Hexokinase inhibitors

• US 5,854,067 A
• Filed: 01/19/1996
• Issued: 12/29/1998
• Est. Priority Date: 01/19/1996
• Status: Expired due to Fees

• Current Assignee: Betagene Incorporated, Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Betagene Incorporated, Board of Regents of the University of Texas System (University of Texas System)
• Inventors: Han, He-Ping, Normington, Karl D., Newgard, Christopher B.
• Primary Examiner(s): LeGuyader, John L.
• Assistant Examiner(s): WANG, ANDREW J
• Application Number:
  US08/588,983
• Time in Patent Office: 1,075 Days
• Field of Search: 435/325, 435/4, 435/6, 435/69.1, 435/320.1, 435/172.3, 424/94.1, 536/23.1, 536/24.5, 514/44, 576/24.31
• US Class Current: 435/366
• CPC Class Codes: C07K 14/62 Insulins
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/121 Hammerhead
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12Y 207/01001 Hexokinase (2.7.1.1)

Abstract

Disclosed are compositions and methods for inhibiting hexokinase enzymes in mammalian cells. Specifically provided are proteins that stimulate the production of trehalose-6-phosphate and their respective genes; hexokinase-specific ribozymes and genes encoding such constructs; and agents that competitively reduce hexokinase activity, e.g., by displacing hexokinase from mitochondria, and their respective genes. The latter group of agents includes inactive hexokinases and fragments thereof that retain mitochondrial binding functions and hexokinase-glucokinase chimeras that further substitute glucokinase activity for hexokinase activity. Mammalian cells including such hexokinase inhibitors, methods of making such cells and various in vitro and in vivo methods of using cells with reduced hexokinase activity are also described herein.

Claims

1. A mammalian host cell comprising an inhibitor of a low K_m hexokinase, the inhibitor selected from:

(a) a trehalose-6-phosphate synthase enzyme that stimulates the production of trehalose-6-phosphate;

or

(b) a ribozyme specific for an RNA transcript of a low K_m hexokinase gene;

wherein said inhibitor is present in an amount effective to reduce the low K_m hexokinase activity of said cell.

2. The cell of claim 1, wherein said cell comprises a trehalose-6-phosphate synthase enzyme that stimulates the production of trehalose-6-phosphate.

3. The cell of claim 1, wherein said cell comprises a ribozyme specific for an RNA transcript of a low K_m hexokinase gene.

4. The cell of claim 1, wherein said cell further comprises a recombinant gene that expresses a selected protein.

5. The cell of claim 1, wherein said cell is a neuroendocrine cell.

6. An engineered host cell that secretes insulin in response to glucose, the cell comprising an inhibitor of a low K_m hexokinase, the inhibitor selected from:

(a) a trehalose-6-phosphate synthase enzyme that stimulates the production of trehalose-6-phosphate;

or

(b) a ribozyme specific for an RNA transcript of a low K_m hexokinase gene;

wherein said inhibitor is present in an amount effective to reduce the low K_m hexokinase activity of said cell.

7. The cell of claim 6, wherein said inhibitor is introduced into said cell by means of a recombinant gene that expresses the inhibitor.

8. The cell of claim 7, wherein said inhibitor is introduced into said cell by means of a recombinant vector comprising a promoter operatively linked to a gene that encodes the inhibitor, the promoter expressing said inhibitor in said cell.

9. The cell of claim 6, wherein said inhibitor reduces the hexokinase I activity of said cell.

10. The cell of claim 6, wherein said inhibitor reduces the hexokinase II activity of said cell.

11. The cell of claim 6, wherein said cell has reduced low K_m hexokinase activity relative to a parent cell from which it was prepared.

12. The cell of claim 6, wherein said low K_m hexokinase activity is reduced to an amount effective to allow insulin secretion in response to an extracellular glucose concentration of between about 1 mM and about 20 mM.

13. The cell of claim 6, wherein the low K_m hexokinase activity of said cell is further reduced by interruption of a low K_m hexokinase gene.

