F sense strand paired with a parental AS strand.

[0265] Figure 2 is a position effect graph showing the effect on the effectiveness of in vitro thermolabile GNA modifications spanning positions 16-18 of the sense strand assessed at 10 nM and 0.1 nM concentrations.

[0266] Figure 3 is a graph showing the effect of modification at position 2 of the antisense strand on the in vitro effectiveness of siRNA targeting mTTR, ApoC3, TTRSC, and TMP, assessed at 10 nM and 0.1 nM concentrations.

[0267] Figure 4 is a graph showing the effect of modification at position 14 of the antisense strand on the in vitro effectiveness of siRNA targeting mTTR, ApoC3, and TTRSC, assessed at 10 nM and 0.1 nM concentrations.

[0268] Figure 5 is a graph showing mTTR silencing in mice after a single SC dose of 2.5 mg / kg.

[0269] Figure 6 Graph showing the dose response of non-F siRNAs AD-61398 and AD-64273 compared to parental 2PS (AD-43527) and 6PS (AD-57727); single SC dose, protein levels measured 96 h post-dose.

[0270] Figure 7 is a graph showing the reduction of mTTR protein in plasma following QW SC dosing of 1 mg / kg siRNA in mice compared to non-FAD-61398 with the parental motif: AD-57727.

[0271] Figure 8 Graph showing silencing of TMPRSS6 mRNA following a single SC dose of 3 mg / kg in mice (n=3 / group): comparison of non-F design with parental motif: AD-60490.

[0272] Figure 9Comparison showing silencing of TMPRSS6 mRNA 7 days after a single SC dose of 3 mg / kg in mice (n=3 / group): Non-F design with parental motif: AD-60490.

[0273] Figure 10 Results are shown for the in vitro activity of two motifs (Motif 1 and Motif 2) compared to the activity of the parent compound AD-57727.

[0274] Figure 11 In vitro evaluation of the silencing activity of this siRNA targeting mTTR is shown.

[0275] Figure 12 Enhanced activity is shown with the chemical stability enhancing conjugate (SEC-C), where activity (mRNA) in the liver was measured 7 days after dosing.

[0276] Figure 13 Depicted is a graph showing an approximately 4-fold improvement in activity with the new motifs (motifs 1 and 2) compared to the parent compound.

[0277] Figure 14 Depicted are graphs showing the clearly improved duration of motif 1 and motif 2 across the three sequences.

[0278] Figure 15 Figure 3 shows the effect of a single 3 mg / kg SC dose of hAAV 1x10 11 Graph showing the results of ApoC3-GalNAc3SAR in GC / mice.

[0279] Figure 16 Schematic diagram illustrating Ago2-loaded siRNA and 5'-vinylphosphonate (5'-VP), a modified phosphate mimic that stabilizes phosphates. This 5'-phosphate is added by the cytosolic Clp1 kinase and serves as a key anchor for Ago2 loading.

[0280] Figure 17 Depicted is a graph showing how the presence of 5'-VP generally enhances activity in vivo. This evaluation was performed on four different ApoB sequences. LDL levels were analyzed 7 days after a single SC dose of 3 mg / kg for the four conjugates (with or without 5'-VP modification).

[0281] Figure 18 Depicted are different chemical modifications that can replace the PS bond and provide more stable chemistries that promote endogenous phosphorylation, including phosphorodithioate (PS2) and methylphosphonate (MePhos).

[0282] Figure 19Graph showing in vitro evaluation of terminal modifications including 2'-OMe-MePhos, 2'-OMe-PS, dN(PS2), and 2'F-PS. Primary mouse hepatocytes (n=4) were transfected with two ApoB conjugates at 10 nM and 0.1 nM.

[0283] Figure 20 It shows that small changes at the antisense 5'-end can significantly improve in vivo efficacy. The left panel shows that 2'F-PS at position 1 of the antisense strand can enhance the activity of 5'P-dependent sequences (F9 activity measured on day 3 using a single 3 mg / kg SC dose). The right panel shows that similar to VP (LDL measured on day 3 against ApoB using a single 10 mg / kg SC dose), dN(PS)2 enhances potency by approximately 3-fold over the parental version.

[0284] Figure 21 AB show SAR analysis of the in vitro and in vivo activity of ApoB siRNAs containing a 5'-OH relative to the 5'-E-VP modification (at the 5'-end of the antisense strand). Figure 21 A shows the results of in vitro transfection of mouse 1° hepatocytes. Figure 21 B shows LDL levels 3 days after a single dose (SC dosing).

[0285] Figure 22 Results showing the in vitro efficacy of 5'-E-VP modification relative to 5'-Z-VP modification on mTTR and F9 siRNA-GalNAc conjugates. These results are from in vitro transfected mouse primary hepatocytes.

[0286] Figure 23 Shown are the results of an in vivo comparison of 5'-E-VP modification versus 5'-Z-VP modification of F9 siRNA-GalNAc conjugate (single SC dose).

[0287] Figure 24 AC are graphs showing dose-response curves for (A) 5'-OH, (B) 5'-C-malonyl, and (C) 5'-phosphate PTEN siRNAs in primary mouse hepatocytes for in vitro PTEN silencing assays. All values ​​are from triplicate experiments.

[0288] Figure 25 Results are shown for the enzymatic stability of 5'-OH, 5'-C-malonyl, and 5'-phosphate siRNAs incubated in rat liver decontaminated lysosomes. The siRNA target sequences are shown in Table 10. Data were normalized to untreated controls.

[0289] Figure 26Results of RISC loading of 5'-OH, 5'-C-malonyl, and 5'-phosphate siRNAs (5'-modification on the antisense strand) as determined by immunoprecipitation of Ago2 from primary mouse hepatocytes and RT-PCR amplification of Ago2-loaded single strands are shown. The levels of endogenous miR122 were measured as a control. The siRNA target sequences are shown in Table 10.

[0290] Figure 27 Figure 2 is a graph showing in vitro knockdown of TTR using siRNA modified with a single (S)-GNA nucleotide. TTR mRNA levels were measured in primary mouse hepatocytes after 24 hours of incubation with 10 nM siRNA. TTR mRNA was measured using RT-qPCR and normalized to PBS-treated cells. All data points are the average of four measurements.

[0291] Figure 28 A is a graph showing in vitro knockdown of TTR modified with a single (S)-GNA base pair using siRNA. TTR mRNA levels were determined in primary mouse hepatocytes after 24 hours of incubation with 10 nM siRNA. TTR mRNA was measured using RT-qPCR and normalized to PBS-treated cells. All data points are the average of four measurements. Figure 28 B shows mixed and matched duplexes in which the sense and antisense strands containing a single (S)-GNA nucleotide are paired as GNA:RNA heterobase pairs.

[0292] Figure 29 Figure 2 is a graph showing in vivo levels of TTR in mouse serum. Animals received a single dose of 2.5 mg / kg siRNA. At the indicated times before or after dosing, animals were bled and serum samples were assayed using a sandwich ELISA assay using an HRP-conjugated antibody and 3,3',5,5'-tetramethylbenzidine for a readout at 450 nm. All samples were measured in duplicate, and each data point is the mean of 3 mice in each group.

[0293] Figure 30 Figure 2 is a graph showing in vivo quantification of TTR mRNA levels. Animals received a single dose of 2.5 mg / kg siRNA. RNA extraction was performed in whole liver homogenates at the indicated times after dosing. TTR mRNA was measured by RT-qPCR as above using the ΔΔCt method with GAPDH as a control transcript and normalized to PBS-treated animals. The dark bar indicates the results for day 21; and the black bar indicates the results for day 7. Detailed Description of the Invention

[0294] The inventors discovered that having 2'-OMe modifications at nucleotide positions 2 and 14 from the 5'-end of the antisense strand inhibited the gene silencing activity of the dsRNA agent. By introducing chemical modifications at the 2' position or at specific positions of the antisense and / or sense strands that provide a non-ribose, acyclic, or equivalent position in the backbone that has a steric volume less than that of the 2'-OMe modification, the dsRNA agent was able to restore the gene silencing activity. The inventors also determined that introducing a thermolabile nucleotide into the sense strand at a position opposite the seed region of the antisense strand (i.e., at positions 2-8 from the 5'-end of the antisense strand) provided better gene silencing activity.

[0295] The sense and antisense strands of the dsRNA agent can be fully modified. The dsRNA agent is optionally conjugated to an asialoglycoprotein receptor (ASGPR) ligand, for example, on the sense strand. The resulting dsRNA agent exhibits effective in vivo gene silencing activity.

[0296] The invention provides a kind of double-stranded RNAi (dsRNA) reagent that can suppress target gene expression.This dsRNA reagent comprises sense strand and antisense strand.Every chain of this dsRNA reagent can have the length in the range of 12-40 nucleotide.For example, every chain can be at 14-40 nucleotide length, 17-37 nucleotide length, 25-37 nucleotide length, 27-30 nucleotide length, 17-23 nucleotide length, 17-21 nucleotide length, 17-19 nucleotide length, 19-25 nucleotide length, 19-23 nucleotide length, 19-21 nucleotide length, 21-25 nucleotide length or 21-23 nucleotide length.

[0297] In some embodiments, the sense strand and antisense strand typically form a duplex dsRNA. The duplex district of dsRNA reagent can have 12-40 nucleotide pairs in length. For example, the duplex district can be 14-40 nucleotide pairs in length, 17-30 nucleotide pairs in length, 25-35 nucleotide pairs in length, 27-35 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length or 21-23 nucleotide pairs in length. In another example, the length in the duplex district is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27 nucleotide pairs.

[0298] In one embodiment, dsRNA reagent of the present invention can comprise one or more overhanging end districts and / or blocking groups of dsRNA reagent at the 3'-end or 5'-end or both ends of a chain.The length of this overhanging end can be 1-10 nucleotide, 1-6 nucleotide, for example, 2-6 nucleotide, 1-5 nucleotide, 2-5 nucleotide, 1-4 nucleotide, 2-4 nucleotide, 1-3 nucleotide, 2-3 nucleotide or 1-2 nucleotide.These overhanging ends can be the result that a chain is longer than another chain, or the result that two chains with the same length are staggered.This overhanging end can form a mispairing with this target mRNA, or it can be complementary to the gene sequence of targeting or can be other sequences.The first chain and the second chain can also be connected to form a hairpin, for example, by other base, or be connected by other non-base joints.

[0299] In one embodiment, the nucleotides in the overhang region of the dsRNA agent of the present invention can each independently be a modified or unmodified nucleotide, including but not limited to 2'-sugar modified, for example, 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. For example, TT can be an overhang sequence at either end of either chain. The overhang can form a mismatch with the target mRNA, or it can be complementary to the targeted gene sequence or can be other sequences.

[0300] The 5'-or 3'-overhang of the sense strand, antisense strand or two chains of the dsRNA reagent of the present invention can be phosphorylated. In certain embodiments, the overhang region contains two nucleotides with a phosphorothioate between the two nucleotides, wherein the two nucleotides can be identical or different. In one embodiment, the overhang is present at the 3' end of the sense strand, antisense strand or two chains. In one embodiment, this 3'-overhang is present in the antisense strand. In one embodiment, this 3'-overhang is present in the sense strand.

[0301] The dsRNA reagent of the present invention can only include a single overhang, which can strengthen the interference activity of the dsRNA without affecting its total stability. For example, the single-stranded overhang is located at the 3' end of the sense strand, or alternatively, at the 3' end of the antisense strand. The dsRNA can also have a flush end located at the 5' end (or 3' end of the sense strand) of the antisense strand, or vice versa. Typically, the antisense strand of the dsRNA has a nucleotide overhang at the 3' end, and the 5' end is flat. Although not bound by theory, the asymmetric flush end at the 5' end of the antisense strand and the 3' end overhang of the antisense strand promote that the guide strand is loaded into the RISC process. For example, the single overhang includes a length of at least two, three, four, five, six, seven, eight, nine, or ten nucleotides.

[0302] In one embodiment, the dsRNA agent of the present invention may also have two blunt ends at both ends of the dsRNA duplex.

[0303] In one embodiment, the dsRNA agent of the invention is a double-ended antibody having a length of 19 nt, wherein the sense strand contains at least one thermolabile nucleotide, wherein at least one thermolabile nucleotide occurs at a site opposite or proximal to the seed region of the antisense strand (i.e., at positions 2-8 of the 5'-end of the antisense strand). For example, the thermolabile nucleotide occurs between positions 14-17 of the 5'-end of the sense strand. The antisense strand contains at least two modified nucleic acids that are smaller than the sterically required 2'-OMe; preferably, the two modified nucleic acids that are smaller than the sterically required 2'-OMe are at positions 2 and 14 of the 5' end of the antisense strand.

[0304] In one embodiment, the dsRNA agent of the invention is a double-ended antibody having a length of 20 nt, wherein the sense strand contains at least one thermolabile nucleotide, wherein at least one thermolabile nucleotide occurs at a site opposite or proximal to the seed region of the antisense strand (i.e., at positions 2-8 of the 5'-end of the antisense strand). For example, the thermolabile nucleotide occurs between positions 14-17 of the 5'-end of the sense strand. The antisense strand contains at least two modified nucleic acids that are smaller than the sterically required 2'-OMe; preferably, the two modified nucleic acids that are smaller than the sterically required 2'-OMe are at positions 2 and 14 of the 5' end of the antisense strand.

[0305] In one embodiment, the dsRNA agent of the invention is a double-ended antibody having a length of 21 nt, wherein the sense strand contains at least one thermolabile nucleotide, wherein at least one thermolabile nucleotide occurs at a site opposite or proximal to the seed region of the antisense strand (i.e., at positions 2-8 of the 5'-end of the antisense strand). For example, the thermolabile nucleotide occurs between positions 14-17 of the 5'-end of the sense strand. The antisense strand contains at least two modified nucleic acids that are smaller than the sterically required 2'-OMe; preferably, the two modified nucleic acids that are smaller than the sterically required 2'-OMe are at positions 2 and 14 of the 5'-end of the antisense strand.

[0306] In one embodiment, the dsRNA agent of the present invention comprises a sense strand of 21 nucleotides (nt) in length and an antisense strand of 23 nucleotides (nt) in length, wherein the sense strand contains at least one thermolabile nucleotide, wherein at least one thermolabile nucleotide occurs at a site opposite or proximal to the seed region of the antisense strand (i.e., at positions 2-8 of the 5'-end of the antisense strand). For example, when the sense strand is 21 nucleotides in length, the thermolabile nucleotide occurs between positions 14-17 of the 5'-end of the sense strand. The antisense strand contains at least two modified nucleic acids that are less than the sterically required 2'-OMe; preferably, the two modified nucleic acids that are less than the sterically required 2'-OMe are at positions 2 and 14 of the 5' end of the antisense strand, wherein one end of the dsRNA is flat, while the other end includes a 2nt overhang. Preferably, the 2nt overhang is at the 3' end of the antisense strand. Optionally, the dsRNA further comprises a ligand (preferably a receptor ligand, ie, an ASGPR ligand).

[0307] In one embodiment, a dsRNA agent of the invention comprises a sense strand and an antisense strand, wherein: the sense strand is 25-30 nucleotide residues in length, wherein positions 1 to 23 of the sense strand, starting from the 5' terminal nucleotide (position 1), comprise at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues in length and, starting from the 3' terminal nucleotide, comprises at least 8 ribonucleotides at positions paired with positions 1-23 of the sense strand to form a duplex; wherein at least the 3' terminal nucleotide of the antisense strand is unpaired with the sense strand, and up to 6 consecutive 3' terminal nucleotides are unpaired with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides; wherein the 5' end of the antisense strand comprises from 10-30 ribonucleotides unpaired with the sense strand. wherein the sense strand comprises a plurality of nucleotides, each of which is a sequence of nucleotides, thereby forming a single-stranded 5' overhang of 10-30 nucleotides; wherein when the sense strand and the antisense strand are aligned for maximum complementarity, at least the 5' and 3' terminal nucleotides of the sense strand base pair with nucleotides of the antisense strand, thereby substantially forming a duplex region between the sense strand and the antisense strand; and when the double-stranded nucleic acid is introduced into a mammalian cell, the antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the length of the antisense strand to reduce target gene expression; and wherein the sense strand contains at least one thermolabile nucleotide, wherein the at least one thermolabile nucleotide occurs at or near a site opposite to the seed region of the antisense strand (i.e., at positions 2-8 of the 5'-end of the antisense strand). For example, the thermolabile nucleotide occurs between positions 14-17 of the 5'-end of the sense strand. The antisense strand contains at least two modified nucleic acids that are smaller than sterically desirable 2'-OMe; preferably, the two modified nucleic acids that are smaller than sterically desirable 2'-OMe are at positions 2 and 14 of the 5' end of the antisense strand.

[0308] In one embodiment, a dsRNA agent of the invention comprises a sense strand and an antisense strand, wherein the dsRNA agent comprises a sense strand having a length of at least 25 and at most 29 nucleotides, and an antisense strand having a length of at most 30 nucleotides, wherein the sense strand comprises a modified nucleotide susceptible to enzymatic degradation at position 11 from the 5' end. The antisense strand comprises two modified nucleic acids at positions 2 and 14 of the 5' end of the antisense strand that are smaller than sterically required 2'-OMe; wherein the 3' end of the sense strand and the 5' end of the antisense strand form a blunt end, and the antisense strand is 1-4 nucleotides longer at its 3' end than the sense strand, wherein the duplex region is at least 25 nucleotides in length and along at least 19 nt of the length of the antisense strand, the antisense strand is sufficiently complementary to a target mRNA to reduce target gene expression when the dsRNA agent is introduced into a mammalian cell, and wherein dicer cleavage of the dsRNA preferably produces an siRNA comprising the 3' end of the antisense strand, thereby reducing target gene expression in the mammal. Optionally, the dsRNA agent further comprises a ligand.

[0309] In one embodiment, the sense strand includes a modified nucleotide susceptible to enzymatic degradation at position 11 from the 5' end. The antisense strand includes two modified nucleic acids smaller than sterically required 2'-OMe at positions 2 and 14 of the 5' end of the antisense strand.

[0310] In one embodiment, the antisense strand includes two modified nucleic acids smaller than the sterically required 2'-OMe at positions 2 and 14 of the 5' end of the antisense strand.

[0311] In one embodiment, each nucleotide in the sense and antisense strands of the dsRNA agent can be modified. Each nucleotide can be modified by the same or different modifications, which can include one or more changes in one or both of the non-linked phosphate oxygens and / or one or more of the linked phosphate oxygens; changes in the composition of the ribose sugar (e.g., the 2' hydroxyl group on the ribose sugar); replacement of all phosphate moieties with "dephospho" linkers; modification or replacement of naturally occurring bases; and replacement or modification of the ribose-phosphate backbone.

[0312] Because nucleic acid is the polymer of subunit, therefore many modifications occur in a position repeated in nucleic acid, such as a kind of base or a kind of phosphate moiety or a kind of modification of a non-connected O of phosphate moiety.In some cases, this modification will occur in all target positions in this nucleic acid, but in many cases it will not be like this.For example, a modification can only occur at 3' or 5' terminal position, can only occur in a terminal area, such as on a terminal nucleotide of a chain or in a position in the last 2, 3, 4, 5 or 10 nucleotides.Modification can occur in double-stranded region, single-stranded region or both.Modification can only occur in the double-stranded region of RNA or can only occur in the single-stranded region of RNA.For example, a phosphorothioate modification of a non-connected O position can only be present in one or two ends, can only be present in a terminal area, such as on a terminal nucleotide of a chain or in a position in the last 2, 3, 4, 5 or 10 nucleotides, or can be present in double-stranded and single-stranded regions, particularly at the end.One or more 5' ends can be phosphorylated.

[0313] In order to enhance stability, it is possible that, for example, specific bases are included in the overhang or modified nucleotides or nucleotide substitutes are included in the single-stranded overhang (for example, in a 5' or 3' overhang or both).For example, it is desirable to include purine nucleotides in the overhang. In certain embodiments, all or some bases in a 3' or 5' overhang can be modified with modifications such as described herein. Modification can include, for example, modification at the 2' position using ribose and modification as known in the art, such as using 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl modified deoxyribonucleotides to replace the ribose of core bases, and modification (such as phosphorothioate modification) in the phosphate group. The overhang need not be homologous to the target sequence.

[0314] In one embodiment, each residue in the sense and antisense strands is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, or 2'-fluoro. These strands may contain more than one modification. In one embodiment, each residue in the sense and antisense strands is independently modified with 2'-O-methyl or 2'-fluoro.

