Secreted and cell surface polypeptides affected by cholesterol and uses thereof

• US 20030170700A1
• Filed: 01/08/2003
• Published: 09/11/2003
• Est. Priority Date: 01/09/2002
• Status: Abandoned Application

• Current Assignee: Lynx Therapeutics, Inc. (Illumina Incorporated)
• Original Assignee: Lynx Therapeutics, Inc. (Illumina Incorporated)
• Inventors: Bowen, Benjamin A., Shang, Jin
• Application Number:
  US10/340,192
• Publication Number:
  US 20030170700A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: G01N 2800/044 Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity
• CPC Class Codes: G01N 33/92 involving lipids, e.g. cholesterol , lipoproteins, or their receptors steroid hormones G01N33/743

Abstract

Polynucleotides, proteins, antibodies, labeled probes, marker sets, and arrays related to secreted and cell surface proteins that are altered in response to cholesterol are provided. Methods of detecting alterations in secreted and cell surface proteins in response to alterations in cholesterol levels (exposure), modulating cholesterol phenotype in cells and for treating a subject with adverse effects of altered levels of cholesterol, e.g., elevated or high levels of cholesterol, are also provided.

Claims

1. A composition comprising at least one expression vector, wherein the at least one expression vector comprises a nucleic acid comprising:

(a) at least one polynucleotide sequence selected from the group consisting of;

SEQ ID NO;

1-SEQ ID NO;

88 or a sequence complementary thereto;

(b) at least one polynucleotide sequence that hybridizes under stringent conditions to a polynucleotide sequence of (a);

(c) at least one polynucleotide sequence that is at least about 70% identical to a polynucleotide sequence of (a);

(d) at least one polynucleotide sequence that encodes a polypeptide or peptide comprising a subsequence encoded by a polynucleotide sequence of (a);

(e) at least one polynucleotide sequence that hybridizes to a nucleic acid that is physically linked in the human genome to a nucleic acid comprising a polynucleotide sequence of (a), (b), (c), or (d);

or,

(f) at least one polynucleotide sequence comprising at least about 10 contiguous nucleotides of a polynucleotide sequence selected from the group consisting of;

SEQ ID NO;

1-SEQ ID NO;

88, or a sequence complementary thereto.

2. The at least one expression vector of claim 1, wherein the at least one expression vector comprises a promoter operably linked to the nucleic acid comprising the polynucleotide of (a), (b), (c), (d), (e) or (f).

3. The at least one expression vector of claim 1, wherein the nucleic acid encodes a polypeptide.

4. The at least one expression vector of claim 1, wherein the nucleic acid encodes a sense or antisense RNA.

5. A method of treating responses to alterations of cholesterol levels in a patient, the method comprising administering to the patient an effective amount of the at least one expression vector of claim 1.

6. A composition comprising the at least one expression vector of claim 1 and an excipient.

7. The composition of claim 6, wherein the excipient is a pharmaceutically acceptable excipient.

8. A cell comprising the at least one expression vector of claim 1.

9. An isolated or recombinant polypeptide comprising one or more amino acid sequences or subsequences encoded by a nucleic acid comprising:

(a) at least one polynucleotide sequence selected from the group consisting of;

SEQ ID NO;

1-SEQ ID NO;

88 or a sequence complementary thereto;

(b) at least one polynucleotide sequence that hybridizes under stringent conditions to a polynucleotide sequence of (a);

(c) at least one polynucleotide sequence that is at least about 70% identical to a polynucleotide sequence of (a);

(d) at least one polynucleotide sequence that hybridizes to a nucleic acid that is physically linked in the human genome to a nucleic acid comprising a polynucleotide sequence of (a), (b), or (c);

or,

(e) at least one polynucleotide sequence comprising at least about 10 contiguous nucleotides of a polynucleotide sequence selected from the group consisting of;

SEQ ID NO;

1-SEQ ID NO;

88, or a sequence complementary thereto.

