Vertebrate embryonic pattern-inducing proteins and uses related thereto

• US 5,789,543 A
• Filed: 12/30/1993
• Issued: 08/04/1998
• 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)
• Inventors: Ingham, Philip W., McMahon, Andrew P., Tabin, Clifford J.
• Primary Examiner(s): Walsh, Stephen
• Assistant Examiner(s): SORENSEN, KENNETH
• Application Number:
  US08/176,427
• Time in Patent Office: 1,678 Days
• Field of Search: 530/350, 530/300, 435/69.1, 424/185.1
• US Class Current: 530/350
• 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 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 67/027 New or modified breeds of vertebrates
• 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: 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: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/46 from vertebrates
• 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 recombinantly produced polypeptide comprising a hedgehog amino acid sequence which is at least 80 percent identical to a sequence selected from the group consisting of SEQ ID. NO. 2, SEQ ID. NO. 4, SEQ ID. NO. 6, SEQ ID. NO. 8, and SEQ ID. NO. 10, which hedgehog amino acid sequence (i) induces expression of a ptc gene (ii) regulates differentiation of neuronal cells, (iii) regulates survival of differentiated neuronal cells, (iv) regulates proliferation of chondrocytes, (v) regulates spermatogenesis, (vi) induces expression of a Hoxd gene, or (vii) functionally replaces drosopholia hedgehog in transgenic drosophila.

23. The polypeptide of any of claims 1, 2, 3, 4 or 15, which polypeptide is a fusion protein.

24. The polypeptide of any of claims 1, 2, 3, 4, 5, 6, 7, 8, 11, or 15 which polypeptide is postranslationally modified.

25. The polypeptide of claim 24, which polypeptide is glycosylated.

26. The polypeptide of any of claims 1, 2, 3, 4, 5, 6, 7, 8, 11, or 15, wherein the polypeptide promotes differentiation of neuronal cells or survival of differentiated neuronal cells.

27. The polypeptide of claim 26, wherein the neuronal cell is a dopaminergic neuron.

28. The polypeptide of claim 27, wherein the neuronal cell is a motorneuron.

29. The polypeptide of any of claims 1, 2, 3, 4, 5, 6, 7, 8, 11, or 15, wherein the polypeptide regulates proliferation of chondrocytes.

30. The polypeptide of any of claims 1, 2, 3, 4, 5, 6, 7, 8, 11, or 15, wherein the polypeptide regulates spermatogenesis.

31. The polypeptide of any of claims 1, 2, 3, 4, 5, 6, 7, 8, 11, or 15, wherein the polypeptide induces expression of a Hoxd gene.

32. The polypeptide of any of claims 1, 2, 3, 4, 5, 6, 7, 8, 11, or 15, wherein the polypeptide induces expression of a ptc gene.

33. The polypeptide of any of claims 1, 2, 3, 4, 5, 6, 7, 8, 11, or 15, which polypeptide ectopically replaces drosopholia hedgehog in a transgenic drosophila fly.

2. A recombinantly produced polypeptide comprising a hedgehog amino acid sequence at least 80 percent identical with a sequence selected from the group consisting of residues 27-189 of SEQ ID. NO. 2, residues 22-187 of SEQ ID NO. 4, residues 1-116 of SEQ ID NO. 6, residues 25-187 of SEQ ID. NO. 8, and residues 24-186 of SEQ ID. NO. 10 which hedgehog amino acid sequence (i) induces expression of a prc gene, (ii) regulates differentiation of neuronal cells, (iii) regulates survival of differentiated neuronal cells, (iv) regulates proliferation of chondrocytes, (v) regulates spermatogenesis, (vi) induces expression of a Hoxd gene, or (vii) functionally replaces drosopholia hedgehog in transgenic drosophila.

