Polynucletides that control delta-6 desaturase genes and methods for identifying compounds for modulating delta-6 desaturase

• US 20040053234A1
• Filed: 03/24/2003
• Published: 03/18/2004
• Est. Priority Date: 03/24/2000
• Status: Abandoned Application

• Current Assignee: Xenon Genetics, Inc.
• Original Assignee: Xenon Genetics, Inc.
• Inventors: Allen, Stephen John, Smith-King, Heidi Lynn, Antueno, Roberto Justo De, Ponton, Andre, Winther, Michael David
• Application Number:
  US10/239,652
• Publication Number:
  US 20040053234A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)

Abstract

The present invention relates to polynucleotides that control desaturase genes and to drug screening assays for identifying pharmaceutially active compounds for use in the treatment of diseases involving abnormal lipid metabolism including diabetic neuropathy, by utilizing fatty acid desaturase enzymes and the genes which encode them as targets for intervention. The drug screening method identifies nucleotides, proteins, compounds and/or other pharmacological agents, which effectively modulate the activity of desaturase enzymes or regulate the level of expression of the desaturase genes.

Claims

1. An isolated polynucleotide segment, comprising a polynucleotide sequence which is selected from the group consisting of:

(a) a sequence comprising SEQ ID NO;

1;

(b) a sequence comprising SEQ ID NO;

2;

(c) a sequence which is at least 80% homologous with a sequence of any of (a) to (b);

(d) a sequence which is at least 90% homologous with a sequence of any of (a) to (b);

(e) a sequence which is at least 95% homologous with a sequence of any of (a) to (b);

(f) a sequence which is at least 98% homologous with a sequence of any of (a) to (b);

(g) a sequence which is at least 99% homologous with a sequence of any of (a) to (b); and

;

(h) a sequence which hybridizes to any of (a) to (g) under stringent conditions.

2. An isolated polynucleotide segment of claim 1, wherein the isolated polynucleotide segment is genomic DNA.

3. A vector comprising a polynucleotide segment of claim 1 in a suitable vector.

4. A host cell comprising a polynucleotide segment of claim 1 in a host cell which is heterogeneous to said segment.

5. A method for producing a polypeptide encoded by a gene operably inked to a polynucleotide segment of claim 1 comprising the step of culturing the host cell of claim 4 under conditions sufficient for the production of said polypeptide.

6. An isolated polynucleotide fragment selected from the group consisting of:

(a) a sequence having at least 15 sequential bases of nucleotides of a segment of claim 1;

(b) a sequence having at least 30 sequential bases of nucleotides of a segment of claim 1;

and

(c) a sequence having at least 50 sequential bases of nucleotides of a segment of claim 1.

7. A vector comprising a polynucleotide segment of claim 6 contained in a vector which is heterogeneous to said segment.

8. An isolated polynucleotide segment, comprising a polynucleotide sequence which retains substantially the same biological function or activity as the polynucleotide encoded by a segment of claim 1.

9. A method for identifying a compound which inhibits or promotes the activity of a polynucleotide segment of claim 1, comprising the steps of:

(a) selecting a control animal having said segment and a test animal having said segment;

(b) treating said test animal using a compound; and

,

(c) determining the relative quantity of an expression product of an operably linked polynucleotide to said segment, as between said control animal and said test animal.

10. A method of claim 9, wherein said animals are mammals.

11. A method of claim 10, wherein said mammals are rats.

12. A method for identifying a compound which inhibits or promotes the activity of a polynucleotide segment of claim 1, comprising the steps of:

(a) selecting a host cell of claim 4;

(b) cloning said host cell and separating said clones into a test group and a control group;

(c) treating said test group using a compound; and

(d) determining the relative quantity of an expression product of a polynucleotide operably linked to said polynucleotide segment, as between said test group and said control group.

13. A method for identifying a compound which inhibits or promotes the activity of a polynucleotide segment of claim 1, comprising the steps of:

(a) selecting a test group having a host cell of claim 4, a part thereof or an isolated polynucleotide thereof and a control group;

(b) treating said test group using a compound; and

(c) determining the relative quantity of an expression product of an operably linked polynucleotide to said segment, as between said test group and said control group.

