Nucleic acids encoding hedgehog proteins

• US 6,664,075 B2
• Filed: 12/13/2000
• Issued: 12/16/2003
• Est. Priority Date: 12/30/1993
• Status: Expired due to Fees

• 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): Spector, Lorraine
• Assistant Examiner(s): Kaufman, Claire M.
• Application Number:
  US09/736,476
• Publication Number:
  US 20030190696A1
• Time in Patent Office: 1,098 Days
• Field of Search: 536/23.1, 536/23.5, 536/23.4, 530/300, 530/350, 435/69.1, 435/325, 435/252.3, 435/69.7, 435/254.11, 435/320.1
• 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 encoding a polypeptide comprising an amino acid sequence at least 80 percent identical to a protein sequence selected from at least one of SEQ ID No:

8, SEQ ID No;

10, SEQ ID No;

11, SEQ ID No;

12, SEQ ID No;

13, SEQ ID No;

14 or an N-terminal fragment of at least 150 contiguous amino acid residues of said protein sequence, which amino acid sequence is not identical to SEQ ID No;

34, and wherein said polypeptide binds to a naturally occurring patched protein.

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

3. The nucleic acid of claim 1, which nucleic acid hybridizes under stringent conditions, including a wash step of 0.2×

SSC at 65°

C., to a nucleic acid probe having a sequence represented by at least 12 consecutive nucleotides of a nucleic acid selected from at least one of SEQ ID No;

1, SEQ ID No;

3, SEQ ID No;

4, SEQ ID No;

5, SEQ ID No;

6 or SEQ ID No;

7.

4. 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.

5. An expression vector, capable of replicating in at least one of a prokaryotic cell and eukaryotic cell, comprising the nucleic acid of claim 1.

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

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

8. The nucleic acid of claim 1, which nucleic acid encodes a polypeptide comprising an amino acid sequence at least 90 percent identical to a protein sequence selected from at least one of SEQ ID No:

8, SEQ ID No;

10, SEQ ID No;

11, SEQ ID No;

12, SEQ ID No;

13, SEQ ID No;

14 or an N-terminal fragment of at least 150 contiguous amino acid residues of said protein sequence.

9. The nucleic acid of claim 1 or 8, which polypeptide comprises an amino acid sequence designated in SEQ ID No:

40.

10. The nucleic acid of claim 8, which polypeptide is a fusion protein.

11. The nucleic acid of claim 1, which nucleic acid encodes a polypeptide comprising an amino acid sequence at least 80 percent identical to an amino acid sequence selected from at least one of SEQ ID No:

8, SEQ ID No;

11, SEQ ID No;

12 or SEQ ID No;

13.

12. The nucleic acid of claim 1, which nucleic acid encodes a polypeptide comprising an amino acid sequence at least 80 percent identical to an amino acid sequence selected from at least one of SEQ ID No:

10 or SEQ ID No;

14.

13. The nucleic acid of claim 12, which nucleic acid encodes a polypeptide comprising an amino acid sequence identical to an amino acid sequence selected from SEQ ID No:

10 or SEQ ID No;

14.

14. The nucleic acid of claim 1, which polypeptide is a mammalian polypeptide.

15. The nucleic acid of claim 1, which nucleic acid encodes a polypeptide that promotes proliferation, differentiation or survival of neuronal cells.

16. The nucleic acid of claim 15, wherein the neuronal cell is a neuronal stem cell.

17. The nucleic acid of claim 15, wherein the neuronal cell is a dopaminergic neuron.

18. The nucleic acid of claim 15, wherein the neuronal cell is a motorneuron.

19. The nucleic acid of claim 1, wherein the nucleic acid encodes a polypeptide that promotes proliferation, differentiation or survival of chondrocytes.

20. An isolated nucleic acid encoding a polypeptide comprising an amino acid sequence which nucleic acid hybridizes under stringent conditions, including a wash step of 0.2×

SSC at 65°

C., to a nucleic acid sequence selected from at least one of SEQ ID No;

1, SEQ ID No;

3, SEQ ID No;

4, SEQ ID No;

5, SEQ ID No;

6 or SEQ ID No;

7, which polypeptide binds to a naturally occurring patched protein.

21. The nucleic acid of claim 20, which nucleic acid encodes a polypeptide that increases at least one of proliferation, differentiation or survival of mesodermal or ectodermal cells.

22. The nucleic acid of claim 20, further comprising a transcriptional regulatory sequence operably linked to said nucleic acid sequence so as to render said nucleic acid suitable for use as an expression vector.

23. An expression vector, comprising the nucleic acid of claim 20, wherein said expression vector is capable of replicating in at least one of a prokaryotic cell and eukaryotic cell.

24. A host cell transfected with the expression vector of claim 23 and expressing said polypeptide.

25. A method of producing a recombinant polypeptide encoded by an isolated nucleic acid comprising culturing the cell of claim 24 in a cell culture medium to express said polypeptide and isolating said polypeptide from said cell culture.

26. The nucleic acid of claim 20, wherein said nucleic acid encodes a polypeptide having a molecular weight of approximately 19 kDa.

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

A represents an amino acid sequence identical to 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 169-221 of SEQ ID No;

40;

wherein A and B together represent a contiguous polypeptide sequence identical to the corresponding sequence represented in SEQ ID No;

40, and said polypeptide binds to a naturally occurring patched protein.

28. The nucleic acid of claim 27, wherein B represents at least five amino acid residues of the amino acid sequence designated by residues 169-221 of SEQ ID No:

40.

29. The nucleic acid of claim 27, wherein B represents at least ten amino acid residues of the amino acid sequence designated by residues 169-221 of SEQ ID No:

40.

30. The nucleic acid of claim 27, which polypeptide is a fusion protein.

31. The nucleic acid of claim 27, which polypeptide agonizes the biological activity of a wild-type sonic hedgehog (Shh) protein.

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

A represents an amino acid sequence identical to 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 at least 80 percent identical to SEQ ID No;

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

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

A represents an amino acid sequence identical to 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 of SEQ ID No;

11;

wherein A and B together represent a contiguous polypeptide sequence at least 80 percent identical to SEQ ID No;

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

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

A represents an amino acid sequence identical to 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 of SEQ ID No;

10;

wherein A and B together represent a contiguous polypeptide sequence at least 80 percent identical to SEQ ID No;

10, and said polypeptide binds a naturally occurring patched protein.

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

A represents an amino acid sequence identical to residues 1-98 of SEQ ID No:

14; and

B represents at least one amino acid residues 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 at least 80 percent identical to SEQ ID No;

14, and said polypeptide binds a naturally occurring patched protein.