10. The isolated or recombinant polypeptide of claim 9, comprising a fusion protein.

11. The isolated or recombinant polypeptide of claim 9, comprising a peptide or polypeptide tag.

12. The isolated or recombinant polypeptide of claim 11, wherein the peptide or polypeptide tag comprises a reporter peptide or polypeptide.

13. The isolated or recombinant polypeptide of claim 11, wherein the peptide or polypeptide tag comprises an epitope.

14. The isolated or recombinant polypeptide of claim 11, wherein the peptide or polypeptide tag comprises a localization signal or sequence.

15. A composition comprising the isolated or recombinant polypeptide of claim 9 and an excipient.

16. The composition of claim 15, wherein the excipient is a pharmaceutically acceptable excipient.

17. A method of treating responses to alterations of cholesterol levels in a patient, the method comprising administering to the patient an effective amount of the isolated or recombinant polypeptide of claim 9.

18. An array of polypeptides comprising two or more different polypeptides of claim 9.

19. An antibody specific for an isolated or recombinant polypeptide of claim 9.

20. The antibody of claim 19, wherein the antibody comprises a monoclonal antibody or polyclonal serum.

21. One or more isolated or recombinant polypeptides that bind to the antibody of claim 19.

22. A labeled probe comprising a nucleic acid sequence comprising:

(a) at least one polynucleotide sequence selected from the group consisting of;

SEQ ID NO;

1-SEQ ID NO;

88 or a sequence complementary thereto;

(b) at least one polynucleotide sequence that hybridizes under stringent conditions to a polynucleotide sequence of (a);

(c) at least one polynucleotide sequence that is at least about 70% identical to a polynucleotide sequence of (a);

(d) at least one polynucleotide sequence that encodes a polypeptide or peptide comprising a subsequence encoded by a polynucleotide sequence of (a);

(e) at least one polynucleotide sequence that hybridizes to a nucleic acid that is physically linked in the human genome to a nucleic acid comprising a polynucleotide sequence of (a), (b), (c), or (d);

or,

(f) at least one polynucleotide sequence comprising at least about 10 contiguous nucleotides of a polynucleotide sequence selected from the group consisting of;

SEQ ID NO;

1-SEQ ID NO;

88, or a sequence complementary thereto.

23. The labeled probe of claim 22, the subsequence comprising at least about 12 nucleotides.

24. The labeled probe of claim 22, the subsequence comprising at least about 14 nucleotides.

25. The labeled probe of claim 22, the subsequence comprising at least about 16 nucleotides.

26. The labeled probe of claim 22, the subsequence comprising at least about 17 nucleotides.

27. The labeled probe of claim 22, comprising an isotopic, fluorescent, fluorogenic or colorimetric label.

28. The labeled probe of claim 22, comprising a DNA or RNA molecule.

29. A labeled probe of claim 22, comprising a cDNA, an amplification product, a transcript, a restriction fragment, or an oligonucleotide.

30. The labeled probe of 22, comprising an oligonucleotide consisting of a polynucleotide sequence selected from SEQ ID NO:

1 to SEQ ID NO;

88.

31. The labeled probe of 22, wherein the labeled probe is a member of an array of probes comprising a plurality of nucleic acids comprising two or more polynucleotide sequences selected from (a), (b), (c), (d), (e) and/or (f).