14. The cell of claim 6, wherein the low K_m hexokinase activity of said cell is further reduced by providing to said cell an antisense RNA molecule that is complementary to and binds to a low K_m hexokinase gene or RNA transcript.

15. The cell of claim 7, wherein said cell comprises a recombinant gene that expresses a trehalose-6-phosphate synthase enzyme.

16. The cell of claim 15, wherein said recombinant gene expresses a yeast trehalose-6-phosphate synthase (TPS1) enzyme.

17. The cell of claim 16, wherein said recombinant gene expresses a yeast TPS1 enzyme having an amino acid sequence as set forth in SEQ ID NO:

2.

18. The cell of claim 17, wherein said recombinant gene has a nucleic acid sequence as set forth in SEQ ID NO:

1.

19. The cell of claim 7, wherein said cell comprises a recombinant gene that expresses a ribozyme specific for an RNA transcript of a low K_m hexokinase gene.

20. The cell of claim 19, wherein said recombinant gene expresses a ribozyme that comprises a ribozyme catalytic domain from a hairpin ribozyme structure, RNase P, hepatitis delta virus, avocado sunblotch viroid virus, lucerne transient streak virus, hammerhead or tobacco ringspot virus.

21. The cell of claim 19, wherein said recombinant gene expresses a ribozyme that comprises a ribozyme catalytic domain linked to a nucleic acid sequence that is complementary to and binds to an RNA transcript of a hexokinase I gene.

22. The cell of claim 19, wherein said recombinant gene expresses a ribozyme that comprises a ribozyme catalytic domain linked to a nucleic acid sequence that is complementary to and binds to an RNA transcript of a hexokinase II gene.

23. The cell of claim 19, wherein said ribozyme comprises a ribozyme catalytic domain linked at each end to a hexokinase I or hexokinase II nucleic acid sequence.

24. The cell of claim 19, wherein said ribozyme comprises a ribozyme catalytic domain linked to a hexokinase I or hexokinase II nucleic acid sequence of between about 6 and about 30 bases in length.

25. The cell of claim 24, wherein said ribozyme comprises a ribozyme catalytic domain linked to a hexokinase I or hexokinase II nucleic acid sequence of between about 10 and about 15 bases in length.

26. The cell of claim 19, wherein said ribozyme comprises a ribozyme catalytic domain linked to a hexokinase I nucleic acid sequence that includes a contiguous nucleic acid sequence from SEQ ID NO:

13 or a hexokinase II nucleic acid sequence that includes a contiguous nucleic acid sequence from SEQ ID NO;

15.

27. The cell of claim 21, wherein said ribozyme comprises the contiguous nucleic acid sequence of SEQ ID NO:

3, SEQ ID NO;

4 or SEQ ID NO;

5.

28. The cell of claim 7, wherein said cell comprises a recombinant gene that expresses a trehalose-6-phosphate synthase enzyme that stimulates the production of trehalose-6-phosphate and a recombinant gene that expresses a ribozyme specific for an RNA transcript of a low K_m hexokinase gene.

29. The cell of claim 6, wherein said cell further comprises a recombinant gene that expresses a selected protein.

30. The cell of claim 6, wherein said cell further comprises a recombinant hexokinase IV gene, a recombinant insulin gene or a recombinant GLUT-2 gene.

31. The cell of claim 30, wherein said recombinant hexokinase IV, insulin or GLUT-2 gene is introduced into the cell by means of a recombinant vector.

32. The cell of claim 30, wherein said cell comprises two recombinant genes selected from the group consisting of hexokinase IV, insulin and GLUT-2.

33. The cell of claim 30, wherein said cell comprises a recombinant hexokinase IV gene, a recombinant insulin gene and a recombinant GLUT-2 gene.

