RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)

• US 20040192626A1
• Filed: 05/23/2003
• Published: 09/30/2004
• Est. Priority Date: 02/20/2002
• Status: Active Grant

• Current Assignee: Sirna Therapeutics, Inc. (Alnylam Pharmaceuticals Incorporated)
• Original Assignee: Sirna Therapeutics, Inc. (Alnylam Pharmaceuticals Incorporated)
• Inventors: Thompson, James, Beigelman, Leonid, Chen, Tongqian, Wang, Weimin, McSwiggen, James, Vargeese, Chandra, Zinnen, Shawn, Haeberli, Peter, Morrissey, David, Usman, Nassim, Jamison, Sharon, Chowrira, Bharat, Macejak, Dennis, Fosnaugh, Kathy, Vaish, Narendra, Pavco, Pamela
• Granted Patent: US 8,202,979 B2
• Time in Patent Office: Days
• US Class Current: 514/44.00A
• CPC Class Codes: A61K 47/544 Phospholipids
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: A61K 47/554 the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
• CPC Class Codes: A61K 9/0019 Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner non-active ingredients are additionally classified in A61K47/00
• CPC Class Codes: C07F 9/65616 containing the ring system having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/317 with an inverted bond, e.g. a cap structure
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/346 having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/351 Conjugate
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2320/51 modulating the chemical stability, e.g. nuclease-resistance
• CPC Class Codes: C12N 2330/30 chemically synthesised
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

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.

Claims

1. A short interfering nucleic acid (siNA) molecule comprising a double-stranded structure that down-regulates expression of a target nucleic acid, wherein said siNA molecule does not require a 2′

-hydroxyl group containing ribonucleotide, each strand of said double-stranded structure of the siNA molecule comprises about 21 nucleotides and the siNA molecule comprises nucleotide sequence having complementarity to nucleotide sequence of said target nucleic acid or a portion thereof.

2. The siNA molecule of claim 1, wherein said siNA molecule comprises a sense region and an antisense region and wherein said antisense region comprises the nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof of the target nucleic acid and said sense region comprises a nucleotide sequence that is complementary to nucleotide sequence of said antisense region or a portion thereof.

3. The siNA molecule of claim 2, wherein said siNA molecule is assembled from two separate oligonucleotide fragments wherein one fragment comprises the sense region and the second fragment comprises the antisense region of said siNA molecule.

4. The siNA molecule of claim 2, wherein said sense region is connected to the antisense region via a linker molecule.

5. The siNA molecule of claim 4, wherein said linker molecule is a polynucleotide linker.

6. The siNA molecule of claim 4, wherein said linker molecule is a non-nucleotide linker.

7. The siNA of claim 1, wherein said target nucleic acid is an endogenous gene.

8. The siNA of claim 1, wherein said target nucleic acid is viral nucleic acid.

9. The siNA of claim 1, wherein said target nucleic acid is an RNA.

10. The siNA molecule of claim 1, wherein said siNA molecule comprises nucleotide sequence having complementarity to nucleotide sequence of RNA or a portion thereof encoded by said target nucleic acid or a portion thereof.

11. The siNA molecule of claim 2, wherein pyrimidine nucleotides when present in the sense region are 2′

-O-methyl pyrimidine nucleotides and purine nucleotides when present in the sense region are 2′

-deoxy purine nucleotides.

12. The siNA molecule of claim 2, wherein the pyrimidine nucleotides when present in the sense region are 2′

-deoxy-2′

-fluoro pyrimidine nucleotides and wherein the purine nucleotides when present in the sense region are 2′

-deoxy purine nucleotides.

13. The siNA molecule of claim 3, wherein the fragment comprising said sense region includes a terminal cap moiety at the 5′

-end, the 3′

-end, or both of the 5′ and

3′

ends.

