Muteins of hypoxia inducible factor alpha and methods of use thereof

• US 20030045686A1
• Filed: 03/19/2002
• Published: 03/06/2003
• Est. Priority Date: 03/20/2001
• Status: Active Grant

• Current Assignee: Dana-Farber Cancer Institute
• Original Assignee: Dana-Farber Cancer Institute
• Inventors: Kaelin, William G. Jr., Ivan, Mircea
• Granted Patent: US 6,849,718 B2
• Time in Patent Office: Days
• US Class Current: 530/350
• CPC Class Codes: A61K 38/1709 from mammals
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: G01N 2333/90245 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: G01N 2500/02 Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes

Abstract

The present invention provides hypoxia inducible factor alpha muteins, DNA sequences encoding these hypoxia inducible factor alpha muteins, recombinant DNA molecules containing those DNA sequences operatively linked to expression control sequences and capable of inducing expression of an hypoxia inducible factor alpha muteins, hosts transformed with those recombinant DNA molecules, pharmaceutical compositions containing hypoxia inducible factor alpha muteins and methods of using those compositions to treat hypoxia and ischemic related tissue damage.

Claims

1. A hypoxia inducible factor (HIF) mutein polypeptide wherein one or more of a hydroxylatable amino acid residue in a wild type HIFα

polypeptide are mutated, said HIF mutein displaying decreased binding to VHL, increased resistance to degradation in the presence of oxygen and comprises both functional wild type HIFα

polypeptide transactivation domains.

2. The HIF mutein polypeptide of claim 1, wherein said hydroxylatable amino acid residue is a proline.

3. The HIF mutein polypeptide of claim 2, wherein said proline is at position 564 in wild type HIFα

, numbered in accordance with wild type HIFα

.

12. A nucleic acid encoding a polypeptide of claim 1-10 or 11.

14. An expression vector comprising a nucleic acid of claim 12 or 13 operably linked to a promoter.

15. A host cell containing the expression vector of claim 14.

16. A pharmaceutical composition comprising the HIFα

mutein any one of claims 1-11 and a pharmaceutically acceptable carrier.

17. A pharmaceutical composition comprising nucleic acid of claim 12 or 13 and a pharmaceutically acceptable carrier.

18. An antibody that specifically binds to the HIFA mutein any one of claims 1-11.

19. A method for increasing the expression of at least one gene in a cell whose expression is activated by a HIF polypeptide comprising contacting the cell with the expression vector or claim 14.

20. The method of claim 16, wherein the cell is provide in vivo, in vitro or ex vivo.

21. A method of reducing, preventing or treating hypoxia or ischemia-related tissue damage in a subject having or at risk of having such damage comprising administering to the subject a therapeutically effective amount of:

(a) the nucleic acid sequence of claim 12 or 13 or

(b) the polypeptide of claim 1-10 or 11

thereby reducing the tissue damage.

22. A method of increasing angiogenesis or vascularization in a subject, comprising administering to the subject a therapeutically effective amount of:

(a) the nucleic acid sequence of claim 12 or 13 or

(b) the polypeptide of claim 1-10 or 11

thereby increasing a angiogenesis or vascularization.

4. A hypoxia inducible factor (HIF) mutein polypeptide wherein the amino acid at position 564 in wild type HIFα

, numbered in accordance with wild type HIFα

is not a proline, said HIF mutein displaying decreased binding to VHL and increased resistance to degradation in the presence of oxygen.

8. The HIF mutein polypeptide of claim 4, 5, 6, or 7 with an additional substitution such that the amino acid at position 402 is not a proline.

9. The HIF mutein polypeptide of claim 4 or 5, wherein the amino acid at position 564 is an alanine.

5. A hypoxia inducible factor (HIF) mutein polypeptide comprising the amino acid sequence at least 85% similar to wild type HIFα

, wherein the amino acid at position 564 in wild type HIFα

, numbered in accordance with wild type HIFα

is not a proline, said HIF mutein displaying decreased binding to VHL and increased resistance to degradation in the presence of oxygen.

6. A hypoxia inducible factor (HIF) mutein polypeptide wherein the amino acid at position 562 in wild type HIFα

, numbered in accordance with wild type HIFα

is not a leucine, said HIF mutein displaying decreased binding to VHL and increased resistance to degradation in the presence of oxygen.

10. The HIF mutein polypeptide of claim 6 or 7, wherein the amino acid at position 562 is an alanine.

7. A hypoxia inducible factor (HIF) mutein polypeptide comprising the amino acid sequence at least 85% similar to wild type HIFα

, wherein the amino acid at position 562 in wild type HIFα

, numbered in accordance with wild type HIFα

is not a leucine, said HIF mutein displaying decreased binding to VHL and increased resistance to degradation in the presence of oxygen.

11. A HIF mutein polypeptide comprising the amino acid sequence of SEQ ID NO:

2 or SEQ ID NO;

4.

13. The nucleic acid comprising the nucleotide sequence of SEQ ID NO:

1 or SEQ ID NO;

3).