RNA interference modulators of hedgehog signaling and uses thereof

• US 8,802,639 B2
• Filed: 03/24/2005
• Issued: 08/12/2014
• Est. Priority Date: 03/26/2004
• Status: Active Grant

• Current Assignee: Curis, Inc., Genentech, Inc. (Roche Holding AG)
• Original Assignee: Curis, Inc., Genentech, Inc. (Roche Holding AG)
• Inventors: Bumcrot, David A.
• Primary Examiner(s): Angell, Jon E
• Application Number:
  US11/088,691
• Publication Number:
  US 20050256076A1
• Time in Patent Office: 3,428 Days
• Field of Search: 514/44.A, 536/24.5
• US Class Current: 514/44.A
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 27/06 Antiglaucoma agents or miotics
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/04 specific for metastasis
• CPC Class Codes: A61P 37/08 Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• 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 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed

Abstract

The instant application relates to methods and reagents for modulating the Hedgehog signaling pathway using RNA interference technology (RNAi). The application provides potential targets of the Hedgehog RNAi, methods to identify additional Hedgehog signaling pathway components, methods to inhibit Hedgehog signaling targets using siRNA, and their uses in the treatment of a number of disease conditions.

Claims

1. A method of inhibiting at least one of unwanted growth, proliferation or survival of a cell, comprising contacting said cell with an effective amount of a hedgehog RNAi antagonist against a Sonic hedgehog (Shh) gene, wherein said RNAi antagonist comprises a sequence that is at least 90% identical to a nucleic acid sequence set forth in any of SEQ ID NOs:

5, 6, 9, or 10, wherein said RNAi antagonist is 19-30 nucleotides in length, wherein contacting said cell with said hedgehog RNAi antagonist decreases at least one of cell growth, proliferation or survival, wherein said cell is a cancer cell, and wherein said cancer cell is a colon cancer cell.

2. The method of claim 1, further comprising determining whether said cell expresses a gli gene, and contacting said cell which expresses a gli gene with an effective amount of the hedgehog RNAi antagonist against the Shh gene.

3. The method of claim 2, wherein said gli gene is gli-1.

4. The method of claim 1, wherein the RNAi antagonist is an siRNA antagonist.

5. The method of claim 4, wherein said siRNA antagonist is an siRNA formed after transcription from a plasmid (RNAi expression vector) or exogenous synthesis.

6. The method of claim 5, wherein said siRNA is a short hairpin siRNA formed after transcription from a single promoter of said plasmid (RNAi expression vector).

7. The method of claim 5, wherein said siRNA is a short dsRNA formed after transcription from two flanking convergent promoters on said plasmid (RNAi expression vector).

8. The method of claim 4, wherein said siRNA is 21-23 nucleotides in length.

9. The method of claim 4, wherein said siRNA is a fragment generated by nuclease dicing of longer double-stranded RNAs at least 25, 50, 100, 200, 300, 400, or 400-800 bases in length.

10. The method of claim 4, wherein said siRNA is double stranded, and includes short overhang(s) at one or both ends.

11. The method of claim 10, wherein said short overhang is 1-6 nucleotides in length at the 3′

end, 2 to 4 nucleotides in length at the 3′

end, or 1-3 nucleotides in length at the 3′

end.

12. The method of claim 11, wherein one strand of said siRNA has a 3′

overhang, and the other strand is blunt-ended, or also has an overhang of the same or different length.

13. The method of claim 10, wherein said 3′

overhang is stabilized against degradation.

14. The method of claim 13, wherein said 3′

overhang is stabilized against degradation by including purine nucleotides adenosine or guanosine.

15. The method of claim 13, wherein said 3′

overhang is stabilized against degradation by substituting pyrimidine nucleotides by modified analogues.

16. The method of claim 4, wherein said siRNA is chemically synthesized.

17. The method of claim 1, wherein said Shh gene is human.

18. The method of claim 1, wherein said Shh gene is hyperactive in the cell.

20. The method of claim 1, wherein the RNAi antagonist comprises the nucleic acid sequence set forth in any of SEQ ID NOs:

5, 6, 9, or 10, wherein said RNAi antagonist is 19-30 nucleotides in length.

22. The method of claim 1, wherein the RNAi comprises a double stranded molecule comprising SEQ ID NO:

5 and SEQ ID NO;

6.

19. A method of inhibiting at least one of unwanted growth, proliferation or survival of a cell, comprising contacting said cell with an effective amount of a hedgehog RNAi antagonist against a Sonic hedgehog (Shh) gene, wherein said RNAi antagonist comprises a sequence that is at least 90% identical to a nucleic acid sequence set forth in any of SEQ ID NOs:

5, 6, 9, or 10, wherein said RNAi antagonist is 19-30 nucleotides in length, wherein contacting said cell with said hedgehog RNAi antagonist decreases at least one of cell growth, proliferation or survival, wherein said cell is a cancer cell, wherein said cancer is selected from the group of cancers consisting of ovarian cancer, colon cancer, salivary cancer, esophageal cancer and endometrial cancer.

21. The method of claim 19, wherein the RNAi antagonist comprises the nucleic acid sequence set forth in any of SEQ ID NOs:

5, 6, 9, or 10, wherein said RNAi antagonist is 19-30 nucleotides in length.