Compositions and methods for inducing gene expression

• US 6,432,927 B1
• Filed: 05/26/2000
• Issued: 08/13/2002
• Est. Priority Date: 12/04/1997
• Status: Expired due to Term

• Current Assignee: Genzyme Corporation (Sanofi )
• Original Assignee: Genzyme Corporation (Sanofi )
• Inventors: Gregory, Richard J., Vincent, Karen
• Primary Examiner(s): NGUYEN, DAVE TRONG
• Application Number:
  US09/579,897
• Time in Patent Office: 809 Days
• Field of Search: 435/320.1, 435/91.4, 435/455, 435/325, 536/23.4, 536/24.1, 424/93.2, 514/44
• US Class Current: 514/44.00R
• 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: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/71 containing domain for transcriptional activaation, e.g. VP16
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2799/022 where the vector is derived from an adenovirus

Abstract

The present invention provides recombinant nucleic acid molecules encoding a chimeric transactivator protein including a DNA binding domain of a DNA binding protein and a protein domain capable of transcriptional activation. The present invention also provides recombinant viral and non-viral vectors that are able to infect and/or transfect and sustain expression of a biologically active chimeric transactivator proteins in mammalian cells. Also provided are host cell lines and non-human transgenic animals capable of expressing biologically active chimeric transactivator proteins. In another aspect, compositions and methods for treating or preventing ischemic damage associated with hypoxia-related disorders are provided.

Claims

1. A nucleic acid molecule encoding a biologically active chimeric transactivator protein comprising:

(a) the DNA binding domain of a hypoxia inducible factor protein; and

(b) a protein domain capable of transcriptional activation which is not derived from a hypoxia inducible factor protein.

2. The nucleic acid molecule according to claim 1, wherein said hypoxia inducible factor protein is HIF-1α

.

3. The nucleic acid molecule according to claim 2, wherein said DNA binding domain of HIF-1α

comprises the amino acid residues that extend from the first amino acid to the Af12 site.

4. The nucleic acid molecule according to claim 1, wherein said protein domain capable of transcriptional activation is derived from a protein selected from the group consisting of:

HSV VP16, NFκ

B, a heat shock factor;

p53;

fos;

v-jun;

factor EF-C;

HIV tat;

HPV E2;

Ad E1A;

Sp1;

AP1;

CTF/NF1;

E2F1;

HAP1;

HAP2;

MCM1;

PHO2;

GAL4, GCN4, and GAL11.

5. The nucleic acid molecule according to claim 4, wherein said hypoxia inducible factor protein is the human hypoxia inducible factor protein HIF-1α

.

6. The nucleic acid molecule according to claim 1, wherein said hypoxia inducible factor protein is HIF-1α

and the protein domain capable of transcriptional activation is a transcriptional activation domain from HSV VP16.

7. The nucleic acid molecule according to claim 6, wherein said DNA binding domain of HIF-1α

comprises the amino acid residues that extend from the first amino acid to the Afl2 site.

8. The nucleic acid molecule according to claim 1, wherein said hypoxia inducible factor protein is HIF-1α

and the protein domain capable of transcriptional activation is a transcriptional activation domain from NFκ

B.

9. The nucleic acid molecule according to claim 8, wherein said DNA binding domain of HIF-1α

comprises the amino acid residues that extend from the first amino acid to the Afl2 site.

10. The nucleic acid molecule according to claim 1, wherein said hypoxia inducible factor protein is the human hypoxia inducible factor protein HIF-1α

.

11. The nucleic acid molecule according to claim 1, wherein said hypoxia inducible factor protein is the human hypoxia inducible factor protein HIF-1α

.

12. An expression vector comprising the nucleic acid molecule according to any one of claims 1-11, operatively linked to an expression control sequence.

13. The expression vector according to claim 12, wherein said expression control sequence comprises an inducible promoter.

14. An isolated host cell comprising the expression vector according to claim 13.

15. A pharmaceutical composition comprising the expression vector according to claim 13 and a pharmaceutically acceptable carrier.

16. A method for reducing ischemic tissue damage in a mammalian subject having a hypoxia-associated disorder comprising the step of administering at the site of said ischemic tissue damage in said subject an effective amount of the pharmaceutical composition according to claim 15.

17. A method for increasing the expression of a hypoxia-inducible gene in a mammalian cell, said method comprising the step of introducing into said mammalian cell in vitro the expression vector according to claim 13 so as to increase the expression of said hypoxia inducible gene in said cell.

18. The expression vector according to claim 12, wherein said expression vector is pcDNA3/HIF/VP16/Afl2.

19. An isolated host cell comprising the expression vector according claim 18.

20. A pharmaceutical composition comprising the expression vector according to claim 18 and a pharmaceutically acceptable carrier.

21. A method for reducing ischemic tissue damage in a mammalian subject having a hypoxia-associated disorder comprising the step of administering at the site of said ischemic tissue damage in said subject an effective amount of the pharmaceutical composition according to claim 20.

22. A method for increasing the expression of a hypoxia-inducible gene in a mammalian cell, said method comprising the step of introducing into said mammalian cell in vitro the expression vector according to claim 18 so as to increase the expression of said hypoxia inducible gene in said cell.

23. The expression vector according to claim 12, wherein said vector is an adenoviral vector.

24. An isolated host cell comprising the expression vector according claim 23.

25. A pharmaceutical composition comprising the expression vector according to claim 23 and a pharmaceutically acceptable carrier.

26. A method for reducing ischemic tissue damage in a mammalian subject having a hypoxia-associated disorder comprising the step of administering at the site of said ischemic tissue damage in said subject an effective amount of the pharmaceutical composition according to claim 25.

27. A method for increasing the expression of a hypoxia-inducible gene in a mammalian cell, said method comprising the step of introducing into said mammalian cell in vitro the expression vector according to claim 23 so as to increase the expression of said hypoxia inducible gene in said cell.

28. An isolated host cell comprising the expression vector according to claim 12.

29. A pharmaceutical composition comprising the expression vector according to claim 12 and a pharmaceutically acceptable carrier.

30. A method for reducing ischemic tissue damage in a mammalian subject having a hypoxia-associated disorder comprising the step of administering at the site of said ischemic tissue damage in said subject an effective amount of the pharmaceutical composition according to claim 29.

31. A method for increasing the expression of a hypoxia-inducible gene in a mammalian cell, said method comprising the step of introducing into said mammalian cell in vitro the expression vector according to claim 12 so as to increase the expression of said hypoxia inducible gene in said cell.