32. An array of probes according to claim 31, wherein the nucleic acids are logically or physically arrayed.

33. A marker set for evaluating a condition or characteristic associated with alterations in cholesterol levels, comprising a plurality of members, which members comprise nucleic acids, polypeptides or peptides comprising:

(a) one or more polynucleotide sequence selected from the group consisting of;

SEQ ID NO;

1-SEQ ID NO;

88 or a sequence complementary thereto;

(b) one or more polynucleotide sequence that hybridizes under stringent conditions to a polynucleotide sequence of (a);

(c) one or more polynucleotide sequence that is at least about 70% identical to a polynucleotide sequence of (a);

(d) one or more polynucleotide sequence that encodes a polypeptide or peptide comprising a subsequence encoded by a polynucleotide sequence of (a);

(e) one or more polynucleotide sequence that hybridizes to a nucleic acid that is physically linked in the human genome to a nucleic acid comprising a polynucleotide sequence of (a), (b), (c), or (d);

(f) one or more polynucleotide sequence comprising at least about 10 contiguous nucleotides of a polynucleotide sequence selected from the group consisting of;

SEQ ID NO;

1-SEQ ID NO;

88, or a sequence complementary thereto;

(g) one or more polypeptides or peptides comprising an amino acid sequence encoded by a polynucleotide of (a), (b), (c), (d), or (e); and

/or,

(h) one or more antibodies specific for a polypeptide or peptide sequence of (g).

34. The marker set of claim 33, wherein the nucleic acids comprise one or more of oligonucleotides, expression products, and amplification products.

35. The marker set of claim 34, wherein the oligonucleotides are synthetic oligonucleotides.

36. The marker set of claim 33, wherein the nucleic acids comprise labeled nucleic acid probes.

37. The marker set of claim 33, comprising a plurality of polypeptides or peptides.

38. The marker set of claim 33, comprising a plurality of antibodies.

39. The marker set of claim 33, wherein the plurality of members comprise nucleic acids and polypeptides.

40. The marker set of claim 33, wherein the plurality of members are logically or physically arrayed.

41. The marker set of claim 40, wherein the array comprises a bead array.

42. The marker set of claim 33, wherein each member of the marker set comprises at least 10 contiguous nucleotides from at least one of SEQ ID NO:

1-SEQ ID NO;

88.

43. The marker set of claim 33, wherein the plurality of members together comprise a plurality of sequences or subsequences selected from a plurality of nucleic acids represented by SEQ ID NO:

1-SEQ ID NO;

88.

44. The marker set of claim 33, comprising a majority of members that together comprise a majority of subsequences from a majority of SEQ ID NO:

1-SEQ ID NO;

88.

45. The marker set of claim 33, wherein a condition or characteristic associated with alterations of cholesterol levels is predicted by hybridizing the nucleic acids of the marker set to a DNA or RNA sample from a cell or a tissue, and detecting at least one expressed expression product.

46. The marker set of claim 33, wherein the condition or characteristic is associated with elevated levels of cholesterol.

47. The marker set of claim 33, wherein the condition or characteristic is selected from among atherosclerosis and heart disease.

48. An array comprising the marker set of claim 33.

49. A method for modulating a physiologic or pathologic response to alterations of cholesterol levels in a cell, tissue or organism, the method comprising:

modulating expression or activity of at least one polypeptide encoded by a nucleic acid comprising;

(a) at least one polynucleotide sequence selected from the group consisting of;

SEQ ID NO;

1-SEQ ID NO;

88 or a sequence complementary thereto;

(b) at least one polynucleotide sequence that hybridizes under stringent conditions to a polynucleotide sequence of (a);

(c) at least one polynucleotide sequence that is at least about 70% identical to a polynucleotide sequence of (a);

(d) at least one polynucleotide sequence that encodes a polypeptide or peptide comprising a subsequence encoded by a polynucleotide sequence of (a);

(e) at least one polynucleotide sequence that hybridizes to a nucleic acid that is physically linked in the human genome to a nucleic acid comprising a polynucleotide sequence of (a), (b), (c), or (d);

or,

(f) at least one polynucleotide sequence comprising at least about 10 contiguous nucleotides of a polynucleotide sequence selected from the group consisting of;

SEQ ID NO;

1-SEQ ID NO;

88, or a sequence complementary thereto.

50. The method of claim 49, comprising modulating expression or activity of at least one polypeptide contributing to a condition selected from atherosclerosis or heart disease.

