Nucleic acids encoding hedgehog proteins

• US 6,165,747 A
• Filed: 06/05/1995
• Issued: 12/26/2000
• 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., Bumcrot, David A., Marti-Gorostiza, Elisa
• Primary Examiner(s): Kunz, Gary L.
• Assistant Examiner(s): Kaufman, Claire M.
• Application Number:
  US08/460,900
• Time in Patent Office: 2,031 Days
• Field of Search: 514/44, 530/350, 530/300, 435/69.1, 435/7.1, 435/320.1, 435/69.7, 435/325, 435/252.3, 435/254.11, 424/185.1, 536/23.1, 536/23.5, 536/23.4
• US Class Current: 435/69.1
• 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 2227/50 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 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 2799/027 where the vector is derived from a retrovirus

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 tissue 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 comprising a coding sequence for a polypeptide comprising a hedgehog 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;

2, SEQ ID No;

3, SEQ ID No;

4, SEQ ID No;

5, SEQ ID No;

6 and SEQ ID No;

7, which hedgehog amino acid sequence binds to a naturally occurring patched receptor.

2. The nucleic acid of claim 1, which nucleic acid encodes a hedgehog polypeptide that promotes at least one of proliferation, differentiation or survival of mesodermal or ectodermal cells.

3. The nucleic acid of claim 1, further comprising a transcriptional regulatory sequence operably linked to said nucleotide sequence so as to render said nucleic acid suitable for use as an expression vector.

4. An expression vector, which replicates in at least one of a prokaryotic cell and eukaryotic cell, comprising the nucleic acid of claim 1.

5. A host cell transfected with the expression vector of claim 4 and expressing said polypeptide.

6. A method of producing a recombinant hedgehog polypeptide comprising culturing the cell of claim 5 in a cell culture medium to express said hedgehog polypeptide, and expressing and isolating said polypeptide from said cell culture.

7. The nucleic acid of claim 1, wherein the coding sequence for the hedgehog amino acid sequence is a coding sequence of a mammalian hedgehog gene.

8. The nucleic acid of claim 7, wherein the hedgehog gene is a human hedgehog gene.

9. The nucleic acid of claim 1, wherein the hedgehog amino acid sequence is an extracellular fragment of a hedgehog protein.

12. The nucleic acid of claim 1 or 10, wherein the hedgehog amino acid sequence comprises an amino acid sequence designated in SEQ ID No:

40.

13. The nucleic acid of claim 1 or claim 10, wherein the hedgehog amino acid sequence mimics a biological activity of a wild-type hedgehog protein.

14. The nucleic acid of claim 1 or 10, wherein the hedgehog amino acid sequence of the polypeptide binds to a naturally occurring patched receptor.

15. The nucleic acid of claim 14, wherein said patched receptor is a patched receptor of a vertebrate organism.

16. The nucleic acid of claim 1 or 10, wherein the hedgehog amino acid sequence of the polypeptide promotes differentiation of neuronal cells or survival of differentiated neuronal cells.

17. The nucleic acid of claim 16, wherein the neuronal cell is a dopaminergic neuron.

18. The nucleic acid of claim 16, wherein the neuronal cell is a motorneuron.

19. The nucleic acid of claim 1 or 10, wherein the hedgehog amino acid sequence of the polypeptide regulates proliferation of chondrocytes.

20. The nucleic acid of claim 1 or 10, wherein the hedgehog amino acid sequence of the polypeptide induces expression of BMP-2, BMP-4, or Hoxd genes.

21. An expression vector, which replicates in at least one of a prokaryotic cell and eukaryotic cell, comprising the nucleic acid of claim 1 or 10.

22. A host cell transfected with the expression vector of claim 21 and expressing said recombinant polypeptide.

23. A method of producing a recombinant hedgehog polypeptide comprising culturing the cell of claim 22 in a cell culture medium to express said hedgehog polypeptide and expressing and isolating said hedgehog polypeptide from said cell culture.

24. An in vitro recombinant transfection system, comprising

(i) a gene construct including the nucleic acid of claim 1 or 10 operably linked to a transcriptional regulatory sequence for causing expression of the hedgehog polypeptide in eukaryotic cells, and

(ii) a gene delivery composition for delivering said gene construct to a cell and causing the cell to be transfected with said gene construct.

25. The recombinant transfection system of claim 24, wherein the gene delivery composition is selected from a group consisting of a recombinant viral particle, a liposome, and a poly-cationic nucleic acid binding agent.

26. The recombinant transfection system of claim 24, wherein the transcriptional regulatory sequence is a conditional transcriptional regulatory sequence.

27. The nucleic acid of claim 1 or claim 10, wherein said hedgehog amino acid sequence is encoded by a naturally occurring hedgehog gene.

28. The nucleic acid of claim 27, wherein said hedgehog gene is a mammalian hedgehog gene.

29. The nucleic acid of claim 28, wherein said hedgehog gene is a human hedgehog gene.

30. The nucleic acid of claim 1 or claim 10, wherein said amino acid sequence includes at least 150 amino acid residues of the N-terminal half of a hedgehog protein.

