Methods and compositions for regulating skeletogenic formation

• US 6,884,775 B1
• Filed: 08/01/1997
• Issued: 04/26/2005
• 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: Vortkamp, Andrea M., Ingham, Philip W., McMahon, Andrew P., Tabin, Clifford J.
• Primary Examiner(s): Spector, Lorraine
• Assistant Examiner(s): Kaufman, Claire M.
• Application Number:
  US08/905,572
• Time in Patent Office: 2,825 Days
• Field of Search: 514/2, 514/12, 424/143.1, 424/130.1, 530/350, 530/300, 536/23.1, 536/23.5
• US Class Current: 514/16.7
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2227/50 Amphibians, e.g. Xenopus
• CPC Class Codes: A01K 2267/03 Animal model, e.g. for test or diseases
• 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 67/0339 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 14/475 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

Abstract

The present application is directed to the discovery that hedgehog proteins, and agents which effect the activities thereof, can be used to control the formation and/or maintenance of cartilage and bone.

Claims

1. A method for promoting proliferation of a chondrocytic cell comprising contacting the cell with an amount of a hedgehog therapeutic effective to promote proliferation of the cell, wherein said hedgehog therapeutic comprises a polypeptide which includes an amino acid sequence at least 80% identical to at least one of SEQ ID NO:

10, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, or at least 80% identical to a 19 kDa N-terminal fragment thereof and wherein said polypeptide which includes said amino acid sequence binds to a patched polypeptide and promotes hedgehog signal transduction.

2. The method of claim 1, wherein said hedgehog therapeutic increases the rate of proliferation of the cell.

3. The method of claim 1, wherein the cell is in culture, and said hedgehog therapeutic is provided as a cell culture additive.

4. The method of claim 1, wherein the cell is in an animal and said hedgehog therapeutic is administered to the animal as a therapeutic composition.

5. The method of claim 1, wherein contacting the chondrocytic cell with an effective amount of said hedgehog therapeutic improves the rate of survival of the chondrocytic cell.

6. The method of claim 1, wherein said polypeptide comprises an amino acid sequence encoded by a nucleic acid at least 80% identical to at least one of SEQ ID NO:

1, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, or SEQ ID NO;

7, and wherein said polypeptide binds to a patched polypeptide and promotes hedgehog signal transduction.

7. The method of claim 1, wherein said polypeptide comprises an amino acid sequence encoded by a nucleic acid at least 90% identical to at least one of SEQ ID NO:

1, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, or SEQ ID NO;

7, and wherein said polypeptide binds to a patched polypeptide and promotes hedgehog signal transduction.

8. The method of claim 1, wherein said polypeptide comprises an amino acid sequence encoded by a nucleic acid at least 95% identical to at least one of SEQ ID NO:

1, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, or SEQ ID NO;

7, and wherein said polypeptide binds to a patched polypeptide and promotes hedgehog signal transduction.

9. The method of claim 1, wherein said polypeptide comprises an amino acid sequence encoded by a nucleic acid at least 98% identical to at least one of SEQ ID NO:

1, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, or SEQ ID NO;

7, and wherein said polypeptidc binds to a patched polypeptide and promotes hedgehog signal transduction.

10. The method of claim 1, wherein said polypeptide comprises an amino acid sequence encoded by a nucleic acid identical to SEQ ID NO:

1, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, or SEQ ID NO;

7, and wherein said polypeptide binds to a patched polypeptide and promotes hedgehog signal transduction.

11. The method of claim 1, wherein said polypeptide 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 designated in at least one of SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, or SEQ ID NO;

7, and wherein said polypeptide binds to a patched polypeptide and promotes hedgehog signal transduction.

12. The method of claim 1, wherein said polypeptide comprises an amino acid sequence at least 90% identical to at least one of SEQ ID NO:

10, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, or at least 90% identical to a 19 kDa N-terminal fragment thereof, and wherein said polypeptide binds to a patched polypeptide and promotes hedgehog signal transduction.