51. The method of claim 49, comprising modulating a physiologic or pathologic response to alterations of cholesterol levels in one or more cell-types selected from the group comprising liver, adipose tissue, gall bladder, pancreas, monocytes, macrophages, foam cells, T cells, endothelia and smooth muscle derived from blood vessels and gut, fibroblasts, glia and nerve cells.

52. The method of claim 49, comprising modulating expression by expressing an exogenous nucleic acid comprising a polynucleotide sequence selected from SEQ ID NO:

1 to SEQ ID NO;

88.

53. The method of claim 49, comprising modulating expression in a cell line or non-human mammal.

54. The method of claim 53, wherein the non-human mammal comprises a mouse, a rat, a dog, a rabbit, a pig, a sheep or a non-human primate.

55. The method of claim 49, comprising modulating expression by inducing or suppressing expression of an endogenous nucleic acid.

56. The method of claim 55, wherein the endogenous nucleic acid encodes a polypeptide comprising a subsequence encoded by a sequence selected from among SEQ ID NO:

1-SEQ ID NO;

88, or homologues thereof.

57. The method of claim 49, comprising modulating expression by expressing an antisense RNA or a ribozyme.

58. The method of claim 49, wherein expression is modulated in response to cholesterol.

59. The method of claim 49, further comprising detecting altered expression or activity of an expression product encoded by a nucleic acid comprising a polynucleotide sequence selected from SEQ ID NO:

1-SEQ ID NO;

88, or conservative variants thereof.

60. The method of claim 49, comprising detecting altered expression or activity in a high throughput assay.

61. The method of claim 60, wherein a plurality of expression products are detected.

62. The method of claim 61, wherein the plurality of expression products are detected in an array.

63. The method,of claim 62, wherein the array comprises a bead array.

64. The method of claim 62, wherein the array comprises a tissue array.

65. The method of claim 49, further comprising detecting altered expression or activity of an expression product encoded by a nucleic acid comprising a polynucleotide sequence selected from SEQ ID NO:

1 to SEQ ID NO;

88.

66. The method of claim 65, comprising detecting altered expression or activity in response to administration of a pharmaceutical agent.

67. The method of claim 65, comprising detecting altered expression or activity in response to diet.

68. The method of claim 65, wherein a data record comprising the altered expression or activity is recorded in a database.

69. The method of claim 68, wherein the database comprises a plurality of character strings recorded on a computer or in a computer readable medium.

70. A method for identifying a gene capable of altering a physiologic or pathologic response to alterations in cholesterol levels, the method comprising:

(i) providing at least one nucleic acid comprising;

(a) at least one polynucleotide sequence selected from the group consisting of;

SEQ ID NO;

1-SEQ ID NO;

88 or a sequence complementary thereto;

(b) at least one polynucleotide sequence that hybridizes under stringent conditions to a polynucleotide sequence of (a);

(c) at least one polynucleotide sequence that is at least about 70% identical to a polynucleotide sequence of (a);

(d) at least one polynucleotide sequence that encodes a polypeptide or peptide comprising a subsequence encoded by a polynucleotide sequence of (a);

(e) at least one polynucleotide sequence that hybridizes to a nucleic acid that is physically linked in the human genome to a nucleic acid comprising a polynucleotide sequence of (a), (b), (c), or (d);

or,

(f) at least one polynucleotide sequence comprising at least about 10 contiguous nucleotides of a polynucleotide sequence selected from the group consisting of;

SEQ ID NO;

1-SEQ ID NO;

88, or a sequence complementary thereto; and

,

(ii) identifying at least one nucleic acid corresponding to a gene capable of altering a physiologic or pathologic response to elevated levels of cholesterol.

71. The method of claim 70, wherein the at least one polynucletode sequence of (f) comprises at least about 12 contiguous nucleotides of SEQ ID NO:

1-SEQ ID NO;

88.