14. A composition for treating a lipid metabolism disorder comprising a compound which modulates a segment according to claim 1 and a pharmaceutically acceptable carrier.

15. A composition as claimed in claim 14, wherein said disorder is selected from the group consisting of atopic eczema, mastalgia, rheumatoid arthritis, Sjö

gren'"'"'syndrome, gastrointestinal disorders, viral infections and postviral fatigue, pre-menstrual syndrome, endometriosis, cystic fibrosis, schizophrenia, alcoholism, congenital liver disease, Alzheimer'"'"'s syndrome, Crohn'"'"'s disease, cardiovascular disease, cancer, diabetes and diabetic complications.

16. A composition as claimed in claim 15, wherein said diabetic complication is selected from the group consisting of diabetic neuropathy, nephropathy and retinopathy.

17. A composition as claimed in claim 16, wherein said compound is selected from the group consisting of small organic molecules, peptides, polypeptides, antisense molecules, oligonucleotides, polynucleotides, fatty acids and derivatives thereof.

18. The use of a composition as claimed in claim 14 for treating a lipid metabolic disorders.

19. The use of claim 18, wherein said disorder is selected from the group consisting of atopic eczema, mastalgia, rheumatoid arthritis, Sjö

gren'"'"'s syndrome, gastrointestinal disorders, viral infections and postviral fatigue, pre-menstrual syndrome, endometriosis, cystic fibrosis, schizophrenia, alcoholism, congenital liver disease, Alzheimer'"'"'s syndrome, Crohn'"'"'s disease, cardiovascular disease, cancer, diabetes and diabetic complications.

20. The use of claim 19, wherein said diabetic complication is selected from the group consisting of diabetic neuropathy, nephropathy and retinopathy.

21. A method for diagnosing the presence of or a predisposition for a lipid metabolic disorder in a subject by detecting a germline alteration in a segment of claim 1 in said subject, comprising comparing the germine sequence of a segment of claim 1 from a tissue sample from said subject with the germline sequence of a wild-type of said segment, wherein an alteration in the germline sequence of said subject indicates the presence of or a predisposition to said lipid metabolic disorder.

22. A method for diagnosing the presence of or a predisposition for a disorder as claimed in claim 21, wherein said disorder is selected from the group consisting of atopic eczema, mastalgia, rheumatoid arthritis, Sjö

gren'"'"'s syndrome, gastrointestinal disorders, viral infections and postviral fatigue, pre-menstrual syndrome, endometriosis, cystic fibrosis, schizophrenia, alcoholism, congenital liver disease, Alzheimer'"'"'s syndrome, Crohn'"'"'s disease, cardiovascular disease, cancer, diabetes and diabetic complications.

23. A method for diagnosing the presence of or a predisposition for a disorder as claimed in claim 22, wherein said diabetic complication is selected from the group consisting of diabetic neuropathy, nephropathy and retinopathy.

24. The method of claims 21 to 23, wherein said comparing is performed by a method selected from the group consisting of immunoblotting, immunocytochemistry, enzyme-linked immunosorbent assay, DNA fingerprinting, in situ hybridization, polymerase chain reaction, reverse transcription polymerase chain reaction, radioimmunoassay, immunoradiometric assay and immunoenzymatic assay.

28. A method for identifying a compound which inhibits or promotes the activity of a mammalian delta-6-desaturase, comprising the steps of:

(a) selecting a host cell of claim 4;

(b) cloning said host cell and separating said clones into a test group and a control group;

(c) treating said test group using a compound; and

(d) determining the relative quantity of an expression product of an expression polynucleotide operably linked to said polynucleotide segment, as between said test group and said control group.

29. A method for identifying a compound which inhibits or promotes the activity of a mammalian delta-6-desaturase, comprising the steps of:

(a) selecting a test group having a host cell of claim 4, a part thereof or an isolated polynucleotide thereof and a control group;

(b) treating said test group using a compound; and

(c) determining the relative quantity or relative activity of a product of said polynucleotide segment or of the said polynucleotide segment, as between said test group and said control group.

