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) agent capable of inhibiting the expression of a target gene, comprising an antisense strand complementary to at least one portion of a mRNA of the target gene and a sense strand, wherein
the sense strand is 14-40 nucleotides in length, the antisense strand is 19-25 nucleotides in length, and the sense and antisense strands together form a duplex region that is 19-25 base pairs in length;
each nucleotide in the sense and antisense strands is a modified nucleotide;
the sense strand is covalently conjugated to one or more ligands, each attached at an internal position of the sense strand, wherein each ligand is conjugated to a 2′-carbon atom of a sugar moiety;
the sense strand contains three consecutive 2′-F modified nucleotides, wherein one of the three consecutive 2′-F modified nucleotides is at either position 11 of the sense strand, counting from the 5′-end of the sense strand, or at a position of the sense strand that is opposite to position 11 of the antisense strand, counting from the 5′ end of the antisense strand;
the antisense strand contains 2′-F modified nucleotides at positions 2 and 14, counting from the 5′ end of the antisense strand; and
each modified nucleotide of the sense strand and antisense strand not conjugated to a ligand has a modification selected from the group consisting of 2′-O-Methyl and 2′-F.
2. The dsRNA agent of claim 1 , wherein each ligand is a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, or polysaccharide conjugated to the 2′-carbon atom of a nucleotide sugar moiety through a monovalent, bivalent, or trivalent branched linker.
3. The dsRNA agent of claim 2 , wherein each ligand is N-acetylgalactosamine (GalNAc) conjugated to the 2′-carbon atom of a nucleotide sugar moiety through a monovalent, bivalent, or trivalent branched linker.
4. The dsRNA agent of claim 2 , wherein each ligand is N-acetylgalactosamine (GalNAc) conjugated to the 2′-carbon atom of a nucleotide sugar moiety through a trivalent branched linker.
5. The dsRNA agent of claim 2 , wherein each ligand is N-acetylgalactosamine (GalNAc) conjugated to the 2′-carbon atom of a nucleotide sugar moiety through a bivalent branched linker.
6. The dsRNA agent of claim 2 , wherein each ligand is N-acetylgalactosamine (GalNAc) conjugated to the 2′-carbon atom of a nucleotide sugar moiety through a monovalent linker.
7. The dsRNA agent of claim 1 , wherein the sense strand is 25-37 nucleotides in length.
8. The dsRNA agent of claim 1 , wherein the duplex region is 19-21 base pairs in length.
9. The dsRNA agent of claim 1 , wherein the antisense strand comprises a 5′-phosphate modification.
10. The dsRNA agent of claim 9 , wherein the antisense strand comprises a 5′-phosphate modification selected from the group consisting of 5′-monophosphate; 5′-diphosphate; 5′-triphosphate; 5′-guanosine cap (7-methylated or non-methylated); 5′-adenosine cap (Appp); 5′-monothiophosphate; 5′-monodithiophosphate; 5′-phosphorothiolate; 5′-phosphoramidates; 5′-alkylphosphonate; 5′-alkenylphosphonates; and 5′-alkyletherphosphonates.
11. The dsRNA agent of claim 1 , wherein the dsRNA agent has a two-nucleotide overhang at the 3′-end of the antisense strand.
12. The dsRNA agent of claim 11 , wherein the two-nucleotide overhang has a phosphorothioate internucleotide linkage between the two overhang nucleotides.
13. The dsRNA agent of claim 12 , wherein the antisense strand has two phosphorothioate internucleotide linkages between the three 3′-terminal nucleotides, in which two of the three 3′-terminal nucleotides are the two overhang nucleotides.
14. The dsRNA agent of claim 1 , wherein the nucleotide at position 1, counting from the 5′-end of the antisense strand in the duplex, is 2′-O-Methyl-modified uridine.
15. The dsRNA agent of claim 1 , wherein the nucleotide at position 1, counting from the 5′-end of the sense strand in the duplex, is 2′-O-Methyl-modified.
16. 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, counting from the 5′-end of the antisense strand, is independently selected from the group consisting of A:U, G:U, I:C, and mismatched pairs.
