METHODS FOR MODULATING DE NOVO HEPATIC LIPOGENESIS BY MODULATING XBP-1 ACTIVITY

• US 20110052669A1
• Filed: 01/14/2009
• Published: 03/03/2011
• Est. Priority Date: 01/14/2008
• Status: Active Grant

• Current Assignee: Cornell University
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Lee, Ann-Hwee, Glimcher, Laurie H.
• Granted Patent: US 8,940,479 B2
• Time in Patent Office: Days
• US Class Current: 424/450
• CPC Class Codes: A61K 31/70 Carbohydrates; Sugars; Derivatives thereof sorbitol A61K31/047
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• 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: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: G01N 2333/4703 Regulators; Modulating activity
• CPC Class Codes: G01N 2500/04 Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
• CPC Class Codes: G01N 2800/044 Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity

Abstract

The invention provides methods and compositions for modulating the expression, processing, post-translational modification, stability and/or activity of XBP-1 protein, or a protein in a signal transduction pathway involving XBP-I to treat dyslipidemias and steatosis disorders. The present invention also pertains to methods for identifying compounds that modulate the expression, processing, post-translational modification, and/or activity of XBP-I protein or a molecule in a signal transduction pathway involving XBP-1.

Claims

1. A method of treating and/or preventing dyslipidemia comprising administering a nucleic acid molecule that downmodulates the activity of XBP-1 to a subject having dyslipidemia in an amount sufficient to downmodulate de novo hepatic lipogenesis, to thereby treat and/or prevent dyslipidemia in the subject.

13. The method of claim 1, wherein the nucleic acid molecule is selected from the group consisting of:

a nucleic acid molecule that is antisense to a XBP-1 molecule, a XBP-1 siRNA molecule, a nucleic acid molecule encoding a dominant negative XBP-1 molecule, or combinations thereof.

14. The method of claim 1, wherein the nucleic acid molecule is a liposome:

XBP-1 siRNA complex.

15. The method of claim 1, wherein the nucleic acid molecule is a cholesterol:

XBP-1 siRNA complex.

19. The method of claim 14, wherein the complex is administered at a dose of about 1-2.5 mg/kg.

20. The method of claim 1, further comprising administering to the subject a hypolipidemic agent.

21. The method of claim 20, wherein the hypolipidemic agent is a statin.

2. A method of treating and/or preventing atherosclerosis comprising administering a nucleic acid molecule that downmodulates the activity of XBP-1 to a subject having atherosclerosis in an amount sufficient to downmodulate de novo hepatic lipogenesis, to thereby treat and/or prevent atherosclerosis.

3. A method of treating and/or preventing hepatic steatosis comprising administering a nucleic acid molecule that downmodulates the activity of XBP-1 to a subject having hepatic steatosis in an amount sufficient to downmodulate de novo hepatic lipogenesis, to thereby treat and/or prevent hepatic steatosis.

4. A method of treating and/or preventing steatohepatitis comprising administering a nucleic acid molecule that downmodulates the activity of XBP-1 to a subject having steatohepatitis in an amount sufficient to downmodulate de novo hepatic lipogenesis, to thereby treat and/or prevent steatohepatitis.

5. A method of treating and/or preventing non-alcoholic hepatic steatohepatitis comprising administering a nucleic acid molecule that downmodulates the activity of XBP-1 to a subject having non-alcoholic hepatic steatohepatitis in an amount sufficient to downmodulate de novo hepatic lipogenesis, to thereby treat and/or prevent non-alcoholic hepatic steatohepatitis.

6. A method of treating and/or preventing dyslipidemia comprising administering a nucleic acid molecule that downmodulates the activity of IRE-1 to a subject having dyslipidemia in an amount sufficient to downmodulate de novo hepatic lipogenesis, to thereby treat and/or prevent dyslipidemia.

16. The method of claim 6, wherein the nucleic acid molecule is selected from the group consisting of:

a nucleic acid molecule that is antisense to an IRE-1 molecule, a IRE-1 siRNA molecule, a nucleic acid molecule encoding a dominant negative IRE-1 molecule, or combinations thereof.

17. The method of claim 6, wherein the nucleic acid molecule is a liposome:

IRE-1 siRNA complex.

18. The method of claim 6, wherein the nucleic acid molecule is a cholesterol:

IRE-1 siRNA complex.