72. The method of claim 70, wherein the at least one polynucletode sequence of (f) comprises at least about 14 contiguous nucleotides of SEQ ID NO:

1-SEQ ID NO;

88.

73. The method of claim 70, wherein the at least one polynucletode sequence of (f) comprises at least about 15 contiguous nucleotides of SEQ ID NO:

1-SEQ ID NO;

88.

74. The method of claim 70, wherein the at least one polynucletode sequence of (f) comprises at least about 17 contiguous nucleotides of SEQ ID NO:

1-SEQ ID NO;

88.

75. The method of claim 70, wherein the polynucleotide sequence in (i) is selected from the group consisting of:

SEQ ID NO;

1-SEQ ID NO;

88, or a conservative variation thereof.

76. The method of claim 70, comprising providing at least one expression vector comprising a polynucleotide sequence selected from among the polynucleotide sequences of (a), (b), (c), (d), (e) or (f).

77. The method of claim 70, comprising providing at least one probe comprising a polynucleotide sequence selected from among the polynucleotide sequences of (a), (b), (c), (d), (e) or (f);

and,

hybridizing the at least one probe to an expression product of a gene capable of altering a physiologic or pathologic response to elevated levels of cholesterol.

78. The method of claim 70, wherein providing the at least one nucleic acid comprises amplifying a target sequence comprising a polynucleotide sequence selected from among the polynucleotide sequences of (a), (b), (c), (d), (e) or (f).

79. The method of claim 78, wherein the amplifying comprises a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR).

80. The method of claim 70, comprising identifying a target sequence that is differentially expressed in response to cholesterol.

81. The method of claim 80, wherein the altered expression or activity of the product is determined by analysis of massively parallel signature sequence data.

82. The method of claim 80, wherein the altered expression or activity is determined to be differentially expressed to a p<

0.01 level of confidence.

83. The method of claim 80, wherein the altered expression or activity is determined to be differentially expressed to a p<

0.001 level of confidence.

84. The method of claim 80, comprising detecting altered expression in response to administration of a pharmaceutical agent.

85. The method of claim 80, comprising detecting altered expression in response to diet.

86. A method of evaluating a condition or characteristic associated with alterations in cholesterol levels in a subject, the method comprising:

(i) providing a subject cell or tissue sample of nucleic acids; and

,

(ii) detecting at least one polymorphic nucleic acid or at least one expression product corresponding to a polynucleotide sequence comprising;

(a) at least one polynucleotide sequence selected from the group consisting of;

SEQ ID NO;

1-SEQ ID NO;

88, or a sequence complementary thereto;

(b) at least one polynucleotide sequence that hybridizes under stringent conditions to a polynucleotide sequence of (a);

(c) at least one polynucleotide that is at least about 70% identical to a polynucleotide sequence of (a)

(d) at least one polynucleotide sequence that encodes a polypeptide or peptide comprising a subsequence encoded by a polynucleotide sequence of (a)

(e) at least one polynucleotide sequence that hybridizes to a nucleic acid that is physically linked in the human genome to a nucleic acid comprising a polynucleotide sequence of (a), (b), (c), or (d);

or,

(f) at least one polynucleotide sequence comprising at least about 10 unique nucleotides of a polynucleotide sequence selected from the group consisting of;

SEQ ID NO;

1-SEQ ID NO;

88, or a sequence complementary thereto;

wherein the polymorphic nucleic acid or expression or activity of the expression product is correlatable to at least one condition or characteristic associated with a physiological or pathologic response to alterations of cholesterol levels.

87. The method of claim 86, wherein the alterations of cholesterol levels comprise an elevated level of cholesterol.

88. The method of claim 86, wherein the expression product comprises an RNA.

89. The method of claim 86, wherein the expression product comprises a protein or polypeptide.

90. The method of claim 86, wherein the detecting step comprises qualitative detection.

91. The method of claim 86, wherein the detecting step comprises quantitative detection.