[0315] At least two different modifications are typically present on the sense and antisense strands. Those two modifications can be 2'-deoxy, 2'-O-methyl or 2'-fluoro modifications, acyclic nucleotides or other modifications.

[0316] In one embodiment, the sense strand and the antisense strand each include two differently modified nucleotides selected from 2'-O-methyl or 2'-deoxy.

[0317] In one embodiment, each residue of the sense and antisense strands is independently modified with 2'-O-methyl nucleotides, 2'-deoxy nucleotides, 2'-deoxyfluoro nucleotides, 2'-O-N-methylacetamido (2'-O-NMA) nucleotides, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotides, 2'-O-aminopropyl (2'-O-AP) nucleotides, or 2'-ara-F nucleotides.

[0318] In one embodiment, the dsRNA agent of the present invention includes an alternating pattern of modifications, particularly in the B1, B2, B3, B1 ', B2 ', B3 ', B4 ' regions as shown in Formula I. As used herein, the term "alternating motif" or "alternating pattern" refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of a chain. Alternating nucleotides can refer to one every other nucleotide or one every three nucleotides, or a similar pattern. For example, if A, B, and C each represent a modification type for a nucleotide, the alternating motif can be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AAABBBAAABBB...", or "ABCABCABCABC...", etc.

[0319] The types of modifications contained in the alternating motif can be the same or different. For example, if A, B, C, D each represent a type of modification on a nucleotide, then the alternating pattern (i.e., the modification on every other nucleotide) can be the same, but each of the sense strand or antisense strand can be selected from several possibilities of modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...", etc.

[0320] In one embodiment, the dsRNA agent of the present invention includes a modification pattern for the alternating motif on the sense strand that is shifted relative to the modification pattern for the alternating motif on the antisense strand. The shift can be such that the modification groups of the nucleotides of the sense strand correspond to different modification groups of the nucleotides of the antisense strand, and vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, the alternating motif in the sense strand can start from "ABABAB" from the 5'-3' of the strand, and the alternating motif in the antisense strand can start from "BABABA" from the 3'-5' of the strand within the duplex region. As another example, the alternating motif in the sense strand can start from "AABBAABB" from the 5'-3' of the strand, and the alternating motif in the antisense strand can start from "BBAABBAA" from the 3'-5' of the strand within the duplex region, so that there is a complete or partial shift of the modification pattern between the sense strand and the antisense strand.

[0321] The dsRNA agents of the present invention may further include at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide at any position in the sense strand or the antisense strand or both strands. For example, the internucleotide linkage modification may occur on every nucleotide on the sense strand and / or the antisense strand; each internucleotide linkage modification may occur on the sense strand or the antisense strand in an alternating pattern; or the sense strand or the antisense strand may include two internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may be the same as or different from that of the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may be shifted relative to the alternating pattern of the internucleotide linkage modification on the antisense strand.

[0322] In one embodiment, the dsRNA reagent includes a phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region includes two nucleotides with a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modification can also be made to allow the overhang nucleotide to be paired with the nucleotide linkage of the end in the duplex region. For example, at least 2, 3, 4 or all of the overhang nucleotides can be connected by a phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there can be another phosphorothioate or methylphosphonate internucleotide linkage that connects the overhang nucleotide to a nucleotide of a pairing next to the overhang nucleotide. For example, there can be at least two phosphorothioate internucleotide linkages between the three nucleotides at the end, wherein two of the three nucleotides are overhang nucleotides, and the third is the nucleotide of the pairing next to the overhang nucleotide. Preferably, these three terminal nucleotides can be at the 3' end of the antisense strand.

[0323] In one embodiment, the sense strand of the dsRNA agent comprises 1-10 blocks having two to ten phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located anywhere in the oligonucleotide sequence, and the sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising a phosphorothioate or methylphosphonate or phosphate linkage.

[0324] In one embodiment, the antisense strand of the dsRNA agent comprises two blocks having two phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising a phosphorothioate or methylphosphonate or phosphate linkage.

[0325] In one embodiment, the antisense strand of the dsRNA agent comprises two blocks having three phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising a phosphorothioate or methylphosphonate or phosphate linkage.

[0326] In one embodiment, the antisense strand of the dsRNA agent comprises two blocks having four phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

[0327] 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, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

[0328] In one embodiment, the antisense strand of the dsRNA agent comprises two blocks of six phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

[0329] In one embodiment, the antisense strand of the dsRNA agent comprises two blocks of seven phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, or 8 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

[0330] In one embodiment, the antisense strand of the dsRNA agent comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, or 6 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

[0331] In one embodiment, the antisense strand of the dsRNA agent comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, or 4 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

[0332] In one embodiment, the dsRNA agent of the invention further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modifications at 1-10 terminal positions of the sense strand and / or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides at one or both ends of the sense strand and / or antisense strand can be linked via phosphorothioate or methylphosphonate internucleotide linkages.

[0333] In one embodiment, the dsRNA agent of the invention further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modifications in 1-10 duplex internal regions of each of the sense strand and / or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides at positions 8-16 of the duplex region, counted from the 5' end of the sense strand, may be linked via phosphorothioate or methylphosphonate internucleotide linkages; the dsRNA agent may optionally further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications in 1-10 terminal positions.

[0334] In one embodiment, the dsRNA agent of the invention further comprises one to five phosphorothioate or methylphosphonate internucleotide linkage modifications in positions 1-5 and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications in positions 18-23 (counting from the 5' end) of the sense strand, and further comprises one to five phosphorothioate or methylphosphonate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and one to five in positions 18-23 (counting from the 5' end).

[0335] In one embodiment, a dsRNA agent of the invention further comprises a phosphorothioate internucleotide linkage modification in positions 1-5 and further comprises a phosphorothioate or methylphosphonate internucleotide linkage modification in positions 18-23 of the sense strand (counting from the 5' end), and further comprises a phosphorothioate internucleotide linkage modification at positions 1 and 2 and further comprises two phosphorothioate or methylphosphonate internucleotide linkage modifications in positions 18-23 of the antisense strand (counting from the 5' end).

[0336] In one embodiment, a dsRNA agent of the invention further comprises two phosphorothioate internucleotide linkage modifications in positions 1-5 and one phosphorothioate internucleotide linkage modification in positions 18-23 of the sense strand (counting from the 5' end), and further comprises one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counting from the 5' end).

[0337] In one embodiment, a dsRNA agent of the invention further comprises two phosphorothioate internucleotide linkage modifications in positions 1-5 and two further phosphorothioate internucleotide linkage modifications in positions 18-23 of the sense strand (counting from the 5' end), and further comprises one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two further phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counting from the 5' end).

[0338] In one embodiment, a dsRNA agent of the invention further comprises two phosphorothioate internucleotide linkage modifications in positions 1-5 and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the sense strand (counting from the 5' end), and further comprises one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification in positions 18-23 of the antisense strand (counting from the 5' end).

[0339] In one embodiment, a dsRNA agent of the invention further comprises one phosphorothioate internucleotide linkage modification in positions 1-5 and one phosphorothioate internucleotide linkage modification in positions 18-23 of the sense strand (counting from the 5' end), and further comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and further comprises two phosphorothioate internucleotide linkage modifications in positions 18-23 (counting from the 5' end).

[0340] In one embodiment, a dsRNA agent of the invention further comprises one phosphorothioate internucleotide linkage modification in positions 1-5 and one phosphorothioate internucleotide linkage modification in positions 18-23 of the sense strand (counting from the 5' end), and further comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and one phosphorothioate internucleotide linkage modification in positions 18-23 (counting from the 5' end).

[0341] In one embodiment, a dsRNA agent of the invention further comprises a phosphorothioate internucleotide linkage modification in positions 1-5 of the sense strand (counting from the 5' end), and further comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and further comprises a phosphorothioate internucleotide linkage modification in positions 18-23 (counting from the 5' end).

[0342] In one embodiment, a dsRNA agent of the invention further comprises two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counting from the 5' end), and further comprises one phosphorothioate internucleotide linkage modification at positions 1 and 2 of the antisense strand and further comprises two phosphorothioate internucleotide linkage modifications in positions 18-23 (counting from the 5' end).

[0343] In one embodiment, a dsRNA agent of the invention further comprises two phosphorothioate internucleotide linkage modifications in positions 1-5 and one phosphorothioate internucleotide linkage modification in positions 18-23 of the sense strand (counting from the 5' end), and further comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification in positions 18-23 of the antisense strand (counting from the 5' end).

[0344] In one embodiment, a dsRNA agent of the invention further comprises two phosphorothioate internucleotide linkage modifications in positions 1-5 and one phosphorothioate internucleotide linkage modification in positions 18-23 of the sense strand (counting from the 5' end), and further comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counting from the 5' end).

[0345] In one embodiment, a dsRNA agent of the invention further comprises two phosphorothioate internucleotide linkage modifications in positions 1-5 and one phosphorothioate internucleotide linkage modification in positions 18-23 of the sense strand (counting from the 5' end), and further comprises one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counting from the 5' end).

[0346] In one embodiment, the dsRNA agent of the invention further comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and further comprises two phosphorothioate internucleotide linkage modifications at positions 20 and 21 of the sense strand (counting from the 5' end), and further comprises one phosphorothioate internucleotide linkage modification at position 1 and further comprises one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5' end).

[0347] In one embodiment, the dsRNA agent of the invention further comprises one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5' end), and further comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and further comprises two phosphorothioate internucleotide linkage modifications at positions 20 and 21 of the antisense strand (counting from the 5' end).

[0348] In one embodiment, the dsRNA agent of the invention further comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and further comprises two phosphorothioate internucleotide linkage modifications at positions 20 and 21 of the sense strand (counting from the 5' end), and further comprises one phosphorothioate internucleotide linkage modification at position 1 and further comprises one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5' end).

[0349] In one embodiment, the dsRNA agent of the invention further comprises one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5' end), and further comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and further comprises two phosphorothioate internucleotide linkage modifications at positions 21 and 22 of the antisense strand (counting from the 5' end).

[0350] In one embodiment, the dsRNA agent of the invention further comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and further comprises two phosphorothioate internucleotide linkage modifications at positions 22 and 23 of the sense strand (counting from the 5' end), and further comprises one phosphorothioate internucleotide linkage modification at position 1 and further comprises one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5' end).

[0351] In one embodiment, the dsRNA agent of the invention further comprises one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5' end), and further comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and further comprises two phosphorothioate internucleotide linkage modifications at positions 23 and 23 of the antisense strand (counting from the 5' end).

[0352] In one embodiment, the dsRNA agent of the invention includes one or more mismatches or combinations thereof with the target within the duplex. The mismatch may be present in the overhang region or the duplex region. Base pairs can be graded based on their tendency to promote dissociation or melting (e.g., for a particular pairing's association or dissociation free energy, the simplest approach is to examine these pairs based on an individual pair, but immediate neighbors or similar analyses may also be used). With respect to promoting dissociation: A:U is preferred to G:C; G:U is preferred to G:C; and I:C is preferred to G:C (I = inosine). Mismatches, such as non-canonical pairings or pairings other than canonical (as described elsewhere herein) are preferred to canonical pairings (A:T, A:U, G:C); and pairings comprising universal bases are preferred to canonical pairings.

[0353] In one embodiment, the dsRNA agent of the invention includes at least one of the first 1, 2, 3, 4 or 5 base pairs within the duplex region from the 5' end of the antisense strand, which can be independently selected from the group consisting of A:U, G:U, I:C, and mismatched pairs (e.g., non-standard or other than standard pairs) or pairs that include a universal base to promote dissociation of the antisense strand at the 5' end of the duplex.

[0354] In one embodiment, the nucleotide at the 1 position from the 5' end of the antisense strand in the duplex region 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 in the duplex region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the duplex region from the 5' end of the antisense strand is an AU base pair.

[0355] The inventors have discovered that introducing 4'-modified and / or 5'-modified nucleotides into the 3'-end of a phosphodiester (PO), phosphorothioate (PS), and / or phosphorodithioate (PS2) linkage of a dinucleotide at any position in a single-stranded or double-stranded oligonucleotide can exert a steric effect on the internucleotide linkage and thereby protect or stabilize the internucleotide linkage against nucleases.

[0356] In one embodiment, the 5'-modified nucleoside is introduced into the 3'-end of the dinucleotide at any position of the single-stranded or double-stranded siRNA. For example, the 5'-alkylated nucleoside is introduced into the 3'-end of the dinucleotide at any position of the single-stranded or double-stranded siRNA. The alkyl group at the 5' position of the ribose can be a racemic or chirally pure R or S isomer. An exemplary 5'-alkylated nucleoside is a 5'-methyl nucleoside. The 5'-methyl can be a racemic or chirally pure R or S isomer.

[0357] In one embodiment, a 4'-modified nucleoside is introduced into the 3'-end of a dinucleotide at any position of a single-stranded or double-stranded siRNA. For example, a 4'-alkylated nucleoside is introduced into 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 can be a racemic or chirally pure R or S isomer. An exemplary 4'-alkylated nucleoside is a 4'-methyl nucleoside. The 4'-methyl group can be a racemic or chirally pure R or S isomer. Alternatively, a 4'-O-alkylated nucleoside can be introduced into the 3'-end of a dinucleotide at any position of a single-stranded or double-stranded siRNA. The 4'-O-alkyl group of the ribose can be a racemic or chirally pure R or S isomer. An exemplary 4'-O-alkylated nucleoside is a 4'-O-methyl nucleoside. The 4'-O-methyl group can be a racemic or chirally pure R or S isomer.

[0358] In one embodiment, 5'-alkylated nucleosides are introduced into any position on the sense or antisense strand of a dsRNA, and such modifications maintain or improve the effectiveness of the dsRNA. The 5'-alkyl group can be a racemic or chirally pure R or S isomer. An exemplary 5'-alkylated nucleoside is a 5'-methyl nucleoside. The 5'-methyl group can be a racemic or chirally pure R or S isomer.

[0359] In one embodiment, 4'-alkylated nucleosides are introduced into any position on the sense or antisense strand of a dsRNA, and such modifications maintain or improve the effectiveness of the dsRNA. The 4'-alkyl group can be a racemic or chirally pure R or S isomer. An exemplary 4'-alkylated nucleoside is a 4'-methyl nucleoside. The 4'-methyl group can be a racemic or chirally pure R or S isomer.

[0360] In one embodiment, 4'-O-alkylated nucleosides are introduced into any position of the sense or antisense strand of a dsRNA, and such modifications maintain or improve the effectiveness of the dsRNA. The 5'-alkyl group can be a racemic or chirally pure R or S isomer. An exemplary 4'-O-alkylated nucleoside is a 4'-O-methyl nucleoside. The 4'-O-methyl group can be a racemic or chirally pure R or S isomer.

[0361] In one embodiment, the sense strand sequence of the dsRNA agent is represented by formula (Is):

[0362]

[0363] in:

[0364] B1, B2, and B3 each independently represent a nucleotide containing a modification selected from the group consisting of 2'-O alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halo, ENA, and BNA / LNA;

[0365] C1 is a thermolabile nucleotide (e.g., an acyclic nucleotide (such as UNA or GNA), a mismatch, an abasic nucleotide, or DNA) located at a position relative to the seed region of the antisense strand (i.e., positions 2-8 of the 5'-end of the antisense strand);

[0366] T1 represents a nucleotide comprising a chemical modification at the 2' position or at an equivalent position in a non-ribose, acyclic or backbone that provides a steric bulk less than that of a 2'-OMe modification; for example, T1 is selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl;

[0367] n 1 or n 3 are independently 4 to 15 nucleotides in length;

[0368] n 5 The length is 1-6 nucleotides;

[0369] n 4 The length is 1-3 nucleotides; alternatively n 4 is 0, and

[0370] n 2 The length is 0-3 nucleotides.

[0371] In one embodiment, the sense strand sequence of the dsRNA agent has a length of 19, 20, 21, or 22 nucleotides, which is represented by formula (Is):

[0372]

[0373] in:

[0374] B1, B2, and B3 each independently represent a nucleotide containing a modification selected from the group consisting of 2'-O alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halo, ENA, and BNA / LNA;

[0375] C1 is a thermolabile nucleotide (e.g., an acyclic nucleotide (such as UNA or GNA), a mismatch, an abasic nucleotide, or DNA) located at a position relative to the seed region of the antisense strand (i.e., positions 2-8 of the 5'-end of the antisense strand);

[0376] T1 represents a nucleotide comprising a chemical modification selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl;

[0377] n 1 or n 3 are independently 4 to 15 nucleotides in length;

[0378] n 5 The length is 1-6 nucleotides;

[0379] n 4 The length is 1-3 nucleotides; alternatively n 4 is 0 and

[0380] n 2 The length is 0-3 nucleotides.

[0381] In one embodiment, the dsRNA agent of Formula (Is) further comprises one or more 3' and / or 5' overhangs of 1-10 nucleotides in length. In one example, the dsRNA agent of Formula (Is) comprises a 5' overhang.

[0382] In one embodiment, C1 comprises a thermolabile nucleotide at position 14, 15, 16 or 17 from the 5'-end of the sense strand. For example, C1 is an acyclic nucleotide (e.g., UNA or GNA), a mismatch, an abasic nucleotide, or DNA. In a specific example, C1 is GNA.

[0383] In one embodiment, T1 comprises DNA, RNA, LNA, 2'-F, or 2'-F-5'-methyl at position 11 from the 5'-end of the sense strand.

[0384] In one embodiment, a dsRNA agent of the invention comprises a sense strand (Is) in which C1 is an acyclic nucleotide (e.g., UNA or GNA), a mismatch, abasic, or DNA; and T1 comprises DNA, RNA, LNA, 2'-F, or 2'-F-5'-methyl at position 11 from the 5'-end of the sense strand.

[0385] In one embodiment, the antisense strand sequence of the dsRNA agent is represented by Formula (Ia):

[0386]

[0387] in:

[0388] 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;

[0389] T1', T2', and T3' each independently represent a nucleotide comprising a chemical modification at the 2' position or at an equivalent position in a non-ribose, acyclic, or backbone that provides a steric bulk less than that of a 2'-OMe modified nucleotide; for example, T1', T2', and T3' are each independently selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl;

[0390] q 1 are independently 4 to 15 nucleotides in length;

[0391] q 3 or q 7 are independently 1-6 nucleotides in length;

[0392] q 2或 q 6 are independently 1-3 nucleotides in length;

[0393] q 4 are independently 0-3 nucleotides in length; and

[0394] q 5 are independently 0-10 nucleotides in length.

[0395] In one embodiment, the antisense strand sequence of the dsRNA agent has a length of 19, 20, 21, 22, 23, 24, or 25 nucleotides, which is represented by formula (Ia):

[0396]

[0397] in:

[0398] 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;

[0399] T1', T2', and T3' each independently represent a nucleotide comprising a chemical modification selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl;

[0400] q 1 are independently 4 to 15 nucleotides in length;

[0401] q 3 or q 7 are independently 1-6 nucleotides in length;

[0402] q 2 or q 6 are independently 1-3 nucleotides in length;

[0403] q 4 are independently 0-3 nucleotides in length; and

[0404] q 5 are independently 0-10 nucleotides in length.

[0405] In one embodiment, the dsRNA of Formula (Ia) further comprises one or more 3' and / or 5' overhangs of 1-10 nucleotides in length. In one example, the dsRNA of Formula (Ia) comprises a 3' overhang.

[0406] In one embodiment, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting target gene expression. The dsRNA agent includes a sense strand and an antisense strand, each strand having 14 to 40 nucleotides:

[0407]

[0408] in:

[0409] 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;

[0410] C1 is an acyclic nucleotide (e.g., UNA or GNA);

[0411] T1, T1', T2', and T3' each independently represent a nucleotide comprising a chemical modification selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl;

[0412] n 1 、n 3 , or q 1 are independently 4 to 15 nucleotides in length;

[0413] n 5 ,q 3 or q 7 are independently 1-6 nucleotides in length;

[0414] n 4 ,q 2 or q 6 The length of is independently 1-3 nucleotides; alternatively n 4 is 0,

[0415] n 2 or q 4 are independently 0-3 nucleotides in length;

[0416] q 5 are independently 0-10 nucleotides in length; and

[0417] The antisense and / or sense strand dsRNA agents may have one or more 3' and / or 5' overhangs of 1-10 nucleotides in length.

