RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
The present invention concerns methods and reagents useful in modulating gene expression in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Specifically, the invention relates to synthetic chemically modified small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against target nucleic acid sequences. The small nucleic acid molecules are useful in the treatment of any disease or condition that responds to modulation of gene expression or activity in a cell, tissue, or organism.
1. A short interfering RNA (siRNA) molecule comprising a sense strand and an antisense strand that mediates RNA interference, wherein:
(a) each strand is 18 to 24 nucleotides in length;
(b) the sense strand comprises 9 or more 2′-O-methyl modified nucleotides;
(c) the antisense strand comprises 9 or more 2′-O-methyl modified nucleotides; and
(d) 10 or more pyrimidine nucleotides of the siRNA molecule are 2′-O-methyl nucleotides.
2. The siRNA molecule of claim 1 , wherein the sense strand and the antisense strand have the same length.
3. The siRNA molecule of claim 1 , wherein the sense strand and the antisense strand are each 19 nucleotides in length.
4. The siRNA molecule of claim 3 , wherein the siRNA molecule has a duplex of 19 base pairs.
5. The siRNA molecule of claim 1 , wherein the siRNA molecule has a duplex of 19 base pairs.
6. The siRNA of claim 4 , wherein the sense strand and the antisense strand are 100% complementary.
7. The siRNA of claim 5 , wherein the sense strand and the antisense strand are 100% complementary.
8. The siRNA molecule of claim 1 , wherein the sense strand comprises one or more phosphorothioate internucleotide linkages.
9. The siRNA molecule of claim 1 , wherein the antisense strand comprises one or more phosphorothioate internucleotide linkages.
10. The siRNA molecule of claim 1 , wherein the sense strand and the antisense strand each comprises one or more phosphorothioate internucleotide linkages.
11. The siRNA molecule of claim 7 , wherein the sense strand comprises one or more phosphorothioate internucleotide linkages.
12. The siRNA molecule of claim 7 , wherein the antisense strand comprises one or more phosphorothioate internucleotide linkages.
13. The siRNA molecule of claim 7 , wherein the sense strand and the antisense strand each comprises one or more phosphorothioate internucleotide linkages.
14. The siRNA molecule of claim 1 , which comprises a blunt end at one end or both ends.
15. The siRNA molecule of claim 1 , wherein the 3′-end of the sense strand is blunt-ended.
16. The siRNA molecule of claim 1 , wherein the 3′-end of the antisense strand is blunt-ended.
17. The siRNA molecule of claim 1 , which further comprises a terminal cap moiety.
18. The siRNA molecule of claim 1 , wherein the sense strand includes a terminal cap moiety at the 5′-end of the sense strand, the 3′-end of the sense strand, or both of the 5′ and 3′ ends of the sense strand.
19. The siRNA molecule of claim 1 , which further comprises a terminal cap moiety at the 5′-end of the sense strand.
20. The siRNA molecule of claim 1 , which comprises one or more ribonucleotides.
21. The siRNA molecule of claim 20 , wherein the ribonucleotide is an unmodified ribonucleotide.
22. The siRNA molecule of claim 1 , which comprises modified nucleotides at about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of nucleotide positions.
23. The siRNA molecule of claim 1 , which comprises ribonucleotides at about 5, 10, 20, 30, 40, or 50% of nucleotide positions.
24. The siRNA molecule of claim 1 , wherein the sense strand comprises no more than nine 2′-O-methyl modified nucleotides.
25. The siRNA molecule of claim 1 , wherein the antisense strand comprises ten 2′-O-methyl modified nucleotides.
26. The siRNA molecule of claim 1 , further comprising a terminal phosphate group.
27. The siRNA molecule of claim 1 , further comprising a terminal 5′ phosphate group.
28. The siRNA molecule of claim 1 , further comprising a terminal 5′ phosphate group on the antisense strand.
29. A short interfering RNA (siRNA) molecule comprising a sense strand and an antisense strand that mediates RNA interference, wherein:
(a) the sense strand is 19 nucleotides in length;
(b) the antisense strand is 19 nucleotides in length;
(c) the sense strand comprises 9 or more 2′-O-methyl modified nucleotides;
(d) the antisense strand comprises 9 or more 2′-O-methyl modified nucleotides;
(e) 10 or more pyrimidine nucleotides of the siRNA molecule are 2′-O-methyl nucleotides; and
(f) the siRNA molecule comprises one or more ribonucleotides.
30. The siRNA molecule of claim 29 , wherein the siRNA molecule has a duplex of 19 base pairs.
31. The siRNA molecule of claim 30 , wherein the sense strand and the antisense strand are 100% complementary.
32. The siRNA molecule of claim 29 , wherein the sense strand and the antisense strand are 100% complementary.
33. The siRNA molecule of claim 29 , wherein the sense strand comprises one or more phosphorothioate internucleotide linkages.
34. The siRNA molecule of claim 29 , wherein the antisense strand comprises one or more phosphorothioate internucleotide linkages.
35. The siRNA molecule of claim 29 , wherein the sense strand and the antisense strand each comprises one or more phosphorothioate internucleotide linkages.
36. The siRNA molecule of claim 31 , wherein the sense strand comprises one or more phosphorothioate internucleotide linkages.
37. The siRNA molecule of claim 31 , wherein the antisense strand comprises one or more phosphorothioate internucleotide linkages.
38. The siRNA molecule of claim 31 , wherein the sense strand and the antisense strand each comprises one or more phosphorothioate internucleotide linkages.
39. The siRNA molecule of claim 29 , which comprises a blunt end at one end or both ends.
40. The siRNA molecule of claim 29 , wherein the 3′-end of the sense strand is blunt-ended.
41. The siRNA molecule of claim 29 , wherein the 3′-end of the antisense strand is blunt-ended.
42. The siRNA molecule of claim 29 , which further comprises a terminal cap moiety.
43. The siRNA molecule of claim 29 , wherein the sense strand includes a terminal cap moiety at the 5′-end of the sense strand, the 3′-end of the sense strand, or both of the 5′ and 3′ ends of the sense strand.
44. The siRNA molecule of claim 29 , which further comprises a terminal cap moiety at the 5′-end of the sense strand.
45. The siRNA molecule of claim 29 , wherein the ribonucleotide is an unmodified ribonucleotide.
46. The siRNA molecule of claim 29 , which comprises modified nucleotides at about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of nucleotide positions.
47. The siRNA molecule of claim 29 , which comprises ribonucleotides at about 5, 10, 20, 30, 40, or 50% of nucleotide positions.
48. The siRNA molecule of claim 29 , wherein the sense strand comprises nine 2′-O-methyl modified nucleotides.
49. The siRNA molecule of claim 29 , wherein the antisense strand comprises ten 2′-O-methyl modified nucleotides.
50. The siRNA molecule of claim 29 , further comprising a terminal phosphate group.
51. The siRNA molecule of claim 29 , further comprising a terminal 5′ phosphate group.
52. The siRNA molecule of claim 29 , further comprising a terminal 5′ phosphate group on the antisense strand.