RNA interference mediated inhibition of adenosine A1 receptor (ADORA1) gene expression using short interfering RNA

• US 20030143732A1
• Filed: 08/20/2002
• Published: 07/31/2003
• Est. Priority Date: 04/05/2001
• Status: Abandoned Application

• Current Assignee: Ribozyme Pharmaceuticals, Inc. (Alnylam Pharmaceuticals Incorporated)
• Original Assignee: Ribozyme Pharmaceuticals, Inc. (Alnylam Pharmaceuticals Incorporated)
• Inventors: Fosnaugh, Kathy, McSwiggen, James A.
• Application Number:
  US10/224,005
• Publication Number:
  US 20030143732A1
• Time in Patent Office: Days
• US Class Current: 435/325
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 15/1138 against receptors or cell surface proteins
• CPC Class Codes: C12N 2310/12 catalytic nucleic acids, e.g. ribozymes
• CPC Class Codes: C12N 2310/121 Hammerhead
• CPC Class Codes: C12N 2310/13 Decoys
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/18 acting by a non-sequence specific mechanism
• 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 2310/332 Abasic residue
• CPC Class Codes: C12N 2310/346 having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/3521 Methyl

Abstract

The present invention concerns methods and reagents useful in modulating adenosine A1 receptor (ADORA1) gene expression in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Specifically, the invention relates to small interfering RNA (siRNA) molecules capable of mediating RNA interference (RNAi) against ADORA1 and related receptors.

Claims

1. A short interfering RNA (siRNA) molecule that down regulates expression of an ADORA1 gene by RNA interference.

2. The siRNA molecule of claim 1, wherein said siRNA molecule is adapted for use to treat asthma.

3. The siRNA molecule of claim 1, wherein said siRNA molecule comprises a sense region and an antisense region and wherein said antisense region comprises sequence complementary to an RNA sequence encoding ADORA1 and the sense region comprises sequence complementary to the antisense region.

4. The siRNA molecule of claim 3, wherein said siRNA molecule is assembled from two nucleic acid fragments wherein one fragment comprises the sense region and the second fragment comprises the antisense region of said siRNA molecule.

5. The siRNA molecule of claim 4, wherein said sense region and antisense region are covalently connected via a linker molecule.

6. The siRNA molecule of claim 5, wherein said linker molecule is a polynucleotide linker.

7. The siRNA molecule of claim 5, wherein said linker molecule is a non-nucleotide linker.

8. The siRNA molecule of claim 3, wherein said antisense region comprises sequence complementary to sequence having any of SEQ ID NOs. 1-161.

9. The siRNA molecule of claim 3, wherein said antisense region comprises sequence having any of SEQ ID NOs. 162-322, 336, 338, 340, 342, 344, or 346.

10. The siRNA molecule of claim 3, wherein said sense region comprises sequence having any of SEQ ID NOs. 1-161, 335, 337, 339, 341, 343, or 345.

11. The siRNA molecule of claim 3, wherein said sense region comprises a sequence of SEQ ID NO. 323 and said antisense region comprises a sequence of SEQ ID NO. 324.

12. The siRNA molecule of claim 3, wherein said sense region comprises a sequence of SEQ ID NO. 325 and said antisense region comprises a sequence of SEQ ID NO. 326.

13. The siRNA molecule of claim 3, wherein said sense region comprises a sequence of SEQ ID NO. 327 and said antisense region comprises a sequence of SEQ ID NO. 328.

14. The siRNA molecule of claim 3, wherein said sense region comprises a sequence of SEQ ID NO. 329 and said antisense region comprises a sequence of SEQ ID NO. 330.

15. The siRNA molecule of claim 3, wherein said sense region comprises a sequence of SEQ ID NO. 331 and said antisense region comprises a sequence of SEQ ID NO. 332.

16. The siRNA molecule of claim 3, wherein said sense region comprises a sequence of SEQ ID NO. 333 and said antisense region comprises a sequence of SEQ ID NO. 334.

17. The siRNA molecule of claim 3, wherein said sense region comprises a 3′

-terminal overhang and said antisense region comprises a 3′

-terminal overhang.

18. The siRNA molecule of claim 17, wherein said 3′

-terminal overhangs each comprise about 2 nucleotides.

19. The siRNA molecule of claim 17, wherein said antisense region 3′

-terminal nucleotide overhang is complementary to RNA encoding ADORA1.

20. The siRNA molecule of claim 3, wherein said sense region comprises one or more 2′

-O-methyl modified pyrimidine nucleotides.

21. The siRNA molecule of claim 3, wherein said sense region comprises a terminal cap moiety at the 5′

-end, 3′

-end, or both 5′ and

3′

ends of said sense region.

22. The siRNA molecule of claim 3, wherein said antisense region comprises one or more 2′

-deoxy-2′

-fluoro modified pyrimidine nucleotides.

23. The siRNA molecule of claim 3, wherein said antisense region comprises a phosphorothioate internucleotide linkage at the 3′

end of said antisense region.

24. The siRNA molecule of claim 3, wherein said antisense region comprises between about one and about five phosphorothioate internucleotide linkages at the 5′

end of said antisense region.

25. The siRNA molecule of claim 17, wherein said 3′

-terminal nucleotide overhangs comprise ribonucleotides that are chemically modified at a nucleic acid sugar, base, or backbone.

26. The siRNA molecule of claim 17, wherein said 3′

-terminal nucleotide overhangs comprise deoxyribonucleotides that are chemically modified at a nucleic acid sugar, base, or backbone.

27. The siRNA molecule of claim 17, wherein said 3′

-terminal nucleotide overhangs comprise one or more universal base ribonucleotides.

28. The siRNA molecule of claim 17, wherein said 3′

-terminal nucleotide overhangs comprise one or more acyclic nucleotides.

29. The siRNA molecule of claim 17, wherein said 3′

-terminal nucleotide overhangs comprise nucleotides comprising internucleotide linkages having Formula I;

30. The siRNA molecule of claim 17, wherein said 3′

-terminal nucleotide overhangs comprise nucleotides or non-nucleotides having Formula II;

31. An expression vector comprising a nucleic acid sequence encoding at least one siRNA molecule of claim 1 in a manner that allows expression of the nucleic acid molecule.

32. A mammalian cell comprising an expression vector of claim 31.

33. The mammalian cell of claim 32, wherein said mammalian cell is a human cell.

34. The expression vector of claim 31, wherein said siRNA molecule comprises a sense region and an antisense region and wherein said antisense region comprises sequence complementary to an RNA sequence encoding ADORA1 and the sense region comprises sequence complementary to the antisense region.

35. The expression vector of claim 34, wherein said siRNA molecule comprises two distinct strands having complementarity sense and antisense regions.

36. The expression vector of claim 34, wherein said siRNA molecule comprises a single strand having complementary sense and antisense regions.