Modified double-stranded rna agents

Abstract

One aspect of the present invention relates to double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene. The sense strand of the dsRNA agent comprises at least one thermally destabilizing nucleotide, and at least one said thermally destabilizing nucleotide occurring at a site opposite to the seed region (positions 2-8) of the antisense strand; and the antisense strand of the dsRNA agent comprises. at least two modified nucleotides that provide the nucleotide a steric bulk that is less than or equal to the steric bulk of a 2′-OMe modification, wherein said modified nucleotides are separated by 11 nucleotides in length. Other aspects of the invention relates to pharmaceutical compositions comprising these dsRNA agents suitable for therapeutic use, and methods of inhibiting the expression of a target gene by administering these dsRNA agents, e.g., for the treatment of various disease conditions.

1 . A double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a sense strand and an antisense strand complementary to at least one portion of a mRNA of the target gene, each strand having 14 to 40 nucleotides, wherein the dsRNA agent is represented by formula (I): 
       
         
           
           
               
               
           
         
       
       wherein:
 B1, B1′, B2′, B3′, and B4′ each independently represent a nucleotide containing a modification selected from the group consisting of 2′-OMethyl and 2′-fluoro; 
 C1 is glycerol nucleic acid (GNA) placed at a site opposite to the seed region (positions 2-8) of the antisense strand; 
 T1′, T2′, and T3′ are each independently selected from DNA, RNA, LNA, 2′-F, and 2′-F-5′-methyl; 
 each n 1 , n 3 , and q 1  is independently 4 to 15 nucleotides in length; 
 each n 5 , q 3 , and q 7  is independently 1-6 nucleotide(s) in length; 
 each q 2  and q 6  is independently 1-3 nucleotide(s) in length; 
 q 5  is independently 0-10 nucleotide(s) in length; 
 each n 4  and q 4  is independently 0-3 nucleotide(s) in length; 
 n 2  is 3 nucleotides in length, and T1 each are 2′-F, wherein T1 is at the cleavage site of the sense strand; 
 B2 each are 2′-OMe; and 
 B3 each are 2′-OMe, 
 
       wherein the dsRNA agent is covalently conjugated to at least one ligand. 
     
     
         2 . The dsRNA agent of  claim 1 , wherein n 4  is 0. 
     
     
         3 . The dsRNA agent of  claim 1 , wherein T1′ and T3′ are separated by 11 nucleotides in length. 
     
     
         4 . The dsRNA agent of  claim 1 , wherein T1′ is at position 14 from the 5′ end of the antisense strand, and q 2  is 1. 
     
     
         5 . The dsRNA agent of  claim 1 , wherein T3′ is at position 2 from the 5′ end of the antisense strand, and q 6  is 1. 
     
     
         6 . The dsRNA agent of  claim 1 , wherein one of the T1 nucleotides is at position 11 from the 5′ end of the sense strand, when the sense strand is 19-22 nucleotides in length. 
     
     
         7 . The dsRNA agent of  claim 1 , wherein T2′ is at positions 6-10 from the 5′ end of the antisense strand. 
     
     
         8 . The dsRNA agent of  claim 1 , wherein B1, B1′, B2′, B3′, and B4′ each contain 2′-OMe modifications. 
     
     
         9 . The dsRNA agent of  claim 1 , wherein the antisense strand comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 16-18 phosphate internucleotide linkages. 
     
     
         10 . The dsRNA agent of  claim 9 , wherein the antisense strand comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand, counting from the 5′-end of the antisense strand. 
     
     
         11 . The dsRNA agent of  claim 1 , wherein the sense strand comprises two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand, counting from the 5′-end of the sense strand. 
     
     
         12 . The dsRNA agent of  claim 1 , wherein the sense strand has 19-22 nucleotides, and the antisense strand has 19-25 nucleotides. 
     
     
         13 . The dsRNA agent of  claim 1 , wherein the sense strand has 21 nucleotides, and the antisense strand has 23 nucleotides. 
     
     
         14 . The dsRNA agent of  claim 1 , wherein the dsRNA agent has a 3′ and/or 5′ overhang(s) of 1-10 nucleotides in length. 
     
     
         15 . The dsRNA agent of  claim 14 , wherein the dsRNA agent has a two-nucleotide overhang at the 3′-end of the antisense strand, and a blunt end at the 5′-end of the antisense strand. 
     
     
         16 . The dsRNA agent of  claim 1 , wherein 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. 
     
     
         17 . The dsRNA agent of  claim 1 , wherein at least one ligand is an ASGPR ligand. 
     
     
         18 . The dsRNA agent of  claim 17 , wherein the ASGPR ligand is attached to the 5′ end or 3′ end of the sense strand. 
     
     
         19 . The dsRNA agent of  claim 18 , wherein the ASGPR ligand is attached to the 3′ end of the sense strand. 
     
     
         20 . The dsRNA agent of  claim 17 , wherein the ASGPR ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker. 
     
     
         21 . The dsRNA agent of  claim 20 , wherein the ASGPR ligand is: 
       
         
           
           
               
               
           
         
       
     
     
         22 . The dsRNA agent of  claim 1 , wherein formula (I) further comprises a 5′-vinyl phosphonate (VP). 
     
     
         23 . The dsRNA agent of  claim 1 , wherein formula (I) further comprises a 2′-deoxythymidine linked via a phosphorodithioate (PS 2 ) linkage at the 5′-end of the antisense strand or sense strand. 
     
     
         24 . A pharmaceutical composition comprising the dsRNA agent according to  claim 1 , in combination with a pharmaceutically acceptable carrier or excipient. 
     
