Compositions comprising hedgehog proteins

• US 6,630,148 B1
• Filed: 10/20/1997
• Issued: 10/07/2003
• Est. Priority Date: 12/30/1993
• Status: Expired due to Fees

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), Imperial Cancer Research Technology, Ltd.
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation), Imperial Cancer Research Technology, Ltd.
• Inventors: Ingham, Philip W., McMahon, Andrew P., Tabin, Clifford J.
• Primary Examiner(s): Spector, Lorraine
• Assistant Examiner(s): O HARA, EILEEN B
• Application Number:
  US08/954,740
• Time in Patent Office: 2,178 Days
• Field of Search: 530/350, 536/23.5, 435/6
• US Class Current: 424/185.1
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/00 Genetically modified animals
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2227/30 Bird
• CPC Class Codes: A01K 2227/40 Fish
• CPC Class Codes: A01K 2227/50 Amphibians, e.g. Xenopus
• CPC Class Codes: A01K 2267/03 Animal model, e.g. for test or diseases
• CPC Class Codes: A01K 2267/0318 Animal model for neurodegenerative disease, e.g. non- Alzheimer's
• CPC Class Codes: A01K 2267/0368 Animal model for inflammation
• CPC Class Codes: A01K 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A01K 67/033 Rearing or breeding invertebrates; New breeds of invertebrates
• CPC Class Codes: A01K 67/0339 Genetically modified insects, e.g. Drosophila melanogaster, medfly
• 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: C07K 14/46 from vertebrates
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C07K 14/475 Growth factors; Growth regulators
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2800/30 : Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Abstract

The present invention concerns the discovery that proteins encoded by a family of vertebrate genes, termed here hedgehog-related genes, comprise morphogenic signals produced by embryonic patterning centers, and are involved in the formation of ordered spatial arrangements of differentiated tissues in vertebrates. The present invention makes available compositions and methods that can be utilized, for example to generate and/or maintain an array of different vertebrate tissue both in vitro and in vivo.

Claims

1. A composition comprising a hedgehog polypeptide that includes a hedgehog amino acid sequence formulated in a pharmaceutically acceptable carrier, wherein said hedgehog amino acid sequence is encoded by a nucleic acid which hybridizes under stringent conditions, including a wash step of 0.2×

SSC at 65°

C., to a nucleic acid sequence selected from SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7, which hedgehog amino acid sequence binds to a naturally occurring patched receptor.

2. The composition of claim 1, wherein said polypeptide comprises a hedgehog amino acid sequence selected from SEQ ID NO:

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13 and SEQ ID NO;

14, or a fragment thereof.

3. The composition of claim 1, wherein said polypeptide comprises a hedgehog amino acid sequence encoded by a naturally occurring vertebrate hedgehog gene.

4. The composition of claim 3, wherein the hedgehog gene is a mammalian hedgehog gene.

5. The composition of claim 4, wherein the hedgehog gene is a human hedgehog gene.

6. The composition of claim 1, A wherein the hedgehog amino acid sequence is represented by the general formula SEQ ID NO:

41.

7. The composition of claim 1, wherein the hedgehog amino acid sequence has a molecular weight of about 19 kD.

8. The composition of claim 1, wherein said hedgehog amino acid sequence includes at least 150 contiguous amino acid residues of the N-terminal half of a hedgehog protein.

9. The composition of claim 1, wherein said patched receptor is a patched receptor of a vertebrate organism.

10. The composition of claim 1, wherein said polypeptide promotes the differentiation of neuronal cells or survival of a differentiated neuronal cells.

11. The composition of claim 10, wherein said neuronal cell is a dopaminergic neuron.

12. The composition of claim 10, wherein said neuronal cell is a motor neuron.

13. The composition of claim 1, wherein said polypeptide promotes proliferation of chondrocytes.

14. The composition of claim 1, wherein said polypeptide induces expression of BMP-2, BMP-4, or Hoxd genes.

15. The composition of claim 1, wherein said polypeptide is provided in a sterile preparation.

16. The composition of claim 1, wherein said pharmaceutically acceptable carrier is selected from the group consisting of water, buffered saline and polyol, or suitable mixtures thereof.

17. The composition of claim 1, wherein said pharmaceutically acceptable carrier iso-osmotic with physiological fluids of an animal.

18. The composition of claim 1, wherein said polypeptide is provided as a freeze-dried powder.

19. The composition of claim 1, wherein said polypeptide is provided in a solution.

20. The composition of claim 1, further comprising at least one trophic factor.

21. The composition of claim 20, wherein said trophic factor is selected from nerve growth factor, cilliary neurotrophic growth factor, schwanoma-derived growth factor, glial growth factor, striatal-derived neuronotrophic factor, platelet-derived growth factor, and scatter factor (HGF-SF).

22. The composition of claim 1, further comprising an antimitogenic agent.

23. The composition of claim 22, wherein said antimitotic agent inhibits the proliferation of glial cells and/or astrocytes.

24. The composition of claim 22, wherein said antimitotic agent is selected from cytosine, arabinoside, 5-fluorouracil, hydroxyurea, and methotrexate.

