Method of promoting chondrocyte differentiation with hedgehog related polypeptides

• US 6,610,656 B1
• Filed: 10/20/1997
• Issued: 08/26/2003
• 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): Kaufman, Claire M.
• Application Number:
  US08/954,128
• Time in Patent Office: 2,136 Days
• Field of Search: 514/2, 514/12, 530/350, 530/300, 435/69.1, 435/325, 435/366, 536/23.1, 536/23.5
• US Class Current: 514/7.6
• 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 2267/0312 Animal model for Alzheimer's disease
• 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 14/475 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 method for promoting differentiation of a chondrocyte, comprising contacting said chondrocyte with an effective amount of a polypeptide, wherein the polypeptide includes a hedgehog polypeptide encoded by a nucleic acid which 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;

4, SEQ ID NO;

5, or SEQ ID NO;

6, and which hedgehog polypeptide binds a patched polypeptide and promotes hedgehog signal transduction.

2. The method of claim 1, wherein said hedgehog polypeptide comprises an amino acid sequence identical to at least 50 contiguous amino acids of at least one of SEQ ID NO:

8, SEQ ID NO;

11, SEQ ID NO;

12, or SEQ ID NO;

13.

3. The method of claim 1, wherein said hedgehog polypeptide is encoded by a nucleic acid identical to at least one of SEQ ID NO:

1, SEQ ID NO;

4, SEQ ID NO;

5, or SEQ ID NO;

6.

4. The method of claim 1, wherein said hedgehog polypeptide is encoded by a nucleic acid which is at least 90% identical to at least one of SEQ ID NO:

1, SEQ ID NO;

4, SEQ ID NO;

5, or SEQ ID NO;

6.

5. The method of claim 1, wherein said hedgehog polypeptide is encoded by a nucleic acid which is at least 95% identical to at least one of SEQ ID NO:

1, SEQ ID NO;

4, SEQ ID NO;

5, or SEQ ID NO;

6.

6. The method of claim 1, wherein said hedgehog polypeptide includes an amino acid sequence encoded by a nucleic acid which hybridizes under stringent conditions, including a wash step of 0.2×

SSC at 65°

C., to a sequence selected from at least one of;

a) residues 310-567 of SEQ ID NO;

1, b) residues 304-561 of SEQ ID NO;

4, or c) residues 301-558 of SEQ ID NO;

5.

7. The method of claim 1, wherein said hedgehog polypeptide includes an amino acid sequence encoded by a nucleic acid which hybridizes under stringent conditions, including a wash step of 0.2×

SSC at 65°

C., to a sequence selected from at least one of;

a) residues 64-567 of SEQ ID NO;

1, b) residues 73-561 of SEQ ID NO;

4, or c) residues 70-558 of SEQ ID NO;

5.

8. The method of claim 1, wherein said hedgehog polypeptide includes an amino acid sequence at least 90 percent identical with a sequence selected from at least one of:

a) residues 104-189 of SEQ ID NO;

8, b) residues 102-187 of SEQ ID NO;

11, or c) residues 101-186 of SEQ ID NO;

12.

9. The method of claim 1, wherein said hedgehog polypeptide includes an amino acid sequence at least 90 percent identical with a sequence selected from at least one of:

a) residues 27-189 of SEQ ID NO;

8, b) residues 25-187 of SEQ ID NO;

11, or c) residues 24-186 of SEQ ID NO;

12.

10. The method of claim 1, wherein said hedgehog polypeptide includes an amino acid sequence at least 90 percent identical with an amino acid sequence selected from at least one of:

a) residues 27-425 of SEQ ID NO;

8, b) residues 25-437 of SEQ ID NO;

11, c) residues 24-418 of SEQ ID NO;

12, d) residues 24-475 of SEQ ID NO;

13, or e) an extracellular fragment of at least 50 amino acids of a, b, c, or d.

11. The method of claim 1, wherein said hedgehog polypeptide includes an amino acid sequence encoded by at least one of:

a) residues 64-567 of SEQ ID NO;

1, b) residues 73-561 of SEQ ID NO;

4, or c) residues 70-558 of SEQ ID NO;

5.

12. The method of claim 1, wherein said hedgehog polypeptide comprises an amino acid sequence represented by the general formula SEQ ID NO:

41.

13. The method of claim 1, wherein said hedgehog polypeptide includes an amino acid sequence represented by the general formula SEQ ID NO:

40.

14. The method of claim 1, wherein said polypeptide includes at least 50 contiguous amino acid residues of the N-terminal half of at least one of SEQ ID NO:

8, SEQ ID NO;

11, SEQ ID NO;

12, or SEQ ID NO;

13.

15. The method of claim 1, wherein said polypeptide includes at least 100 contiguous amino acid residues of the N-terminal half of at least one of SEQ ID NO:

8, SEQ ID NO;

11, SEQ ID NO;

12, or SEQ ID NO;

13.

16. The method of claim 1, wherein said patched polypeptide is a vertebrate patched polypeptide.

17. The method of claim 1, wherein said fragment has an approximate molecular weight of 19 kD.

21. The method of claim 1, or 18, wherein said hedgehog polypeptide is a mammalian hedgehog polypeptide.

22. The method of claim 21, wherein said hedgehog polypeptide is a human hedgehog polypeptide.

23. The method of any one of claim 1 or 18, or wherein said hedgehog polypeptide is administered in combination with at least one additional factor that promotes differentiation of a chondrocyte.

24. The method of any one of claim 1 or 18, wherein said hedgehog polypeptide is administered in combination with at least one additional factor that promotes osteogenesis.

25. The method of claim 24, wherein the additional factor that promotes osteogenesis is selected from at least one of TGFβ

, BMP-2, BMP-4 or activin.

26. The method of any one of claim 1 or 18, wherein said hedgehog polypeptide is administered in combination with at least one inhibitor of bone resorption.

27. The method of claim 26, wherein the inhibitor is selected from at least one of estrogen, bisphosphonate, sodium fluoride, calcitonin or tamoxifen.

18. A method for promoting differentiation of mammalian chondrocytes responsive to hedgehog induction, comprising treating the chondrocytes with an amount of a polypeptide effective to promote the differentiation of the chondrocytes, wherein the polypeptide includes a hedgehog polypeptide greater than 90% identical to at least one of SEQ ID NO:

8, SEQ ID NO;

11, SEQ ID NO;

12, or SEQ ID NO;

13, and which hedgehog polypeptide binds a patched polypeptide and promotes hedgehog signal transduction.

19. The method of claim 18, wherein the hedgehog polypeptide is greater than 95% identical to at least one of SEQ ID NO:

8, SEQ ID NO;

11, SEQ ID NO;

12, or SEQ ID NO;

13.

20. The method of claim 18, wherein the hedgehog polypeptide is greater than 98% identical to at least one of SEQ ID NO:

8, SEQ ID NO;

11, SEQ ID NO;

12, or SEQ ID NO;

13.