13. The method of claim 1, wherein said polypeptide comprises an amino acid sequence at least 95% identical to at least one of SEQ ID NO:

10, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, or at least 95% identical to a 19 kDa N-terminal fragment thereof, and wherein said polypeptide binds to a patched polypeptide and promotes hedgehog signal transduction.

14. The method of claim 1, wherein said polypeptide comprises an amino acid sequence at least 98% identical to at least one of SEQ ID NO:

10, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, or at least 98% identical to a 19 kDa N-terminal fragment thereof, and wherein said polypeptide binds to a patched polypeptide and promotes hedgehog signal transduction.

15. The method of claim 1, wherein said polypeptide comprises an amino acid sequence identical to SEQ ID NO:

10, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, or a 19 kDa N-terminal fragment thereof, and wherein said polypeptide binds to a patched polypeptide and promotes hedgehog signal transduction.

16. The method of claim 1, wherein said polypeptide comprises:

a) amino acid residues 24-197 of SEQ ID NO;

15 or b) amino acid residues 28-202 of SEQ ID NO;

16.

17. The method of claim 1, wherein said polypeptide comprises:

a) amino acid residues 1-168 of SEQ ID NO;

21, b) amino acid residues 24-193 of SEQ ID NO;

15, c) amino acid residues 25-193 of SEQ ID NO;

13, or d) amino acid residues 28-197 of SEQ ID NO;

12.

18. The method of claim 5, wherein said polypeptide comprises an amino acid sequence encoded by a non-synthetic vertebrate hedgehog gene.

19. The method of claim 18, wherein said hedgehog gene is a mammalian hedgehog gene.

20. The method of claim 19, wherein said hedgehog gene is a human hedgehog gene.

21. The method of claim 5, wherein said polypeptide comprises an amino acid sequence represented by the general formula SEQ ID NO:

21.

22. A method for promoting survival of a chondrocytic cell comprising contacting the cell with an amount of a hedgehog therapeutic effective to promote survival of the cell, wherein said hedgehog therapeutic comprises a polypeptide which includes an amino acid sequence at least 80% identical to at least one of SEQ ID NO:

10, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ

     1) NO 16, or at least 80% identical to a 19 kDa N-terminal fragment thereof, and wherein said polypeptide which includes said amino acid sequence binds to a patched polypeptide and promotes hedgehog signal transduction.

23. The method of claim 22, wherein said polypeptide comprises an amino acid sequence encoded by a nucleic acid at least 80% identical to at least one of SEQ ID NO:

1, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, or SEQ ID NO;

7, and wherein said polypeptide which includes said amino acid sequence binds to a patched polypeptide and promotes hedgehog signal transduction.

24. The method of claim 22, wherein said polypeptide comprises an amino acid sequence encoded by a nucleic acid at least 90% identical to at least one of SEQ ID NO:

1, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, or SEQ ID NO;

7, and wherein said polypeptide which includes said amino acid sequence binds to a patched polypeptide and promotes hedgehog signal transduction.

25. The method claim 22, wherein said polypeptide comprises an amino acid sequence encoded by a nucleic acid at least 95% identical to at least one of SEQ ID NO:

1, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, or SEQ ID NO;

7, and wherein said polypeptide which includes said amino acid sequence binds to a patched polypeptide and promotes hedgehog signal transduction.

26. The method of claim 22, wherein said polypeptide comprises an amino acid sequence encoded by a nucleic acid at least 98% identical to at least one of SEQ ID NO:

1, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, or SEQ D) NO;

7, and wherein said polypeptide which includes said amino acid sequence binds to a patched polypeptide and promotes hedgehog signal transduction.

27. The method of claim 22, wherein said polypeptide comprises an amino acid sequence encoded by a nucleic acid identical to SEQ ID NO:

1, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, or SEQ ID NO;

7, and wherein said polypeptide which includes said amino acid sequence binds to a patched polypeptide and promotes hedgehog signal transduction.

