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

• US 20070004665A1
• Filed: 08/04/2006
• Published: 01/04/2007
• Est. Priority Date: 02/20/2002
• Status: Abandoned Application

• 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
• Application Number:
  US11/499,633
• Publication Number:
  US 20070004665A1
• Time in Patent Office: Days
• US Class Current: 514/44.A
• 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 chemically synthesized double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a target RNA via RNA interference (RNAi), wherein:

(a) each strand of said siNA molecule is 19 to 23 nucleotides in length; and

(b) one strand of said siNA molecule comprises nucleotide sequence having sufficient complementarity to said target RNA for the siNA molecule to direct cleavage of the target RNA via RNA interference and wherein said siNA molecule comprises modified nucleotides at 20% or more of the nucleotide positions; and

(c) said siNA molecule possesses similar or improved capacity to mediate RNAi compared to its native double stranded siRNA counterpart.

2. The siNA molecule of claim 1, wherein said siNA molecule comprises no ribonucleotides.

3. The siNA molecule of claim 1, wherein said siNA molecule comprises ribonucleotides.

4. The siNA molecule of claim 1, wherein each strand of the siNA molecule comprises 19 to 23 nucleotides, and wherein each strand comprises at least 19 nucleotides that are complementary to the nucleotides of the other strand.

5. The siNA molecule of claim 4, 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.

6. The siNA molecule of claim claim 5, wherein said sense region is connected to the antisense region via a linker molecule.

7. The siNA molecule of claim 6, wherein said linker molecule is a polynucleotide linker.

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

9. The siNA molecule of claim 4, wherein pyrimidine nucleotides in the sense region are 2′

-O-methyl pyrimidine nucleotides and wherein purine nucleotides in the sense region are 2-deoxy purine nucleotides.

10. The siNA molecule of claim 5, wherein the pyrimidine nucleotides present in the sense region are 2′

-deoxy-2′

-fluoro pyrimidine nucleotides and wherein the purine nucleotides present in the sense region are selected from the group consisting of 2′

-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′

-methoxyethyl nucleotides, 4′

-thionucleotides, 2′

-O-methyl nucleotides and purine ribonucleotides.

11. The siNA molecule of claim 5, 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 of the fragment comprising said sense region.

12. The siNA molecule of claim 11, wherein said terminal cap moiety is an inverted deoxy abasic moiety.

13. The siNA molecule of claim 5, wherein the pyrimidine nucleotides of said antisense region are 2′

-deoxy-2′

-fluoro pyrimidine nucleotides and wherein the purine nucleotides of said antisense region are 2′

-O-methyl purine nucleotides.

14. The siNA molecule of claim 5, wherein the purine nucleotides present in said antisense region comprise 2′

-deoxy-purine nucleotides and wherein the pyrimidine nucleotides of said antisense region are 2′

-deoxy-2′

-fluoro pyrimidine nucletides.

15. The siNA molecule of claim 13, wherein said antisense region comprises a phosphorothioate internucleotide linkage at the 3′

end of said antisense region.

16. The siNA molecule of claim 5, wherein said antisense region comprises a glyceryl modification at the 3′

end of said antisense region.

17. The siNA molecule of claim 5, wherein each of the two fragments of said siNA molecule comprise 21 nucleotides.

18. The siNA molecule of claim 17, wherein 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.

19. The siNA molecule of claim 18, wherein each of the two 3′

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

-deoxy-pyrimidines.

20. The siNA molecule of claim 19, wherein said 2′

-deoxy-pyrimidine is 2′

-deoxy-thymidine.

21. The. siNA molecule of claim 17, 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.

22. The siNA molecule of claim 17, wherein 19 nucleotides of the antisense region are base-paired to the nucleotide sequence of the RNA encoded by a target gene or a portion thereof

23. The siNA molecule of claim 17, wherein 21 nucleotides of the antisense region are base-paired to the nucleotide sequence of the RNA encoded by a target gene or a portion thereof.

24. The siNA molecule of claim 5, wherein the 5′

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

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

26. The siNA molecule of claim 1, wherein, said modified nucleotide is a 2′

-sugar modified nucleotide.

27. The siNA molecule of claim 26, wherein said 2′

-sugar modification is selected from the group consisting of 2′

-H, 2′

-O-alkyl, 2′

-O-CF3 and 2′

-deoxy-2′

-Fluoro.

28. The siNA of claim 1, wherein said modified nucleotides are present in one strand or both strands of the siNA molecule.