7. A method of treating and/or preventing atherosclerosis comprising administering a nucleic acid molecule that downmodulates the activity of IRE-1 to a subject having atherosclerosis in an amount sufficient to downmodulate de novo hepatic lipogenesis, to thereby treat and/or prevent atherosclerosis.

8. A method of treating and/or preventing hepatic steatosis comprising administering a nucleic acid molecule that downmodulates the activity of IRE-1 to a subject having hepatic steatosis in an amount sufficient to downmodulate de novo hepatic lipogenesis, to thereby treat and/or prevent hepatic steatosis.

9. A method of treating and/or preventing steatohepatitis comprising administering a nucleic acid molecule that downmodulates the activity of IRE-1 to a subject having steatohepatitis in an amount sufficient to downmodulate de novo hepatic lipogenesis, to thereby treat and/or prevent steatohepatitis.

10. A method of treating and/or preventing non-alcoholic hepatic steatohepatitis comprising administering a nucleic acid molecule that downmodulates the activity of IRE-1 to a subject having non-alcoholic hepatic steatohepatitis in an amount sufficient to downmodulate de novo hepatic lipogenesis, to thereby treat and/or prevent non-alcoholic hepatic steatohepatitis.

11. A method of treating and/or preventing dyslipidemia comprising administering an agent that upmodulates the activity of XBP-1 to a subject that would benefit from increased de novo hepatic lipogenesis in an amount sufficient to upmodulate de novo hepatic lipogenesis, to thereby treat and/or prevent dyslipidemia in the subject.

12. A method of treating and/or preventing dyslipidemia comprising administering an agent that upmodulates the activity of IRE-1 to a subject a subject that would benefit from increased de novo hepatic lipogenesis in an amount sufficient to upmodulate de novo hepatic lipogenesis, to thereby treat and/or prevent dyslipidemia in the subject.

22. A method of identifying a compound that is useful in treating and/or preventing dyslipidemia comprising,

a) providing a cell comprising an XBP-1 polypeptide;

b) contacting the cell with each member of a library of compounds;

c) determining the effect of the compound on de novo hepatic lipogenesis; and

d) selecting a compound of interest that downmodulates de novo hepatic lipogeneis as compared to an appropriate control, thereby identifying the compound as useful in treating and/or preventing dyslipidemia.

24. The method of claim 22, wherein the effect of the compound on de novo hepatic lipogenesis is determined by determining the effect on the compound on the production of cholesterol and/or triglyceride.

26. The method of claim 22, wherein determining the ability of the compound to modulate de novo hepatic lipogenesis comprises determining the expression of SREBP and/or chREBP.

27. The method of claim 22, wherein the ability of the compound to modulate de novo hepatic lipogenesis comprises determining the expression of a lipogenic gene.

28. The method of claim 27, wherein the lipogenic gene is selected from the group consisting of:

fasn, coA desaturase, diacyl glycerol acetyltransferase 2, acetyl coA carboxylase 2, and proprotein convertase subtilisin/kexin type 9 (PCSK9).

29. The method of claim 28, wherein the expression of the lipogeneic gene is determined by a method selected from the group consisting of:

microarray analysis, RT-PCR, and Northern blot analysis.

30. The method of claim 22, wherein:

the cell further comprises a DNA molecule to which XBP-1 binds, wherein the DNA molecule comprises a promoter selected from the group consisting of a Dgat2 promoter, a Scd1 promoter, an Acc2 promoter; and

a PCSK9 promoter; and

the effect of the test compound on de novo hepatic lipogenesis is determined by evaluating the binding of XBP-1 to the DNA molecule in the presence and absence of the test compound.

31. The method of claim 30, wherein the binding of XBP-1 to the promoter is determined by chromatin immunoprecipitation analysis.

32. The method of claim 22, wherein:

the cell further comprises a reporter gene responsive to the XBP-1 polypeptide; and

the effect of the test compound on de novo hepatic lipogenesis is determined by evaluating the expression of the reporter gene in the presence and absence of the test compound.

33. The method of claim 22, wherein the compound is present in a small molecule microarray.

34. The method of claim 22, wherein the cell has been engineered to express the XBP-1 polypeptide by introducing into the cell an expression vector encoding the polypeptide.