28. The method of claim 22, wherein said polypeptide 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 designated in at least one of SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, or SEQ ID NO;

7, and wherein said polypeptide which includes said amino acid sequence binds to a patched polypeptide and promotes hedgehog signal transduction.

29. The method of claim 22, wherein said polypeptide comprises an amino acid sequence at least 90% identical to at least one of SEQ ID NO:

10, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO 16, or at least 90% identical to a 19 kDa N-terminal fragment thereof, and wherein said polypeptide which includes said amino acid sequence binds to a patched polypeptide and promotes hedgehog signal trasduction.

30. The method of claim 22, wherein said polypeptide comprises an amino acid sequence at least 95% identical to at least one of SEQ ID NO:

10, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO 16, or at least 95% identical to a 19 kDa N-terminal fragment thereof, and wherein said polypeptide which includes said amino acid sequence binds to a patched polypeptide and promotes hedgehog signal transduction.

31. The method of claim 22, wherein said polypeptide comprises an amino acid sequence at least 98% identical to at least one of SEQ ID NO:

10, SEQ ID NO;

12, SEQ ID NO;

13, SEQ

     1) NO;

14, SEQ ID NO;

15, SEQ ID NO 16, or at least 98% identical to a 19 kDa N-terminal fragment thereof, and wherein said polypeptide which includes said amino acid sequence binds to a patched polypeptide and promotes hedgehog signal transduction.

32. The method of claim 22, wherein said polypeptide comprises an amino acid sequence identical to SEQ ID NO:

10, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO 16, or a 19 kDa N-terminal fragment thereof, and wherein said polypeptide which includes said amino acid sequence binds to a patched polypeptide and promotes hedgehog signal transduction.

33. The method of claim 22, wherein said polypeptide comprises:

a) amino acid residues 24-197 of SEQ ID NO;

15 or b) amino acid residues 28-202 of SEQ ID NO;

16.

34. The method of claim 22, wherein said polypeptide comprises:

a) amino acid residues 1-168 of SEQ ID NO;

21, b) amino acid residues 24-193 of SEQ ID NO;

15, c) amino acid residues 25-193 of SEQ ID NO;

13, or d) amino acid residues 28-197 of SEQ ID NO;

12.

35. A method for promoting proliferation of a chondrocytic cell comprising contacting the cell with an amount of a hedgehog therapeutic effective to promote proliferation of the cell, wherein said hedgehog therapeutic comprises a polypeptide including an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions, including a wash step of 0.2×

SSC at 65°

C., to at least one of SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, or SEQ ID NO;

7, and wherein said polypeptide which includes said amino acid sequence binds to a patched polypeptide and promotes hedgehog signal transduction.

36. A method of promoting survival of a chondrocytic cell comprising contacting the cell with an amount of a hedgehog therapeutic effective to promote survival of the cell, wherein said hedgehog therapeutic comprises a polypeptide including an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions, including a wash step of 0.2×

SSC at 65°

C., to at least one of SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, or SEQ ID NO;

7, and wherein said polypeptide which includes said amino acid sequence binds to a patched polypeptide and promotes hedgehog signal transduction.

37. A method for promoting proliferation of a chondrocytic cell comprising contacting the cell with an amount of a hedgehog therapeutic effective to promote proliferation of the cell, wherein said hedgehog therapeutic comprises a polypeptide including an amino acid sequence identical to SEQ ID NO:

21, and wherein said polypeptide which includes said amino acid sequence binds to a patched polypeptide and promotes hedgehog signal transduction.

38. A method of promoting survival of a chondrocytic cell comprising contacting the cell with an amount of a hedgehog therapeutic effective to promote survival of the cell, wherein said hedgehog therapeutic comprises a polypeptide including an amino acid sequence identical to SEQ ID NO:

21, and wherein said polypeptide which includes said amino acid sequence binds to a patched polypeptide and promotes hedgehog signal transduction.