Inducing insulin gene expression in pancreas cells expressing recombinant PDX-1

• US 6,448,045 B1
• Filed: 03/10/2000
• Issued: 09/10/2002
• Est. Priority Date: 03/10/2000
• Status: Expired due to Fees

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Dufayet, Dominique, Levine, Fred
• Primary Examiner(s): Yucel, Remy
• Assistant Examiner(s): LOEB, BRONWEN
• Application Number:
  US09/522,376
• Time in Patent Office: 914 Days
• Field of Search: 435/70.1, 435/455, 435/366, 435/375, 435/69.1, 530/303
• US Class Current: 435/70.1
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• 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: C12N 2501/335 Glucagon; Glucagon-like peptide [GLP]; Exendin
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2510/04 Immortalised cells
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: G01N 2500/10 involving cells

Abstract

The present invention provides methods for inducing insulin gene expression in cultured pancreas cells, the method comprising contacting a culture of endocrine pancreas cells expressing a PDX-1 gene with a GLP-1 receptor agonist, wherein the cells have been cultured under conditions such that the cells are in contact with other cells in the culture, thereby inducing insulin gene expression in the cells. The invention also provides high throughput screening methods for modulators of β-cell function, stable cultures of cells made by the methods of the invention, and methods of treating a human subject using the methods of the invention.

Claims

1. A method,for inducing insulin gene expression in cultured endocrine pancreas cells which initially express no detectable pancreatic hormones, the method comprising the steps of:

(i) expressing a recombinant polynucleotide encoding PDX-1 endocrine pancreas in cells that have been cultured under conditions such that the cells are in cell-to-cell contact in the culture; and

(ii) contacting the cells with a GLP-1 receptor agonist, thereby inducing insulin gene expression in the cells.

2. The method of claim 1, wherein the GLP- 1 receptor agonist is a GLP-1 analog.

3. The method of claim 1, wherein the GLP-1 receptor agonist has an amino acid sequence of a naturally occurring peptide.

4. The method of claim 1, wherein the cells are cultured as aggregates in suspension.

5. The method of claim 1, wherein the cells are human cells.

6. The method of claim 1, wherein the cells further express a recombinant oncogene.

7. The method of claim 1, wherein the cells further express a recombinant polynucleotide encoding telomerase.

8. The method of claim 1, wherein the cells are β

lox5 cells.

9. The method of claim 3, wherein the GLP-1 receptor agonist is GLP-1, exendin-3, or exendin-4.

10. The method of claimed 6, wherein the cells express more than one recombinant oncogene.

11. A stable culture of endocrine pancreas cells which initially expresses no detectable pancreatic hormones, wherein the cells are in contact with other cells in the culture, the cells express a recombinant polynucleotide encoding PDX-1, and insulin gene expression is stimulated in the cells when exposed to an effective amount of a GLP-1 receptor agonist.

12. The culture of claim 11, wherein the GLP-1 receptor agonist is a GLP-1 analog.

13. The culture of claim 11, wherein the GLP-1 receptor agonist has an amino acid sequence of a naturally occurring peptide.

14. The culture of claim 11, wherein the cells are cultured as aggregates in suspension.

15. The culture of claim 11, wherein the cells are human cells.

16. The culture of claim 11, wherein the cells further express a recombinant oncogene.

17. The culture of claim 11, wherein the cells further express a recombinant polynucleotide encoding telomerase.

18. The culture of claim 11, wherein the cells are β

lox5 cells.

19. The culture of claim 13, wherein the GLP-1 receptor agonist is GLP-1, exendin-3, or exendin-4.

20. The culture of claim 16, wherein the cells express more than one recombinant oncogene.