14. The siNA molecule of claim 13, wherein said terminal cap moiety is an inverted deoxy abasic moiety.

15. The siNA molecule of claim 13, wherein said terminal cap moiety is an inverted deoxy thymidine moiety.

16. The siNA molecule of claim 13, wherein said terminal cap moiety is a thymidine moiety.

17. The siNA molecule of claim 2, wherein the pyrimidine nucleotides when present in said antisense region are 2′

-deoxy-2′

-fluoro pyrimidine nucleotides and the purine nucleotides when present in said antisense region are 2′

-O-methyl purine nucleotides.

18. The siNA molecule of claim 2, wherein the pyrimidine nucleotides when present in said antisense region are 2′

-deoxy-2′

-fluoro pyrimidine nucleotides and wherein the purine nucleotides when present in said antisense region comprise 2′

-deoxy-purine nucleotides.

19. The siNA molecule of claim 17, wherein said antisense region comprises a phosphate backbone modification at the 3′

end of said antisense region.

20. The siNA molecule of claim 19, wherein said phosphate backbone modification is a phosphorothioate modification.

21. The siNA molecule of claim 2, wherein said antisense region comprises a glyceryl modification at the 3′

end of said antisense region.

22. The siNA molecule of claim 2, wherein each of sense and the antisense regions of said siNA molecule comprise 21 nucleotides.

23. The siNA molecule of claim 3, wherein each of the two fragments of said siNA molecule comprise 21 nucleotides.

24. The siNA molecule of claim 23, wherein about 19 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule and wherein at least two 3′

terminal nucleotides of each fragment of the siNA molecule are not base-paired to the nucleotides of the other fragment of the siNA molecule.

25. The siNA molecule of claim 24, wherein each of the two 3′

terminal nucleotides of each fragment of the siNA molecule are 2′

-deoxy-pyrimidines.

26. The siNA molecule of claim 25, wherein the 2′

-deoxy-pyrimidine is thymidine.

27. The siNA molecule of claim 23, wherein all 21 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule.

28. The siNA molecule of claim 23, wherein about 19 nucleotides of the antisense region of the siNA molecule are base-paired to the nucleotide sequence or a portion thereof of the the target nucleic acid.

29. The siNA molecule of claim 23, wherein 21 nucleotides of the antisense region of the siNA molecule are base-paired to the nucleotide sequence or a portion thereof of the target nucleic acid.

30. The siNA molecule of claim 3, wherein the 5′

-end of the fragment comprising said antisense region optionally includes a phosphate group.

31. The siNA molecule of claim 1, wherein said target nucleic acid is a mammalian gene.

32. The siNA molecule of claim 1, wherein said target nucleic acid is a plant gene.

33. The siNA molecule of claim 1, wherein said target nucleic acid is a bacterial gene.

34. The siNA molecule of claim 1, wherein said target nucleic acid is a fungal gene.

35. The siNA molecule of claim 1, wherein said target nucleic acid is an exogenous gene.

36. The siNA molecule of claim 31, wherein said mammalian gene is a human gene.

37. The siNA molecule of claim 8, wherein said viral nucleic acid is a mammalian viral nucleic acid.

38. The siNA molecule of claim 8, wherein said viral nucleic acid is a plant viral nucleic acid.

39. The siNA molecule of claim 37, wherein said mammalian viral nucleic acid is from hepatitis C virus.

40. The siNA molecule of claim 37, wherein said mammalian viral nucleic acid is from human immunodeficiency virus.

41. The siNA molecule of claim 37, wherein said mammalian viral nucleic acid is from hepatitis B virus.

42. The siNA molecule of claim 37, wherein said mammalian viral nucleic acid is from herpes simplex virus.

43. The siNA molecule of claim 37, wherein said mammalian viral nucleic acid is from cytomegalovirus.

44. The siNA molecule of claim 37, wherein said mammalian viral nucleic acid is from human papilloma virus.

45. The siNA molecule of claim 37, wherein said mammalian viral nucleic acid is from respiratory syncytial virus.

46. The siNA molecule of claim 37, wherein said mammalian viral nucleic acid is from influenza virus.

47. The siNA molecule of claim 37, wherein said mammalian viral nucleic acid is from severe acute respiratory syndrome virus.

48. A pharmaceutical composition comprising the siNA molecule of claim 1, in an acceptable carrier or diluent.