Vertebrate embryonic pattern-inducing proteins

• US 6,384,192 B1
• Filed: 10/20/1997
• Issued: 05/07/2002
• Est. Priority Date: 12/30/1993
• Status: Expired due to Term

• 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:
  US08/957,874
• Time in Patent Office: 1,660 Days
• Field of Search: 530/300, 530/350, 530/395, 536/23.1, 536/23.5, 435/7.1, 514/2, 514/12
• US Class Current: 530/350
• 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 2227/40 Fish
• 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 67/0275 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. An isolated and/or recombinantly produced polypeptide, comprising a hedgehog amino acid sequence encoded by a nucleic acid sequence which hybridizes under highly 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, or SEQ ID NO. 6, which hedgehog amino acid sequence binds to a naturally occurring patched receptor, or a fragment of said polypeptide which binds said patched receptor.

5. The polypeptide of claim 1 wherein said hedgehog amino acid sequence is encoded by a naturally occurring Sonic hedgehog gene.

6. The polypeptide of claim 5, wherein said Sonic hedgehog gene is a mammalian Sonic hedgehog gene.

7. The polypeptide of claim 5, wherein said Sonic hedgehog gene is a human Sonic hedgehog gene.

8. The polypeptide of claim 1, wherein the hedgehog amino acid sequence mimics a biological activity of a wild-type polypetide.

9. The polypeptide of claim 1, wherein said patched receptor is apatched protein of a vertebrate organism.

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

11. The polypeptide of claim 10, wherein said neuromal cells are dopaminergic neurons.

12. The polypeptide of claim 10, wherein said neturonal cells are motor neurons.

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

14. The polypeptide of claim 1, wherein said polypeptide is post-translationally modified.

15. The polypeptide of claim 1, wherein said polypeptide is post-translationally modified with one or more of glycosyl groups, lipids, phosphate groups and acetyl groups.

16. The polypeptide of claim 1, wherein said polypeptide is purified to at least 95% by dry weight.

17. The polypeptide of claim 1, wherein said polypeptide is purified to at least 80% by dry weight.

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

19. The polypeptide of claim 1 or 2, wherein said hedgehog amino acid sequence includes at least 50 contiguous amino acid residues of the N-terminal half of one of SEQ ID Nos. 8, 11, 12, or 13.

20. The polypeptide of claim 19, wherein said hedgehog amino acid sequence includes at least 100 contiguous amino acids of the N-terminal half of one of SEQ ID Nos. 8, 11, 12, or 13.

21. The polypeptide of claim 19, wherein said hedgehog amino acid sequence includes at least 150 contiguous amino acids of the N-terminal half of one of SEQ ID Nos. 8, 11, 12, or 13.

22. The polypeptide of any one of claims 1, 2, 3, or 4, wherein said polypeptide further comprises a heterologous amino acid sequence.

23. The polypeptide of claim 22, wherein the heterologous amino acid sequence is (i) a detectable label or (ii) a matrix-binding domain.

24. The polypeptide of any one of claims 1, 2, 3, or 4, which polypeptide promotes at least one of proliferation, differentiation or survival of mesodermal or ectodermal cells.

2. An isolated and/or recombinantly produced polypeptide comprising a hedgehog amino acid sequence which is at least 80 percent identical to a sequence selected from SEQ ID NO. 8, SEQ ID NO. 11, SEQ ID NO. 12, or SEQ ID NO. 13, which hedgehog amino acid sequence binds to a naturally occurring patched receptor.

25. The polypeptide of claim 2, wherein said hedgehog amino acid sequence is at least 80 percent identical to an amino acid sequence selected from residues 27-425 of SEQ ID NO:

8, residues 25-437 of SEQ ID NO;

11, residues 24-418 of SEQ ID NO;

12, or residues 24-475 of SEQ ID NO;

13.

26. The polypeptide of claim 2 or 25, wherein said hedgehog amino acid sequence is at least 90 percent identical to a sequence selected from SEQ ID NO. 8, SEQ ID NO. 11, SEQ ID NO. 12, or SEQ ID NO. 13.

27. The polypeptide of claim 2 or 25, wherein said hedgehog amino acid sequence is at least 95 percent identical to a sequence selected from SEQ ID NO. 8, SEQ ID NO. 11, SEQ ID NO. 12, or SEQ ID NO. 13.

28. The polypeptide of claim 2 or 25, wherein said hedgehog amino acid sequence is identical to a sequence selected from SEQ ID NO. 8, SEQ ID NO. 11, SEQ ID NO. 12, or SEQ ID) NO. 13.

3. An isolated and/or recombinantly produced polypeptide, comprising a hedgehog amino acid sequence encoded by a nucleic acid sequence which hybridizes under highly stringent conditions, including a wash step of 0.2×

SSC at 65°

C., to a nucleic acid sequence selected from SEQ ID NO. 3 or SEQ ID NO. 7, which hedgehog amino acid sequence binds to a naturally occurring patched receptor.

29. The polypeptide of claim 3, wherein said hedgehog amino acid sequence is encoded by a naturally occurring Indian hedgehog gene.

30. The polypeptide of claim 3, wherein the hedgehog amino acid sequence mimics a biological activity of a wild-type Indian hedgehog polypeptide.

31. The polypeptide of claim 30, wherein said Indian hedgehog gene is a mammalian Indian hedgehog gene.

32. The polypeptide of claim 30, wherein said Indian hedgehog gene is a human Indian hedgehog gene.

33. The polypeptide of claim 3, wherein said polypeptide promotes proliferation of chondrocytes.

34. The polypeptide of claim 3, wherein said polypeptide is post-translationally modified.

35. The polypeptide of claim 34, wherein said polypeptide is post-translationally modified with one or more of glycosyl groups, lipids, phosphate groups and acetyl groups.

36. The polypeptide of claim 3 or 4, wherein said hedgehog amino acid sequence includes at least 50 contiguous amino acid residues of the N-terminal half of one of SEQ ID Nos. 8, 11, 12, or 13.

37. The polypeptide of claim 36, wherein said hedgehog amino acid sequence includes at least 100 contiguous amino acids of the N-terminal half of one of SEQ ID Nos. 8, 11, 12, or 13.

38. The polypeptide of claim 36, wherein said hedgehog amino acid sequence includes at least 150 contiguous amino acids of the N-terminal half of one of SEQ ID Nos. 8, 11, 12, or 13.

4. An isolated and/or recombinantly produced polypeptide comprising a hedgehog amino acid sequence which is at least 80 percent identical to a sequence selected from SEQ ID NO. 10 or SEQ ID NO. 14, which hedgehog amino acid sequence binds to a naturally occurring patched receptor.