     
         25 . A double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a sense strand and an antisense strand complementary to at least one portion of a mRNA of the target gene, wherein:
 the sense strand has 19-22 nucleotides,   the antisense strand has 19-25 nucleotides; and   the dsRNA agent is represented by formula (I):   
       
         
           
           
               
               
           
         
         wherein:
 B1, B1′, B2′, B3′, and B4′ each independently represent a nucleotide containing a modification selected from the group consisting of 2′-OMethyl and 2′-fluoro; 
 each B2 and B3 is 2′-OMe; 
 C1 is glycerol nucleic acid (GNA) placed at a site opposite to the seed region (positions 2-8) of the antisense strand; 
 T1′, T2′, and T3′ are each 2′-F, wherein:
 T1′ is at position 14 from the 5′ end of the antisense strand, and q 2  is 1; and 
 T3′ is at position 2 from the 5′ end of the antisense strand, and q 6  and q 7  are 1; 
 
 each n 1 , n 3 , and q 1  is independently 4 to 15 nucleotides in length; 
 each n 5  and q 3  is independently 1-6 nucleotide(s) in length; 
 q 5  is independently 0-10 nucleotide(s) in length; 
 each n 4  and q 4  is independently 0-3 nucleotide(s) in length; 
 n 2  is 3 nucleotides in length, and T1 each are 2′-F, wherein T1 is at the cleavage site of the sense strand, and 
 
       
       wherein the dsRNA agent is covalently conjugated to at least one ligand. 
     
     
         26 . The dsRNA agent of  claim 25 , wherein n 4  is 0. 
     
     
         27 . The dsRNA agent of  claim 25 , wherein one of the T1 nucleotides is at position 11 from the 5′ end of the sense strand, when the sense strand is 19-22 nucleotides in length. 
     
     
         28 . The dsRNA agent of  claim 25 , comprising at least one phosphorothioate or methylphosphonate internucleotide linkage. 
     
     
         29 . The dsRNA agent of  claim 25 , wherein the antisense strand comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 16-18 phosphate internucleotide linkages, and the sense strand comprises any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages. 
     
     
         30 . The dsRNA agent of  claim 29 , wherein the antisense strand comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand, counting from the 5′-end of the antisense strand. 
     
     
         31 . The dsRNA agent of  claim 25 , wherein at least one ligand is an ASGPR ligand, and wherein the ASGPR ligand is attached to the 5′ end or 3′ end of the sense strand. 
     
     
         32 . The dsRNA agent of  claim 31 , wherein the ASGPR ligand is attached to the 3′ end of the sense strand. 
     
     
         33 . The dsRNA agent of  claim 31 , wherein the ASGPR ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker. 
     
     
         34 . The dsRNA agent of  claim 33 , wherein the branched linker comprises a linking group of the general formula —NHCHR A C(O)NHCR B C(O)—, wherein R A  and R B  are the R groups of the two adjacent amino acids. 
     
     
         35 . The dsRNA agent of  claim 1 , wherein 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 is chosen independently from the group consisting of: A:U, G:U, I:C, and mismatched pairs. 
     
     
         36 . The dsRNA agent of  claim 35 , wherein one of the first 1, 2, 3, 4, or 5 base pairs within the duplex region from the 5′-end of the antisense strand is I:C. 
     
     
         37 . The dsRNA agent of  claim 1 , wherein 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. 
     
     
         38 . The dsRNA agent of  claim 1 , wherein B4′ is 2′-OMe. 
     
     
         39 . The dsRNA agent of  claim 1 , wherein n 1  is 8. 
     
     
         40 . The dsRNA agent of  claim 1 , wherein the antisense strand of the dsRNA agent comprises a 5′-phosphate modification selected from the group consisting of 5′-monophosphate, 5′-diphosphate, 5′-triphosphate, 7-methylated or non-methylated 5′-guanosine cap, 5′-adenosine cap, a modified or unmodified nucleotide cap structure,5′-monothiophosphate, 5′-monodithiophosphate, 5′-phosphorothiolate, 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, 5′-phosphoramidate, 5′-alkylphosphonates, 5′-alkenylphosphonate, (OH) 2 (O)P-5′-CH 2 —, and 5′-alkyletherphosphonate. 
     
     
         41 . The dsRNA agent of  claim 1 , wherein the dsRNA agent is covalently conjugated to two ligands. 
     
     
         42 . The dsRNA agent of  claim 41 , wherein the ligands are conjugated to the sense strand. 
     
     
         43 . The dsRNA agent of  claim 41 , wherein the ligands are conjugated to the antisense strand. 
     
     
         44 . The dsRNA agent of  claim 1 , wherein the dsRNA agent is covalently conjugated to one or more ligands or moieties that improve nuclease resistance. 
     
     
         45 . The dsRNA agent of  claim 44 , wherein the sense strand is conjugated to the one or more ligands or moieties that improve nuclease resistance at the 3′ end, the 5′ end, or both ends of the sense strand. 
     
     
         46 . The dsRNA agent of  claim 44 , wherein the sense strand is conjugated to the one or more ligands or moieties that improve nuclease resistance at the 3′ end. 
     
     
         47 . The dsRNA agent of  claim 45 , wherein the ligand or moiety that improves nuclease resistance comprises a nuclease resistant monomer containing nuclease resistance promoting modifications. 
     
     
         48 . The dsRNA agent of  claim 47 , wherein the nuclease resistant monomer is an abasic monomer. 
     
     
         49 . The dsRNA agent of  claim 47 , wherein the nuclease resistant monomer comprises a 3′-3′ or 5′-5′ linkage.