Vertebrate embryonic pattern-inducing proteins, and uses related thereto

• US 5,844,079 A
• Filed: 12/14/1994
• Issued: 12/01/1998
• Est. Priority Date: 12/30/1993
• Status: Expired due to Term

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), Harvard University, 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:
  US08/356,060
• Time in Patent Office: 1,448 Days
• Field of Search: 435/7.1, 435/65.1, 435/252.3, 435/320.1, 435/325, 530/300, 530/390, 536/23.1, 536/23.5
• 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 polypeptide which polypeptide comprises a hedgehog amino acid sequence comprising an C-terminal portion of a vertebrate hedgehog protein having an approximate molecular weight of 27 kd.

2. The polypeptide of claim 1, which hedgehog amino acid sequence comprises a C-terminal portion of a vertebrate hedgehog protein comprising Asn-189 through Ala-475 of SEQ ID No. 13.

3. An isolated and/or recombinantly produced hedgehog polypeptide comprising an amino acid sequence at least 80 percent identical with a hedgehog amino acid sequence selected from a group consisting of residues 27-425 of SEQ ID No:

8, residues 22-396 of SEQ ID No;

9, 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, or a soluble extracellular fragment thereof of at least 150 contiguous amino acids, which polypeptide (i) binds to a patched protein, (ii) regulates differentiation of neuronal cells, (iii) regulates survival of differentiated neuronal cells, (iv) regulates proliferation of chondrocytes, (v) regulates proliferation of testicular germ line cells, or (vi) functionally replaces drosopholia hedgehog in transgenic drosophila fly.

26. The polypeptide of any of claims 6, 3, 4, or 5, which polypeptide is postranslationally modified.

27. The polypeptide of claim 26, which polypeptide is glycosylated.

4. An isolated or recombinantly produced hedgehog polypeptide having an amino acid sequence crossreactive with an antibody which specifically binds a hedgehog protein having an amino acid sequence selected from the group consisting of SEQ ID No:

8, SEQ ID No;

9, SEQ ID No;

10, SEQ ID No;

11, SEQ ID No;

12, SEQ ID No;

13 and SEQ ID No;

14, or a soluble extracellular fragment thereof of at least 150 contiguous and acids, which polypeptide (i) binds to a patched protein, (ii) regulates differentiation of neuronal cells, (iii) regulates survival of differentiated neuronal cells, (iv) regulates proliferation of chondrocytes, (v) regulates proliferation of testicular germ line cells, or (vi) functionally replaces drosopholia hedgehog in transgenic drosophila fly.

5. An isolated extracellular domain of a hedgehog polypeptide having an amino acid sequence encoded by a nucleic acid which hybridizes under highly stringent conditions to a nucleic acid sequence selected from the group consisting of SEQ ID No:

1, SEQ ID No 2, SEQ ID No;

3, SEQ ID No;

4, SEQ ID No;

5, SEQ ID No;

6 and SEQ ID No;

7, and which polypeptide (i) binds to a patched protein, (ii) regulates differentiation of neuronal cells (iii) regulates survival of differentiated neuronal cells, (iv) regulates proliferation of chondrocytes, (v) regulates proliferation of testicular germ line cells, (vi) functionally replaces drosopholia hedgehog in transgenic drosophila fly.

6. An isolated and/or recombinantly produced polypeptide comprising a hedgehog amino acid sequence which is at least 80 percent identical to a hedgehog protein selected from the group consisting of SEQ ID No:

8, SEQ ID No;

9, SEQ ID No;

10, SEQ ID No;

11, SEQ ID No;

12, SEQ ID No;

13 and SEQ ID No;

14, or a fragment thereof, which hedgehog amino acid sequence (i) binds to a patched protein, (ii) regulates differentiation of neuronal cells, (iii) regulates survival of differentiated neuronal cells, (iv) regulates proliferation of chondrocytes, (v) regulates proliferation of testicular germ line cells, or (vi) functionally replaces drosopholia hedgehog in transgenic drosophila fly.

7. The polypeptide of claim 6, which polypeptide is a fusion protein.

8. The polypeptide of claim 7, wherein fusion protein further includes (i) a detectable label for detecting the presence of said fusion protein, or (ii) a matrix-binding domain for immobilizing said fusion protein.

9. The polypeptide of claim 6, wherein the hedgehog amino acid sequence which is at least 90 percent identical to a hedgehog protein selected from the group consisting of SEQ ID No:

8, SEQ ID No;

9, SEQ ID No;

10, SEQ ID No;

11, SEQ ID No;

12, SEQ ID No;

13 and SEQ ID No;

14.

10. The polypeptide of claim 6, wherein the hedgehog amino acid sequence which is at least 95 percent identical to a hedgehog protein selected from the group consisting of SEQ ID No:

8, SEQ ID No;

9, SEQ ID No;

10, SEQ ID No;

11, SEQ ID No;

12, SEQ ID No;

13 and SEQ ID No;

14.