[0418] In one embodiment, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting target gene expression. The dsRNA agent includes a sense strand and an antisense strand, each strand having 14 to 40 nucleotides:

[0419]

[0420] in:

[0421] 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;

[0422] C1 is an acyclic nucleotide (e.g., UNA or GNA);

[0423] T1, T1', T2', and T3' each independently represent a nucleotide comprising a chemical modification selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl;

[0424] n 1 、n 3 , or q 1 are independently 4 to 15 nucleotides in length;

[0425] n 5 ,q 3 or q 7 are independently 1-6 nucleotides in length;

[0426] n 4 ,q 2 or q 6 The length of is independently 1-3 nucleotides; alternatively n 4 is 0,

[0427] n 2 or q 4 are independently 0-3 nucleotides in length;

[0428] q 5 are independently 0-10 nucleotides in length; and

[0429] The dsRNA agent has a 3' overhang of 2 nucleotides at the 3'-end of the antisense strand.

[0430] In one embodiment, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting target gene expression. The dsRNA agent includes a sense strand and an antisense strand, each strand having 15-30 nucleotides:

[0431]

[0432] in:

[0433] B1, B2, B3, B1', B2', B3', and B4' each independently represent a nucleotide containing a 2'-OMe modification;

[0434] C1 is an acyclic nucleotide GNA;

[0435] T1, T2, T2′, and T3′ are each independently DNA or RNA;

[0436] n 1 、n 3 , or q 1 are independently 4 to 15 nucleotides in length;

[0437] n5 ,q 3 or q 7 are independently 1-6 nucleotides in length;

[0438] N4, q2 or q6 are independently 1-3 nucleotides in length; alternatively, n4 4 ,q 2 or q 6 The length of n is independently 1-3 nucleotides; alternatively, n 4 is 0,

[0439] n 2 or q 4 are independently 0-3 nucleotides in length;

[0440] q 5 are independently 0-10 nucleotides in length; and

[0441] The dsRNA agent has a 3' overhang of 1-6 nucleotides in length at the 3'-end of the antisense strand.

[0442] In one embodiment, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting target gene expression. The dsRNA agent includes a sense strand and an antisense strand, each strand having 19-23 nucleotides:

[0443]

[0444] in:

[0445] B1, B2, B3, B1', B2', B3', and B4' each independently represent a nucleotide containing a 2'-OMe modification;

[0446] C1 is an acyclic nucleotide GNA;

[0447] T1, T1', T2', and T3' are independently DNA or RNA;

[0448] n 1 、n 3 ,q 1 , or q 3 are independently 4 to 15 nucleotides in length;

[0449] n 5 ,q 3 or q 7 are independently 1-6 nucleotides in length;

[0450] n 4 ,q 2 or q 6 The length of n is independently 1-3 nucleotides; alternatively n4 is 0,

[0451] n 2 ,q 4 or q 5 are independently 0-3 nucleotides in length;

[0452] q 5 are independently 0-10 nucleotides in length; and

[0453] The dsRNA agent has a 3' overhang of 2 nucleotides at the 3'-end of the antisense strand.

[0454] In one embodiment, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting target gene expression. The dsRNA agent includes a sense strand and an antisense strand, each strand having 14 to 40 nucleotides:

[0455]

[0456] in:

[0457] 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;

[0458] C1 is an acyclic nucleotide (e.g., UNA or GNA);

[0459] T1, T1', T2', and T3' each independently represent a nucleotide comprising a chemical modification selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl;

[0460] n 1 、n 3 , or q 1 are independently 4 to 15 nucleotides in length;

[0461] n 5 ,q 3 or q 7 are independently 1-6 nucleotides in length;

[0462] n 4 ,q 2 or q 6 The length of is independently 1-3 nucleotides; alternatively n 4 is 0,

[0463] n 2 or q4 are independently 0-3 nucleotides in length;

[0464] q 5 are independently 0-10 nucleotides in length; and

[0465] Wherein at the 5'-end of the sense strand, the dsRNA agent has a 5' overhang of 1-10 nucleotides in length.

[0466] In one embodiment, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting target gene expression. The dsRNA agent includes a sense strand and an antisense strand, each strand having 14 to 40 nucleotides:

[0467]

[0468] in:

[0469] 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;

[0470] C1 is an acyclic nucleotide (e.g., UNA or GNA);

[0471] T1, T1', T2', and T3' each independently represent a nucleotide comprising a chemical modification selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl;

[0472] n 1 、n 3 , or q 1 are independently 4 to 15 nucleotides in length;

[0473] n 5 ,q 3 or q 7 are independently 1-6 nucleotides in length;

[0474] n 4 ,q 2 or q 6 The length of is independently 1-3 nucleotides; alternatively n 4 is 0,

[0475] n 2 or q 4 are independently 0-3 nucleotides in length;

[0476] q 5are independently 0-10 nucleotides in length; and

[0477] Wherein at the 5'-end of the sense strand, the dsRNA agent has a 5' overhang of 1-6 nucleotides in length.

[0478] In one embodiment, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting target gene expression. The dsRNA agent includes a sense strand and an antisense strand, each strand having 14 to 40 nucleotides:

[0479]

[0480] in:

[0481] 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;

[0482] C1 is an acyclic nucleotide (e.g., UNA or GNA);

[0483] T1, T1', T2', and T3' each independently represent a nucleotide comprising a chemical modification selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl;

[0484] n 1 、n 3 , or q 1 are independently 4 to 15 nucleotides in length;

[0485] n 5 ,q 3 or q 7 are independently 1-6 nucleotides in length;

[0486] n 4 ,q 2 or q 6 The length of is independently 1-3 nucleotides; alternatively n 4 is 0,

[0487] n 2 or q 4 are independently 0-3 nucleotides in length;

[0488] q 5 are independently 0-10 nucleotides in length; and

[0489] wherein the dsRNA agent has a 5' overhang of 1-10 nucleotides in length at the 5'-end of the sense strand and a 3' overhang of 1-10 nucleotides in length at the 5'-end of the antisense strand.

[0490] Thermal instability modifications.

[0491] By introducing a thermolabile modification into the sense strand at a site opposite the seed region of the antisense strand (i.e., at positions 2-8 of the 5'-end of the antisense strand), dsRNA agents can be optimized for RNA interference by increasing the propensity of the dsRNA duplex to dissociate or melt (reducing the free energy of duplex association). This modification can increase the propensity of the duplex to dissociate or melt in the seed region of the antisense strand.

[0492] These thermolabile modifications can include abasic modifications; mismatches with the opposite nucleotide in the opposite strand; and sugar modifications, such as 2'-deoxy modifications or acyclic nucleotides, e.g., unlocked nucleic acids (UNA) or glycerol nucleic acids (GNA).

[0493] Exemplary abasic modifications are:

[0494]

[0495] Exemplary sugar modifications are:

[0496]

[0497] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, e.g., wherein any linkage between the ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') is absent from the nucleotide and / or at least one ribose carbon or oxygen (e.g., C1', C2', C3', C4', or O4') is absent, independently or in combination, from the nucleotide. In some embodiments, an acyclic nucleotide is Wherein B is a modified or unmodified nucleobase, R 1 and R 2is independently H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar). The term "UNA" refers to an unlocked acyclic nucleic acid in which any bond to the sugar is removed to form an unlocked "sugar" residue. In one example, UNA also includes monomers having a bond between C1'-C4' removed (i.e., a covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another example, the C2'-C3' bond of the sugar (i.e., a covalent carbon-carbon bond between the C2' and C3' carbons) is removed. (See Mikhailov et al., Tetrahedron Letters, 26(17):2059 (1985); and Fluiter et al., Mol. Biosyst., 10:1039 (2009), which are incorporated herein by reference in their entireties). Acyclic derivatives provide greater backbone flexibility without affecting Watson-Crick pairing.The acyclic nucleotides can be linked via 2'-5' or 3'-5' linkages.

[0498] The term 'GNA' refers to a glycol nucleic acid, which is a polymer similar to DNA or RNA, but differs in the composition of the "backbone" in that it is composed of repeating glycerol units connected by phosphodiester bonds:

[0499]

[0500] The thermolabile modification can be a mismatch (i.e., a non-complementary base pair) between a thermolabile nucleotide and an opposing nucleotide in the opposite strand of 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 a combination thereof. Other mismatch base pairs known in the art are also subject to the present invention. Mismatches can be present between nucleotides of naturally occurring or modified nucleotides, i.e., mismatched base pairing can be present between nucleobases from each nucleotide, regardless of the modification on the ribose of the nucleotide. In certain embodiments, the dsRNA agent contains at least one nucleobase in the mismatched pairing that is a 2'-deoxynucleobase; for example, a 2'-deoxynucleobase in the sense strand.

[0501] Further examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications have been described in detail in WO 2011 / 133876, which is incorporated herein by reference in its entirety.

[0502] Thermolabile modifications can also include universal bases with reduced or lost activity in forming hydrogen bonds with opposing bases, as well as phosphate modifications.

[0503] Nucleobase modifications that impair or completely eliminate the ability to form hydrogen bonds with bases in the opposite strand have been evaluated for destabilization of the central region of the dsRNA duplex, as described in WO 2010 / 0011895, which is incorporated herein by reference in its entirety. Exemplary nucleobase modifications are:

[0504]

[0505] Exemplary phosphate modifications known to reduce the thermal stability of dsRNA duplexes compared to native phosphodiester linkages are:

[0506]

[0507] In one embodiment, the dsRNA agents of the invention can include 2'-5' linkages (with 2'-H, 2'-OH, and 2'-OMe, as well as with P=O or P=S). For example, 2'-5' linkage modifications can be used to promote nuclease resistance or inhibit ligation of the sense strand to the antisense strand, or can be used to prevent activation of the sense strand by RISC at the 5' end of the sense strand.

[0508] In another embodiment, the dsRNA agents of the present invention can include L sugars (e.g., L ribose, L-arabinose with 2'-H, 2'-OH, and 2'-OMe). For example, these L sugar modifications can be used to promote nuclease resistance or inhibit the ligation of the sense strand to the antisense strand, or can be used to prevent activation of the sense strand by RISC at the 5' end of the sense strand.

[0509] In one embodiment, the dsRNA agent is a polymer comprising at least two duplexes represented by formula (I), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the polymer further comprises a ligand. Each dsRNA agent can target the same gene or two different genes; or each dsRNA agent can target the same gene at two different target sites.

[0510] In one embodiment, the dsRNA agent is a polymer comprising three, four, five, six or more duplexes represented by formula (I), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the polymer further comprises a ligand. Each dsRNA agent can target the same gene or two different genes; or each dsRNA agent can target the same gene at two different target sites.

[0511] In one embodiment, the two dsRNA agents represented by formula (I) are linked to each other at the 5' end, and one or both of the 3' ends are optionally conjugated to a ligand. Each dsRNA can target the same gene or two different genes; or each dsRNA can target the same gene at two different target sites.

[0512] Various publications describe multimeric siRNAs and all can be used with the dsRNA of the present invention. Such publications include WO 2007 / 091269, U.S. Patent No. 7858769, WO 2010 / 141511, WO 2007 / 117686, WO 2009 / 014887, and WO 2011 / 031520, the entire contents of which are hereby incorporated.

[0513] The dsRNA agent comprising one or more carbohydrate moieties combined with a dsRNA agent can optimize one or more properties of the dsRNA agent. In many cases, the carbohydrate moiety will be connected to a modified subunit of the dsRNA agent. For example, the ribose of one or more ribonucleotide subunits of a dsRNA agent can be replaced by another part (e.g., a carbohydrate ligand is attached to a non-carbohydrate (preferably cyclic) carrier thereon). The ribonucleotide subunit in which the ribose of the subunit has been replaced is referred to as a ribose replacement modified subunit (RRMS) herein. A cyclic carrier can be a carbocyclic ring system, i.e., all annular atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more annular atoms can be a heteroatom, such as nitrogen, oxygen, sulfur. The cyclic carrier can be a monocyclic ring system, or can contain two or more rings, such as fused rings. The cyclic carrier can be a fully saturated ring system, or it can contain one or more double bonds.

[0514] The ligand can be attached to the polynucleotide via a carrier. These carriers include (i) at least one "backbone attachment point", preferably two "backbone attachment points", and (ii) at least one "tether attachment point". As used herein, a "backbone attachment point" refers to a functional group (e.g., a hydroxyl group), or generally, a bond (e.g., a phosphate or modified phosphate) that is available and suitable for binding the carrier to the backbone (e.g., a sulfur-containing backbone) of a ribonucleic acid. In some embodiments, a "tether attachment point" (TAP) refers to a constituent ring atom of the cyclic carrier, such as a carbon atom or a heteroatom (different from the atom that provides the backbone attachment point), to which a selected moiety is attached. The moiety can be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, and polysaccharide. Optionally, the selected moiety is connected to the cyclic carrier via an intervening tether. Thus, the cyclic carrier will often include a functional group (e.g., an amino group), or generally provide a bond suitable for binding or tethering another chemical entity (e.g., a ligand) to the constituent ring.

[0515] In one embodiment, the dsRNA agent of the present invention can be conjugated to a ligand via a carrier, wherein the carrier can be a cyclic group or a non-cyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinone, tetrahydrofuranyl and decahydronaphthalene; preferably, the non-cyclic group is selected from a serinol backbone or a diethanolamine backbone.

[0516] The double-stranded RNA (dsRNA) agents of the present invention may optionally be conjugated to one or more ligands. The ligand may be attached to the sense strand, the antisense strand, or both strands at the 3' end, the 5' end, or both ends. For example, the ligand may be conjugated to the sense strand, particularly at the 3' end of the sense strand.

[0517] In one embodiment, the dsRNA agents of the invention are 5' phosphorylated or include a phospho group analog at the 5' major end. 5'-Phosphate modifications include those that are compatible with RISC-mediated gene silencing. Suitable modifications include: 5'-monophosphate ((HO)2(O)PO-5'); 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-monothioate (phosphorothioate; (HO)2(S)PO-5'); 5'-monodithioate (phosphorodithioate; ( HO)(HS)(S)PO-5'), 5'-thiophosphate ((HO)2(O)PS-5'); any additional combination of oxygen / sulfur substituted mono-, di-, and triphosphates (e.g., 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-phosphoramidate ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'), 5'-alkyl phosphates (R = alkyl = methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)-O-5'-, 5'-alkenyl phosphates (i.e., vinyl, substituted vinyl), (OH)2(O)P-5'-CH2-), 5'-alkyl ether phosphates (R = alkyl ether = methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5'-). In one example, the modification can be on the antisense strand of the dsRNA agent.

[0518] ligand

[0519] A wide variety of entities can be coupled to the oligonucleotides of the invention. Preferred moieties are ligands which are preferably covalently coupled directly or indirectly via an intervening tether.

[0520] In preferred embodiments, a ligand alters the distribution, targeting, or lifetime of a molecule into which it is incorporated. In preferred embodiments, a ligand provides enhanced affinity for a selected target, such as a molecule, cell or cell type, compartment, receptor, such as a cellular compartment or organ compartment, tissue, organ, or region of the body, compared to a species lacking such a ligand. Ligands that provide enhanced affinity for a selected target are also referred to as targeting ligands.

[0521] Some ligands may have endosomal lytic properties. The endosomal lytic ligand promotes the dissolution of endosomes and / or the transport of the composition of the invention or its components from endosomes to the cytoplasm of cells. The endosomal lytic ligand may be a polyanionic peptide or peptide mimetic that exhibits pH-dependent membrane activity and fusogenicity. In one embodiment, the endosomal lytic ligand assumes its active conformation at endosomal pH. An "active" conformation is one in which the endosomal lytic ligand promotes the dissolution of endosomes and / or the transport of the composition of the invention or its components from endosomes to the cytoplasm of cells. Exemplary endosomolytic ligands include GALA peptide (Subbarao et al., Biochemistry, 1987, 26:2964-2972), which is incorporated herein by reference in its entirety, EALA peptide (Vogel et al., J. Am. Chem. Soc., 1996, 118:1581-1586, which is incorporated herein by reference in its entirety), and derivatives thereof (Turk et al., Biochem. Biophys. Acta, 2002, 1559:56-68, which is incorporated herein by reference in its entirety). In one embodiment, the endosomolytic component can comprise a chemical group (e.g., an amino acid) that undergoes a charge change or protonation in response to a pH change. The endosomolytic component can be linear or branched.

[0522] Ligands can improve the transport, hybridization, and specificity properties and can also improve the nuclease resistance of the resulting natural or modified oligoribonucleotide or a polymeric molecule comprising any combination of monomers described herein and / or natural or modified ribonucleotides.

[0523] Typical ligands may include, for example, therapeutic modulators for enhancing uptake; diagnostic compounds or reporter groups for monitoring distribution; cross-linking agents; and moieties that confer nuclease resistance. Typical examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptide mimetics.

[0524] The ligand can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); a carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, for example, a synthetic polyamino acid, an oligonucleotide (e.g., an aptamer). Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha helical peptides.

[0525] The part can also include a targeting group, such as a cell or tissue targeting agent that is combined with a specified cell type such as nephrocytes, such as a lectin, glycoprotein, lipid or protein, such as an antibody. The targeting group can be thyrotropin, melanocyte stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent trehalose, glycosylated polyamino acids, multivalent galactose, transferrins, bisphosphonates, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, biotin, RGD peptide, RGD peptide mimetics or aptamers. Table 2 shows some examples of targeting ligands and their associated receptors.

[0526] Other examples of ligands include dyes, intercalating agents (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases or a chelating agent (e.g., EDTA), lipophilic molecules such as cholesterol, bile acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl, hexadecylglycerol, camphenol, menthol, 1,3-propylene glycol, heptadecanol, palmitic acid, myristic acid, O3-(oleoyl)glycerol, ) lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphates, amino groups, sulfhydryl groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), delivery / adsorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP or AP.

[0527] The ligand can be a protein, such as a glycoprotein, or a peptide, such as a molecule with a specific affinity for a co-ligand, or an antibody, such as an antibody that binds to a specific cell type (such as a cancer cell, endothelial cell, or bone cell). Ligands can also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, or aptamers. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

[0528] The ligand can be a substance, such as a drug, that can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cytoskeleton of the cell (e.g., by disrupting cellular microtubules, microfilaments, and / or intermediate filaments). The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, limnid A, phalloidin, swinholide A, indanocine, or myoservin.

[0529] The ligand can increase the uptake of the oligonucleotide into the cell by, for example, activating an inflammatory response. Exemplary ligands with this effect include tumor necrosis factor alpha (TNF-α), interleukin-1β, or interferon gamma.

[0530] In one aspect, the ligand is a lipid or lipid-based molecule. The lipid or lipid-based molecule preferably binds to a serum protein, such as human serum albumin (HSA). A ligand that binds to HSA allows 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 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 (a) increase the resistance of the conjugate to degradation, (b) increase targeting or transport to target cells or cell membranes, and / or (c) can be used to modulate binding to serum proteins (e.g., HSA).

[0531] Lipid-based ligands can be used to modulate (e.g., control) the binding of the conjugate to the target tissue. For example, a lipid or lipid-based ligand that binds more strongly to HSA will be less likely to target the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds less strongly to HSA can be used to target the conjugate to the kidney.

[0532] In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, it binds to HSA with sufficient affinity so that the conjugate will preferentially distribute to non-renal tissues. However, it is preferred that this affinity is not so strong that the HSA-ligand binding cannot be reversed.

[0533] In another preferred embodiment, the lipid-based ligand binds poorly or not at all to HSA, such that the conjugate will preferentially distribute to the kidney.Other moieties that target kidney cells may be used instead of or in addition to lipid-based ligands.

[0534] On the other hand, the part is a part taken in by target cells (e.g., cells being proliferated), such as vitamins. These are particularly useful for treating conditions characterized by unwanted cell proliferation (e.g., with malignant or non-malignant types, such as cancer cells). Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken in by cancer cells. Also included are HSA, low-density lipoprotein (LDL), and high-density lipoprotein (HDL).

[0535] In another aspect, the ligand is a cell permeating agent, preferably a helical cell permeating agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide, such as tat or antennapedia. If the agent is a peptide, it can be modified, including the use of peptidyl mimetics, retromers, non-peptide linkages or pseudopeptide linkages, and D-amino acids. The helical agent is preferably an α-helical agent, which preferably has a lipophilic phase and a lipophobic phase.