3. An isolated polypeptide comprising a hedgehog amino acid sequence of at least 150 amino acid residues encoded by a nucleic acid which hybridizes under highly stringent conditions to a sequence selected from the group consisting of SEQ ID. NO. 1, SEQ ID. NO 3, SEQ ID. NO 5, SEQ ID. NO. 7, and SEQ ID. NO, 9, which hedgehog amino acid sequence (i) induces expression of a prc gene, (ii) regulates differentiation of neuronal cells (iii) regulates survival of differentiated neuronal cells, (iv) regulates proliferation of chondrocytes, (v) regulates spermatogenesis. (vi) induces expression of a Hoxd gene, or (vii) functionally replaces drosopholia hedgehog in transgenic drosophila.

9. The polypeptide of any of claims 3, 4, 5, 6 or 7, wherein said hedgehog amino acid sequence is at least 90 percent identical with a sequence selected from the group consisting SEQ ID. NO. 2, SEQ ID. NO. 4, SEQ ID. NO. 6, SEQ ID. NO. 8, and SEQ ID. NO. 10.

18. The polypeptide of claim 9, wherein said hedgehog amino acid sequence is at least 95 percent identical with a sequence selected from the group consisting SEQ ID. NO. 2, SEQ ID. NO. 4, SEQ ID. NO. 6, SEQ ID. NO. 8, and SEQ ID. NO. 10.

19. The polypeptide of any of claims 3, 5, 6 or 7, wherein said hedgehog amino acid sequence is identical to a sequence selected from the group consisting SEQ ID. NO. 2, SEQ ID. NO. 4, SEQ ID. NO. 6, SEQ ID. NO. 8, and SEQ ID. NO. 10.

20. The polypeptide of claims 3 or 5, encoded by a nucleic acid which hybridizes under highly stringent conditions to a sequence selected from the group consisting of SEQ ID. NO. 3, SEQ ID. NO. 5 and SEQ ID. NO. 7.

21. The polypeptide of any of claims 3 or 5, wherein said hedgehog amino acid sequence comprising an N-terminal portion of a mature vertebrate hedgehog protein selected from the group consisting of SEQ ID. NO. 2, SEQ ID. NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, and SEQ ID. NO. 10.

4. An isolated polypeptide comprising a hedgehog amino acid sequence at least 80 percent identical to a sequence selected from the group consisting of SEQ ID. NO. 2, SEQ ID. NO. 4, SEQ ID. NO. 6, SEQ ID. NO. 8, and SEQ ID. NO. 10, which hedgehog amino acid sequence (i) induces expression of a ptc gene, (ii) regulates differentiation of neuronal cells, (iii) regulates survival of differentiated neuronal cells, (iv) regulates proliferation of chondrocytes (v) regulates spermatogenesis, (vi) induces expression of a Hoxd gene, or (vii) functionally replaces drosopholia hedgehog in transgenic drosophila.

5. A recombinantly produced polypeptide comprising a hedgehog amino acid sequence of at least 150 amino acid residues encoded by a nucleic acid which hybridizes under highly stringent conditions to a sequence selected from the group consisting of SEQ ID. NO. 1, SEQ ID. NO. 3, SEQ ID. NO. 5, SEQ ID. NO 7, and SEQ ID. NO. 9, which hedgehog amino acid sequence (i) induces expression of a pct genes (ii) regulates differentiation of neuronal cells (iii) regulates survival of differentiated neuronal cells, (iv) regulates proliferation of chondrocytes, (v) regulates spermatogenesis, (vi) induces expression of a Hoxd gene, or (vii) functionally replaces drosopholia hedgehog in transgenic drosophila.

6. An isolated polypeptide comprising a hedgehog amino acid sequence including an N-terminal portion of a mature hedgehog protein, said hedgehog amino acid sequence encoded by a nucleic acid which hybridizes under highly stringent conditions to a sequence selected from the group consisting of SEQ ID. NO. 1, SEQ ID. NO. 3, SEQ ID. NO. 5, SEQ ID. NO. 7, and SEQ ID. NO 9.