31. The nucleic acid of claim 1 or claim 10, wherein said polypeptide is post-translationally modified.

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

33. The nucleic acid of claim 1 or claim 10, wherein said polypeptide is purified to at least 95% by dry weight.

34. The nucleic acid of claim 1 or claim 10, wherein said polypeptide is purified to at least 80% by dry weight.

35. The nucleic acid of claim 1 or claim 10 wherein the amino acid sequence includes at least 100 contiguous amino acids of the N-terminal half of the hedgehog protein.

36. The nucleic acid of claim 1 or claim 10, wherein the amino acid sequence includes at least 50 contiguous amino acids of the N-terminal half of the hedgehog protein.

39. The nucleic acid of claim 1, 10, 37, or 38, wherein the polypeptide further comprises a heterologous amino acid sequence.

40. The nucleic acid of claim 39, wherein the heterologous amino acid sequence is (i) a detectable label or (ii) a matrix-binding domain.

10. An isolated nucleic acid encoding a polypeptide, which polypeptide comprises a hedgehog amino acid sequence comprising at least 150 contiguous amino acids residues the N-terminal half of a polypeptide 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, which hedgehog amino acid sequence binds to a naturally occurring patched receptor.

11. The nucleic acid of claim 10, wherein the hedgehog amino acid sequence is encoded by a naturally occurring hedgehog gene of a vertebrate organism.

37. A nucleic acid comprising:

(A) a coding sequence for a polypeptide comprising a hedgehog 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;

2, 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; and

(B) a transcriptional regulatory sequence operably linked to said coding sequence and heterologous thereto.

38. A nucleic acid comprising:

(A) a coding sequence for a polypeptide comprising 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 an extracellular fragment thereof of at least 150 amino acids, which amino acid sequence binds to a naturally occurring patched receptor; and

(B) a transcriptional regulatory sequence operably linked to said coding sequence and heterologous thereto.

41. An isolated nucleic acid encoding a fragment of the N-terminal half of a vertebrate polypeptide, which fragment binds to a naturally occurring patched receptor and comprises at least 50 contiguous amino acid residues of a polypeptide 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, wherein said nucleic acid hybridizes under stringent conditions, including a wash step of 0.5 x SSC at 60°

C., 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.

42. An isolated nucleic acid encoding a polypeptide, which polypeptide comprises an amino acid sequence represented by the formula A-B wherein,

A represents

residues 1-168 of SEQ ID No:

40; and

B represents at least one amino acid residue of the amino acid sequence designated by residues

16- 221of SEQ ID No;

40;

wherein A and B together represent a contiguous polypeptide sequence represented by SEQ ID No. 40, and said polypeptide binds a naturally occurring patched receptor.

43. The nucleic acid of claim 42, wherein B represents at least five amino acid contiguous residues of the amino acid sequence designated by residues 169-221 of SEQ ID No:

40.

44. The nucleic acid of claim 42, wherein B represents at least ten contiguous amino acid residues of the amino acid sequence designated by residues 169-221 of SEQ ID No:

40.

45. The nucleic acid of claim 42, which polypeptide further comprises a heterologous amino acid sequence.

46. The nucleic acid of claim 42, which polypeptide agonizes the biological activity of a wild-type sonic hedgehog (Shh) protein.

47. An isolated nucleic acid encoding a polypeptide comprising an amino acid sequence represented by the formula A-B wherein,

A represents

residues 24-193 of SEQ ID No:

13; and

B represents at least one amino acid residue of the amino acid sequence designated by residues 194-250 of SEQ ID No;

13;

wherein A and B together represent a contiguous polypeptide sequence designated in SEQ ID No;

13, and said polypeptide binds to a naturally occurring patched receptor.

48. An isolated nucleic acid encoding a polypeptide comprising an amino acid sequence represented by the formula A-B wherein,

A represents

residues 25-193 of SEQ ID No:

11; and

B represents at least one amino acid residue of the amino acid sequence designated by residues 194-250, or analogous residues thereof, of a vertebrate polypeptide identical or homologous to SEQ ID No;

11;

wherein A and B together represent a contiguous polypeptide sequence identical or homologous to SEQ ID No;

11, and said polypeptide binds to a naturally occurring patched receptor.

49. An isolated nucleic acid encoding a polypeptide comprising an amino acid sequence represented by the formula A-B wherein

A represents residues 28-197 of SEQ ID No:

10; and

B represents at least one amino acid residue of the amino acid sequence designated by residues 198-250, or analogous residues thereof, of a vertebrate polypeptide identical or homologous to SEQ ID No;

10;

wherein A and B together represent a contiguous polypeptide sequence identical or homologous to SEQ ID No;

10, and said polypeptide binds to a naturally occurring patched receptor.

50. An isolated nucleic acid encoding a polypeptide comprising an amino acid sequence represented by the formula A-B wherein,

A represents residues 1-98 of SEQ ID No:

14; and

B represents at least one amino acid residue of the amino acid sequence designated by residues 99-150 of SEQ ID No;

14;

wherein A and B together represent a contiguous polypeptide sequence designated in SEQ ID No;

14, and said polypeptide binds to a naturally occurring patched receptor.