[0536] The part can be a peptide or peptide mimetic. Peptide mimetic (also referred to as oligopeptide mimetic herein) is a molecule that can fold into a limited three-dimensional structure similar to a natural peptide. The peptide or peptide mimetic portion can be about 5-50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids long. The peptide or peptide mimetic can be, for example, a cell permeable peptide, a cationic peptide, an amphipathic peptide or a hydrophobic peptide (for example, mainly composed of Tyr, Trp or Phe). The peptide portion can be a dendritic peptide, a constrained peptide or a cross-linked peptide. In another alternative, the peptide portion can include a hydrophobic membrane translocation sequence (MTS). An exemplary peptide containing hydrophobic MTS is the RFGF with the amino acid sequence AAVALLPAVLLALLAP. A RFGF analog containing a hydrophobic MTS (for example, the amino acid sequence AALLPVLLAAP) can also be a targeting moiety. The peptide portion can be a "delivery" peptide that can carry large polar molecules, including peptides, oligonucleotides, and proteins that span cell membranes. For example, sequences from HIV Tat protein (GRKKRRQRRRPPQ) and Drosophila antenna foot protein (RQIKIWFQNRRMKWKK) have been found to be capable of serving as delivery peptides. A peptide or peptide mimetic can be encoded by a random DNA sequence (e.g., a peptide identified by a phage display library or a one-bead-one-compound (OBOC) combinatorial library) (Lam et al., Nature, 354:82-84, 1991, which is incorporated herein by reference in its entirety). Preferably, the peptide or peptide mimetic tethered to an iRNA agent via an incorporated monomeric unit is a cell targeting peptide, such as an arginine-glycine-aspartic acid (RGD) peptide or RGD mimetic. The length of the peptide portion can be in the range of from about 5 amino acids to about 40 amino acids. These peptide portions can have structural modifications, such as to increase stability or guide conformational properties. Any of the structural modifications described below can be utilized. An RGD peptide portion can be used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002, which is incorporated herein by reference in its entirety). An RGD peptide can facilitate targeting of an iRNA agent to tumors of various other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001, which is incorporated herein by reference in its entirety). Preferably, the RGD peptide will facilitate targeting of the iRNA agent to the kidney. The RGD peptide can be linear or cyclic and can be modified (e.g., glycosylated or methylated) to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver an iRNA agent to a cell expressing αV β3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001, which is incorporated herein by reference in its entirety). Peptides that target markers that are enriched in proliferating cells can be used. For example, peptides and peptide mimetics containing RGD can target cancer cells, specifically cells that display an 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 parts of integrin-targeting ligands can be used. In general, such ligands can be used to control proliferating cells and angiogenesis. Preferred conjugates of this type of ligand target PECAM-1, VEGF, or other oncogenes (e.g., an oncogene described herein).

[0537] "Cell penetrating peptides" are capable of penetrating cells such as microbial cells (e.g., bacteria or fungal cells) or mammalian cells (e.g., human cells). A microbial cell penetrating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a peptide containing a disulfide bond (e.g., α-defensin, β-defensin or bacteriocin) or a peptide containing only one or two major amino acids (e.g., PR-39 or indolicidin). Cell penetrating peptides can also include a nuclear localization signal (NLS). For example, a cell penetrating peptide can be a dual amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of the SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31: 2717-2724, 2003, which is incorporated herein by reference in its entirety).

[0538] In one embodiment, a targeting peptide can be an amphipathic α-helical peptide. Exemplary amphipathic α-helical peptides include, but are not limited to, cecropin, lycotoxin, paradaxin, buforin, CPF, bombesin-like peptide (BLP), cathelicidin, ceratotoxin, S. clava peptide, hagfish intestinal antimicrobial peptide (HFIAP), magainin, brevinins-2, dermaseptin, melittin, pleurocidin, H2A peptide, clawed frog peptide, esculentinis-1 and caerins. Many factors will preferably be considered to maintain the integrity of spiral stability. For example, the maximum number of spiral stabilizing residues (e.g., leu, ala or lys) will be used, and the minimum number of spiral destabilizing residues (e.g., proline or cyclic monomer units) will be used. Capping residues will be considered (e.g., Gly is an exemplary N-capping residue) and / or C-terminal amidation can be used to provide an additional H-bond to stabilize the helix. The formation of salt bridges between residues of opposite charge separated by i±3 or i±4 positions can provide stability. For example, cationic residues such as lysine, arginine, homoarginine, ornithine, or histidine can form salt bridges with anionic residues glutamate or aspartate.

[0539] Peptide and peptidomimetic ligands include those with naturally occurring or modified peptides, e.g., D or L peptides; α, β or γ peptides; N-methyl peptides; azapeptides; peptides with one or more amides, i.e., peptides with linkages replaced with one or more urea, thiourea, carbamate or sulfonylurea linkages; or cyclic peptides.

[0540] The targeting ligand can be any ligand capable of targeting a specific receptor. Examples are: folate, GalNAc, galactose, mannose, mannose-6P, sugar clusters (such as GalNAc clusters, mannose clusters, galactose clusters) or an aptamer. A cluster is a combination of two or more sugar units. These targeting ligands also include integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands. These ligands can also be based on nucleic acids, such as an aptamer. The aptamer can be unmodified or have any combination of modifications disclosed herein.

[0541] Endosomal release agents include imidazoles, polyimidazoles or oligoimidazoles, PEI, peptides, fusion peptides, polycarboxylates, polycations, masked oligo- or polycations or anions, acetals, polyacetals, ketals / polyketals, orthoesters, polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges.

[0542] PK regulator represents a pharmacokinetic regulator. PK regulators include lipophiles, cholic acid, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, etc. Exemplary PK regulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkyl glycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, etc. Oligonucleotides comprising many thiophosphate linkages are also known to bind to serum proteins, so short oligonucleotides comprising multiple thiophosphate linkages in the backbone, for example, oligonucleotides with about 5 bases, 10 bases, 15 bases, or 20 bases, are also subject to the present invention as ligands (for example, as PK regulating ligands).

[0543] Additionally, aptamers that bind to serum components (eg, serum proteins) as PK modulating ligands are also subject to the present invention.

[0544] Other ligand conjugates of the present invention are described in the following U.S. patent applications: USSN: 10 / 916,185 filed on August 10, 2004; USSN: 10 / 946,873 filed on September 21, 2004; USSN: 10 / 833,934 filed on August 3, 2007; USSN: 11 / 115,989 filed on April 27, 2005 and USSN: 11 / 944,227 filed on November 21, 2007; the entire contents of these patent applications are incorporated by reference for all purposes.

[0545] When two or more ligands are present, these ligands may all have the same properties, all have different properties, or some ligands may have the same properties while others may have different properties. For example, one ligand may have targeting properties, endosomal lytic activity, or PK modulating properties. In a preferred embodiment, all of these ligands have different properties.

[0546] The ligand can be coupled to different positions of the oligonucleotide, such as the 3' end, the 5' end and / or at an internal position. In preferred embodiments, the ligand is attached to the oligonucleotides via an intervening tether (e.g., a carrier described herein). When a monomer is incorporated into a growing chain, the ligand or ligand of the tether can be present on the monomer. In some embodiments, the ligand can be incorporated via coupling to a "precursor" monomer after it has been incorporated into the growing chain. For example, a monomer (e.g., TAP-(CH2)) with, for example, an amino-terminated tether (i.e., without an associated ligand) can be incorporated into the growing chain. n In a subsequent operation, i.e., after the precursor monomer has been incorporated into the chain, a ligand having an electrophilic group (e.g., a pentafluorophenyl ester or aldehyde group) can then be attached to the precursor monomer by coupling the electrophilic group of the ligand to the terminal nucleophilic group of the precursor monomer's tether.

[0547] In another example, a monomer with a chemical group suitable for participating in click chemistry reactions, such as an azide or alkyne-terminated tether / linker, can be incorporated. In a subsequent operation, i.e., after the precursor monomer is incorporated into the chain, a ligand with a complementary chemical group (e.g., an alkyne or azide) can be attached to the precursor monomer by coupling the alkyne and the azide together.

[0548] For double-stranded oligonucleotides, a ligand can be attached to one or both strands. In some embodiments, a double-stranded iRNA agent comprises a ligand conjugated to the sense strand. In other embodiments, a double-stranded iRNA agent comprises a ligand conjugated to the antisense strand.

[0549] In certain embodiments, the ligand can be conjugated to the core base, sugar moiety or internucleoside linkage of the nucleic acid molecule. Conjugation with a purine core base or its derivative can occur at any position (including endocyclic and exocyclic atoms). In certain embodiments, the 2-, 6-, 7- or 8-position of a purine core base is attached to a conjugate moiety. Conjugates with pyrimidine core bases or its derivatives can also occur at any position. In certain embodiments, the 2-, 5- and 6-positions of a pyrimidine core base can be replaced with a conjugate moiety. Conjugation with the sugar moiety of a nucleoside can occur at any carbon atom. Exemplary carbon atoms of a sugar moiety that can be attached to a conjugate moiety include 2', 3' and 5' carbon atoms. 1' position can also be attached to a conjugate moiety, such as in an abasic residue. Internucleoside linkage can also have a conjugate moiety. For phosphorus-containing linkages (e.g., phosphodiester, thiosulfate, phosphorodithioate, phosphoramidate, etc.), the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom bound to the phosphorus atom. For amine- or amide-containing internucleoside linkages (e.g., PNA), the conjugate moiety can be attached to the nitrogen atom or an adjacent carbon atom of the amine or amide.

[0550] Any suitable ligand in the art of RNA interference can be used, but the ligand is typically a carbohydrate, such as a monosaccharide (eg, GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, or a polysaccharide.

[0551] Linkers that bind the ligand to the nucleic acid include those discussed above. For example, the ligand can be one or more GalNAc (N-acetylglucosamine) derivatives attached via a monovalent, divalent, or trivalent branched linker.

[0552] In one embodiment, the dsRNA of the invention is conjugated to a divalent and trivalent branched linker comprising the structure shown in any one of Formulas (IV)-(VII):

[0553]

[0554] in:

[0555] q 2A ,q 2B ,q 3A ,q 3B 、q4 A ,q 4B ,q 5A ,q 5B and q 5C represents independently for each occurrence 0-20 and wherein the repeating units may be the same or different;

[0556] P 2A 、P 2B、P 3A 、P 3B 、P 4A 、P 4B 、P 5A 、P 5B 、P 5C 、T 2A 、T 2B 、T 3A 、T 3B 、T 4A 、T 4B 、T 5A 、T 5B 、T 5C for each occurrence, independently: CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CHO;

[0557] Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C is, independently for each occurrence, absent, alkylene, substituted alkylene, wherein one or more methylene groups may be interrupted or terminated by one or more of: O, S, S(O), SO2, N(R N ), C(R')=C(R"), C≡C or C(O);

[0558] R 2A 、R 2B 、R 3A 、R 3B 、R 4A 、R 4B 、R 5A 、R 5B 、R 5C For each occurrence, independently: absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-、CO、CH=NO、 or a heterocyclic group;

[0559] L 2A 、L 2B 、L 3A 、L 3B 、L 4A 、L 4B 、L 5A 、L 5Band L 5C represents a ligand; that is, independently for each occurrence represents a monosaccharide (such as GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, or a polysaccharide; and

[0560] R a is H or an amino acid side chain.

[0561] Trivalently conjugated GalNAc derivatives are particularly useful with RNAi agents for inhibiting target gene expression, such as those having formula (VII):

[0562]

[0563] Among them L 5A 、L 5B and L 5C Represents a monosaccharide, such as a GalNAc derivative.

[0564] Examples of suitable divalent and trivalent branched linker groups for conjugation to GalNAc derivatives include, but are not limited to, the following compounds:

[0565]

[0566]

[0567] definition

[0568] As used herein, the terms "dsRNA," "siRNA," and "iRNA agent" are used interchangeably for agents that can mediate the silencing of a target RNA (e.g., mRNA, e.g., a transcript of a protein-encoding gene). For convenience, such mRNA is also referred to herein as the mRNA to be silenced. Such a gene is also referred to as a target gene. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNAs other than mRNA (e.g., tRNAs) and viral RNAs can also be targeted.

[0569] As used herein, the phrase "mediates RNAi" refers to the ability to silence a target RNA in a sequence-specific manner. Although not wishing to be bound by theory, it is believed that silencing utilizes the RNAi mechanism or process and a guide RNA, such as an siRNA agent having 21 to 23 nucleotides.

[0570] As used herein, "specifically hybridizable" and "complementary" are terms used to indicate a sufficient degree of complementarity to allow stable and specific binding to occur between a compound of the invention and a target RNA molecule. Specific binding requires a sufficient degree of complementarity to prevent nonspecific binding of the oligomeric compound to non-target sequences under conditions where specific binding is desired, i.e., under physiological conditions in the case of an assay or therapeutic treatment, or under the conditions under which such assays are performed in the case of in vitro assays. These non-target sequences typically differ by at least 5 nucleotides.

[0571] In one embodiment, the dsRNA agent of the present invention is "sufficiently complementary" to a target RNA (e.g., a target mRNA) so that the dsRNA agent silences the production of a protein encoded by the target mRNA. In another embodiment, the dsRNA agent of the present invention is "completely complementary" to a target RNA, e.g., the target RNA and the dsRNA duplex agent anneal, e.g., to form a hybrid consisting exclusively of Watson-Crick base pairs in a region of complete complementarity. A "sufficiently complementary" target RNA may include an internal region (e.g., having at least 10 nucleotides) that is completely complementary to a target RNA. In addition, in some embodiments, the dsRNA agent of the present invention is distinguished by a single nucleotide difference. In this case, the dsRNA agent will mediate RNAi only if complete complementarity is found in a region (e.g., within 7 nucleotides of the single nucleotide difference).

[0572] As used herein, the term "oligonucleotide" refers to a nucleic acid molecule (RNA or DNA) having, for example, fewer than 100, 200, 300, or 400 nucleotides in length.

[0573] The term 'BNA' refers to a bridged nucleic acid, and generally refers to a constrained or inaccessible RNA. BNAs can contain 5-, 6-, or even 7-membered bridge structures with a "fixed" C3'-endo sugar condensation. The bridge is typically incorporated into the 2'-, 4'-position of the ribose to provide a 2', 4'-BNA nucleotide (e.g., LNA, or ENA). Examples of BNA nucleotides include the following nucleosides:

[0574]

[0575] The term 'LNA' refers to locked nucleic acid and generally refers to constrained or inaccessible RNA. LNA is a modified RNA nucleotide. The ribose portion of an LNA nucleotide is modified with an additional bridge (e.g., a methylene bridge or an ethylene bridge) linking the 2' hydroxyl group to the 4' carbon of the same ribose.

[0576] For example, the bridge can "lock" the ribose in a 3'-endo (North) conformation:

[0577]

[0578] The term 'ENA' refers to ethylene-bridged nucleic acid, and generally refers to constrained or inaccessible RNA.

[0579] " Cleavage site " herein refers to the backbone linkage in the target gene or in the sense strand that is cracked by the RISC mechanism using an iRNA reagent. And the targeted cleavage site region includes at least one or at least two nucleotides on both sides of the cleavage site. For the sense strand, the cleavage site is the backbone linkage in the sense strand, and if the sense strand itself is a target for cleavage by the RNAi mechanism, it will be cleaved. Methods known in the art can be used to determine the cleavage site, such as the 5'-RACE assay described in detail in Soutschek et al., Nature (2004) 432,173-178, which is incorporated herein by reference in its entirety. As is known in the art, the cleavage site region for conical double-stranded RNAi reagents includes two 21-nucleotide long chains (wherein the chain forms a double-stranded region of 19 continuous base pairs with an overhang of a 2-nucleotide single chain at the 3' end), and the cleavage site region corresponds to position 9-12 from the 5'-end of the sense strand.

[0580] The term "halo" refers to any group containing fluorine, chlorine, bromine or iodine. The term "alkyl" refers to a saturated or unsaturated non-aromatic hydrocarbon chain (such alkyl groups include but are not limited to propyl, allyl or propargyl) that may be linear or branched and contain the specified number of carbon atoms, which may be optionally interrupted by N, O or S. For example, C1-C 10 Indicates that the group may have from 1 to 10 (inclusive) carbon atoms. The term "alkoxy" refers to an -O-alkyl group. The term "alkylene" refers to a divalent alkyl group (i.e., -R-). The term "alkylenedioxo" refers to a divalent species having the structure -ORO-, where R represents an alkylene group. The term "aminoalkyl" refers to an alkyl group substituted with an amino group. The term "mercapto" refers to an -SH group. The term "thioalkoxy" refers to an -S-alkyl group.

[0581] The term "aryl" refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system, wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted with a substituent. Examples of aryl groups include phenyl, naphthyl, and the like. The term "arylalkyl" or "aralkyl" refers to an alkyl group substituted with an aryl group. The term "arylalkoxy" refers to an alkoxy group substituted with an aryl group.

[0582] As used herein, the term "cycloalkyl" includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons (e.g., 3 to 8 carbons and, for example, 3 to 6 carbons), wherein the cycloalkyl group may additionally be optionally substituted. Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0583] The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms (if monocyclic), 1-6 heteroatoms (if bicyclic), or 1-9 heteroatoms (if tricyclic), wherein the heteroatoms are selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 N, O, or S heteroatoms, respectively, if monocyclic, bicyclic, or tricyclic), wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted with one substituent. Examples of heteroaryl groups include pyridyl, furyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl, thienyl, quinolinyl, indolyl, thiazolyl, and the like. The term "heteroarylalkyl" or the term "heteroaralkyl" refers to an alkyl group substituted with a heteroaryl group. The term "heteroarylalkoxy" refers to an alkoxy group substituted with a heteroaryl group.

[0584] The term "heterocyclyl" refers to a non-aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms (if monocyclic), 1-6 heteroatoms (if bicyclic), or 1-9 heteroatoms (if tricyclic), wherein the heteroatoms are selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 N, O, or S heteroatoms, respectively, if monocyclic, bicyclic, or tricyclic), wherein 0, 1, 2, or 3 atoms of each ring may be substituted with one substituent. Examples of heterocyclyl groups include triazolyl, tetrazolyl, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.

[0585] The term "oxo" refers to an oxygen atom that forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.

[0586] The term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted with a substituent.

[0587] The term "substituted" refers to the replacement of one or more hydrogen radicals in a given structure with a group of a specified substituent, including but not limited to halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkyloxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkyloxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclyl, and aliphatic compounds. It should be understood that the substituents may be further substituted.

[0588] Cleavable linker

[0589] A cleavable linking group is sufficiently stable outside the cell, but is cleaved upon entry into the target cell to release the two parts of the linker that bind the two parts together. According to preferred embodiments of the dsRNA agent of the present invention, the cleavable linking group is cleaved at least 10 times or more preferably at least 100 times faster in the target cell or under first reference conditions (which can be selected to simulate or represent intracellular conditions) than in the blood of a subject or under second reference conditions (which can be selected to simulate or represent conditions found in blood or serum).

[0590] Cleavable linking groups are susceptible to the influence of cleavage factors (e.g., pH, redox potential, or the presence of degradation molecules). Typically, cleavage factors are more prevalent or found at higher levels or activity in cells than in serum or blood. Examples of such degradation factors include redox factors that are selected for specific substrates or have no substrate specificity, including, for example, oxidases or reductases or reducing factors present in cells such as thiols (which can degrade a redox-cleavable linking group by reduction); esterases; endosomes or factors that can create an acidic environment, such as those that form a pH of five or less; enzymes that can hydrolyze or degrade an acid-cleavable linking group by acting as a generalized acid, peptidases (which can be substrate-specific), and phosphatases.

[0591] A cleavable linkage group (e.g., a disulfide bond) can be sensitive to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers will have a cleavable linker that is cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand inside the cell or releasing it into the desired cellular compartment.

[0592] The linker can include a cleavable linking group that can be cleaved by a specific enzyme. The type of cleavable linking group incorporated into the linker can depend on the cell to be targeted. For example, a liver targeting ligand can be linked to a cationic lipid via a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be more effectively cleaved in liver cells 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.

[0593] When targeting cell types rich in peptidases, such as hepatocytes and synoviocytes, linkers containing peptide bonds can be used.

[0594] Typically, the suitability of a candidate's cleavable linking group can be assessed by testing the ability of the candidate's linking group to be cracked by a degradation agent (or condition). It is also desirable to test the candidate's cleavable linking group in blood or when in contact with other non-target tissues to resist the ability to cut. Therefore, it is possible to determine the relative sensitivity of cracking between a first condition and a second condition, wherein the first condition is selected as the cracking indicated in a target cell and the second condition is selected as the cracking indicated in other tissues or biofluids (e.g., blood or serum). These assessments can be carried out in a cell-free system, in a cell, in a cell culture, in an organ or tissue culture, or in a whole animal. It may be useful to make a preliminary evaluation under cell-free or culture conditions and to confirm by further evaluating in a complete animal. In a preferred embodiment, compared with blood or serum (or under the in vitro conditions selected to simulate extracellular conditions), available candidate compounds can be cracked at least 2, 4, 10, or 100 times in a cell (or under the in vitro conditions selected to simulate intracellular conditions).