35. The method of claim 34, wherein the cell is a hepatocyte.

36. The method of claim 34, wherein the hepatocyte is an adult hepatocyte.

37. The method of claim 34, wherein the cell is a HeLa cell.

38. The method of claim 34, wherein the cell is a hepatoma cell.

41. The method of any one of claims 22, 23, 39, and 40 further comprising determining the effect of a test compound identified as useful in modulating dyslipidemia on the production of cholesterol and triglycerides in a non-human animal, comprising administering the test compound to the animal, measuring the production of cholesterol and triglycerides in the presence and absence of the test compound, and selecting a compound that modulates the production of cholesterol and/or triglycerides.

42. The method of claim 41, wherein the production of cholesterol and triglycerides is determined by measuring the amount of at least one of:

total cholesterol, LDL, VLDL, IDL, and HDL.

43. The method of any one of claims 22, 23, 39, and 41, further comprising determining the effect of the identified compound on obesity and/or insulin resistance in a non-human animal.

44. The method of claim 43, wherein the step of determining comprises

providing the non-human animal with a high carbohydrate diet;

administering the test compound to the non-human animal;

determining the effect of the compound on at least one of body weight, serum triglycerides, total cholesterol, blood glucose, glucose tolerance, insulin tolerance, and glucose-stimulated insulin secretion in the presence and absence of the test compound, and

selecting a compound that decreases obesity and/or insulin resistance in the non-human animal.

45. The method of any one of claims 22, 23, 39, and 41, further comprising determining the effect of a identified compound on non-alcoholic hepatosteatosis in a non-human animal.

46. The method of claim 45, wherein the step of determining comprises providing the

non-human animal with a methionine and choline deficient diet;

administering the test compound to the non-human animal;

determining the effect of the compound on at least one of aspartate aminotransferase level, alanine aminotransferase level, liver cell inflammation, and presence of triglyceride vacuoles in a liver cell in the presence and absence of the test compound, and

selecting a compound that decreases non-alcoholic hepatosteatosis in the non-human animal.

47. The method of claim 45, wherein the step of determining comprises providing the

non-human animal with an atherogenic diet;

administering the test compound to the non-human animal;

determining the effect of the compound on at least one of liver cell inflammation, and presence of triglyceride vacuoles in a liver cell in the presence and absence of the test compound, and

selecting a compound that decreases non-alcoholic hepatosteatosis in the non-human animal.

48. The method of any one of claims 22, 23, 39, and 41, further comprising determining the effect of a identified compound on hypercholesterolemia and/or atherosclerosis in a non-human animal.

49. The method of claim 48, wherein the step of determining comprises

providing the non-human animal model with a high fat diet;

administering the test compound to the non-human animal;

determining the effect of the compound on at least one of serum triglycerides, total cholesterol, distribution of cholesterol among HDL, IDL, and LDL, and presence of atherosclerotic lesions, and

selecting a compound that decreases hypercholesterolemia and/or atherosclerosis in the non-human animal.

23. A method of identifying a compound that is useful in treating and/or preventing dyslipidemia comprising,

a) providing a cell comprising an IRE-1 and an XBP-1 polypeptide;

b) contacting the cell with each member of a library of compounds;

c) determining the effect of the compound on de novo hepatic lipogenesis; and

d) selecting a compound of interest that downmodulates de novo hepatic lipogeneis as compared to an appropriate control, thereby identifying the compound as useful in treating and/or preventing dyslipidemia.

25. The method of 24, wherein the determination of the production of cholesterol is determined by determining the amount of at least one of:

total cholesterol, LDL, IDL, VLDL, and HDL.

39. A method of identifying a compound that is useful in treating and/or preventing dyslipidemia comprising,

a) providing a cell-free composition comprising an XBP-1 polypeptide and a DNA molecule to which XBP-1 binds, wherein the DNA molecule comprises the regulatory region of a lipogenic gene;

b) contacting the polypeptide with each member of a library of compounds;

c) selecting a compound that downmodulates the binding of the polypeptide to the DNA molecule as compared to an appropriate control, thereby identifying the compound as useful in treating and/or preventing dyslipidemia.

40. A method of identifying a compound that is useful in treating and/or preventing dyslipidemia comprising,

a) providing a cell comprising an XBP-1 polypeptide and a DNA molecule comprising a transcriptional regulatory site responsive to the DNA binding domain operatively linked a reporter gene, wherein the transcriptional regulatory site is selected from the group consisting of Dgat2 promoter, a Scd1 promoter, an Acc2 promoter;

and a PCSK9 promoter;

b) contacting the polypeptide with each member of a library of compounds;

c) determining the effect of the compound on the level of expression of the reporter gene in the indicator cell in the presence and absence of the test compound; and

d) selecting a compound of interest that downmodulates the level of expression of the reporter gene responsive to the XBP-1 protein as compared to an appropriate control, thereby identifying the compound as useful in treating and/or preventing dyslipidemia.

