Vertebrate embryonic pattern-inducing proteins and uses related thereto

• US 6,607,913 B1
• Filed: 11/23/1999
• Issued: 08/19/2003
• 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: 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:
  US09/448,188
• Time in Patent Office: 1,365 Days
• Field of Search: 536/23.1, 536/23.5, 536/23.4, 435/69.1, 435/320.1, 435/325, 435/252.3, 435/254.11, 435/69.4, 530/300, 530/350, 514/2, 514/12
• US Class Current: 435/325
• 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 2227/30 Bird
• 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 2267/0312 Animal model for Alzheimer's disease
• 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 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 polypeptide comprising a hedgehog amino acid sequence at least 80% identical to an amino acid sequence selected from SEQ ID No:

8, SEQ ID No;

11, SEQ ID No;

12, or SEQ ID No;

13, or a fragment of said hedgehog amino acid sequence, wherein said hedgehog amino acid sequence, or fragment thereof, 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 polypeptide comprising a hedgehog amino acid sequence and expressing and isolating said polypeptide from said cell.

8. The nucleic acid of claim 1 or 7, wherein the hedgehog amino acid sequence comprises an amino acid sequence designated in SEQ ID No:

40.

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

11. The nucleic acid of claim 1, wherein the hedgehog amino acid sequence is encoded by a mammalian hedgehog gene.

12. The nucleic acid of claim 11, wherein the hedgehog gene is a human hedgehog gene.

13. The nucleic acid of any one of claims 1 or 7, wherein said patched receptor is a patched receptor of a vertebrate organism.

14. The nucleic acid of claim 1 or 7, wherein the hedgehog amino acid sequence of the polypeptide promotes differentiation of a neuronal cells or survival of a differentiated neuronal cell.

15. The nucleic acid of claim 14, wherein the neuronal cell is a dopaminergic neuron.

16. The nucleic acid of claim 14, wherein the neuronal cell is a motor neuron.

17. The nucleic acid of claim 1 or 7, wherein the hedgehog amino acid sequence regulates proliferation of chondrocytes.

18. The nucleic acid of claim 1 or 7, wherein the hedgehog amino acid sequence induces expression of BMP-2, BMP-4, or Hoxd genes.

19. An expression vector, which replicates in at least one of a prokaryotic cell and eukaryotic cell, comprising the nucleic acid of claim 1 or 7.

20. A host cell transfected with the expression vector of claim 19 and expressing said polypeptide.

21. A method of producing a polypeptide comprising a hedgehog amino acid sequence, comprising culturing the cell of claim 20 in a cell culture medium to express said polypeptide comprising a hedgehog amino acid sequence and expressing and isolating said polypeptide comprising a hedgehog amino acid sequence from said cell culture.

22. An in vitro recombinant transfection system, comprising

(i) a gene construct including the nucleic acid of claim 1 or 7 operably linked to a transcriptional regulatory sequence for causing expression of said polypeptide encoded by said nucleic acid 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.

23. The recombinant transfection system of claim 22, wherein the gene delivery composition is selected from a recombinant viral particle, a liposome, and a poly-cationic nucleic acid binding agent.

24. The recombinant transfection system of claim 22, wherein the transcriptional regulatory sequence is a conditional transcriptional regulatory sequence.

25. The nucleic acid of claim 1 or 7, wherein said hedgehog amino acid sequence is encoded by a naturally occurring hedgehog gene.

26. The nucleic acid of claim 25, wherein said hedgehog gene is a mammalian hedgehog gene.

27. The nucleic acid of claim 26, wherein said hedgehog gene is a human hedgehog gene.

28. The nucleic acid of claim 1 or 7, wherein said amino acid sequence includes at least 150 amino acid residues of the N-terminal half of a hedgehog protein.

29. The nucleic acid of claim 1 or 7, wherein the amino acid sequence includes at least 100 contiguous amino acids of the N-terminal half of the hedgehog protein.

30. The nucleic acid of claim 1 or 7, wherein the amino acid sequence includes at least 50 contiguous amino acids of the N-terminal half of the hedgehog protein.

31. The nucleic acid of claim 1, which nucleic acid encodes a polypeptide comprising a hedgehog amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID No:

8, SEQ ID No;

11, SEQ ID No;

12, or SEQ ID No;

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

32. The nucleic acid of claim 1, which nucleic acid encodes a polypeptide comprising a hedgehog amino acid sequence at least 95% identical to an amino acid sequence selected from SEQ ID No:

8, SEQ ID No;

9, SEQ ID No;

10, SEQ ID No;

11, SEQ ID No;

12, or SEQ ID No;

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

33. The nucleic acid of claim 1, which nucleic acid encodes a polypeptide comprising a hedgehog amino acid sequence at least 98% identical to an amino acid sequence selected from SEQ ID No:

8, SEQ ID No;

9, SEQ ID No;

10, SEQ ID No;

11, SEQ ID No;

12, or SEQ ID No;

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

35. The nucleic acid of claim 1, 7, or 34, wherein the encoded polypeptide further comprises a heterologous amino acid sequence.

36. The nucleic acid of claim 35, wherein the heterologous amino acid sequence is (i) a detectable label or (ii) a matrix-binding domain.

7. An isolated nucleic acid encoding a polypeptide, which polypeptide comprises a hedgehog amino acid sequence or a fragment thereof, which sequence or fragment comprises at least 150 contiguous amino acid residues from the N-terminal half of a polypeptide selected from SEQ ID No:

8, SEQ ID No;

11, SEQ ID No;

12, or SEQ ID No;

13, wherein said hedgehog amino acid sequence or fragment thereof binds to a naturally occurring patched receptor.

9. The nucleic acid of claim 7, wherein the hedgehog amino acid sequence is encoded by a naturally occurring hedgehog gene of a vertebrate organism.

34. A nucleic acid comprising:

(A) a coding sequence for a polypeptide comprising a hedgehog amino acid sequence at least 80% identical to an amino acid sequence selected from SEQ ID No;

8, SEQ ID No;

11, SEQ ID No;

12, or SEQ ID No;

13, 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.