[0595] Redox-cleavable linker

[0596] A class of cleavable linking groups is a redox cleavable linking group, which can be used in dsRNA agents according to the present invention. It is cleaved when reduced or oxidized. An example of a linking group that can be reduced and cleaved is a disulfide linking group (-SS-). In order to determine whether a candidate's cleavable linking group is a suitable "reduction-cleavable linking group", or whether it is suitable for use with a specific iRNA portion and a specific targeting agent, reference can be made to the methods described herein. For example, a candidate can be evaluated by incubating with dithiothreitol (DTT) or other reagents known in the art using reducing agents, which simulate the cleavage rate observed in cells (e.g., target cells). These candidates can also be evaluated under conditions selected to simulate blood or serum conditions. In a preferred embodiment, the candidate compound is cleaved up to 10% in blood. In a preferred embodiment, the candidate compound can be degraded at least 2, 4, 10, or 100 times in cells (or under in vitro conditions selected to simulate intracellular conditions) compared to blood (or under in vitro conditions selected to simulate extracellular conditions). The rate of cleavage of a candidate compound can be determined using standard enzyme kinetic assays under conditions chosen to mimic intracellular media and compared to the rate under conditions chosen to mimic extracellular media.

[0597] Phosphate-based cleavable linking groups

[0598] Phosphate-based cleavable linking groups can be used in dsRNA agents according to the present invention, which are cleaved by an agent that degrades or hydrolyzes the phosphate group. An example of an agent that cleaves a phosphate group in a cell is an enzyme, such as a phosphatase in the cell. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. Methods similar to those described above can be used to evaluate these candidates.

[0599] Acid-cleavable linking group

[0600] Acid-cleavable linking groups can be used for dsRNA reagents according to the present invention, and are linking groups that crack under acid conditions. In a preferred embodiment, acid-cleavable linking groups are cracked under an acidic environment of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0 or lower) or cracked by a reagent (such as an enzyme) that can serve as a broad acid. In cells, specific low pH organelles (such as endosomes or lysosomes) can provide a cracking environment for acid-cleavable linking groups. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and amino acid esters. Acid-cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). A preferred embodiment is when the carbon attached to the oxygen of an ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group (such as dimethylpentyl or tert-butyl). These candidates can be evaluated using methods similar to those described above.

[0601] Ester-based linking groups

[0602] Ester-based cleavable linking groups can be used for dsRNA agents according to the present invention, which are cleaved by, for example, esterases and amidases in cells. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester-cleavable linking groups have the general formula -C(O)O- or -OC(O)-. Methods similar to those described above can be used to evaluate these candidates.

[0603] Peptide-based cleavage groups

[0604] The cleavable linking group based on peptide can be used in the dsRNA reagent according to the present invention, and is cleaved by enzymes such as peptidases and proteases in cells. The cleavable linking group based on peptide is a peptide bond formed between amino acids to produce oligopeptides (such as dipeptides, tripeptides, etc.) and polypeptides. The cleavable group based on peptide does not include an amide group (-C (O) NH-). The amide group can be formed between any alkylene, alkenylene or alkynylene groups. The peptide bond is an amide bond formed between amino acids to produce a specific type of peptide and protein. The cleavable group based on peptide is generally limited to the peptide bond (i.e., amide bond) formed between amino acids to produce peptides and proteins, and does not include the entire amide functional group. The cleavable linking group based on peptide has the general formula -NHCHR A C(O)NHCHR B C(O)-, where R A and R B is the R group of the two adjacent amino acids. Methods similar to those described above can be used to evaluate these candidates. As used herein, "carbohydrate" refers to a compound that is a carbohydrate (which can be linear, branched or cyclic) consisting of one or more monosaccharide units with at least 6 carbon atoms, wherein oxygen, nitrogen or sulfur atoms are bound to each carbon atom; or it is a compound having a carbohydrate portion as part of it, which is composed of one or more monosaccharide units with at least six carbon atoms (which can be linear, branched or cyclic), wherein oxygen, nitrogen or sulfur atoms are bound to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides and oligosaccharides containing about 4-9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (preferably C5-C8) sugars; disaccharides and trisaccharides, including sugars with two or three monosaccharide units (preferably C5-C8).

[0605] The present invention further relates to the use of a dsRNA agent as described herein for inhibiting target gene expression. In one embodiment, the present invention further relates to the use of a dsRNA agent for inhibiting target gene expression in vitro.

[0606] The present invention further relates to a dsRNA agent as described herein for inhibiting target gene expression in a subject. The subject can be any animal, such as a mammal, for example, a mouse, rat, sheep, cow, dog, cat, or human.

[0607] In one embodiment, the dsRNA agents of the invention are administered in a buffer.

[0608] In one embodiment, the siRNA compound described herein can be formulated for administration to a subject. A formulated siRNA composition can take a variety of states. In some instances, the composition is at least partially crystalline, uniformly crystalline and / or anhydrous (e.g., less than 80%, 50%, 30%, 20% or 10% water). In another example, the siRNA is in an aqueous phase, for example, in a solution comprising water.

[0609] The aqueous or crystalline compositions can be incorporated into a delivery vehicle, such as a liposome (particularly for aqueous phases) or a particle (e.g., such as a microparticle that can be adapted for a crystalline composition). Typically, as described herein, the siRNA composition is formulated in a manner compatible with the intended method of administration. For example, in particular embodiments, the composition is prepared by at least one of the following methods: spray drying, freeze drying, vacuum drying, evaporation, fluidized bed drying, or a combination of these techniques; or sonication, freeze drying, condensation, and other self-assembly with a lipid.

[0610] An siRNA formulation 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 complexes with the siRNA to form an iRNP. Still other agents include chelating agents such as EDTA (e.g., to remove divalent cations such as Mg 2+ )), salts, RNase inhibitors (e.g., a broad specificity RNase inhibitor, such as RNAsin), and the like.

[0611] In one embodiment, the siRNA formulation includes another siRNA compound, for example, a second siRNA that mediates RNAi against a second gene or against the same gene. Still other formulations 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.

[0612] In one embodiment, the siRNA formulation includes at least one second therapeutic agent (e.g., an agent other than an RNA or a DNA). For example, an siRNA composition for treating a viral disease (e.g., HIV) may include a known antiviral agent (e.g., a protease inhibitor or a reverse transcriptase inhibitor). In another example, an siRNA composition for treating cancer may further include a chemotherapeutic agent.

[0613] Exemplary formulations of dsRNA agents according to the invention that can be used for administration are discussed below.

[0614] Liposome. For ease of explanation, formulations, compositions and methods have been discussed in this part to a great extent relative to unmodified siRNA compounds. However, it is understood that these formulations, compositions and methods can be practiced with other siRNA compounds (e.g., modified siRNA), and such practice belongs to the present invention. A siRNA compound formulation can be formulated for delivery in a membranous molecular assembly (e.g., liposome or micelle), wherein the siRNA compound is, for example, a double-stranded siRNA compound or ssiRNA compound (e.g., a precursor, such as a larger siRNA compound that can be processed into an ssiRNA compound or a DNA encoding an siRNA compound (e.g., a double-stranded siRNA compound or ssiRNA compound or its precursor)). As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one double layer (e.g., a double layer or multiple double layers). Liposomes include unilamellar or multilamellar vesicles having a film formed from a lipophilic material and an aqueous interior. The aqueous portion comprises the siRNA composition. This lipophilic material separates this aqueous interior from an aqueous exterior, and this aqueous exterior typically does not include this siRNA composition, but in some instances, it may include.Liposome is useful for active ingredient transfer and delivery to site of action. Because liposome membrane is similar in structure to biomembrane, when liposome is applied to a kind of tissue, the double layer of this liposome merges with the double layer of cell membrane. Along with the fusion of liposome and cell, the internal aqueous inclusion comprising this siRNA is delivered to this cell, wherein this siRNA can be specifically bound to a kind of target RNA and can mediate RNAi. In some cases, these liposomes are also specifically targeted to, for example, direct this siRNA to specific cell type.

[0615] Liposomes comprising a siRNA can be prepared by a variety of methods. In one example, the lipid component of the liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic 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 formulation is then added to these micelles comprising the lipid component. The cationic group on the lipid interacts with the siRNA and condenses around the siRNA to form a liposome. After condensation, the detergent is removed by, for example, dialysis to produce a siRNA liposome formulation.

[0616] If necessary, a carrier compound that assists condensation can be added during the condensation reaction, for example, by controlled addition. For example, the carrier compound can be a polymer other than the nucleic acid (e.g., spermine or spermidine). The pH can also be adjusted to promote condensation.

[0617] Additional descriptions of methods for producing stable polynucleotide delivery vehicles incorporating a polynucleotide / cationic lipid complex as a structural component of the delivery vehicle are described, for example, in WO96 / 37194. Liposome formation may also include one or more aspects of the exemplary methods described in Felgner, PL 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, which are incorporated herein by reference in their entirety. Common techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer et al., Biochim. Biophys. Acta 858:161, 1986, which is incorporated herein by reference in its entirety). When uniformly small (50 nm to 200 nm) and relatively uniform aggregates are desired, microfluidization can be used (Mayhew et al., Biochimica et Biophysica Acta 775:169, 1984, which is incorporated herein by reference in its entirety). These methods are readily adapted for packaging siRNA formulations into liposomes.

[0618] pH-sensitive or negatively charged liposomes entrap nucleic acid molecules rather than complexing with them. Because the nucleic acid molecules and lipids are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid molecules are embedded in the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of exogenous genes has been detected in target cells (Zhou et al., Journal of Controlled Release, 19, (1992) 269-274, which is incorporated herein by reference in its entirety).

[0619] A main type of liposome composition comprises the phospholipids except the phosphatidylcholine of natural origin.For example neutral liposome composition can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC).Anionic liposome composition can be formed from dimyristoylphosphatidylglycerol usually, and anionic fusogenic liposome is mainly formed from dioleoylphosphatidylethanolamine (DOPE).Another type of liposome composition is formed from phosphatidylcholine (PC), for example, as soybean PC and egg PC.Another type is formed from a mixture of phosphatidylcholine and / or phosphatidylcholine and / or cholesterol.

[0620] Examples of other methods for introducing liposomes into cells in vitro include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94 / 00569; WO 93 / 24640; WO 91 / 16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss, EMBO J. 11:417, 1992.

[0621] In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse to the cell membrane. Although non-cationic liposomes cannot fuse effectively with the plasma membrane, they are absorbed by macrophages in vivo and can be used to deliver siRNA to macrophages.

[0622] Additional advantages of liposomes include: liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water- and lipid-soluble drugs; liposomes can protect encapsulated siRNA in their internal compartments from metabolism and degradation (Rosoff, "Pharmaceutical Dosage Forms," ​​Lieberman, Rieger, and Banker (eds.), 1988, vol. 1, p. 245). Important considerations in preparing liposome formulations are the lipid surface charge, vesicle size, and the aqueous volume of the liposomes.

[0623] A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), can be used to form small liposomes that spontaneously interact with nucleic acids to form lipid-nucleic acid complexes that are capable of fusing with negatively charged lipids of the cell membranes of tissue culture cells, thereby resulting in the delivery of siRNA (for a description of DOTMA and its use with DNA, see, e.g., Felgner, PL et al., Proc. Natl. Acad. Sci., 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355, which are incorporated herein by reference in their entirety).

[0624] A DOTMA analog, 1,2-bis(oleoyloxy)-3-(trimethylamino)propane (DOTAP), can be used in combination with phospholipids to form DNA-complexed vesicles. TM (Bethesda Research Laboratories, Gaithersburg, Md.), is an effective agent for delivering highly anionic nucleic acids to living tissue culture cells that contain positively charged DOTMA liposomes, which spontaneously interact with negatively charged polynucleotides to form complexes. When sufficient positively charged liposomes are used, the net charge of the resulting complex is also positive. Positively charged complexes prepared in this manner spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and effectively deliver functional nucleic acids to, for example, tissue culture cells. Another commercially available cationic liposome, 1,2-bis(oleoyloxy)-3,3-(trimethylamino)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Indiana), differs from DOTMA in that the oleoyl moiety is ester-linked rather than ether-linked.

[0625] Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties, including, for example, carboxyspermine, which has been combined with one of two types of lipids and includes compounds such as 5-carboxysperminylglycine dioctaoleoylamide ("DOGS") (Transfectam TM , Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxysperminyl-amide ("DPPES") (see, e.g., U.S. Patent No. 5,171,678).

[0626] Another cationic lipid conjugate includes derivatization of the lipid with cholesterol ("DC-Chol"), which has been formulated into a combination of liposomes and DOPE (see Gao, X and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, prepared 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, which is incorporated herein by reference in its entirety). For certain cell lines, these liposomes containing conjugated cationic lipids are said to show lower toxicity and provide more efficient transfection than compositions containing DOTMA. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, CA) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, MD). Other cationic lipids suitable for delivery of oligonucleotides are described in WO 98 / 39359 and WO 96 / 37194.

[0627] Liposomal formulations are particularly suitable for topical administration, and liposomes present several advantages over other formulations. Such advantages include that the side effects associated with the high systemic absorption of the administered drug are reduced, the accumulation of the administered drug at the desired target is increased, and the ability of siRNA to be administered to the skin. In some implementations, liposomes are used to deliver siRNA to epidermal cells and also enhance siRNA penetration into skin tissue, such as into the skin. For example, these liposomes can be applied topically. The topical delivery of drugs formulated as liposomes to the skin has been documented (see, for example, Weiner et al., Journal of Drug Targeting, 1992, Vol. 2, 405-410 and du Plessis et al., Antiviral Research. 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., Methods in Enzymology 149: 157-176, 1987; Straubinger, RM and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, CY and Huang, Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987), which are incorporated herein by reference in their entirety.

[0628] Nonionic liposome systems have also been tested to determine their use in delivering drugs to the skin, specifically systems comprising nonionic surfactants and cholesterol. Nonionic liposome formulations including Novasome I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with siRNA are suitable for treating a skin disorder.

[0629] Liposomes comprising siRNA can be made highly deformable. Such deformation can enable liposomes to penetrate through holes smaller than the average radius of the liposome. For example, a transfersome is a type of deformable liposome. A transfersome can be made by adding a surface edge activator (usually a surfactant) to a standard liposome composition. A transfersome comprising siRNA can be delivered, for example, subcutaneously by injection, thereby delivering siRNA to the keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of pores under the influence of a suitable transdermal gradient, each having a diameter less than 50nm. In addition, due to these lipid properties, these transfersomes can be self-optimizing (adapting to the shape of the pores in the skin, for example), self-repairing, and can frequently reach their targets without fragmentation, and are typically self-loading.

[0630] Other formulations according to the present invention are described in the following U.S. Provisional Application Serial Nos. 61 / 018,616, filed January 2, 2008, 61 / 018,611, filed January 2, 2008, 61 / 039,748, filed March 26, 2008, 61 / 047,087, filed April 22, 2008, and 61 / 051,528, filed May 8, 2008. PCT Application No. PCT / US2007 / 080331, filed October 3, 2007, also describes formulations according to the present invention.

[0631] Surfactants. For ease of explanation, formulations, compositions and methods are discussed in this section to a large extent relative to unmodified siRNA compounds. However, it is understood that these formulations, compositions and methods can be practiced with other siRNA compounds (e.g., modified siRNA compounds), and such practice is within the scope of the present invention. Surfactants are widely used in formulations such as emulsions (including microemulsions) and liposomes (see above). siRNA (or a precursor, such as a larger dsiRNA that can be processed into an siRNA, or a DNA encoding an siRNA or precursor) composition can include a surfactant. In one embodiment, siRNA is formulated as an emulsion including a surfactant. The most common method for classifying and rating the properties of many different types of surfactants (both natural and synthetic) is by using a hydrophilic / lipophilic balance (HLB). The properties of the hydrophilic group provide the most useful means for classifying the different surfactants used in the formulation (Rieger, "Drug Formulations", Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0632] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants are widely used in pharmaceutical products and are available over a wide pH range. In general, their HLB values ​​range from 2 to about 18, depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glycerol esters, polyglycerol 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 category. Polyoxyethylene surfactants are the most commonly used members of the nonionic surfactant class.

[0633] If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates (e.g., soaps), acyl lactylates, acyl amides of amino acids, sulfates (e.g., alkyl sulfates and ethoxylated alkyl sulfates), sulfonates (e.g., alkylbenzenesulfonates, acyl isethionates, acyl taurates, and sulfosuccinates), and phosphates. The most important members of the anionic surfactant class are alkyl sulfates and soaps.

[0634] If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. These quaternary ammonium salts are the most commonly used members of this class.

[0635] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phospholipids.

[0636] The use of surfactants in pharmaceuticals, formulations, and in emulsions has been reviewed (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0637] Micelles and other membranous formulations. For ease of explanation, micelles and other formulations, compositions and methods are discussed in this section to a great extent relative to unmodified siRNA compounds. However, it is understood that these micelles and other formulations, compositions and methods can be practiced with other siRNA compounds (e.g., modified siRNA compounds), and such practice belongs to the present invention. A siRNA compound composition can be provided as a micellar formulation, wherein the siRNA compound is, for example, a double-stranded siRNA compound or ssiRNA compound (e.g., a precursor, such as a larger siRNA compound that can be processed into an ssiRNA compound or a DNA encoding an siRNA compound (e.g., a double-stranded siRNA compound or ssiRNA compound or its precursor)). "Micelles" are defined herein as a specific type of molecular aggregate, wherein amphiphilic molecules are arranged in a spherical structure so that all hydrophobic portions of these molecules are oriented inwardly, and the hydrophilic portion is in contact with the surrounding water. If the environment is hydrophobic, there is an opposite arrangement.

[0638] Mixed micellar formulations suitable for transdermal membrane delivery can be prepared by mixing an aqueous solution of the siRNA composition, an alkali metal C8-C 22 Alkyl sulfate and micelle forming compound are prepared. Exemplary micelle forming compound includes lecithin, hyaluronic acid, pharmaceutically acceptable salt of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleate, monolaurate, borage oil, evening primrose oil, menthol, trihydroxyoxycholesterol alkyl glycine and its pharmaceutically acceptable salt, glycerol, polyglycerol, lysine, polylysine, triolein, polyoxyethylene ether and its analogue, polydocanol alkyl ether and its analogue, chenodeoxycholate, deoxycholate, and its mixture. Micelle forming compound can be added at the same time as or after adding alkali metal alkyl sulfate. Mixed micelles can be formed with the mixing (but vigorous mixing) of these ingredients of substantially any kind to provide micelles of smaller size.

[0639] In one method, a first micellar composition is prepared comprising the siRNA composition and at least the alkali metal alkyl sulfate. The first micellar composition is then mixed with at least three micelle-forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the siRNA composition, the alkali metal alkyl sulfate, and at least one micelle-forming compound, and then adding the remaining micelle-forming compound (under vigorous mixing).

[0640] Phenol and / or m-cresol can be added to the mixed micellar composition to stabilize the formulation and prevent bacterial growth. Alternatively, phenol and / or m-cresol can be added along with the micelle-forming ingredients. An isotonicity agent, such as glycerol, can also be added after the mixed micelle composition is formed.

[0641] For the delivery of micellar formulations as sprays, the formulation can be loaded into an aerosol dispenser and the dispenser is filled with propellant. The propellant (which is under pressure) is in liquid form in the dispenser. The ratio of the components is adjusted so that the aqueous phase and the propellant phase become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser before distributing the portions of the contents (e.g., through a metering valve). The dispensed amount of the pharmaceutical agent is propelled from the metering valve as a fine mist.

[0642] Propellants may include hydrochlorofluorocarbons, hydrofluorocarbons, dimethyl ether, and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2-tetrafluoroethane) may also be used.

[0643] The specific concentrations of these essential ingredients can be determined by relatively simple experimentation.For oral absorption, it is generally desirable to increase the dose by, for example, at least two-fold or three-fold, that for injection or administration via the gastrointestinal tract.

