Claims
- 1. A GAD65 polypeptide comprising an E517P mutation, said polypeptide characterized by decreased specific binding to an antibody selected from the group consisting of GAD6, MICA-1, MICA-3, MICA-4, and MICA-6, wherein the decreased binding is relative to a corresponding GAD65 polypeptide not having the E517P mutation.
- 2. The mutant GAD65 polypeptide of claim 1, further characterized by decreased specific binding to MICA-2 antibody, wherein the decreased binding is relative to a corresponding GAD65 polypeptide not having the E517P mutation
- 3. The mutant GAD65 polypeptide of claim 1 which is a full-length GAD65 polypeptide.
- 4. The mutant GAD65 polypeptide of claim 1, which comprises a sequence at position 515-525 that is selected from group consisting of SEQ ID NO: 15 and SEQ ID NO:16.
- 5. A method for detecting the presence of or risk of type 1 diabetes in a subject, the method comprising:
1) isolating from the subject a first serum sample and a second serum sample; 2) contacting the first serum sample with a mutant GAD65 polypeptide according to claim 1;3) contacting the second serum sample with a control GAD65 polypeptide that is immunologically cross-reactive with C-terminal conformational epitopes of wild-type GAD65, said control GAD65 polypeptide not having the E517P mutation and having substantially the same GAD6-, MICA-1-, MICA-3-, MICA-4-, or MICA-6-specific binding activity as the corresponding GAD65 polypeptide; 4) determining the degree of GAD65 autoantibody binding activity in the first and second serum samples; and 5) comparing the degree of GAD65 autoantibody binding activity in the first and second serum samples to detect the presence of or risk of type 1 diabetes in the subject.
- 6. The method of claim 5, wherein the control GAD65 polypeptide is the corresponding GAD65 polypeptide not having the E517P mutation.
- 7. The method of claim 5, wherein the control GAD65 polypeptide is wild-type GAD65.
- 8. The method of claim 5, wherein the control GAD65 polypeptide has the amino acid sequence of SEQ ID NO: 12 at position 515-525.
- 9. The method of claim 5, wherein the mutant GAD65 polypeptide is full-length.
- 10. The method of claim 5, wherein the mutant GAD65 polypeptide consists of the GAD65 wild-type amino acid sequence at positions other than position 517.
- 11. An isolated nucleic acid selected from the group consisting of:
(a) nucleic acids which encode the rat Ian5(+) polypeptide of SEQ ID NO: 3; (b) nucleic acids which encode the rat Ian5(lyp) polypeptide of SEQ ID NO: 4; and (c) full length complements of the nucleic acids of (a) or (b).
- 12. The isolated nucleic acid of claim 11, which comprises a nucleotide sequence selected from the group consisting of (a) SEQ ID NO: 1 and (b) nucleotides 1-312 of SEQ ID NO: 2.
- 13. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 3.
- 14. An isolated polypeptide consisting essentially of amino acids 1-84 of SEQ ID NO: 4.
- 15. The isolated polypeptide of claim 14, further consisting of 1-40 random amino acids adjacent and carboxy terminal to amino acid 84.
- 16. The isolated polypeptide of claim 15, which is the polypeptide having the amino acid sequence of SEQ ID NO: 4.
- 17. An isolated polypeptide consisting essentially of amino acids 1-85 of SEQ ID NO: 6.
- 18. The isolated polypeptide of claim 17, further consisting of 1-40 random amino acids adjacent and carboxy terminal to amino acid 85.
- 19. An antibody that specifically binds to rat Ian5(+) polypeptide, wherein said antibody is not immunologically cross-reactive with human Ian5 or mouse Ian5 polypeptide.
- 20. The antibody of claim 19, which is a monoclonal antibody, a polyclonal antibody, a single chain antibody, a heavy chain antibody, an F(ab′)2, F(ab′), or Fv fragment.
- 21. An antibody that specifically binds to rat Ian5(lyp) polypeptide, wherein said antibody is not immunologically cross-reactive with rat Ian5(+), human Ian5, or mouse Ian5 polypeptide.
- 22. The antibody of claim 19, which is a monoclonal antibody, a polyclonal antibody, a single chain antibody, a heavy chain antibody, an F(ab′)2, F(ab′), or Fv fragment.
- 23. An expression construct comprising the following elements linked in operable combination:
a transcriptional promoter; a nucleic acid according to claim 11, and a transcriptional terminator.
- 24. The expression construct of claim 23, wherein the nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and nucleotides 1-312 of SEQ ID NO: 2.
- 25. A prokaryotic or eukaryotic cell transformed or transfected with the expression construct according to claim 23.
- 26. The prokaryotic or eukaryotic cell of claim 25, which is selected from the group consisting of a bacterial cell, a yeast cell, and a mammalian cell.
- 27. A vector comprising the expression construct according to claim 23.
- 28. An isolated host cell comprising the vector of claim 27.
- 29. A method for producing an Ian5 polypeptide, the method comprising:
growing cells transformed or transfected with the vector of claim 27; and isolating the Ian5 polypeptide from the cells.
- 30. The method of claim 29, wherein the cells are selected from the group consisting of bacterial cells, yeast cells, and mammalian cells.
