Glucose-dependent insulin-secreting cells transfected with a nucleotide sequence encoding GLP-1

• US 20030186436A1
• Filed: 03/12/2002
• Published: 10/02/2003
• Est. Priority Date: 03/12/2002
• Status: Active Grant

• Current Assignee: Cedars-Sinai Medical Center (Cedars Sinai Health System)
• Original Assignee: Cedars-Sinai Medical Center (Cedars Sinai Health System)
• Inventors: Hui, Hongxiang, Perfetti, Riccardo
• Granted Patent: US 7,141,240 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: A61K 38/26 Glucagons
• 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: A61K 48/0058 Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
• CPC Class Codes: C07K 14/605 Glucagons

Abstract

Disclosed herein are cells that secrete insulin in a glucose-dependent manner. The cell line comprises insulin-secreting cells that have been transfected with a minigene construct comprising a nucleotide sequence encoding for glucagon-like peptide-1 (GLP-1). In preferred embodiments, the minigene construct is operatively associated with a promoter. The cell line may be used to treat diabetes or other conditions in which delivering insulin in a glucose-dependent manner would be advantageous, to investigate the function and development of pancreatic cells, and to test the efficacy of drugs that stimulate insulin secretion. The cells may be implanted in a mammal, or may be included in a device that resides exterior to the mammal, yet which delivers insulin to the mammal in response to the glucose level of a body fluid in contact therewith. The minigene construct may also be implemented in conjunction with an in vivo gene transfer approach.

Claims

1. An insulin-secreting cell, comprising an insulin-secreting cell transfected with a nucleotide sequence encoding a protein selected from the.group consisting of glucagon-like peptide-1 (GLP-1) and an analog of GLP-1.

2. The insulin-secreting cell of claim 1, wherein the nucleotide sequence encodes GLP-1.

3. The insulin-secreting cell of claim 2, wherein the nucleotide sequence encodes SEQ ID NO. 2.

4. The insulin-secreting cell of claim 2, wherein the nucleotide sequence comprises the coding region for human GLP-1, a partial fragment of an IP sequence at the 5′

-end, and a fragment of a GLP-2 sequence at the 3′

-end.

5. The insulin-secreting cell of claim 4, wherein the nucleotide sequence comprises SEQ ID NO. 6.

6. The insulin-secreting cell of claim 1, wherein the nucleotide sequence encodes an analog of GLP-1.

7. The insulin-secreting cell of claim 6, wherein the nucleotide sequence encodes a protein selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5.

8. The insulin-secreting cell of claim 1, wherein the nucleotide sequence is operatively associated with a promoter.

9. The insulin-secreting cell of claim 8, wherein the promoter is selected from the group consisting of a human promoter that has a glucose-responsive element and an insulin promoter of an animal species.

10. The insulin-secreting cell of claim 8, wherein the promoter is selected from the group consisting of rat insulin II promoter (RIP) and rat insulin 7 promoter.

21. The method of claim 8, wherein the minigene construct is inserted at the Hind-III and Pst-I sites of the plasmid.

11. A method for constructing an insulin-dependent glucose-secreting cell, the method comprising the steps of:

providing an insulin-secreting cell;

isolating from proglucagon a minigene construct comprising a nucleotide sequence comprising (a) the coding region for a protein selected from the group consisting of glucagon-like peptide-1 (GLP-1) and an analog of GLP-1;

(b) a partial fragment of an IP sequence at the 5′

-end; and

(c) a partial fragment of a glucagon-like peptide-2 (GLP-2) sequence at the 3′

-end;

providing a plasmid;

transfecting the plasmid with the minigene construct; and

including the plasmid in the insulin-secreting cell.

12. The method of claim 11, wherein the protein is GLP-1.

13. The method of claim 12, wherein the nucleotide sequence comprises SEQ ID NO. 6.

14. The method of claim 12, wherein the GLP-1 comprises SEQ ID NO. 2.

15. The method of claim 11, wherein the protein is an analog of GLP-1.

16. The method of claim 15, wherein the protein is selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5.

17. The method of claim 11, the method further comprising the steps of transfecting the plasmid with a promoter and inserting the minigene construct into the plasmid at a point such that the minigene construct is operatively associated with the promoter.