[0644] Particles. For ease of explanation, particles, formulations, compositions and methods are discussed in this section to a great extent with respect to modified siRNA compounds. However, it is understood that these particles, formulations, compositions and methods can be practiced with other siRNA compounds (e.g., unmodified siRNA compounds), and such practice belongs to the present invention. In another embodiment, a siRNA compound formulation can be incorporated into a particle (e.g., microparticle), which is, for example, a double-stranded siRNA compound or ssiRNA compound (e.g., a precursor, such as a larger siRNA compound that can be processed into an ssiRNA compound or a DNA encoding an siRNA compound (e.g., a double-stranded siRNA compound or ssiRNA compound or its precursor)). Microparticles can be produced by spray drying, but can also be produced by other methods including freeze drying, evaporation, fluidized bed drying, vacuum drying or a combination of these techniques.

[0645] Pharmaceutical composition

[0646] The iRNA agents 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 comprises a therapeutically effective amount of one or more of the dsRNA agents of any of the above embodiments, alone or in combination with one or more pharmaceutically acceptable carriers (additives), excipients, and / or diluents.

[0647] The pharmaceutical compositions can be specifically formulated for administration in solid or liquid form, including forms suitable for: (1) oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., tablets targeted for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection, as, for example, a sterile solution or suspension or sustained-release formulation; (3) topical administration, such as in the form of a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, such as in the form of a pessary, cream, or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally. Delivery using subcutaneous or intravenous methods can be particularly advantageous.

[0648] As used herein, the phrase "therapeutically effective amount" means an amount of a compound, material, or composition including a compound of the invention effective to produce some desired therapeutic effect in at least a subpopulation of cells in an animal, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0649] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0650] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable substance, composition, or vehicle that participates in carrying or transporting the subject compound from one organ or part of the body to another organ or part of the body, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating 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 carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants such as magnesium stearate, 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 propylene glycol; (11) Polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances used in pharmaceutical formulations.

[0651] These formulations can be conveniently presented in unit dosage form and can be prepared by any method well known in the art of pharmaceutics. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the compound that produces the therapeutic effect. Typically, in one hundred percent, this amount will be in the range of from about 0.1 percent to about 99 percent, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent active ingredient.

[0652] In certain embodiments, a formulation of the present invention comprises an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle formers (e.g., bile acids), and polymeric carriers (e.g., polyesters and polyanhydrides), and a compound of the present invention. In certain embodiments, the aforementioned formulation renders a compound of the present invention orally bioavailable.

[0653] iRNA agent formulations 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 complexes with the iRNA to form an iRNP. Still other agents include chelating agents such as EDTA (e.g., to remove divalent cations such as Mg 2+ )), salts, RNase inhibitors (e.g., a broad specificity RNase inhibitor, such as RNAsin), and the like.

[0654] The methods for preparing these formulations or compositions include the step of bringing into association a compound of the invention with a carrier and, optionally, one or more auxiliary ingredients. In general, these formulations are prepared by uniformly and intimately bringing into association a compound of the invention with a liquid carrier or a finely divided solid carrier or both and then, if necessary, shaping the product.

[0655] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of crystalline or amorphous material with poor water solubility. The absorption rate of the drug, in turn, depends on its dissolution rate, which in turn can depend on the crystal size and crystalline form. Alternatively, delayed absorption of parenteral drug forms is achieved by dissolving or suspending the drug in an oily vehicle.

[0656] The compounds according to the invention may be formulated for administration in any convenient way by analogy with other drugs for use in human or veterinary medicine.

[0657] The term "treatment" is intended to also encompass prevention, therapy, and cure. Patients receiving such treatment are generally any animal in need, including primates (particularly humans) and other mammals such as horses, cattle, pigs, and sheep; as well as poultry and pets.

[0658] Double-stranded RNAi agents are produced in vivo in cells, for example, from exogenous DNA templates delivered to the cells. For example, these DNA templates can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, topical administration (U.S. Patent No. 5,328,470, which is incorporated herein by reference in its entirety), or by targeted injection (see, for example, Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3054-3057, which is incorporated herein by reference in its entirety). Pharmaceutical formulations of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can include a sustained-release matrix in which the gene delivery vehicle is embedded. These DNA templates can, for example, include two transcription units, one producing a transcript comprising the top strand of the dsRNA agent, and one producing a transcript comprising the bottom strand of the dsRNA agent. When these templates are transcribed, the dsRNA agent is produced and processed into siRNA agent fragments that mediate gene silencing.

[0659] Delivery route

[0660] A dsRNA agent as defined herein or a pharmaceutical composition comprising a dsRNA agent as defined herein can be administered to a patient using various routes of delivery. A composition comprising an iRNA can be delivered to a subject via a variety of routes. Exemplary routes include intravenous, subcutaneous, topical, rectal, anal, vaginal, nasal, pulmonary, and ocular.

[0661] The iRNA molecules and / or dsRNA agents of the present invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include one or more iRNAs and a pharmaceutically acceptable carrier. As used herein, the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, their use in these compositions is encompassed. Supplementary active compounds can also be incorporated into the composition.

[0662] 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 topical (including ocular, vaginal, rectal, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous infusion, subcutaneous, intraperitoneal, or intramuscular injection, or intrathecal or intraventricular administration.

[0663] The route and location of administration can be selected to enhance targeting. For example, to target myocytes, intramuscular injection into the muscle of interest would be a logical choice. Pneumocytes can be targeted by administering the iRNA in an aerosol form. Vascular endothelial cells can be targeted by coating a balloon catheter with the iRNA and mechanically introducing the DNA.

[0664] dose

[0665] In one aspect, the invention features a method of administering a dsRNA agent (e.g., an siRNA agent) to a subject (e.g., a human subject). In another aspect, the invention relates to a dsRNA agent as defined herein for inhibiting expression of a target gene in a subject. The method or medical use comprises administering a unit dose of a dsRNA agent, e.g., an siRNA agent, e.g., a double-stranded siRNA agent: (a) having a double-stranded portion of 14-30 nucleotides (nt) in length, e.g., 21-23 nt, (b) being complementary to a target RNA (e.g., an endogenous or pathogen target RNA), and optionally, (c) comprising at least one 3' overhang of 1-5 nucleotides in length. In one embodiment, the unit dose is less than 10 mg / kg body weight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001 mg / kg body weight, and less than 200 nmoles of RNA agent (e.g., about 4.4 x 10 16 copies) / kg body weight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of RNA reagent / kg body weight.

[0666] The determined amount can be an amount effective to treat or prevent a disease or disorder (e.g., a disease or disorder associated with a target RNA). The unit dose can be administered, for example, by injection (e.g., intravenous, subcutaneous, or intramuscular), an inhaled dose, or a topical administration. In some embodiments, the dose can be less than 10, 5, 2, 1, or 0.1 mg / kg of body weight.

[0667] In some embodiments, the unit dose is administered less frequently than once a day, e.g., less than once every 2, 4, 8, or 30 days. In another embodiment, the unit dose is not administered at a frequency (e.g., an irregular frequency). For example, the unit dose can be administered in a single dose.

[0668] In one embodiment, the effective dose is administered in other conventional therapeutic modalities. In one embodiment, the subject suffers from a viral infection and the modality is an antiviral agent rather than a dsRNA agent, e.g., rather than an siRNA agent. In another embodiment, the subject suffers from arteriosclerosis, and an effective dose of a dsRNA agent (e.g., an siRNA agent) is administered in combination, e.g., after surgical intervention (e.g., angioplasty).

[0669] In one embodiment, a subject is given an initial dose of a dsRNA agent and one or more maintenance doses of a dsRNA agent, such as an siRNA agent (e.g., a precursor, such as a larger dsRNA agent that can be processed into an siRNA agent, or a DNA encoding a dsRNA agent (e.g., an siRNA agent or its precursor)). The maintenance dose or doses can be the same or lower than the initial dose, such as half the initial dose. A maintenance regimen can include treating the subject with one or more doses ranging from 0.01 μg to 15 mg / kg of body weight per day, such as 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg / kg of body weight per day. For example, the maintenance dose is administered once every 2, 5, 10, or 30 days at most. In addition, the treatment regimen can continue for a period of time that will vary depending on the nature of the specific disease, its severity, and the overall condition of the patient. In certain embodiments, the dosage can be delivered at most once a day, e.g., at most once every 24, 36, 48 or more hours, e.g., at most once every 5 or 8 days. After treatment, the patient can be monitored for changes in the patient's condition and for alleviation of the symptoms of the disease state. The dosage of the compound can be increased if the patient does not significantly respond to the current dosage level, or the dosage can be reduced when alleviation of the symptoms of the disease state is observed, when the disease state has been eliminated, or when undesirable side effects are observed.

[0670] The effective dose can be administered in a single dose or in two or more doses, as is appropriate or contemplated under the particular circumstances. If it is desired to facilitate repeated or frequent infusions, it may be advisable to implant a delivery device, such as a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal, or intracapsular), or reservoir.

[0671] In one embodiment, the composition comprises a plurality of dsRNA agent species. In another embodiment, the dsRNA agent species has a sequence that is non-overlapping and non-adjacent to another species relative to a naturally occurring target sequence. In another embodiment, the plurality of dsRNA agent species are specific for different naturally occurring target genes. In another embodiment, the dsRNA agent is allele-specific.

[0672] The dsRNA agents of the invention described herein can be administered to mammals, particularly large mammals, such as non-human primates or humans, in a variety of ways.

[0673] In one embodiment, the administration of the dsRNA agent (e.g., an siRNA agent) composition is parenteral, such as intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intracapsular, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, transpulmonary, intranasal, urethral, ​​or ocular. Administration can be provided by the experimenter or by another person (e.g., a medical service person). The medicament can be provided in a measured dose or with a dispenser that delivers a metered dose. Selected delivery modes are discussed in more detail below.

[0674] The present invention provides methods, compositions, and kits for rectal administration or delivery of the dsRNA agents described herein.

[0675] In specific embodiments, the present invention relates to dsRNA agents of the present invention for use in the methods described above.

[0676] Methods for inhibiting target gene expression

[0677] Embodiments of the present invention also relate to methods for inhibiting target gene expression. The method comprises the step of administering a dsRNA agent as described in any of the above embodiments in an amount 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 target gene expression in a target cell. In a preferred embodiment, the present invention further relates to the use of a dsRNA agent for inhibiting target gene expression in a target cell in vitro.

[0678] On the other hand, the present invention relates to a method for regulating target gene expression in a cell, the method comprising providing 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, hepciden, activated protein C, cyclin D gene, VEGF gene mutations in the p53 tumor suppressor gene, and mutations in the p53 tumor suppressor gene.

[0679] In specific embodiments, the present invention relates to dsRNA agents of the present invention for use in the methods described above.

[0680] The present invention is further illustrated by the following examples, which should not be construed as further limiting.The contents of all references, pending patent applications, and published patents cited throughout this application are expressly hereby incorporated by reference.

[0681] Examples

[0682] Example 1. In vitro screening of siRNA duplexes

[0683] Cell culture and transfection:

[0684] Human Hep3B cells or rat H.II.4.E cells (ATCC, Manassas, VA) were grown to near confluence in RPMI (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) at 37°C in a 5% CO2 atmosphere and subsequently released from the plates by trypsinization. Transfection was performed by adding 14.8 μl Opti-MEM plus 0.2 μl Lipofectamine RNAiMax / well (Invitrogen, Carlsbad, CA, catalog number 13778-150) to 5 μl siRNA duplex / well into a 96-well plate and incubated at room temperature for 15 minutes. Then, a 50 μl siRNA duplex containing approximately 2×10 siRNAs was added without antibiotics. 4 80 μl of complete growth medium from Hep3B cells was added to the siRNA mixture. Cells were incubated for 24 or 120 hours prior to RNA purification. Single dose experiments were performed at 10 nM and 0.1 nM final duplex concentrations, and dose-response experiments were performed using 8- and 4-fold serial dilutions with a maximum dose of 10 nM final duplex concentration.

[0685] Total RNA was isolated using the DYNABEADS mRNA Isolation Kit (Invitrogen, part number: 610-12):

[0686] The cells are harvested and dissolved in 150 μl dissolution / binding buffer, then mixed at 850 rpm for 5 minutes (the stirring speed throughout the processing is the same) using an Eppendorf thermomixer. 10 microliters of magnetic beads and 80 μl dissolution / binding buffer mixture are added to a round bottom plate and mixed for 1 minute. Magnetic beads are captured using a magnetic stand and the supernatant is removed without disturbing the beads. After removing the supernatant, the lysed cells are added to the remaining magnetic beads and mixed for 5 minutes. After removing the supernatant, the magnetic beads are washed 2 times with 150 μl wash buffer A and mixed for 1 minute. The beads are captured again and the supernatant is removed. The beads are then washed with 150 μl wash buffer B, captured, and the supernatant is removed. The beads are then washed with 150 μl elution buffer, captured, and the supernatant is removed. Allow the beads to dry for 2 minutes. After drying, 50 μl elution buffer is added and mixed at 70°C for 5 minutes. Capture the beads with a magnet for 5 minutes. 40 μl of supernatant was removed and added to another 96-well plate.

[0687] Using ABI High Capacity c DNA reverse transcription kit (Applied Biosystems, Foster Foster City, California, catalog number 4368813) cDNA synthesis:

[0688] The master mix of each reaction (1 μl 10X buffer, 0.4 μl 25X dNTP, 1 μl random primer, 0.5 μl reverse transcriptase, 0.5 μl RNase inhibitor and 1.6 μl of H O) was added to 5 μl of total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, CA) by the following steps: 25°C for 10 min, 37°C for 120 min, 85°C for 5 sec, and 4°C hold.

[0689] Real-time PCR:

[0690] 2 μl of cDNA was added to a master mix containing 0.5 μl of GAPDH TaqMan probe (Applied Biosystems catalog number 4326317E (human) catalog number 4308313 (rodent)), 0.5 μl of TTR TaqMan probe (Applied Biosystems catalog number HS00174914_m1 (human) catalog number Rn00562124_m1 (rat)) and 5 μl of Lightcycler 480 probe master mix (Roche catalog number 04887301001) in a 384-well plate (Roche catalog number 04887301001). Real-time PCR was performed in a Roche LC 480 real-time PCR machine (Roche). Unless otherwise stated, each duplex was tested in at least two independent transfections, and each transfection was assayed in duplicate.

[0691] To calculate relative fold changes, real-time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955 or mock-transfected cells. IC values ​​were calculated using XLFit using a 4-parameter fitting model. 50 , and the IC 50 Normalize to cells transfected with AD-1955 within the same dose range or to naive cells or to its own lowest dose. Calculate IC for each individual transfection and combination. 50 , which converts a single IC 50 Fitted to data from two transfections.

[0692] The gene silencing results of exemplary siRNA duplexes with different motif modifications of the present invention are shown in the table below.

[0693] Example 2. RNA synthesis and duplex annealing

[0694] 1. Oligonucleotide synthesis:

[0695] All oligonucleotides were synthesized on an AKTA oligopilot synthesizer or an ABI 394 synthesizer. Unless otherwise stated, the following were used for oligonucleotide synthesis: commercially available controlled pore glass solid supports (dT-CPG, Prime Synthetics RNA phosphoramidites with standard protecting groups, 5'-O-dimethoxytrityl N6-benzoyl-2'-tert-butyldimethylsilyl-adenosine-3'-ON,N'-diisopropyl-2-cyanoethyl phosphoramidite, 5'-O-dimethoxytrityl-N4-acetyl-2'-tert-butyldimethylsilyl-cytidine-3'-ON,N'-diisopropyl-2-cyanoethyl phosphoramidite, 5'-O-dimethoxytrityl-N2-isobutyl-2'-tert-butyldimethylsilyl-guanosine-3'-ON,N'-diisopropyl-2-cyanoethyl phosphoramidite, and 5'-O-dimethoxytrityl-2'-tert-butyldimethylsilyl-uridine-3'-ON,N'-diisopropyl-2-cyanoethyl phosphoramidite (Pierce Nucleic Acid Technologies, Inc.) were used. Acids Technologies). 2'-F phosphoramidite, 5'-O-dimethoxytrityl-N4-acetyl-2'-fluoro-cytidine-3'-ON,N'-diisopropyl-2-cyanoethyl-phosphoramidite, and 5'-O-dimethoxytrityl-2'-fluoro-uridine-3'-ON,N'-diisopropyl-2-cyanoethyl-phosphoramidite were purchased from Promega. All phosphoramidites were used at a concentration of 0.2 M in acetonitrile (CH3CN), except for guanosine, which was used at a concentration of 0.2 M in 10% THF / ANC (v / v). A coupling / recycle time of 16 minutes was used. The activator was 5-ethylthiotetrazolium (0.75 M, American International Chemicals) for PO-oxidation using iodine / water / pyridine and for PS-oxidation using PADS (2%) in 2,6-lutidine / ACN (1:1 v / v).

[0696] The ligand-conjugated chain is synthesized using a solid support containing the corresponding ligand. For example, a carbohydrate moiety / ligand (e.g., GalNAc) is introduced at the 3' end of a sequence by starting synthesis with the corresponding carbohydrate solid support. Similarly, a cholesterol moiety is introduced at the 3' end by starting synthesis on a cholesterol support. Typically, the ligand moiety is tethered to trans-4-hydroxyprolinol via a selected tether as described in the previous example to obtain a hydroxyprolinol-ligand moiety. The hydroxyprolinol-ligand moiety is then coupled to a solid support via a succinate linker or converted to a phosphoramidite via standard phosphitylation conditions to obtain the desired carbohydrate conjugate building block. Fluorophore-labeled siRNA is synthesized from the corresponding phosphoramidite or solid support (purchased from Biosearch Technologies). The internally manufactured load is 38.6 micromoles / gram of oleyllithocholic acid (GalNAc)3 polymer support. Also manufactured in-house was a polymer support loaded with 42.0 μmol / g of mannose (Man) 3 .

[0697] Unless otherwise specified, binding of the selected ligand at the desired position, for example at the 5' end of the sequence, was achieved by coupling the corresponding phosphoramidite to the growing chain under standard phosphoramidite coupling conditions. A 0.1 M solution of phosphoramidite in anhydrous CH3CN was coupled to the solid-bound oligonucleotide for an extended 15 min in the presence of 5-(ethylthio)-1H-tetrazole activator. Internucleotide phosphite oxidation to phosphate was performed as reported (1) using standard iodine-water or the oligonucleotide was conjugated by oxidation with tert-butyl hydroperoxide / acetonitrile / water (10:87:3) with a 10 min wait time. Phosphothioates were introduced by oxidation of phosphites to phosphorothioates using a sulfur transfer reagent such as DDTT (available from AM Chemicals), PADS, and / or Beaucage reagent. Cholesterol phosphoramidites were synthesized in-house and used at a concentration of 0.1 M in dichloromethane. The coupling time for cholesterol phosphoramidites was 16 min.

[0698] 2. Deprotection-I (nucleobase deprotection)

[0699] After completing the synthesis, support is transferred to a 100ml glass bottle (VWR). Oligonucleotide is cracked from this support, and at 55 ℃, with the mixture [ammonia: ethanol (3: 1)] of 80mL ethanolamine, base and phosphate group are deprotected for 6.5h simultaneously. Bottle is simply cooled on ice, and then the ethanolization ammonia mixture is filtered into a new 250ml bottle. CPG is washed with the ethanol / water (1: 1v / v) of 2x40mL part. Then by rotary evaporation, the volume of mixture is reduced to about 30ml. Then this mixture is frozen on dry ice, and on a speed vac, dry under vacuum.

[0700] 3. Deprotection II (Removal of 2'-TBDMS group)

[0701] The dried 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 and the pH was adjusted to 6.5 and stored in a freezer until purification.

[0702] 4. Analysis

[0703] Oligonucleotides are analyzed by high performance liquid chromatography (HPLC) and subsequently purified, with the choice of buffer and column depending on the sequence and or the nature of the bound ligand.

[0704] 5.HPLC purification

[0705] The oligonucleotide of ligand binding is passed through reverse phase preparative HPLC purification.Unconjugated oligonucleotide is purified by anion exchange HPLC on the TSK gel column of an inner filling.Buffer is 20mM sodium phosphate (pH 8.5) (buffer A) and 10%CH among the CN 20mM sodium phosphate (pH 8.5) (buffer B) among the CN, 1MNaBr.The elution fraction that contains full-length oligonucleotide is gathered, desalination, and lyophilizing.The oligonucleotide of the desalination of about 0.15OD is diluted to 150 μ l in water, and is drawn into then and is used for CGE and LC / MS analysis in the special bottle.Finally pass through LC-ESMS and CGE analytical compound.

[0706] 6. siRNA Preparation

[0707] For siRNA preparation, 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 duplex was confirmed by HPLC analysis.