10. The polypeptide of claims 6 or 7, comprising an amino acid sequence encoded by a nucleic acid which hybridizes under stringent conditions to a sequence selected from the group consisting of residues 64-567 of SEQ ID. NO. 1, residues 64-561 of SEQ ID. NO. 3, residues 1-348 of SEQ ID NO. 5, residues 73-561 of SEQ ID NO. 7, and residues 70-558 of SEQ ID NO:

10.

17. The polypeptide of claim 6 or 9, wherein the hedgehog gene is a mammalian hedgehog gene.

7. A recombinantly produced polypeptide comprising a hedgehog amino acid sequence including an N-terminal portion of a mature hedgehog protein, said hedgehog amino acid sequence encoded by a nucleic acid which hybridizes under highly stringent conditions to a sequence selected from the group consisting of SEQ ID. NO 1, SEQ ID. NO. 3, SEQ ID NO. 5, SEQ ID. NO. 7, and SEQ ID. NO. 10.

8. An isolated hedgehog polypeptide encoded by a hedgehog gene of a vertebrate organism.

11. An isolated hedgehog polypeptide having at least one biological activity of a vertebrate hedgehog protein, said polypeptide comprising an amino acid sequence at least 80 percent identical with a sequence selected from the group consisting of residues 27-189 of SEQ ID. NO. 2, residues 22-187 of SEQ ID. NO. 4, residues 1-116 of SEQ ID. NO. 6, residues 25-187 of SEQ ID. NO. 8, and residues 24-186 of SEQ ID. NO. 10.

12. The polypeptide of any of claims 2 or 11, wherein said polypeptide includes a hedgehog amino acid sequence at least 90 percent identical with a sequence selected from the group consisting of residues 27-189 of SEQ ID. NO. 2, residues 22-187 of SEQ ID. NO. 4, residues 1-116 of SEQ ID. NO. 6, residues 25-187 of SEQ ID. NO. 8, and residues 24-186 of SEQ ID. NO. 10.

13. The polypeptide of claim 12, wherein said polypeptide includes a hedgehog amino acid sequence at least 95 percent identical with a sequence selected from the group consisting of residues 27-189 of SEQ ID. NO. 2, residues 22-187 of SEQ ID. NO. 4, residues 1-116 of SEQ ID. NO. 6, residues 25-187 of SEQ ID. NO. 8, and residues 24-186 of SEQ ID. NO. 10.

14. The polypeptide of claim 12, wherein said polypeptide includes a hedgehog amino acid sequence identical to a sequence selected from the group consisting of residues 27-189 of SEQ ID. NO. 2, residues 22-187 of SEQ ID. NO. 4, residues 1-116 of SEQ ID. NO. 6, residues 25-187 of SEQ ID. NO.8, and residues 24-186 of SEQ ID. NO. 10.

16. The polypeptide of any of claims 2, 4, or 11, wherein the hedgehog amino acid sequence is encoded by a nucleic acid which hybridizes under highly stringent conditions to a sequence selected from the group consisting of SEQ ID. NO. 1, SEQ ID. NO. 3, SEQ ID. NO. 5, SEQ ID. NO. 7, and SEQ ID. NO. 9.

34. The polypeptide of any of claims 3, 4, 5, 8 or 11, wherein the polypeptide is purified to at least 80% by dry weight.

35. The polypeptide of claim 34, wherein the polypeptide is purified to at least 95% by dry weight.

15. An isolated hedgehog polypeptide comprising an amino acid sequence is encoded by at least a portion of a hedgehog gene of vertebrate origin comprising residues 64-561 of SEQ ID. NO. 3, residues 1-348 of SEQ ID. NO. 5 and residues 73-561 of SEQ ID NO. 7.

22. An hedgehog protein represented in SEQ ID. NO. 2, SEQ ID. NO. 4, SEQ ID. NO. 6, SEQ ID. NO. 8, and SEQ ID. NO. 10.