11. The polypeptide of claim 6, wherein the hedgehog amino acid sequence is identical to a hedgehog protein selected from the group consisting of SEQ ID No:

8, SEQ ID No;

9, SEQ ID No;

10, SEQ ID No;

11, SEQ ID No;

12, SEQ ID No;

13 and SEQ ID No;

14.

12. The polypeptide of claim 6, wherein the hedgehog amino acid sequence is encoded by a naturally occurring hedgehog gene of a mammal.

13. The polypeptide of claim 6, wherein the hedgehog amino acid sequence comprises an extracellular fragment of a hedgehog protein.

14. The polypeptide of claim 13, which fragment has an approximate molecular weight of 19 kd.

15. The polypeptide of claim 6, wherein the polypeptide binds to a patched protein.

16. The polypeptide of claim 15, wherein the patched protein is a patched protein of a vertebrate organism.

17. The polypeptide of claim 6, wherein the polypeptide promotes differentiation of neuronal cells or survival of differentiated neuronal cells.

18. The polypeptide of claim 17, wherein the neuronal cell is a dopaminergic neuron.

19. The polypeptide of claim 17, wherein the neuronal cell is a motorneuron.

20. The polypeptide of claim 6, wherein the polypeptide regulates proliferation of chondrocytes.

21. The polypeptide of claim 6, wherein the polypeptide induces expression BMP-2, BMP-4, Islet1, Pax1, or Hoxd genes.

22. The polypeptide of claim 6, which hedgehog amino acid sequence comprises an N-terminal portion of a vertebrate hedgehog protein comprising Cys-23 through Asp 189 of SEQ ID No. 9.

23. The polypeptide of claim 6, which hedgehog amino acid sequence comprises an N-terminal portion of a vertebrate hedgehog protein comprising Cys-24 through Glu-188 of SEQ ID No. 13.

24. The polypeptide of any of claims 14, 6, 13, 15, 17, 20, or 21, which polypeptide comprises an amino acid sequence represented by SEQ ID No. 40.

25. The polypeptide of any of claims 14, 6, 13, 15, 17, 20, or 21, which polypeptide comprises an amino acid sequence represented by SEQ ID No. 41.

28. The polypeptide of any of claims 6, 23, 9, 10, 12, 13, 15, 17, 20, 21, or 22, wherein the hedgehog amino acid sequence is encoded by a nucleic acid sequence that is hybridizes under highly stringent conditions with a nucleic acid molecule selected from the group consisting of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6 and SEQ ID NO;

7.

29. The polypeptide of any of claims 6 or 15, wherein the hedgehog amino acid sequence includes at least 50 contiguous amino acids of an extracellular domain of the hedgehog protein.

30. The polypeptide of claim 29, wherein the hedgehog amino acid sequence includes at least 100 contiguous amino acids of an extracellular domain of the hedgehog protein.

31. The polypeptide of claim 29, wherein the hedgehog amino acid sequence includes at least 150 contiguous amino acids of an extracellular domain of the hedgehog protein.

32. The polypeptide of any of claims 6 or 13, wherein the polypeptide is purified to have less than 20% by dry weight of extracellular proteins.

33. The polypeptide of claim 32, wherein the polypeptide is purified to have less than 5% by dry weight of extracellular proteins.

34. The polypeptide of any of claims 6 or 32, wherein the polypeptide is recombinantly produced.

35. The polypeptide of claim 28, which polypeptide includes an amino acid sequence of a Desert hedgehog protein encoded by a nucleic acid which hybridizes under highly stringent conditions to the nucleic acid sequence of SEQ ID No. 2.

36. The polypeptide of claim 35, wherein the hedgehog amino acid sequence is encoded by a nucleic acid which hybridizes under highly stringent conditions to the nucleic acid of SEQ ID No. 2.

37. The polypeptide of claim 28, which polypeptide includes an amino acid sequence of an Indian hedgehog protein encoded by a nucleic acid which hybridizes under highly stringent conditions to a nucleic acid sequence selected from the group consisting of SEQ ID No. 3 and SEQ ID No. 7.

38. The polypeptide of claim 6, wherein the hedgehog amino acid sequence is encoded by a nucleic acid which hybridizes under highly stringent conditions to the nucleic acid of SEQ ID No. 7.

39. The polypeptide of claim 28, which polypeptide includes an amino acid sequence of a Sonic hedgehog protein encoded by a nucleic acid which hybridizes under highly stringent conditions to a nucleic acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6.

40. The polypeptide of claim 39, wherein the hedgehog amino acid sequence is encoded by a nucleic acid which hybridizes under highly stringent conditions to the nucleic acid of SEQ ID No. 6.

41. The polypeptide of claim 40, wherein the hedgehog amino acid sequence is encoded by a naturally occurring hedgehog gene of a human.