30. A method for identifying a compound which inhibits or promotes the activity of a mammalian delta-6-desaturase, comprising the steps of:

(a) selecting a control animal having a polypeptide segment of claim 1 and a test animal having said polypeptide segment;

(b) treating said test animal using a compound;

(c) determining the relative quantity or relative activity of an expression product of said polypeptide segment or of the said polypeptide segment, as between said control animal and said test animal.

31. A method of claim 30, wherein said animals are mammals.

32. A method of claim 31, wherein said mammals are rats.

46. A method for diagnosing the presence of or a predisposition for a lipid metabolism disorder in a subject by detecting a germline alteration in a polynucleotide of claim 1 in said subject, wherein the polynucleotide encodes a mammalian delta-6-desaturasecomprising comparing the germline sequence of said polynucleotide from a tissue sample from said subject with the germline sequence of a wild-type of said polynucleotide, wherein an alteration in the germline sequence of said subject indicates the presence of or a predisposition to said lipid metabolism disorder.

47. A method for diagnosing the presence of or a predisposition for a disorder as claimed in claim 46, wherein said disorder is selected from the group consisting of atopic eczema, mastalgia, rheumatoid arthritis, Sjö

gren'"'"'s syndrome, gastrointestinal disorders, viral infections and postviral fatigue, pre-menstrual syndrome, endometriosis, cystic fibrosis, schizophrenia, alcoholism, congenital liver disease, Alzheimer'"'"'s syndrome, Crohn'"'"'s disease, cardiovascular disease, cancer, diabetes and diabetic complications.

48. A method for diagnosing the presence of or a predisposition for a disorder as claimed in claim 47, wherein said diabetic complication is selected from the group consisting of diabetic neuropathy, nephropathy and retinopathy.

49. The method of claims 46 to 48, wherein said comparing is performed by a method selected from the group consisting of immunoblotting, immunocytochemistry, enzyme-linked immunosorbent assay, DNA fingerprinting, in situ hybridization, polymerase chain reaction, reverse transcription polymerase chain reaction, radioimmunoassay, immunoradiometric assay and immunoenzymatic assay.

62. A compound identified by the methods of any one of claims 9 to 13 or 25 to 35.

63. The use of a compound as claimed in claim 62 for treating a lipid metabolism disorder.

64. The use of claim 63, wherein said disorder is selected from the group consisting of atopic eczema, mastalgia, rheumatoid arthritis, Sjö

gren'"'"'s syndrome, gastrointestinal disorders, viral infections and postviral fatigue, pre-menstrual syndrome, endometriosis, cystic fibrosis, schizophrenia, alcoholism, congenital liver disease, Alzheimer'"'"'s syndrome, Crohn'"'"'s disease, cardiovascular disease, cancer, diabetes and diabetic complications.

65. The use of claim 64, wherein said diabetic complication is selected from the group consisting of diabetic neuropathy, nephropathy and retinopathy.

66. A host cell as claimed in claim 4, wherein said host cell is a spheroplast

67. A spheroplast as claimed in claim 66, wherein said spheroplast is a Saccharomyces cerevisiae.

68. A method as claimed in any one of claims 5, 12, 13, 28, 29 or 54, wherein said host cell is a spheroplast.

69. A method as claimed in claim 68, wherein said spheroplast is a Saccharomyces cerevisiae.

70. A composition for treating a lipid metabolism disorder comprising a compound identified by any one of the methods of claims 68 or 69 and a pharmaceutically acceptable carrier.

71. A composition as claimed in claim 70, wherein said disorder is selected from the group consisting of atopic eczema, mastalgia, rheumatoid arthritis, Sjö

gren'"'"'s syndrome, gastrointestinal disorders, viral infections and postviral fatigue, pre-menstrual syndrome, endometriosis, cystic fibrosis, schizophrenia, alcoholism, congenital liver disease, Alzheimer'"'"'s syndrome, Crohn'"'"'s disease, cardiovascular disease, cancer, diabetes and diabetic complications.

72. A composition as claimed in claim 71, wherein said diabetic complication is selected from the group consisting of diabetic neuropathy, nephropathy and retinopathy.