[0708] Example 3: In vitro silencing activity of different chemical modifications on ANGPTL3 siRNA

[0709] Cell culture and transfection

[0710] Hep3B cells (ATCC, Manassas, VA) were grown to near confluence at 37°C in an atmosphere of 5% CO2 in RPMI (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) and subsequently released from the plates by trypsinization. Transfection was performed by adding 14.8 μl Opti-MEM plus 0.2 μl Lipofectamine RNAiMax / well (Invitrogen, Carlsbad, CA, catalog number 13778-150) to 5 μl siRNA duplex / well in a 96-well plate and incubated at room temperature for 15 minutes. The cells were then incubated with approximately 2×10 siRNA duplexes containing 1% siRNA in a 96-well plate without antibiotics. 4 80 μl of complete growth medium from Hep3B cells was added to the siRNA mixture. Cells were incubated for 24 or 120 hours prior to RNA purification. Unless otherwise stated, single dose experiments were performed at 10 nM and 0.1 nM final duplex concentrations, and dose-response experiments were performed at 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, and 0.00001 nM final duplex concentrations.

[0711] cDNA synthesis using the ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, catalog number 4368813)

[0712] A master mix of 2 μl 10X buffer, 0.8 μl 25X dNTPs, 2 μl random primers, 1 μl reverse transcriptase, 1 μl RNase inhibitor, and 3.2 μl H O was added to 10 μl of total RNA per reaction. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, CA) by the following steps: 25° C. for 10 minutes, 37° C. for 120 minutes, 85° C. for 5 seconds, and 4° C. hold.

[0713] Real-time PCR

[0714] 2 μl of cDNA was added to a master mix containing 0.5 μl of GAPDH TaqMan probe (Applied Biosystems, catalog number 4326317E), 0.5 μl of ANGPTL TaqMan probe (Applied Biosystems, catalog number Hs00205581_m1) and 5 μl of Lightcycler 480 probe master mix (Roche, catalog number 04887301001) per well in a 384-well plate (Roche, catalog number 04887301001). Real-time PCR was performed using the ΔΔCt (RQ) assay on an ABI 7900HT Real-Time PCR System (Applied Biosystems). Unless otherwise indicated in the summary table, each duplex was tested in two independent transfections, and each transfection was assayed in duplicate.

[0715] To calculate relative fold changes, real-time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955 or mock-transfected cells. IC values ​​were calculated using XLFit using a 4-parameter fitting model. 50 , and the IC 50 Normalization was performed relative to cells transfected with AD-1955 within the same dose range or to naive cells or relative to its own lowest dose. The AD-1955 sequence used as a negative control targets luciferase and has the following sequence:

[0716] Sense strand: cuuAcGcuGAGuAcuucGAdTsdT;

[0717] Antisense strand: UCGAAGuACUcAGCGuAAGdTsdT.

[0718] The different embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, foreign patents, foreign patent applications, and non-patent applications cited in this specification are incorporated herein by reference in their entirety. Aspects of these embodiments can be modified, if necessary, to provide yet further embodiments utilizing concepts from the various patents, applications, and publications.

[0719] These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in this specification, but rather should be construed to include all possible embodiments, along with the full scope of equivalents to which such claims are entitled. Therefore, the claims are not limited by this disclosure.

[0720] Example 4: Chemical modifications on siRNA and in vitro silencing of modified siRNA.

[0721] Sense chain design

[0722] Ligand design and conjugation site

[0723] The sense strand was conjugated to a GalNAc ligand at the 3'-position, identical to the parent compound.

[0724] Meaningful position 11

[0725] The sense strand position 11 is modified with a nuclease-sensitive modification (e.g., DNA) at the putative cleavage site (opposite AS position 11 when the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length). Data from statistical analysis of many different conjugates indicate the importance of this position.

[0726] Thermal instability of the 3'-region of the sense strand (positions 16-18):

[0727] This region is modified with thermolabile modifications such as GNA or mismatches to the opposite AS strand. Modifications at positions 16 or 17 appear to be most impactful. Figure 1 Table 1 highlights the impact of this position / region and thermal instability on in vitro efficacy. Efficient knockdown comparable to the parental template design was achieved using GNA or other thermally labile modifications (e.g., abasic (Y34) or mismatches with the antisense strand). On the other hand, reduced silencing was generally observed with 2'-OMe or DNA modifications complementary to the opposite AS strand.

[0728]

[0729]

[0730] Figure 2 The position effect of the thermolabile modified GNA at the 3'-region (positions 16-18) is shown in Table 2. The results indicate that the GNA modifications at positions 16 and 17 showed good efficacy similar to the parent design, whereas the GNA at position 18 showed a decrease in activity.

[0731]

[0732] AS position 2

[0733] This position has been identified as sensitive to sterically desirable 2'-modifications (including 2'-OMe) by statistical analysis of large datasets of conjugates and by positional analysis of the AS chain. However, the inventors found that several modifications (including DNA, and in some cases RNA, and other modifications that are not sterically bulky at the 2'-position) can be well tolerated in the context of non-F designs. The results from the in vitro silencing studies are summarized in Figure 3 and Table 3, indicating that DNA as well as RNA at position 2 generally maintains activity similar to the non-F designs of the parental template design, however 2'-OME is generally not well tolerated and causes decreased activity.

[0734] AS position 14

[0735] This position has been identified as sensitive to sterically desirable 2'-modifications (including 2'-OMe) by statistical analysis of large datasets of conjugates and by positional analysis of the AS chain. However, it has been found that several modifications (including DNA, and in some cases RNA, and other modifications that are not sterically bulky at the 2'-position) can be well tolerated in the context of non-F designs. The results from the in vitro silencing studies are summarized in Figure 4 and Table 4, indicating that DNA as well as RNA at position 14 generally maintained activity similar to the non-F designs of the parental template design, however 2'-OME was generally not well tolerated and caused a decrease in activity.

[0736]

[0737] siRNA targeting mTTR

[0738] Animals (n=3 / group) were given a single siRNA dose of 2.5 mg / kg, and FVII serum protein levels were measured before and on days 4, 7, 13, 22, 29, and 36 after dosing. Figure 5 Shown is a plot of FVII protein concentration versus time for two non-F siRNAs AD-61398 and 64273 compared to the parent compound AD-57727. Figure 6 In Figure 5, the reduction of mTTR protein at 96 h post-dose is shown for two non-F siRNAs at three different dose levels compared to the parent compound. Figure 7 , a graph showing the reduction in mTTR serum protein for the repeat dose regimen (1 mg / kg, QW) up to day 42 (6 doses total) is shown.

[0739] Overall, this study demonstrates that the non-F siRNAs AD-61398 and AD-642733 exhibited similar efficacy and potency in vivo as the parental template design.

[0740] siRNA targeting TMPRSS6

[0741] Table 6. Sequences and chemistries of siRNAs targeting TMPRSS6.

[0742]

[0743] The results indicate that the different in vivo potencies of the non-F designs depend on the exact position of the modification and the combination of the sense and AS strands. Although the in vitro data indicate that the non-F compounds have similar potency / efficacy to the parent, the most active non-F compound AD-64604 in vivo was found to still have significantly lower potency than the parent AD-60940 (see Figure 8 ).

[0744] Further improvements to the non-F design were made and evaluated as summarized in Table 7. Figure 9 TMPRSS6 mRNA was shown to be silenced in the liver 7 days after a single SC dose of 3 mg / kg.

[0745]

[0746] like Figure 9 As shown, this improvement resulted in at least one non-F compound (AD-65105) with comparable in vivo efficacy 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.

[0747] Motif design

[0748] When designing a motif, the sense strand was conjugated to a GalNAc ligand at the 3'-position using the same method as the parent compound. Additional motifs were designed according to the examples of the present invention. Representative sequences are listed in Table 8.

[0749] Table 8. Representative sequences

[0750]

[0751] In vitro results

[0752] like Figure 10As shown, across ten sequences representing three targets and two motifs, these sequences were found to have statistically significant improvements in activity compared to the parent compound AD-57727, including motif 1 (six phosphorothioate internucleotide linkage modifications on both the sense and antisense strands; four 2'-F modifications at positions 7 and 9-11 of the sense strand from the 5'-end of the sense strand, and four 2'-F modifications at positions 2, 6, 14, and 16 of the antisense strand from the 5'-end of the antisense strand) and motif 2 (six phosphorothioate internucleotide linkage modifications on both the sense and antisense strands; four 2'-F modifications at positions 7 and 9-11 of the sense strand from the 5'-end of the sense strand, and six 2'-F modifications at positions 2, 6, 8-9, 14, and 16 of the antisense strand from the 5'-end of the antisense strand).

[0753] In vivo evaluation

[0754] Target silencing of siRNA was assessed by qPCR. The performance of this motif was assessed to target mTTR. Animals (n=3 / group) were given a single siRNA dose of 3 mg / kg and liver levels were measured (first before dosing and then on days 7 and 22 after dosing). Figure 11 shown.

[0755] Figure 12 Enhanced activity was demonstrated with the chemical stability enhancing conjugate (SEC-C), where liver activity (mRNA) was measured 7 days after administration. Animals received a single dose of 3 mg / kg (sc). This data demonstrates the effect of this motif on in vivo activity.

[0756] Figure 13 The data evaluated at day 7 post-dose showed that the new motifs (motifs 1 and 2) had enhanced activity (approximately 4-fold increase in activity) compared to the parent compound. This data demonstrates the impact of this motif on in vivo activity. The fold improvement was consistent between sequences.

[0757] Figure 14 Significantly improved durations were shown for all three sequences, indicating that the new motif exhibits enhanced duration.

[0758] Figure 15 A single 3 mg / kg SC dose of hAAV 1x10 11 Results of ApoC3-GalNAc3 SAR in GC / mice.

[0759] Example 5: In the antisense strand 5' end VP and PS2 Modification

[0760] The following is an exemplary scheme for the synthesis of oligonucleotides containing 5'-vinyl phosphate (VP) and the synthesis of oligonucleotides containing 2'-deoxythymidine (which is connected by phosphorodithioate (PS2) linkages at the 5'-end of the oligonucleotide). It will be understood by those skilled in the art that these same or similar techniques can be used to synthesize similar oligonucleotides. Other synthetic techniques known to those skilled in the art can also be used to synthesize and prepare these and similar oligonucleotides and modifications, including, but not limited to, those described in 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 49:6984-87 (2008); and Zhang et al., "Synthesis and Preliminary Biochemical Studies with 5'-Deoxy-5'-methylidyne Phosphonate Linked Thymidine Oligonucleotides." Tetrahedron Letters 49:6984-87 (2008). Letters) 37(35):6239-42 (1996); and the synthetic techniques disclosed in U.S. Patent Application Publication No. 2013 / 0084576, all of which are incorporated herein by reference in their entirety.

[0761] Synthesis protocol for oligonucleotides containing 5'-vinyl phosphate

[0762] Introduction of pivaloyloxymethyl-(POM)-protected VP

[0763]

[0764] Coupling and Oxidation: Amidite coupling was performed under standard synthetic conditions using 0.25 M 5-(ethylthio)-1H-tetrazole in acetonitrile for activation. The phosphite triester was converted to the phosphorothioate linkage using a standard thiolation protocol using 3-(dimethylaminomethylene)amino-3H-1,2,4-dithiazole-5-thione (DDTT) or phenylacetyl disulfide (PADS). Because the vinylphosphonate building block does not contain a DMT protecting group at the 5'-position, the final detritylation step was omitted.

[0765] Deprotection and cleavage: After synthesis of oligonucleotides containing vinylphosphonates, deprotection was performed in a 3:1 mixture of aqueous NH3 and EtOH by adding 1%-2.5% by volume of a 40% methylamine solution at 60°C for 5 hours or at 35°C for 16 hours.

[0766] Introduction of ethyl-protected VP

[0767]

[0768] Coupling and Oxidation: Amidite coupling was performed under standard synthetic conditions using 0.25 M 5-(ethylthio)-1H-tetrazole in acetonitrile for activation. Standard thiolation protocols were used with 3-(dimethylaminomethylene)amino-3H-1,2,4-dithiazole-5-thione (DDTT) or phenylacetyl disulfide (PADS) to oxidize the triester phosphite and introduce the phosphorothioate linkage. Because the vinylphosphonate building block does not contain a DMT protecting group at the 5'-position, the final detritylation step was omitted.

[0769] Deprotection and cleavage: Prepare a solution of acetonitrile (ACN) and pyridine (Pyr) 50:1 (v / v) and add Molecular sieves to keep the mixture as dry 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 must be freshly prepared and have a maximum shelf life of one day. Then, a 0.5 M solution of mercaptoethanol in 1:1 (v / v) acetonitrile-triethylamine was prepared and added. Molecular sieve. Using the oligonucleotide containing 5'-VP on the resin and in the synthesis column, the TMSI solution is slowly added at about 5-10 μl and allowed to react for 15 min. This step is repeated twice, resulting in a total exposure time of approximately 45 minutes. Subsequently, the resin is thoroughly washed with ACN, and then about 5-10 column volumes of mercaptoethanol solution are flowed over the column and allowed to react for 10 min. This step is repeated once, resulting in a total exposure time of 20 minutes. After another thorough wash with ACN, the support-bound oligonucleotide is deprotected and cleaved from the support using standard conditions.

[0770] Scheme for the synthesis of oligonucleotides containing 2'-deoxythymidine linked via a phosphorodithioate linkage at the 5'-end of the oligonucleotide

[0771]

[0772] 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 performed under standard 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 the synthesis cycle. Oxidation (sulfhydration) was performed using 3-(dimethylaminomethylene)amino-3H-1,2,4-dithiazole-5-thione (DDTT) by extending the reagent delivery and reaction time to 3x10 minutes. Final detritylation was performed using standard synthesis conditions.

[0773] Deprotection and cleavage: The solid support (on the column) was washed with 0.5 M piperidine in ACN (2 x 15 min exposure time), then the resin was transferred to a suitable container and treated under standard conditions (e.g., 3:1 NH:EtOH aqueous solution at 60°C for 5 hours or 35°C for 16 hours) to cleave from the solid support and deprotect from the oligonucleotide.

[0774] The remaining steps for the oligonucleotide synthesis method were similar to those described in Example 2.

[0775] Figure 16 Schematic diagram illustrating Ago2-loaded siRNA. In general, 5'-phosphate-functionalized siRNAs (ESC chemistry) showed improved in vitro activity. For example, approximately 80% of the sequences tested showed improved intrinsic potency when transfected in vitro, and approximately 30% showed approximately 10-fold higher IC 50 However, in vivo, the 5'-phosphate is rapidly lost in the endo / lysosomal compartment. Figure 16Also shown in Figure 1 is a modified phosphate, a simulated stable phosphate, 5'-vinylphosphonate (5'-VP), attached to the 5' end of the modified oligonucleotide. This phosphate was originally designed by Merck.

[0776] One embodiment of the present invention is directed to 5'-terminal modification for efficacy improvement (RISC loading). The terminal modification provides a stable phosphate mimetic and promotes endogenous phosphorylation.

[0777] Figure 17 Depicted is a graph showing how the presence of 5'-VP based on four different ApoB sequences generally improves activity in vivo. LDL levels were analyzed 7 days after a single SC dose of 3 mg / kg for the four conjugates (with or without 5'-VP modification). As can be seen from the graph, ED 50 The ApoB sequences are listed in Table 9.

[0778] Table 9

[0779]

[0780]

[0781] Figure 18 Describes different chemical modifications that can replace the PS bond, including dithiophosphate (PS2) and methylphosphonate (MePhos), which promote endogenous phosphorylation. Modified siRNA is generally not a good substrate for Clp1 kinase, probably due to interference from the 2'OMe modification at the first nucleotide of the AS chain. However, 2'-OMe modification together with phosphorothioate linkage is required for exonuclease protection. Replacing the 2'-OMe modification with, for example, 2'F and modifying the PS bond can promote exonuclease protection while maintaining metabolic stability.

[0782] Figure 19 Figure showing in vitro evaluation of terminal modifications including 2'-OMe-MePhos, 2'-OMe-PS, dN(PS2), and 2'F-PS. As shown in the graph, the dn(PS2) linkage and 2'F-PS exhibited improved in vitro activity relative to the parent (2'OMe-PS). In particular, dN(PS)2 was stable in an in vitro lysosomal decontamination assay, whereas 2'F-PS exhibited metabolic propensity. Primary mouse hepatocytes were transfected with both ApoB conjugates at 10 nM and 0.1 nM (n=4).

[0783] Figure 20 It was shown that small changes at the antisense 5'-end can significantly improve effectiveness in vivo. Figure 20 A shows that 2'F-PS at position 1 of the antisense strand can enhance the activity of 5'P-dependent sequences, and Figure 20 B shows that similar to 5'-VP, the efficacy was improved approximately 3-fold by dN(PS)2 over the parent.

[0784] Example 6: 5'-VP modification and evaluation of siRNA activity

[0785] Synthesis of 5'-vinylphosphonate phosphoramidite with pivaloyloxymethyl protecting group:

[0786]

[0787] Kit reaction conditions for Scheme 1: (a) Dess-Martin periodinane, DCM, 0°C; (b) NaH, tetrakis(pivaloyloxymethyl)bisphosphate, THF, -78°C, followed by stirring at 0°C, 70% (E and Z isomers); (c) formic acid: water, 1:1, 24 hours, E and Z isomers separated by silica column chromatography or by RP-HPLC (reverse phase HPLC); (d) 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite, 5-(ethylthio)-1H-tetrazole, ACN, 6 hours, room temperature, 65%.

[0788] Synthesis of Tetrakis(pivaloyloxymethyl)-bis-phosphate (X)

[0789]

[0790] Tetramethyl methylene bisphosphate (120g, 0.51mol), NaI (308g, 2mol), chloromethyl pivalate (387g, 2.5mol) and acetonitrile (400ml) are mixed, and it is refluxed and spent the night.TLC (thin layer chromatography) confirms the formation of product with 5% methyl alcohol in EtOAc.This reaction mixture is diluted with ether (1000ml), and washed with water (2 × 1000ml), use Na SO Be dried, and evaporate.Wash this solid residue with ice-cold hexane, and dry in a vacuum to provide 148g (45%) X that is light yellow solid.

[0791] 1 H NMR (500MHz, CDCI3): δ5.73-5.63(m,8H),2.65(t,2H),1.22(s,36H); 31 P NMR (500MHz, CDCI3): δ18.61.

[0792] Compound 2 Preparation

[0793]

[0794] To a solution of compound 1 (3.0 g, 8 mmol) in 150 mL of anhydrous dichloromethane cooled with ice was added Dess-Martin periodinane (DMP) (1.4 equivalents; 4.7 g, 11.2 mmol). The reaction mixture was stirred at 0°C for 1 hour and then at room temperature for 3 hours. TLC confirmed the formation of the product. The reaction mixture was then added to a 200 ml solution of 10% Na2S2O3 and saturated NaHCO3 (1:1), followed by the addition of 200 ml of ethyl acetate. Under reduced pressure, the crude aldehyde was extracted into ethyl acetate, dried, and concentrated. The crude aldehyde was used directly in the next step without purification.

[0795] Yield = 2.87 gm (97%); purity by NMR was approximately 70%; LC-MS: m / z 371.

[0796] Preparation of compound 3

[0797]

[0798] To a suspension of NaH (0.58 g, 24 mmol) in 20 mL of THF at -78°C a solution of tetraoxometalate (POM)-bisphosphonate sodium salt was added bisphosphate (X) in 14 ml of THF (12.6 gm, 20 mmol) and stirred for 15 minutes.

[0799] At -78 ° C, aldehyde 2 (2.86 g) was added dropwise in 40 mL of anhydrous THF solution to the tetrakis (POM) bisphosphate sodium salt solution prepared above. The reaction mixture was stirred at -78 ° C for 1 hour, stirred at 0 ° C for another 1 hour, and then stirred at room temperature for 1 hour. TLC confirmed the formation of the product (EtOAc: hexane 7: 3). The crude reaction mixture was added to 300 ml of saturated ammonium chloride and extracted with 300 ml of ethyl acetate. The organic layer was washed with brine and dried over sodium sulfate. The solution was then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (EtOAc in hexane = 20%-100%) to give compound 3 (4.0 g) of a mixture of E / Z isomers (88 / 12) in 72% yield.

[0800] Preparation of compound 4

[0801]

[0802] A solution of compound 3 (4 g, 5.7 mmol) in 200 mL of HCOOH / H2O (1:1, v:v) was stirred at room temperature for 24 hours. TLC confirmed the formation of the product (MeOH:CH2Cl2=5:95).

