Nucleic acids encoding hedgehog proteins

• US 6,271,363 B1
• Filed: 10/20/1997
• Issued: 08/07/2001
• 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:
  US08/954,698
• Time in Patent Office: 1,387 Days
• Field of Search: 536/23.1, 536/23.5, 536/23.51, 530/300, 530/350, 435/69.1
• US Class Current: 536/23.5
• 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 nucleic acid encoding a hedgehog polypeptide comprising an amino acid sequence at least 80 percent identical to an amino acid sequence selected from the group consisting of residues 27-425 of SEQ ID NO:

8, residues 1-336 of SEQ ID NO;

10, residues 25-437 of SEQ ID NO;

11, residues 24-418 of SEQ ID NO;

;

12, residues 24-475 of SEQ ID NO;

13 and residues 1-312 of SEQ ID NO;

14, which polypeptide binds to a naturally occurring patched receptor.

2. An isolated nucleic acid encoding a polypeptide selected from the group consisting of Sonic hedgehog polypeptide and Indian hedgehog polypeptide, wherein said hedgehog polypeptide comprises 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 nucleic acid sequence selected from the group consisting of SEQ ID. NO. 1, SEQ ID. NO. 3, SEQ ID. NO. 4, SEQ ID. NO. 5, SEQ ID. NO. 6, and SEQ ID. NO. 7, which amino acid sequence binds to a naturally occurring patched receptor.

4. The nucleic acid of any one of claims 2 or 3, which nucleic acid encodes a fragment of the N-terminal half of a Sonic hedgehog polypeptide, which polypeptide promotes proliferation of chondrocytes.

5. The nucleic acid of any one of claims 2 or 3, which nucleic acid encodes a fragment of the N-terminal half of a Indian hedgehog polypeptide, which polypeptide promotes proliferation of testicular germ line cells.

6. The nucleic acid of any one of claims 2 or 3, wherein said hedgehog polypeptide promotes at least one of proliferation, survival, or differentiation of mesodermal tissue.

7. The nucleic acid of claim 6, wherein said mesodermal tissue is the dorsal mesoderm.

8. The nucleic acid of any one of claims 2 or 3, wherein said hedgehog polypeptide promotes at least one of proliferation, survival or differentiation of ectodermal tissue.

9. The nucleic acid of claim 8, wherein said hedgehog polypeptide is from a tissue selected from the group consisting of neural tube, neural crest or head mesenchyme.

10. The nucleic acid of any one of claims 2 or 3, wherein said hedgehog polypeptide promotes at least one of proliferation, survival or differentiation of endodermal tissue.

11. The nucleic acid of claim 10, wherein said hedgehog polypeptide is from the primitive gut.

12. The nucleic acid of any one of claims 2 or 3, wherein said hedgehog polypeptide can induce expression of secondary signaling molecules selected from the group consisting of members of the TGFβ

family and members of the FGF family.

13. The nucleic acid of any one of claims 2 or 3, wherein said hedgehog nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:

1, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6 and SEQ ID NO;

7.

14. The nucleic acid of any one of claims 2 or 3, wherein said hedgehog nucleic acid encodes a sonic hedgehog (Shh) polypeptide as set forth in SEQ ID NO:

40.

15. The nucleic acid of any one of claims 2 or 3, wherein said hedgehog nucleic acid encodes an Indian hedgehog (Ihh) polypeptide as set forth in SEQ ID NO:

10.

16. The nucleic acid of any one of claims 2 or 3, wherein said hedgehog polypeptide further comprises a heterologous amino acid sequence.

17. The nucleic acid of any one of claims 2 or 3, wherein said amino acid sequence is encoded by a naturally occurring vertebrate Sonic hedgehog gene.

18. The nucleic acid of claim 17, wherein said Sonic hedgehog gene is a mammalian Sonic hedgehog gene.

19. The nucleic acid of claim 18, wherein said Sonic hedgehog gene is a human Sonic hedgehog gene.

20. The nucleic acid of any one of claims 2 or 3, wherein said amino acid sequence includes at least 150 amino acid residues of the N-terminal half of a hedgehog protein.

21. The nucleic acid of any one of claims 2 or 3, wherein said amino acid sequence is encoded by a naturally occurring vertebrate Indian hedgehog gene.

22. The nucleic acid of claim 21, wherein said Indian hedgehog gene is a mammalian Indian hedgehog gene.

23. The nucleic acid of claim 21, wherein said Indian hedgehog gene is a human Indian hedgehog gene.

24. The nucleic acid of any one of claims 2 or 3, wherein said patched receptor is a patched receptor of a vertebrate organism.

25. The nucleic acid of any one of claims 2 or 3, wherein said polypeptide promotes differentiation of neuronal cells or survival of differentiated neuronal cells.

26. The nucleic acid of claim 25, wherein said neuronal cell is a dopaminergic neuron.

27. The nucleic acid of claim 26, wherein said neuronal cell is a motorneuron.

28. The nucleic acid of any one of claims 2 or 3, wherein said polypeptide promotes proliferation of chondrocytes.

29. The nucleic acid of any one of claims 2 or 3, wherein said polypeptide induces expression of BMP-2, BMP-4, or Hoxd genes.

30. The nucleic acid of any one of claims 2-3, wherein said polypeptide is post-translationally modified.

31. The polypeptide of claim 30, wherein said polypeptide is post-translationally modified with one or more of glycosyl groups, lipids, phosphate groups and acetyl groups.

32. The nucleic acid of any one of claims 2-3, wherein said polypeptide is purified to at least 95% by dry weight.

33. The nucleic acid of any one of claims 2-3, wherein said polypeptide is purified to at least 80% by dry weight.

34. The nucleic acid of any one of claims 2-3, wherein said amino acid sequence includes at least 150 contiguous amino acids of the N-terminal half of the hedgehog polypeptide.

35. The nucleic acid of any one of claims 2-3, wherein said amino acid sequence includes at least 100 contiguous amino acids of the N-terminal half of the hedgehog polypeptide.

36. The nucleic acid of any one of claims 2-3, wherein said amino acid sequence includes at least 50 contiguous amino acids of the N-terminal half of the hedgehog polypeptide.

3. An isolated nucleic acid encoding a polypeptide selected from the group consisting of Sonic hedgehog polypeptide and Indian hedgehog polypeptide, wherein said hedgehog polypeptide comprises an amino acid sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID. NO. 8, SEQ ID. NO. 10, SEQ ID. NO. 11, SEQ ID. NO. 12, SEQ ID. NO. 13, and SEQ ID. NO. 14, which amino acid sequence binds to a naturally occurring patched receptor.

37. An isolated nucleic acid of claim 3, wherein said hedgehog polypeptide comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID. NO. 8, SEQ ID. NO. 10, SEQ ID. NO. 11, SEQ ID. NO. 12, SEQ ID. NO. 13, and SEQ ID. NO 14.

38. An isolated nucleic acid of claim 3, wherein said hedgehog polypeptide comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID. NO. 8, SEQ ID. NO. 10, SEQ ID. NO. 11, SEQ ID. NO. 12, SEQ ID. NO. 13, and SEQ ID. NO. 14.