73. A composition as claimed in any one of claims 70 to 72, wherein said compound is selected from the group consisting of small organic molecules, peptides, polypeptides, antisense molecules, oligonucleotides, polynucleotides, fatty acids and derivatives thereof.

74. The use of a composition as claimed in any one of claims 70 to 73 for treating lipid metabolism disorders.

75. The use of claim 74, wherein said disorder is selected from the group consisting of atopic eczema, mastalgia, rheumatoid arthritis, Sjö

gren'"'"'s syndrome, gastrointestinal disorders, viral infections and postviral fatigue, pre-menstrual syndrome, endometriosis, cystic fibrosis, schizophrenia, alcoholism, congenital liver disease, Alzheimer'"'"'s syndrome, Crohn'"'"'s disease, cardiovascular disease, cancer, diabetes and diabetic complications.

76. The use of claim 75, wherein said diabetic complication is selected from the group consisting of diabetic neuropathy, nephropathy and retinopathy.

25. A method for identifying a compound which inhibits or promotes the activity of a polynucleotide, wherein the polynucleotide encodes a mammalian delta-6-desaturase, comprising the steps of:

(a) selecting a control animal having said polynucleotide and a test animal having said polynucleotide;

(b) treating said test animal using a compound; and

,

(c) determining the relative quantity of an expression product of said polynucleotide, as between said control animal and said test animal.

26. A method of claim 25, wherein said animals are mammals.

27. A method of claim 26, wherein said mammals are rats.

33. A method for identifying a compound according to any one of the claims 25 to 32, wherein said relative activity of said expression product is determined by assaying for a conversion of 18:

2n6 to 22;

5n6.

34. A method for identifying a compound according to any one of the claims 25 to 32, wherein said relative activity of said expression product is determined by assaying for a conversion of 18:

3n3 to 22;

6n3.

35. A method for identifying a compound according to any one of the claims 25 to 32, wherein said relative activity of said expression product is determined by assaying for a conversion of 16:

0 to 22;

4n9.

36. A use of a method according to any one of the claims 25 to 34 for identifying a modulator that modulates lipid metabolism disorders.

37. A use according to claim 36 for identifying a modulator that modulates a disorder selected from the group consisting of atopic eczema, mastalgia, rheumatoid arthritis, Sjö

gren'"'"'s syndrome, gastrointestinal disorders, viral infections and postviral fatigue, pre-menstral syndrome, endometriosis, cystic fibrosis, schizophrenia, alcoholism, congenital liver disease, Alzieimer'"'"'s syndrome, Crohn'"'"'s disease, cardiovascular disease, cancer, diabetes and diabetic complications.

38. A use according to claims 37, wherein said diabetic complication is selected from the group consisting of diabetic neuropathy, diabetic nephropathy and diabetic retinopathy.

39. A composition for treating a lipid metabolism disorder comprising a compound identified by any one of the methods of claims 25 to 35 and a pharmaceutically acceptable carrier.

40. A composition as claimed in claim 39, wherein said disorder is selected from the group consisting of atopic eczema, mastalgia, rheumatoid arthritis, Sjö

gren'"'"'s syndrome, gastrointestinal disorders, viral infections and postviral fatigue, pre-menstrual syndrome, endometriosis, cystic fibrosis, schizophrenia, alcoholism, congenital liver disease, Alzheimer'"'"'s syndrome, Crohn'"'"'s disease, cardiovascular disease, cancer, diabetes and diabetic complications.

41. A composition as claimed in claim 40, wherein said diabetic complication is selected from the group consisting of diabetic neuropathy, nephropathy and retinopathy.

42. A composition as claimed in any one of claims 39 to 41, wherein said compound is selected from the group consisting of small organic molecules, peptides, polypeptides, antisense molecules, oligonucleotides, polynucleotides, fatty acids and derivatives thereof.