18. The method of claim 11, wherein the promoter is selected from the group consisting of a human promoter that has a glucose-responsive element and an insulin promoter of an animal species.

19. The method of claim 11, wherein the promoter is selected from the group consisting of rat insulin II promoter (RIP) and rat insulin 7 promoter.

20. The method of claim 11, wherein the plasmid is a pSecTag2 plasmid, the method further comprising the steps of:

deleting from the plasmid the cytomegalovirus (CMV) sequence;

transfecting the plasmid with a promoter; and

inserting the minigene construct into the plasmid at a point such that the minigene construct is operatively associated with the promoter.

22. A method of determining the ability of a drug to stimulate cells to secrete insulin, the method comprising the steps of:

providing an insulin-secreting cell transfected with a nucleotide sequence encoding a protein selected from the group consisting of glucagon-like peptide-1 (GLP-1) and an analog of GLP-1;

exposing the cell to the drug; and

measuring the amount of insulin secreted by the cell.

23. The method of claim 22, wherein the nucleotide sequence encodes GLP-1.

24. The method of claim 23, wherein the nucleotide sequence encodes SEQ ID NO. 2.

25. The method of claim 23, wherein the nucleotide sequence comprises the coding region for human GLP-1, a partial fragment of an IP sequence at the 5′

-end, and a fragment of a GLP-2 sequence at the 3′

-end.

26. The method of claim 25, wherein the nucleotide sequence comprises SEQ ID NO. 6.

27. The method of claim 22, wherein the nucleotide sequence encodes an analog of GLP-1.

28. The method of claim 27, wherein the nucleotide sequence encodes a protein selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5.

29. The method of claim 22, wherein the nucleotide sequence is operatively associated with a promoter.

30. The method of claim 29, wherein the promoter is selected from the group consisting of a human promoter that has a glucose-responsive element and an insulin promoter of an animal species.

31. The method of claim 29, wherein the promoter is selected from the group consisting of rat insulin II promoter (RIP) and rat insulin 7 promoter.

32. The method of claim 22, wherein the step of exposing the cells to the drug comprises exposing the cells to a sulfonylurea.

33. The method of claim 22, wherein the step of exposing the cells to the drug comprises exposing the cells to a drug selected from the group consisting of metformin, acarbose, troglitazone, and repaglinide.

34. A method of supplying insulin to a mammal, the method comprising the steps of:

providing insulin-secreting cells transfected with a nucleotide sequence encoding a protein selected from the group consisting of glucagon-like peptide-1 (GLP-1) and an analog of GLP-1;

exposing the cells to a body fluid of the mammal, the body fluid providing an indication of glucose level; and

transferring to the mammal insulin secreted from the cells.

35. The method of claim 34, wherein the nucleotide sequence encodes GLP-1.

36. The method of claim 35, wherein the nucleotide sequence encodes SEQ ID NO. 2.

37. The method of claim 35, wherein the nucleotide sequence comprises the coding region for human GLP-1, a partial fragment of an IP sequence at the 5′

-end, and a fragment of a GLP-2 sequence at the 3′

-end.

38. The method of claim 37, wherein the nucleotide sequence comprises SEQ ID NO. 6.

39. The method of claim 34, wherein the nucleotide sequence encodes a n analog of GLP-1.

40. The method of claim 39, wherein the nucleotide sequence encodes a protein selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5.

41. The method of claim 34, wherein the nucleotide sequence is operatively associated with a promoter.

42. The method of claim 41, wherein the promoter is selected from the group consisting of a human promoter that has a glucose-responsive element and an insulin promoter of an animal species.

43. The method of claim 34, wherein the step of exposing the cell to the body fluid further comprises:

providing a cell-containing device;

including the cells in the cell-containing device; and

introducing the body fluid to the cell-containing device such that the fluid and the cell operably contact one another.

44. The method of claim 43, wherein the cell-containing device resides exterior to the mammal.

45. The method of claim 43, wherein the cell-containing device is implanted in the mammal.

46. The method of claim 34, wherein the body fluid is selected from the group consisting of interstitial fluid in the skin of the mammal and blood.