Modified double-stranded RNA agents
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) comprising a sense strand having 19-22 nucleotides and an antisense strand having 19-25 nucleotides that is at least 85% complementary to a portion of a target mRNA of a target gene, wherein:
(a) the sense strand and the antisense strand form a duplex region of 19-21 base pairs in length;
(b) each nucleotide of the sense strand and the antisense strand is independently modified with a modification selected from the group consisting of 2′-O-methyl and 2′-deoxy-2′-fluoro (2′-F), wherein the sense strand and the antisense strand contain at least two different modifications;
(c) the antisense strand has 2′-F modifications only at positions 2, 6, 14, and 16 of the antisense strand, counting from the 5′-end of the antisense strand;
(d) the 5′-end or 3′-end of the sense strand is conjugated to a ligand; and
(e) the dsRNA has a blunt end at the 5′-end of the antisense strand.
2. The dsRNA of claim 1 , having at least 2 nucleotides linked through a phosphorothioate internucleotide linkage at one end or at each end of the sense strand, the antisense strand, or both strands.
3. The dsRNA of claim 1 , wherein the dsRNA contains one overhang region consisting of two nucleotides at the 3′-end of the antisense strand, wherein the antisense strand comprises two phosphorothioate internucleotide linkages between the three 3′-terminal nucleotides, in which two of the three nucleotides are the overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide.
4. The dsRNA of claim 1 , having three nucleotides linked through phosphorothioate internucleotide linkages at one end or at each end of the sense strand, the antisense strand, or both strands.
5. The dsRNA of claim 4 , having three nucleotides linked through phosphorothioate internucleotide linkages at each end of the sense strand and the antisense strand.
6. The dsRNA of claim 1 , wherein
(a) the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides, or
(b) the sense strand has 21 nucleotides and the antisense strand has 21 nucleotides; and
wherein the duplex region is 21 base pairs in length.
7. The dsRNA of claim 1 , wherein
(a) the sense strand has 19 nucleotides and the antisense strand has 21 nucleotides, or
(b) the sense strand has 19 nucleotides and the antisense strand has 19 nucleotides; and
wherein the duplex region is 19 base pairs in length.
8. The dsRNA of claim 1 , wherein the first base pair from the 5′-end of the antisense strand is an AU base pair.
9. The dsRNA of claim 1 , wherein the ligand is a cell targeting agent that binds to a specific cell type, a protein, a lipophilic molecule, or an endosomolytic ligand.
10. The dsRNA of claim 9 , wherein the ligand is a cell targeting agent, wherein the cell targeting is an asialoglycoprotein receptor (ASGPR) ligand, and wherein the asialoglycoprotein receptor (ASGPR) ligand is one or more GalNAc (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker.
11. The dsRNA of claim 10 , wherein the ASGPR ligand comprises:
12. The dsRNA of claim 9 , wherein the ligand is a cell targeting agent comprising an antibody.
13. A double-stranded RNA (dsRNA) comprising a sense strand having 19-21 nucleotides and an antisense strand having 21-23 nucleotides that is at least 90% complementary to a portion of a target mRNA of a target gene, wherein:
(a) the sense strand and the antisense strand form a duplex region of 19-21 base pairs in length;
(b) each nucleotide of the sense strand and the antisense strand is independently modified with a modification selected from the group consisting of 2′-O-methyl and 2′-deoxy-2′-fluoro (2′-F), wherein the sense strand and the antisense strand each contain at least two different modifications;
(c) the antisense strand has 2′-F modifications only at positions 2, 6, 14, and 16 of the antisense strand, counting from the 5′-end of the antisense strand;
(d) the 5′ end or 3′ end of the sense strand is conjugated to a ligand; and
(e) the dsRNA has a blunt end at the 5′-end of the antisense strand.
14. The dsRNA of claim 13 , wherein the ligand is a cell targeting agent that binds to a specific cell type, a protein, a lipophilic molecule, or an endosomolytic ligand.
15. The dsRNA of claim 14 , wherein the ligand is a cell targeting agent, wherein the cell targeting agent is an asialoglycoprotein receptor (ASGPR) ligand, and wherein the ASGPR ligand is one or more GalNAc (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker.