25. The composition of claim 1, wherein the hedgehog amino acid sequence is represented by the general formula SEQ ID NO:

40.

26. The composition of claim 1, wherein the hedgehog amino acid sequence includes at least 150 contiguous amino acids of the extracellular domain of said polypeptide.

27. The composition of claim 1, wherein the hedgehog amino acid sequence includes at least 100 contiguous amino acids of the extracellular domain of said polypeptide.

28. The composition of claim 1, wherein the hedgehog amino acid sequence includes at least 50 contiguous amino acids of the extracellular domain of said polypeptide.

29. The composition of claim 1, wherein said polypeptide is post-translationally modified with one or more glycosyl groups.

30. The composition of claim 1, which polypeptide is post-translationally modified with one or more lipids.

31. The composition of claim 1, which polypeptide is post-translationally modified with one or more phosphate groups.

32. The composition of claim 1, which polypeptide is post-translationally modified with one or more and acetyl groups.

33. The composition of claim 1, which hedgehog amino acid sequence comprises an N-terminal portion of a vertebrate hedgehog protein comprising Cys-24 through Glu-188 of SEQ ID-NO:

13.

34. The composition of claim 1, which hedgehog amino acid sequence comprises an N-terminal portion of a vertebrate hedgehog protein comprising Cys-23 through Asp 189 of SEQ ID NO:

9.

35. The composition of claim 1, which hedgehog amino acid sequence comprises a C-terminal portion of a vertebrate hedgehog protein comprising Asn-189 through Ala-475 of SEQ ID NO:

13.

36. The composition of claim 1, wherein the polypeptide is recombinantly produced.

37. The composition of claim 1, wherein the hedgehog amino acid sequence is encoded by a nucleic acid which hybridizes under stringent conditions, including a wash step of 0.2×

SSC at 65°

C., to a nucleic acid sequence selected from SEQ ID NO;

1, SEQ ID NO;

4, SEQ ID NO;

5 and SEQ ID NO;

6.

38. The composition of claim 37, wherein the hedgehog amino acid sequence is encoded by a nucleic acid which hybridizes under stringent conditions, including a wash step of 0.2×

SSC at 65°

C., to the nucleic acid of SEQ ID NO;

6.

39. The composition of claim 37, wherein the hedgehog amino acid sequence is encoded by a nucleic acid which hybridizes under stringent conditions, including a wash step of 0.2×

SSC at 65°

C., to the nucleic acid of SEQ ID NO;

7.

40. The composition of claim 1, wherein the hedgehog amino acid sequence is encoded by a nucleic acid which hybridizes under stringent conditions, including a wash step of 0.2×

SSC at 65°

C., to the nucleic acid sequence of SEQ ID NO;

2.

41. The composition of claim 1, wherein the hedgehog amino acid sequence is encoded by a nucleic acid which hybridizes under stringent conditions, including a wash step of 0.2×

SSC at 65°

C., to a nucleic acid sequence selected from SEQ ID NO;

3 and SEQ ID NO;

7.

42. The composition of claim 1, wherein the protein is purified to have less than 20% by dry weight of contaminating proteins.

43. The composition of claim 1, wherein the protein is purified to have less than 5% by dry weight of contaminating proteins.

44. The composition of claim 1 or 15, wherein said polypeptide is formulated for topical administration to an animal.

45. The composition of claim 1 or 15, wherein said polypeptide is formulated for systemic administration to an animal.

46. The composition of claim 1 or 15, wherein said polypeptide is formulated for intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, intraventricular, intrathecal or intranasal delivery to an animal.

47. The composition of claim 1 or 15, wherein said polypeptide is provided in a rechargeable or biodegradable implantable device.

48. The composition of claim 47, wherein said polypeptide is encapsulated in an implantable polymer.

49. A composition comprising a polypeptide that includes a hedgehog amino acid sequence, formulated in a pharmaceutically acceptable carrier, wherein said hedgehog amino acid sequence is at least 80% identical to a polypeptide selected from SEQ ID NO:

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13 and SEQ ID NO;

14, or a fragment thereof of at least 50 contiguous amino acid residues, wherein said hedgehog amino acid sequence binds to a naturally occurring patched receptor.

50. A composition of claim 49, comprising a polypeptide that includes a hedgehog amino acid sequence at least 90% identical to a polypeptide selected from SEQ ID NO:

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13 and SEQ ID NO;

14, or a fragment thereof of at least 50 contiguous amino acid residues, wherein said hedgehog amino acid sequence binds to a naturally occurring patched receptor.

51. A composition of claim 49, comprising a polypeptide that includes a hedgehog amino acid sequence at least 95% identical to a polypeptide selected from SEQ ID NO:

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13 and SEQ ID NO;

14, or a fragment thereof of at least 50 contiguous amino acid residues, wherein said hedgehog amino acid sequence binds to a naturally occurring patched receptor.

52. A composition of claim 49, comprising a polypeptide that includes a hedgehog amino acid sequence at least 98% identical to a polypeptide selected from SEQ ID NO:

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13 and SEQ ID NO;

14, or a fragment thereof of at least 50 contiguous amino acid residues, wherein said hedgehog amino acid sequence binds to a naturally occurring patched receptor.