RNA interference modulators of hedgehog signaling and uses thereof

• US 20050256076A1
• Filed: 03/24/2005
• Published: 11/17/2005
• Est. Priority Date: 03/26/2004
• Status: Active Grant

• Current Assignee: Curis, Inc., Genentech, Inc. (Roche Holding AG)
• Original Assignee: Curis, Inc.
• Inventors: Bumcrot, David A.
• Granted Patent: US 8,802,639 B2
• Time in Patent Office: Days
• 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 target sequence of the hedgehog pathway;

wherein said target sequence is a positive regulator of the hedgehog pathway, and wherein contacting said cell with said hedgehog RNAi antagonist decreases at least one of cell growth, proliferation or survival.

3. 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 a hedgehog RNAi antagonist against a target sequence of the hedgehog pathway.

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

5. The method of claim 1, wherein said unwanted cell proliferation is cancer.

6. The method of claim 1, wherein said unwanted cell proliferation is benign hyperplasia.

7. The method of claim 5, wherein said cancer is urogenital cancer.

8. The method of claim 5, wherein said cancer is cancer of the neuronal system including malignant glioma, meningioma, medulloblastoma, neuroectodermal tumor, and ependymoma.

9. The method of claim 5, wherein said cancer is associated with one or more of lung, prostate, breast, ovary, uterus, muscle, bladder, colon, kidney, pancreas, and liver tissues.

10. The method of claim 9, wherein said form of cancer associated with breast tissue is selected from inferior ductal carcinoma, inferior lobular carcinoma, intraductal carcinoma, medullary carcinoma and tubular carcinoma.

11. The method of claim 9, wherein said cancer associated with lung tissue is selected from adenocarcinoma, broncho-alveolar adenocarcinoma and small cell carcinoma.

12. The method of claim 9, wherein said cancer associated with the prostate is adenocarcinoma.

13. The method of claim 1, wherein said unwanted cell proliferation is unwanted angiogenesis.

14. The method of claim 13, wherein said unwanted angiogenesis occurs in tumor growth, tumor metastases, or abnormal growths by endothelial cells, including neovascular disease, age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, Sjogren'"'"'s syndrome, acne rosacea, phylctenulosis, syphilis, Mycobacteria infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections, Kaposi'"'"'s sarcoma, Mooren'"'"'s ulcer, Terrien'"'"'s marginal degeneration, mariginal keratolysis, rheumatoid arthritis, systemic lupus, polyarteritis, trauma, Wegener'"'"'s granulomatosis, sarcoidosis, scleritis, Stevens-Johnson syndrome, pemphigoid radial keratotomy, corneal graph rejection, rheumatoid arthritis, osteoarthritis chronic inflammation (e.g., ulcerative colitis or Crohn'"'"'s disease), hemangioma, Osler-Weber-Rendu disease, and hereditary hemorrhagic telangiectasia.

15. The method of claim 13, wherein said unwanted angiogenesis occurs in normal physiological processes including wound healing, ovulation, and implantation of the blastula after fertilization.

16. The method of claim 1, wherein said unwanted growth, proliferation or survival of said cell occurs in normal hair growth, in trichosis, hypertrichosis, hirsutism, or folliculitis including folliculitis decalvans, folliculitis ulerythematosa reticulata, keloid folliculitis, and pseudofolliculitis.

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

27. The method of claim 26, wherein said siRNA antagonist is an siRNA formed after transcription from a plasmid (RNAi expression vector) or exogenous synthesis.

28. The method of claim 27, wherein said siRNA is a short hairpin siRNA formed after transcription from a single promoter of said plasmid (RNAi expression vector).

29. The method of claim 27, wherein said siRNA is a short dsRNA formed after transcription from two flanking convergent promoters on said plasmid (RNAi expression vector).

30. The method of claim 1, wherein said siRNA is around 19-30 nucleotides in length.

31. The method of claim 30, wherein said siRNA is 21-23 nucleotides in length.

32. The method of claim 26, 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.

33. The method of claim 26, wherein said siRNA is double stranded, and includes short overhang(s) at one or both ends.

34. The method of claim 33, 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.

35. The method of claim 34, 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.

36. The method of claims 33, wherein said 3′

overhang is stabilized against degradation.

37. The method of claim 36, wherein said 3′

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

38. The method of claim 36, wherein said 3′

overhang is stabilized against degradation by substituting pyrimidine nucleotides by modified analogues, e.g., substitution of uridine nucleotide 3′

overhangs by 2′

-deoxythymidine.

39. The method of claim 26, wherein said siRNA is chemically synthesized.

40. The method of claim 1, wherein said RNAi comprise either long stretches of double stranded RNA identical or substantially identical to said target nucleic acid sequence, or short stretches of double stranded RNA identical to substantially identical to only a region of said target nucleic acid sequence.

41. The method of claim 1, wherein said target sequence is a positive HH signaling component listed in Table X.

43. The method of claim 41, wherein said target sequence is a human sequence.

44. The method of claim 41, wherein said target sequence is a non-human sequence.

45. The method of claim 1, wherein said target sequence is a homolog of any one of the sequences listed in Table X or Y, but is not itself listed in Table X or Y.

46. The method of claim 1, wherein said RNAi antagonist is specific for one member of several homologs of the same HH signaling component.

47. The method of claim 45, wherein said HH signaling component is a mammalian hedgehog, and said RNAi antagonist is specific for Shh.

48. The method of claim 45, wherein said RNAi antagonist is at least 1.5-fold, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold more selective for one member over all other members of several homologs of the same HH signaling component

49. The method of claim 1, wherein said RNAi antagonist is specific for the HH signaling pathway and does not significantly affect other cell signaling pathways.

50. The method of claim 48, wherein said other cell signaling pathway is a wingless pathway.

2. (canceled)

17-25. -25. (canceled)

42. (canceled)

51-53. -53. (canceled)

54. A method for treating a tumor in a patient, comprising administering to said patient an amount of a hedgehog RNAi antagonist sufficient to decrease at least one of the growth, proliferation or survival of the tumor, wherein the tumor expresses at least one of a hedgehog gene or a gli gene.

55. The method of claim 54, wherein said hedgehog RNAi antagonist is administered as part of a cancer treatment regimen.

56. A method of inhibiting at least one of unwanted growth, proliferation or survival of a cell, comprising

(a) determining whether said cell expresses a hedgehog gene, and

(b) contacting said cell which expresses said hedgehog gene with an effective amount of a hedgehog RNAi antagonist;

wherein contacting said cell with said hedgehog RNAi antagonist decreases at least one of cell growth, proliferation or survival.

57. The method of claim 56, wherein said hedgehog gene is Sonic hedgehog.

58. The method of claim 56, wherein said unwanted cell growth, proliferation or survival of a cell is cancer.

59-65. -65. (canceled)

66. A method of decreasing signal transduction in a hedgehog-mediated pathway in a cell, comprising contacting said cell with an effective amount of a hedgehog RNAi antagonist, wherein said hedgehog-mediated pathway is independent of Ptc, Smo, and/or Gli.

67. The method of claim 66, wherein said hedgehog RNAi antagonist is an Shh siRNA antagonist.

68-71. -71. (canceled)