16. The dsRNA of claim 15 , wherein the ASGPR ligand comprises:
17. The dsRNA of claim 14 , wherein the ligand is a cell targeting agent comprising an antibody.
18. The dsRNA of claim 17 , wherein the ligand is conjugated to the 5′ end of the sense strand.
19. The dsRNA of claim 13 , wherein:
(a) the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides, or
(b) the sense strand has 21 nucleotides and the antisense strand has 21 nucleotides; and
wherein the duplex region is 21 base pairs in length.
20. The dsRNA of claim 13 , wherein the sense strand has 19 nucleotides and the antisense strand has 21 nucleotides, and the duplex region is 19 base pairs in length.
21. The dsRNA of claim 13 , having at least 2 nucleotides linked through a phosphorothioate internucleotide linkage at one end or at each end of the sense strand, the antisense strand, or both strands.
22. The dsRNA of claim 13 , wherein the dsRNA contains one overhang region consisting of two nucleotides at the 3′-end of the antisense strand, wherein the antisense strand comprises two phosphorothioate internucleotide linkages between the three 3′-terminal nucleotides, in which two of the three nucleotides are the overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide.
23. The dsRNA of claim 13 , having 3 nucleotides linked through phosphorothioate internucleotide linkages at one end or at each end of the sense strand, the antisense strand, or both strands.
24. The dsRNA of claim 23 , having 3 nucleotides linked through phosphorothioate internucleotide linkages at each end of the antisense strand.
25. The dsRNA of claim 13 , wherein the first base pair from the 5′-end of the antisense strand is an AU base pair.
26. The dsRNA of claim 13 , wherein the sense strand has 19 nucleotides represented by formula (Is):
wherein
each B1 is independently a 2′-OMe or 2′-F modified nucleotide and n 1 is 6;
each T1 is a 2′F modified nucleotide and n 2 is 3;
each B2 is a 2′-OMe modified nucleotide and n 3 is 7;
n 4 is 0; and
each B3 is a 2′OMe modified nucleotide and n 5 is 3.
27. The dsRNA of claim 26 , having 3 nucleotides linked through phosphorothioate internucleotide linkages at each end of the sense strand.
28. The dsRNA of claim 26 , wherein the antisense strand is at least 95% complementary to a portion of the target mRNA.
29. The dsRNA of claim 13 , wherein the sense strand has 21 nucleotides represented by formula (Is):
wherein
each B1 is independently a 2′-OMe or 2′-F modified nucleotide and n 1 is 8;
each T1 is a 2′F modified nucleotide and n 2 is 3;
each B2 is a 2′-OMe modified nucleotide and n 3 is 7;
n 4 is 0; and
each B3 a is 2′OMe modified nucleotide and n 5 is 3.
30. The dsRNA of claim 29 , wherein the sense strand comprises four, and only four, 2′-F modifications at positions 7, 9, 10, and 11, counting from the 5′-end of the sense strand.
31. The dsRNA of claim 1 , wherein the antisense strand contains an overhang region, consisting of 1-5 nucleotides in length; and wherein the antisense strand contains one or more mismatches to the target mRNA occurring at one or more nucleotides of the overhang region.
32. The dsRNA of claim 1 , wherein the antisense strand contains an overhang region consisting of 1-2 nucleotides in length at the 3′-end of the antisense strand; and wherein the antisense strand contains one or more mismatches to the target mRNA occurring at the overhang region.
33. The dsRNA of claim 1 , wherein the antisense strand contains one or more mismatches to the target mRNA occurring at one or more of the following nucleotides: the first 5 nucleotides from the 5′ end of the antisense strand, and the first 5 nucleotides from the 3 ′ end of the antisense strand.
34. The dsRNA of claim 1 , wherein the antisense strand contains one or more mismatches to the target mRNA occurring at one or more of the following nucleotides: first nucleotide from the 5′ end of the antisense strand, and the first 5 nucleotides from the 3′ end of the antisense strand.
35. The dsRNA of claim 28 , wherein the first base pair from the 5′-end of the antisense strand is an AU base pair.