43. The use of a composition as claimed in claims 39 to 40 for treating lipid metabolism disorders.

44. The use of claim 43, wherein said disorder is selected from the group consisting of atopic eczema, mastalgia, rheumatoid arthritis, Sjö

gren'"'"'s syndrome, gastrointestinal disorders, viral infections and postviral fatigue, premenstrual syndrome, endometriosis, cystic fibrosis, schizophrenia, alcoholism, congenital liver disease, Alzheimer'"'"'s syndrome, Crohn'"'"'s disease, cardiovascular disease, cancer, diabetes and diabetic complications.

45. The use of claim 44, wherein said diabetic complication is selected from the group consisting of diabetic neuropathy, nephropathy and retinopathy.

50. A method for diagnosing the presence of or a predisposition for a lipid metabolic disorder in a subject, comprising comparing the polypeptide sequence of a control region of delta-6desaturase from a tissue sample from said subject with the sequence of a wild-type of said delta-6-desaturase, wherein an alteration in the sequence of said subject as compared to said wild-type indicates the presence of or a predisposition to said lipid metabolic disorder.

51. A method as claimed in claim 50, wherein said disorder is selected from the group consisting of atopic eczema, mastalgia, rheumatoid arthritis, Sjö

gren'"'"'s syndrome, gastrointestinal disorders, viral infections and postviral fatigue, pre-menstrual syndrome, endometriosis, cystic fibrosis, schizophrenia, alcoholism, congenital liver disease, Alzheimer'"'"'s syndrome, Crohn'"'"'s disease, cardiovascular disease, cancer, diabetes and diabetic complications.

52. A method as claimed in any one of claims 50 to 51, wherein said diabetic complication is selected from the group consisting of diabetic neuropathy, nephropathy and retinopathy.

53. The method of any one of claims 50 to 52, wherein said comparing is performed by a method selected from the group consisting of immunoblotting, immunocytochemistry, enzyme-linked immunosorbent assay, DNA fingerprinting, radioimmunoassay, immunoradiometric assay, immunoenzymatic assay and polypeptide microarrays.

54. A method for identifying a compound which inhibits or promotes the activity of control regions of mammalian delta-6- and/or delta-5-desaturases, comprising the steps of:

(a) selecting one or more host cells comprising said polynucleotides, wherein such host cells are heterogeneous to said polynucleotides;

(b) cloning said host cells and separating said clones into a test group and a control group;

(c) treating said test group using a compound; and

(d) determining the relative quantities of expression products of operably linked polynucleotides to said control regions, as between said test group and said control group.

55. A composition for treating a lipid metabolism disorder comprising a compound identified by a method of claim 54 and a pharmaceutically acceptable carrier.

56. A composition as claimed in claim 55, wherein said disorder is selected from the group consisting of atopic eczema, mastalgia, rheumatoid arthritis, Sjö

gren'"'"'s syndrome, gastrointestinal disorders, viral infections and postviral fatigue, pre-menstrual syndrome, endometriosis, cystic fibrosis, schizophrenia, alcoholism, congenital liver disease, Alzheimer'"'"'s syndrome, Crohn'"'"'s disease, cardiovascular disease, cancer, diabetes and diabetic complications.

57. A composition as claimed in claim 56, wherein said diabetic complication is selected from the group consisting of diabetic neuropathy, nephropathy and retinopathy.

58. A composition as claimed in any one of claims 55 to 57, wherein said compound is selected from the group consisting of small organic molecules, peptides, polypeptides, antisense molecules, oligonucleotides, polynucleotides, fatty acids and derivatives thereof.

59. The use of a composition as claimed in any one of claims 55 to 58 for treating lipid metabolism disorders.

60. The use of claim 59, wherein said disorder is selected from the group consisting of atopic eczema, mastalgia, rheumatoid arthritis, Sjö

gren'"'"'s syndrome, gastrointestinal disorders, viral infections and postviral fatigue, premenstrual syndrome, endometriosis, cystic fibrosis, schizophrenia, alcoholism, congenital liver disease, Alzheimer'"'"'s syndrome, Crohn'"'"'s disease, cardiovascular disease, cancer, diabetes and diabetic complications.

61. The use of claim 60, wherein said diabetic complication is selected from the group consisting of diabetic neuropathy, nephropathy and retinopathy.