- 31. An in vitro method of identifying agonists or antagonists of an Ian5 pathway to identify candidates for type 1 diabetes drug development, the method comprising:
administering a candidate compound to a first cell that expresses the Ian5 polypeptide; administering the candidate compound to a second cell that does not express the polypeptide; and determining whether the candidate compound produces a physiological change in the first cell relative to the second cell.
- 32. The method of claim 31, wherein the first and second cells are mammalian cells.
- 33. The method of claim 32, wherein the mammalian cells are mammalian hematopoietic cells.
- 34. The method of claim 32, wherein the Ian5 polypeptide is selected from the group consisting of rat Ian5(+), rat Ian(lyp), human Ian5, and mouse lan5.
- 35. The method of claim 31, wherein the candidate compound stimulates or inhibits cell proliferation.
- 36. A method for developing gene therapy for type 1 diabetes, the method comprising:
(1) administering a nucleic acid comprising an Ian5 polynucleotide to a non-human mammal having a frameshift mutation in the Ian5 gene locus, wherein said frameshift mutation results in a truncated mutant Ian5 polypeptide consisting essentially of amino acids corresponding to amino acids 1-84 of SEQ ID NO: 4; and wherein said non-human animal exhibits at least one clinical symptom of type 1 diabetes; and (2) determining whether the nucleic acid encoding the Ian5 polynucleotide produces an amelioration of at least one clinical symptom of diabetes.
- 37. The method of claim 36, wherein the nucleic acid comprising the Ian5 polynucleotide is selected from the group consisting of:
(a) a vector comprising an Ian5 polynucleotide that encodes a wild-type Ian5 polypeptide, said wild-type Ian5-encoding polynucleotide flanked by regions that promote intrachromosomal homologous recombination; (b) a vector comprising, linked in operative combination, a transcription promoter, the wild-type Ian5-encoding polynucleotide as in (a), and a transcription terminator; (c) an antisense Ian5 polynucleotide hybridizable within a cell to a polynucleotide encoding the truncated mutant Ian5 polypeptide; and (d) a vector comprising, linked in operative combination, a transcription promoter, the antisense Ian5 polynucleotide as in (c), and a transcription terminator.
- 38. The method of claim 36, wherein the non-human mammal is a genetically modified mammal having an exogenous mutant Ian5 gene.
- 39. The method of claim 36, wherein the non-human mammal having the frameshift mutation is a DR.lyp/lyp rat.
- 40. A method for developing gene therapy for type 1 diabetes, the method comprising:
(1) administering a vector comprising an Ian5 polynucleotide to a non-human mammal having a knockout mutation in the Ian5 gene locus, wherein said non-human animal exhibits at least one clinical symptom of type 1 diabetes; and (2) determining whether the vector produces an amelioration of at least one clinical symptom of diabetes.
- 41 The method of claim 40, wherein the vector is selected from the group consisting of:
(a) a vector comprising an Ian5 polynucleotide that encodes a wild-type Ian5 polypeptide, said wild-type Ian5-encoding polynucleotide flanked by regions that promote intrachromosomal homologous recombination; and (b) a vector comprising, linked in operative combination, a transcription promoter, the wild-type Ian5-encoding polynucleotide as in (a), and a transcription terminator.
- 42. The method of claim 40, wherein the non-human animal having the Ian5 knockout mutation is a transgenic knockout mouse.
- 43. A method for detecting in a subject the presence of or risk of developing type 1 diabetes, the method comprising:
detecting the presence of a mutation at one or more nucleotide positions in the Ian5 gene in a sample from the subject; and therefrom identifying the presence or risk of developing type 1 diabetes.
- 44. The method of claim 43, wherein the mutation is a frameshift mutation resulting is a truncated mutant Ian5 polypeptide.
- 45. The method of claim 44, wherein the mutation is a mutation in codon 85 of the human Ian5 coding sequence.
- 46. The method according to claims 43, wherein the presence of the mutation is detected by a technique that is selected from the group consisting of direct sequencing, hybridization with oligonucleotide probes, a ligation reaction, a polymerase chain reaction, and single nucleotide primer-guided extension assays.
- 47. A method for identifying a genetic mutation that correlates with type 1 diabetes, the method comprising:
(a) determining the sequence of the Ian5 gene from a plurality of humans known to have diabetes; (b) comparing the sequence to the wild-type human Ian5 gene sequence; and (c) identifying mutations in the human Ian5 genes that correlate with the presence of type 1 diabetes.
- 48. A method for detecting in a human subject the presence of or the risk of developing type 1 diabetes, the method comprising:
(a) obtaining from the subject a biological sample containing or derived from lymphocytes; (b) obtaining a control sample containing or derived from lymphocytes; (c) determining the level of Ian5 gene expression in the subject sample and the control sample; and (d) comparing the level of Ian5 gene expression in the subject sample and the control sample to detect the presence of or the risk of developing type 1 diabetes.
- 49. The method of claim 48, wherein the level of Ian5 gene expression is determined with a nucleic acid probe.
- 50. The method of claim 49, wherein the level of Ian5 gene expression is determined with an anti-Ian5 antibody.
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional application No. 60/383,913, filed on May 29, 2002, incorporated by reference herein in its entirety for all purposes.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This work was supported in part by grants A142380 and DK26190-14 from the National Institutes of Health. The U.S. Governnent may have certain rights in the invention.
Provisional Applications (1)
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Number |
Date |
Country |
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60383913 |
May 2002 |
US |