34. The cell of claim 30, comprising a recombinant hexokinase IV gene.

35. The cell of claim 34, wherein said recombinant hexokinase IV gene is an islet isoform hexokinase IV gene.

36. The cell of claim 30, comprising a recombinant insulin gene.

37. The cell of claim 30, comprising a recombinant GLUT-2 gene.

38. The cell of claim 37, wherein said recombinant GLUT-2 gene is an islet isoform GLUT-2 gene.

39. The cell of claim 6, further defined as a cell derived from a cell that forms secretory granules.

40. The cell of claim 39, further defined as a cell derived from a neuroendocrine cell.

41. The cell of claim 39, further defined as a cell derived from an AtT-20 cell.

42. The cell of claim 39, further defined as a cell derived from a GH-1 cell or a GH-3 cell.

43. The cell of claim 39, further defined as a cell derived from a pancreatic β

cell.

44. The cell of claim 43, further defined as a cell derived from a β

TC, HIT or RIN cell.

45. The cell of claim 44, further defined as a cell derived from a RIN cell.

46. The cell of claim 6, wherein said cell secretes recombinant insulin.

47. The cell of claim 6, wherein said cell comprises a glutamic acid decarboxylase gene.

48. The cell of claim 47, wherein said cell comprises a recombinant glutamic acid decarboxylase gene.

49. The cell of claim 6, wherein said cell is formulated in a pharmaceutically acceptable medium.

50. The cell of claim 6, wherein said cell is encapsulated in a biocompatible coating.

59. The cell of claim 15, wherein said recombinant gene expresses an E coli trehalose-6-phosphate synthase enzyme.

51. An engineered mammalian host cell derived from a cell that forms secretory granules, the cell secreting insulin in response to glucose and comprising a first recombinant gene selected from the group consisting of hexokinase IV, insulin and GLUT-2 and a second recombinant gene selected from the group consisting of:

(a) a recombinant gene that expresses a trehalose-6-phosphate synthase enzyme that stimulates the production of trehalose-6-phosphate; and

(b) a recombinant gene that expresses a ribozyme specific for an RNA transcript of a low K_m hexokinase gene.

52. A composition comprising an inhibitor of a low K_m hexokinase, characterized as:

(a) a recombinant vector comprising a promoter operably linked to a gene that encodes a trehalose-6-phosphate synthase enzyme that stimulates the production of trehalose-6-phosphate, the promoter expressing said trehalose-6-phosphate synthase enzyme in a mammalian host cell;

or

(b) a ribozyme specific for an RNA transcript of a low K_m hexokinase gene or a recombinant gene or vector that expresses said ribozyme.

53. The composition of claim 52, comprising a recombinant vector that comprises a promoter operably linked to a gene that encodes a trehalose-6-phosphate synthase enzyme that stimulates the production of trehalose-6-phosphate, the promoter expressing said trehalose-6-phosphate synthase enzyme in a mammalian host cell.

54. The composition of claim 53, wherein said recombinant vector comprises a gene that encodes a yeast trehalose-6-phosphate synthase enzyme.

55. The composition of claim 53, wherein said recombinant vector comprises a promoter that expresses said trehalose-6-phosphate synthase enzyme in a human cell.

56. The composition of claim 53, wherein said recombinant vector comprises a promoter that expresses said trehalose-6-phosphate synthase enzyme in a pancreatic β

cell.

57. The composition of claim 52, comprising a ribozyme specific for an RNA transcript of a low K_m hexokinase gene or a recombinant gene or vector that expresses said ribozyme.

58. The composition of claim 57, comprising a recombinant gene or vector that expresses a ribozyme specific for an RNA transcript of a low K_m hexokinase gene.

60. The composition of claim 54, wherein said recombinant vector comprises a gene that encodes a yeast TPS1 enzyme having an amino acid sequence as set forth in SEQ ID NO:

2.

61. The composition of claim 60, wherein said recombinant vector comprises a gene having a nucleic acid sequence from SEQ ID NO:

1.

62. The composition of claim 53, wherein said recombinant vector comprises a gene that encodes an E. coli trehalose-6-phosphate synthase enzyme.

63. The composition of claim 58, wherein said recombinant gene or vector comprises a promoter that expresses said ribozyme in a human cell.

64. The composition of claim 63, wherein said recombinant gene or vector comprises a promoter that expresses said ribozyme in a pancreatic β

cell.