[0803] Under reduced pressure, the solution was concentrated, and the residue was purified by silica gel column chromatography (MeOH:CH2Cl2=7:93v / v). Fractions were tested on RP-HPLC (C18 column, buffer A=0.05% TFA in water, buffer B=0.05% TFA in ACN; gradient 5%-95%, over 25 minutes) to determine 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 initial fraction from silica gel chromatography contained a mixture of E and Z isomers, and the remaining fraction was the E isomer. The fraction containing the E and Z isomer mixture was purified on RP-HPLC. The 4-E isomer (2.3 g, 71% yield) obtained.

[0804] E isomer:

[0805] 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.

[0806] Z Isomer:

[0807] 1H NMR (500MHz, acetonitrile-d3): δ9.50 (s, 1H), 7.44 (d, J = 8.1Hz, 1H), 6.69 (ddd, J = 54.4, 13.3, 8.7Hz, 1H), 5.93 (ddd, J = 17.8, 13.3, 1.3Hz, 1H), 5.80 (d, J=2.9Hz,1H),5.69-5.58(m,5H),5.22(m,1H),4.01(dd,J=7.1,5.3Hz,1H),3.88(dd,J=5.3,2.9Hz,1H),3.49(s,3H),1.19(d,J=5.8Hz,18H); 31 P NMR (202 MHz, acetonitrile-d3): δ 18.75.

[0808] Compound 5 Preparation

[0809]

[0810] To compound 4-E isomer (2.1g, 3.62mmol) and ethylthio tetrazole (0.46g, 3.62mmol) in ACN (40mL) solution, 2-cyanoethyl N, N, N ', N '-tetraisopropyl, phosphorodiamidite (1.311g, 4.35mmol) was added. The mixture was stirred at room temperature for 2 hours. TLC confirmed the formation of the product 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% to 100% EtOAc and TEA (0.15%) solution in hexane to obtain compound 5 (1.75g, 62%) as white foam.

[0811] E isomer:

[0812] 1 H NMR (400 MHz, acetonitrile-d3): δ9.09 (s, 1H), 7.38 (d, J=8.1 Hz, 1H), 6.89 (m, 1H), 6.10 (dddd, J=21.4, 17.1, 2.8, 1.7 Hz, 1H), 5.86 (t, J=3.8 Hz, 1H), 5.67-5.55 (m, 5H), 4.66-4.50 (m, 1H), 4.40-4.20 (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); 31P NMR (162 MHz, acetonitrile-d3): δ 151.79 (d, J = 71.3 Hz), 18.07 (d, J = 54.0 Hz).

[0813] Z Isomer:

[0814] 1 H NMR (400MHz, acetonitrile-d3): δ9.02 (s, 1H), 7.41 (dd, J=8.1, 1.6Hz, 1H), 6.62 (dddd, J= 53.7,13.1,9.7,7.0Hz,1H),5.97(dd,J=17.4,13.1Hz,1H),5.80(dd,J=7.0,3. 5Hz,1H),5.70-5.52(m,5H),5.41(m,1H),4.40-4.10(m,1H),4.06-3.98(m,1H) ,3.93-3.56(m,4H),3.47(s,3H),2.68(m,2H),2.14(s,1H),1.33-1.11(m,30H); 31 P NMR (202 MHz, acetonitrile-d3): δ 150.81 (d, J = 141.4 Hz), 15.17.

[0815] Synthesis protocol for oligonucleotides containing 5'-vinyl phosphate

[0816] The method for synthesizing vinylphosphonate monomers and 5'-VP modified oligonucleotides is similar to that in the literature (WO 2008 / 100447 to Chen et al.; Lima et al., "Single-Stranded siRNAs Activate RNAi in Animals," Cell 150:883-894 (2012); Prakash et al., "Identification of metabolically stable 5-phosphate analogs that supports single-stranded siRNA activity," Nucleic Acids Research 43:2993-3011 (2015), which are incorporated herein by reference in their entirety). Briefly, the 5'-phosphate was protected with ether, and then the ether-protected phosphate was deprotected in two steps: 1) TMS-1 on a solid support under anhydrous conditions, and 2) standard oligonucleotide deprotection to obtain a 5'-VP modified oligonucleotide. This process is also discussed in Example 5.

[0817] Effects of metabolically stable (E-) and (Z-) 5′-vinylphosphonates on siRNA activity

[0818] Double-stranded small interfering RNA (siRNA) with a 5′-phosphorylated antisense strand facilitates efficient loading onto the RNA-induced silencing complex (RISC) to elicit robust RNAi-mediated gene silencing. Endogenous 5′-phosphorylation of Clp1 kinase by synthetic siRNA is therefore critical for RISC loading and strand selection (Weitzer et al., “The human RNA kinase hClp1 is active on 3′ transfer RNA exons and short interfering RNAs,” Nature 447:222-226 (2007)). Phosphate mimetics with metabolically stable linkages have been used for nucleoside modification as antiviral agents (WO 2008 / 100447 to Chen et al.), because modification of the 5′ end of siRNA enhances gene silencing activity over corresponding non-phosphorylated siRNAs, particularly single-stranded siRNAs (Lima et al., “Single-Stranded siRNAs Activate RNAi in Animals,” Cell 150:883-894 (2012); Prakash et al., “Identification of metabolically stable 5-phosphate analogs that support single-stranded siRNA activity,” Nucleic Acids Research 43:2993-3011 (2015)).

[0819] In this example, the effects of phosphate mimetics in double-stranded siRNA were evaluated in vitro and in vivo.

[0820] The siRNA sequences used in this example are shown in the table below.

[0821]

[0822]

[0823] u=2'OMe,5'OH U

[0824] Vpu=2'OMe,5'VP U

[0825] VPUf=2'F,5'VP U

[0826] VP(Tam)=2'N-methylacetamide, 5'VP T

[0827]

[0828] The effects of 5'-vinylphosphonate (VP) with E- and Z-geometries on double-stranded siRNA activity were compared. The results showed that in vivo efficacy of chemically modified siRNAs with 5'-trans-(E-)VP, which closely mimics the natural phosphate, could be improved, whereas 5'-cis-(Z-)VP showed no improvement in efficacy, suggesting that the Z-isomer does not mimic the natural phosphate well.

[0829] Figure 21 AB show SAR analysis of the in vitro and in vivo activity of ApoB siRNAs containing a 5'-OH relative to the 5'-E-VP modification (at the 5'-end of the antisense strand). Figure 21 A shows the results of in vitro transfection of mouse 1° hepatocytes. Figure 21 B shows LDL levels 3 days after a single dose (SC dosing). Figure 21 The results in B show that ApoB siRNA modified with 5'-E-VP exhibits enhanced activity.

[0830] Figure 22 Results showing the in vitro efficacy of 5'-E-VP modification versus 5'-Z-VP modification for mTTR and F9 siRNA-GalNAc conjugates. These results are from in vitro transfected mouse primary hepatocytes. As shown, siRNA conjugates modified with 5'-E-VP showed maintained or improved efficacy, whereas siRNA conjugates modified with 5'-Z-VP showed decreased efficacy.

[0831] Figure 23 Results are shown for an in vivo comparison of 5'-E-VP modification versus 5'-Z-VP modification of F9 siRNA-GalNAc conjugates (single SC dose). The results indicate that siRNA conjugates modified with 5'-E-VP exhibited improved gene silencing activity over the 5'-OH control, whereas siRNA conjugates modified with 5'-Z-VP exhibited similar activity to the 5'-OH control.

[0832] The results of these figures show that 5'-phosphorylation of the antisense strand is required for efficient RNAi-mediated gene silencing.The effectiveness of chemically modified siRNA can be enhanced by using 5'-trans-vinylphosphonate (5'-E-VP), which mimics natural phosphates.

[0833] Example 7: 5'-C - Malonyl modification and evaluation of siRNA activity

[0834] Synthesis and incorporation of 5′-C-malonyl nucleotides into the 5′ end of siRNA:

[0835] General Experimental Conditions: All moisture sensitive reactions were performed under anhydrous conditions under an argon atmosphere. Flash chromatography was performed on a Teledyne ISCO (Lincoln, NE) Combi flash system using pre-packed ReadySep Teledyne ISCO silica gel columns. Electrospray ionization-high resolution mass spectrometry (ESI-HRMS) spectra were recorded on a Waters (Milford, MA) QT API-US spectrometer using direct flow injection in positive mode (capillary voltage = 3000 kV, cone = 35, source temperature = 120°C, and desolvation temperature = 350°C). The spectra were recorded at room temperature on a Varian spectrometer (Palo Alto, CA) at 400 MHz ( 1 H) and 126MHz( 13 C) Record 1 H and 13 C NMR spectra, and chemical shifts are in ppm referenced to the residual solvent peak. Coupling constants are given in Hertz. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), broad (br), or multiplet (m). Recorded at 162 MHz in proton decoupling mode. 31 P NMR spectra, and chemical shifts were referenced to external H3PO4 (80%). LC / ESI-MS was performed at 60°C on an Agilent (Santa Clara, CA) 6130 Single Quadrupole LC / MS system using an XBridge C8 column (2.1×50 mm, 2.5 μm). Buffer A consisted of 100 mM 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and 16.3 mM triethylamine (TEA) in H2O, and buffer B was 100% methanol.

[0836]

[0837] Reagents and conditions for Scheme 2: (a) benzyloxymethyl acetal (BOM) chloride, DBU, DMF, 30 min, 0° C., quantitative (Kurosu et al., “Synthetic studies towards the identification of novel capuramycin analogs with mycobactericidal activity,” Heterocycles 77:217-225 (2009); Kurosu et al., “Concise Synthesis of capuramycin”). Capuramycin,” Org. Lett. 11:2393-2396 (2009), which is incorporated herein by reference in its entirety); (b) methyltriphenoxyphosphonium iodide, DMF, 15 min, rt, 92%; (c) sodium methoxide, dimethyl malonate, 1,2-DME, 24 h, reflux, 92%; (d) 10% Pd / C, H2 atm, i-PrOH / H2O (10:1, v / v), 0.05 eq. formic acid, 12 h, rt, 98% (Aleiwi et al., “Areliable Pd-mediated hydrogenolytic deprotection of BOM group of uridine ureido nitrogen,” Tetrahedron (E) NEt3-3HF, THF, 48 hours, room temperature, 88%; (D) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, DIEA, DCM, 18 hours, room temperature, 56%; (G) 1 M piperidine in water, 24 hours, room temperature; then 30% ammonia / ethanol in water (3:1, v / v), 36 hours, room temperature, quantitative, Z + =piperidinium.

[0838] N 3 Synthesis of Benzyloxymethyl-2′-O-methyl-3′-O-tert-butyldimethylsilyluridine (2)

[0839] 2'-O-Methyl-3'-O-tert-butyldimethylsilyl-uridine (1, 20 g, 53.7 mmol) was converted to 2 (26.5 g, quantitative) following a variation of a previously reported method.

[0840] N 3 -Benzyloxymethyl-5′-deoxy-5′-iodo-2′- O -methyl-3′- O - tert-butyl Dimethylsilyluridine (3) Synthesis

[0841] Compound 2 (10 g, 20.3 mmol) was dissolved in 100 mL of anhydrous DMF and 20 g (40.6 mmol) of methyl triphenoxyphosphonium iodide was added. The mixture was stirred at room temperature for 15 minutes. Methanol (200 mL) was added to the reaction, and the mixture was stirred for another 15 minutes. The solvent was evaporated to dryness; the residue was dissolved in dichloromethane (DCM) and washed with 5% Na2S2O3 solution, then washed with water. The organic layer was collected, dried over Na2SO4, filtered, and evaporated to dryness. The crude residue was purified by column chromatography using 0-50% ethyl acetate (EtOAc) in hexane as eluent to obtain 3 (11.2 g, 92%) as white foam.

[0842] 1 H NMR (400MHz, DMSO-d6): δ7.77(d,J=8.2Hz,1H),7.30(m,5H),5.90(d,J=5.2Hz,1H),5. 85(d,J=8.2Hz,1H),5.33(d,J=13.0Hz,1H),5.30(d,J=13.0Hz,1H),4.58(s,2H),4.23( t,J=4.5Hz,1H),4.07(t,J=5.1Hz,1H),3.87(q,J=6.1Hz,1H),3.55(dd,J=10.6,6.3Hz, 1H), 3.39 (dd, J = 10.6, 6.3Hz, 1H), 3.32 (s, 3H), 0.89 (s, 9H), 0.14 (s, 3H), 0.12 (s, 3H). 13 C NMR (126MHz, DMSO-d6): δ161.7,150.7,140.2,138.0,128.2,127.4,127.3,1 01.6,87.9,83.3,80.8,72.7,71.0,70.1,57.6,25.6,17.7,6.2,-4.7,-4.8.

[0843] HRMS-ESI for C 24 H 35 IN2NaO6Si(M+Na) + Calculated: 625.1207; Found: 625.1205.

[0844] N 3 -Benzyloxymethyl-5′-deoxy-5′-C-(dimethylmalonyl)-2′-O-methyl-3′-O-tert-butyldimethyl Synthesis of methylsilyl uridine (4)

[0845] Sodium methoxide (2g, 33mmol) is placed in a dry round-bottom flask, dimethyl malonate (12mL, 100mmol) and anhydrous 1,2-dimethoxyethane (DME, 100mL) are added, and the mixture is refluxed. After compound 3 (10g, 16.5mmol) is co-evaporated twice with anhydrous acetonitrile, it is dissolved in 70mL of anhydrous DME, and it is added into the reflux solution of dimethyl malonate and sodium methoxide. Reflux lasts 24 hours. The reaction mixture is cooled to room temperature, and methanol (50mL) is added to quench the reaction. Solvent and volatiles are evaporated in vacuo. The crude residue is purified by column chromatography, using 0-100% EtOAc in hexane as eluent to obtain compound 4 (9.2g, 92%) as colorless oil.

[0846] 1 H NMR (400MHz, DMSO-d6): δ7.66(d,J=8.2Hz,1H),7.30(m,5H),5.80(d,J=8.2Hz ,1H),5.76(d,J=4.0Hz,1H),5.33(d,J=13.4Hz,1H),5.30(d,J=13.4Hz,1H),4. 58(s,2H),4.14(t,J=5.4Hz,1H),3.91(m,1H),3.76(m,1H),3.64(m,4H),3.60( s,3H),3.33(s,3H),2.37-2.09(m,2H),0.87(s,9H),0.09(s,3H),0.08(s,3H). 13 C NMR (126MHz, DMSO-d6): δ169.1,168.8,161.9,150.6,140.4,138.0,128.1,127.4,127.3,101.3, 88.6,88.5,81.3,80.9,73.1,71.0,70.0,59.7,57.5,52.5,48.0,31.5,25.6,17.7,-4.76,-5.06.

[0847] HRMS-ESI for C 29 H 42 N2NaO 10 Si(M+Na) + Calculated: 629.2506; Found: 629.2508.

[0848] 5′-Deoxy-5′-C-(dimethylmalonyl)-2′-O-methyl-3′-O-tert-butyldimethylsilyluridine Synthesis of (5)

[0849] Compound 4 (8.7 g, 14.3 mmol) was dissolved in 660 mL of isopropanol / water (10: 1, v / v), and 0.9 g of 10% Pd / C was added, followed by 27 mL (0.7 mmol) of formic acid. The air in the flask was removed under vacuum; the reaction flask was flushed with hydrogen, and stirred at room temperature for 12 hours under a hydrogen atmosphere, at normal pressure. The reaction mixture was filtered through diatomaceous earth and rinsed with ethanol. The filtrate was collected and evaporated to dryness. The crude residue was purified by silica gel chromatography using 0-5% MeOH in DCM as eluent. The applicable fractions were combined and evaporated to dryness to obtain 5 (6.7 g, 98%) as a white foam.

[0850] 1 H NMR (400MHz, DMSO-d6): δ11.38(d,J=1.8Hz,1H),7.61(d,J=8.1Hz,1H),5.71(d,J=4.3Hz,1H),5.65(dd,J=8.0Hz,J=2.1Hz,1H),4.16(t,J=5.3 Hz,1H),3.91(t,J=4.8Hz,1H),3.73(m,1H),3.63(m,4H),3.61(s,3H), 3.31(s,3H),2.24-2.07(m,2H),0.87(s,9H),0.08(s,3H),0.08(s,3H). 13 C NMR (126MHz, DMSO-d6): δ169.2,168.9,163.0,150.4,141.2,141.2,102.1,87. 7,81.2,80.9,73.1,57.5,52.5,52.4,52.3,48.0,31.6,25.6,17.7,-4.8,-5.1.

[0851] HRMS-ESI for C 21 H 34 N2NaO9Si(M+Na) + Calculated: 509.1931; Found: 509.1929.

[0852] Synthesis of 5′-deoxy-5′-C-(dimethylmalonyl)-2′-O-...

Claims

1. A double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a sense strand and an antisense strand, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, The sense strand and the antisense strand form a duplex region of 21 nucleotide pairs in length, and The dsRNA agent has a 3' overhang at the 3' end of the antisense strand and a blunt end at the 5'-end of the antisense strand, The dsRNA agent is represented by formula (I): in: B1, B2, B3, B1', B2', B3' and B4' each independently represent a nucleotide containing a modification selected from the group consisting of: 2'-O methyl and 2'-fluoro; C1 is a thermolabile nucleotide selected from unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA) and located at a site opposite to positions 2-8 of the 5'-end of the antisense strand; T1, T1′, T2′, and T3′ each independently represent a 2′-fluoro-modified nucleotide; T1' is located at position 14 from the 5' end of the antisense strand, and q 2 is 1; T3' is located at position 2 from the 5' end of the antisense strand, and q 6 and q 7 is 1; Each n 1 、n 3 and q 1 are independently 4 to 15 nucleotides in length; Each n 5 and q 3 are independently 1-6 nucleotides in length; q 5 are independently 0-10 nucleotides in length; n 4 is 0; and Each n 2 and q 4 are independently 0-3 nucleotides in length, in (a) the sense strand comprises at least one phosphorothioate linkage; (b) one of the T1 nucleotides is at the cleavage site of the sense strand; and (c) the sense strand has only four 2'-F modifications at positions 7, 9, 10, and 11, counted from the 5' end of the sense strand; and the antisense strand has only four 2'-F modifications at antisense strand positions 2, 6, 14, and 16, or only six 2'-F modifications at antisense strand positions 2, 6, 8-9, 14, and 16, counted from the 5' end of the antisense strand.

2. The dsRNA agent of claim 1, wherein T2' is located at position 6-10 from the 5' end of the antisense strand, and q 4 It is 1.

3. The dsRNA agent of claim 1, wherein each of B1, B2, B3, B1', B2', B3', and B4' contains a 2'-OMe modification.

4. The dsRNA agent of claim 1, wherein the sense strand and / or antisense strand comprises one or more blocks of phosphorothioate or methylphosphonate internucleotide linkages.

5. The dsRNA agent of claim 4, wherein the sense strand comprises a block of two phosphorothioate or methylphosphonate internucleotide linkages.

6. The dsRNA agent of claim 4, wherein the sense strand comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 14, 15, 16, 17, or 18 phosphate internucleotide linkages.

7. The dsRNA agent of claim 4, wherein the antisense strand comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 16-18 phosphate internucleotide linkages.

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

9. The dsRNA agent of 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.

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

11. The dsRNA agent of claim 10, wherein at least one ligand increases nuclease resistance of the dsRNA agent.

12. The dsRNA agent of claim 10, comprising at least one ASGPR ligand.

13. The dsRNA agent of claim 12, wherein the ASGPR ligand is attached to the 5' end or the 3' end of the sense strand. The dsRNA agent of claim 13 , wherein the ASGPR ligand is attached to the 3′ end of the sense strand.

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

16. The dsRNA agent of claim 15, wherein the ASGPR ligand is:

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

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

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

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

21. The dsRNA agent of claim 1, wherein at least one of the first 1, 2, 3, 4 or 5 base pairs at the 5' end of the antisense strand in the duplex region is a mismatched pair.

22. The dsRNA agent of claim 1, wherein formula (I) further comprises a 5'-vinyl phosphate (VP).

23. The dsRNA agent of claim 1, wherein formula (I) further comprises a 2'-deoxythymidine linked via a phosphorodithioate (PS2) linkage at the 5'-end of the antisense strand or the sense strand.

24. The dsRNA agent of claim 1, wherein B4' is 2'-OMe.

25. A pharmaceutical composition comprising the dsRNA agent according to any one of claims 1 to 24, alone or in combination with a pharmaceutically acceptable carrier or excipient.

26. Use of the dsRNA agent according to any one of claims 1 to 24 for inhibiting target gene expression in cells in vitro.

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