50. A method of identifying a compound that is useful in treating and/or preventing dyslipidemia comprising, providing an indicator composition comprising an XBP-1 polypeptide;

a) contacting the indicator cell with each member of a library of test compounds;

b) determining the activity of the XBP-1 polypeptide in the presence and absence of the test compound;

c) selecting from the library of test compounds a compound of interest that downmodulates the activity of the XBP-1 polypeptide;

d) determining the effect of the compound on de novo hepatic lipogeneis;

e) selecting a compound of interest that downmodulates de novo hepatic lipogeneis as compared to an appropriate control, thereby identifying a compound that is useful in treating and/or preventing dyslipidemia.

51. The method of claim 50, wherein the effect of the compound on XBP-1 activity is determined by measuring the binding of XBP-1 to IRE-1.

52. The method of claim 50, wherein the effect of the compound on XBP-1 activity is determined by assaying the activity of IRE-1.

53. The method of claim 52, wherein the activity of IRE-1 is a kinase activity.

54. The method of claim 52, wherein the activity of IRE-1 is an endoribonuclease activity.

55. The method of claim 50, wherein the effect of the compound on XBP-1 activity is determined by assaying XBP-1 and/or IRE-1 protein levels.

56. The method of claim 50, wherein the XBP-1 polypeptide comprises a transactivation domain and the indicator composition further comprises a vector comprising a regulatory element responsive to the XBP-1 transactivation domain operatively linked to a reporter gene and the effect of the compound on the activity of the polypeptide is determined by evaluating the expression of the reporter gene in the presence and absence of the test compound.

57. The method of claim 50, wherein the indicator composition comprises a recombinant expression vector encoding a spliced XBP-1 protein;

and a vector comprising a regulatory element responsive to the XBP-1 protein operatively linked a reporter gene and the effect of the compound on the activity of the polypeptide is determined by evaluating the expression of the reporter gene in the presence and absence of the test compound.

58. The method of claim 50, wherein the indicator composition is a cell and comprises, a recombinant expression vector comprising a regulatory element responsive to XBP-1 spliced protein operatively linked a reporter gene, wherein the cell is contacted with an agent that induces ER stress, and the effect of the compound on the activity of the polypeptide is determined by evaluating the expression of the reporter gene in the presence and absence of the test compound.

59. The method of claim 56, 57, or 58, further comprising determining the effect of the identified compound on the ratio of unspliced XBP-1 to spliced XBP-1 mRNA and/or protein.

60. The method of claim 56, 57, or 58, further comprising determining the effect of the identified compound on the production of XBP-1 protein.

61. The method of claim 50, further comprising providing an indicator cell comprising a glucose-dependent promoter operably linked to a reporter gene, wherein the cell in cultured in the presence of glucose, and the effect of the compound on de novo hepatic lipogenesis is determined by evaluating the expression of the reporter gene in the presence and absence of the test compound.

62. The method of claim 50, wherein the effect of the compound on de novo hepatic lipogeneis is determined by determining the effect on the compound on the production of cholesterol and/or triglyceride.

63. The method of claim 50, wherein determining the ability of the compound to modulate de novo hepatic lipogenesis comprises determining the expression of SREBP and/or chREBP.

64. The method of claim 50, wherein the ability of the compound to modulate de novo lipogenesis comprises determining the expression of a lipogenic gene.

65. The method of claim 50, wherein:

the indicator composition further comprises a DNA molecule to which XBP-1 binds, wherein the DNA molecule comprises a promoter selected from the group consisting of a Dgat2 promoter, a Scd1 promoter, an Acc2 promoter; and

a PCSK9 promoter; and

the effect of the test compound on de novo hepatic lipogenesis is determined by evaluating the binding of XBP-1 to the DNA molecule in the presence and absence of the test compound.

66. The method of claim 50, further comprising determining the effect of the identified compound on obesity and/or insulin resistance in a non-human animal.

67. The method of claim 50, further comprising determining the effect of a identified compound on non-alcoholic hepatosteatosis in a non-human animal.

68. The method of claim 50, further comprising determining the effect of an identified compound on hypercholesterolemia and/or atherosclerosis in a non-human animal.