17. The dsRNA agent of claim 1 , wherein the sense strand comprises at least one phosphorothioate internucleotide linkage.
18. The dsRNA agent of claim 1 , wherein the antisense strand comprises two blocks of two phosphorothioate internucleotide linkages separated by 16-18 phosphate internucleotide linkages.
19. The dsRNA agent of claim 1 , wherein the sense strand and antisense strand comprise six phosphorothioate internucleotide linkages within the 4 nucleotides at the ends of the sense strand and antisense strand.
20. The dsRNA agent of claim 1 , wherein the sense strand contains no more than four 2′-F modifications.
21. The dsRNA agent of claim 20 , wherein the sense strand contains only four 2′-F modifications.
22. The dsRNA agent of claim 21 , wherein the only four 2′-F modifications comprise three consecutive 2′-F modifications at positions 9, 10, and 11 of the sense strand, counting from the 5′-end of the sense strand.
23. The dsRNA agent of claim 1 , wherein the antisense strand contains no more than six 2′-F modifications.
24. The dsRNA agent of claim 23 , wherein the antisense strand contains only four 2′-F modifications.
25. The dsRNA agent of claim 23 , wherein the antisense strand contains only six 2′-F modifications.
26. The dsRNA agent of claim 1 , wherein at least one ligand is conjugated to the dsRNA agent via a cyclic group or acyclic group.
27. The dsRNA agent of claim 26 , wherein the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalinyl.
28. The dsRNA agent of claim 26 , wherein the acyclic group is selected from the group consisting of a serinol backbone and a diethanolamine backbone.
29. The dsRNA agent of claim 1 , wherein at least one ligand is conjugated to the dsRNA agent, directly or indirectly, via an intervening tether.
30. The dsRNA agent of claim 1 , wherein the intervening tether comprises a cleavable linking group selected from the group consisting of a redox cleavable linking group, a phosphate-based cleavable linking group, an acid cleavable linking group, an ester-based linking group, a peptide-based cleaving group, and combinations thereof.
31. The dsRNA agent of claim 30 , wherein the intervening tether comprises an acid cleavable linking group contain a formula —C═NN—, —C(O)O—, or —OC(O)—.
32. The dsRNA agent of claim 1 , wherein at least one ligand is conjugated to an internucleosidic linkage containing an amine- or amide group.
33. The dsRNA agent of claim 1 , wherein at least one ligand comprises a lipid, steroid, vitamin, protein, peptide, or combinations thereof.
34. The dsRNA agent of claim 1 , wherein at least one ligand comprises a lipophile, PEG, fatty acid, lithocholic acid, dialkylglyceride, diacylglyceride, alkyl, substituted alkyl, cholesterol, cholic acid, adamantane acetic acid, or combinations thereof.
35. The dsRNA agent of claim 1 , wherein at least one ligand comprises an integrin-targeting moiety.
36. The dsRNA agent of claim 35 , wherein integrin-targeting moiety targets integrin αvβ 3 .
37. The dsRNA agent of claim 1 , wherein at least one ligand comprises a transferrin-receptor targeting moiety.
38. A pharmaceutical composition comprising the dsRNA agent of claim 1 , and one or more pharmaceutically acceptable carriers, excipient and/or diluents.
39. A method for inhibiting the expression of a target gene comprising the step of administering the dsRNA agent according to claim 1 , in an amount sufficient to inhibit expression of the target gene.
40. The method of claim 39 , wherein the dsRNA agent is administered through subcutaneous or intravenous administration.
41. A method for delivering polynucleotide to a specific target in a subject by administering the dsRNA agent according to claim 1 .
42. The method of claim 41 , wherein said administering step is carried out by an administration means comprising intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, intravenous, subcutaneous, cerebrospinal, intrathecal, intraventricular, intradermal, intracranial, transmucosal, buccal, sublingual, endoscopic, rectal, vaginal, topical, pulmonary, intranasal, urethral, ocular, oral, or combinations thereof.
43. The dsRNA agent of claim 1 , wherein the duplex region is 19-23 base pairs in length.