Claims
- 1. An isolated small interfering RNA (siRNA) polynucleotide, comprising at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, 104-105, and 108-109.
- 2. The small interfering RNA polynucleotide of claim 1 that comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 83-86, 88-91, 93-96, 98-101 and the complementary polynucleotide thereto.
- 3. A small interfering RNA polynucleotide of either claim 1 or claim 2 that is capable of interfering with expression of a PTP1B polypeptide, wherein the PTP-1B polypeptide comprises an amino acid sequence as set forth in a sequence selected from the group consisting of GenBank Ace. Nos. M31724, NM—002827, and M33689.
- 4. The siRNA polynucleotide of either claim 1 or claim 2 wherein the nucleotide sequence of the siRNA polynucleotide differs by one, two, three or four nucleotides at any of positions 1-19 of a sequence selected from the group consisting of the sequences set forth in SEQ ID NOS: 83-86, 88-91, 93-96, and 98-101, or at any position of a sequence selected from the group consisting of the sequences set forth in 104, 105, 108, and 109.
- 5. The siRNA polynucleotide of either claim 1 or claim 2 wherein the nucleotide sequence of the siRNA polynucleotide differs by at least two, three or four nucleotides at any of positions 1-19 of a sequence selected from the group consisting of the sequences set forth in SEQ ID NOS: 83-86, 88-91, 93-96, and 98-101, or at any position of a sequence selected from the group consisting of the sequences set forth in 104, 105, 108, and 109.
- 6. An isolated siRNA polynucleotide comprising a nucleotide sequence according to SEQ ID NO: 83, or the complement thereof.
- 7. An isolated siRNA polynucleotide comprising a nucleotide sequence according to SEQ ID NO: 88, or the complement thereof.
- 8. An isolated siRNA polynucleotide comprising a nucleotide sequence according to SEQ ID NO: 93, or the complement thereof.
- 9. An isolated siRNA polynucleotide comprising a nucleotide sequence according to SEQ ID NO: 98, or the complement thereof.
- 10. An isolated siRNA polynucleotide comprising a nucleotide sequence according to SEQ ID NO: 104 or 105.
- 11. An isolated siRNA polynucleotide comprising a nucleotide sequence according to SEQ ID NO: 108 or 109.
- 12. The siRNA polynucleotide of claim 1 or claim 2 wherein the polynucleotide comprises at least one synthetic nucleotide analogue of a naturally occurring nucleotide.
- 13. The siRNA polynucleotide of claim 1 or claim 2 wherein the polynucleotide is linked to a detectable label.
- 14. The siRNA polynucleotide of claim 13 wherein the detectable label is a reporter molecule.
- 15. The siRNA of claim 14 wherein the reporter molecule is selected from the group consisting of a dye, a radionuclide, a luminescent group, a fluorescent group, and biotin.
- 16. The siRNA polynucleotide of claim 15 wherein the fluorescent group is fluorescein isothiocyanate.
- 17. The siRNA polynucleotide of claim 13 wherein the detectable label is a magnetic particle.
- 18. A pharmaceutical composition comprising the siRNA polynucleotide of either claim 1 or claim 2 and a physiologically acceptable carrier.
- 19. The pharmaceutical composition of claim 18 wherein the carrier comprises a liposome.
- 20. A recombinant nucleic acid construct comprising a polynucleotide that is capable of directing transcription of a small interfering RNA (siRNA), the polynucleotide comprising:
(i) a first promoter; (ii) a second promoter; and (iii) at least one DNA polynucleotide segment comprising at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, or a complement thereto, wherein each DNA polynucleotide segment and its complement are operably linked to at least one of the first and second promoters, and wherein the promoters are oriented to direct transcription of the DNA polynucleotide segment and its reverse complement.
- 21. A recombinant nucleic acid construct comprising a polynucleotide that is capable of directing transcription of a small interfering RNA (siRNA), the polynucleotide comprising a promoter operably linked to at least one DNA polynucleotide segment comprising at least one nucleotide sequence that is selected from the group consisting of SEQ ID NOs: 102, 103, 106, and 107.
- 22. The recombinant nucleic acid construct of either claim 20 or 21, comprising at least one enhancer that is selected from a first enhancer operably linked to the first promoter and a second enhancer operably linked to the second promoter.
- 23. The recombinant nucleic acid construct of claim 20, comprising at least one transcriptional terminator that is selected from (i) a first transcriptional terminator that is positioned in the construct to terminate transcription directed by the first promoter and (ii) a second transcriptional terminator that is positioned in the construct to terminate transcription directed by the second promoter.
- 24. The recombinant nucleic acid construct of either claim 20 or claim 21 wherein the siRNA is capable of interfering with expression of a PTP1B polypeptide, wherein the PTP1B polypeptide comprises an amino acid sequence as set forth in a sequence selected from the group consisting of GenBank Acc. Nos. M31724, NM—002827, and M33689.
- 25. A recombinant nucleic acid construct comprising a polynucleotide that is capable of directing transcription of a small interfering RNA (siRNA), the polynucleotide comprising at least one promoter and a DNA polynucleotide segment, wherein the DNA polynucleotide segment is operably linked to the promoter, and wherein the DNA polynucleotide segment comprises (i) at least one DNA polynucleotide that comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, or a complement thereto; (ii) a spacer sequence comprising at least 4 nucleotides operably linked to the DNA polynucleotide of (i); and (iii) the reverse complement of the DNA polynucleotide of (i) operably linked to the spacer sequence.
- 26. The recombinant nucleic acid construct of claim 25 wherein the siRNA comprises an overhang of at least one and no more than four nucleotides, the overhang being located immediately 3′ to (iii).
- 27. The recombinant nucleic acid construct of claim 25 wherein the spacer sequence comprises at least 9 nucleotides.
- 28. The recombinant nucleic acid construct of claim 25 wherein the spacer sequence comprises two uridine nucleotides that are contiguous with (iii).
- 29. The recombinant nucleic acid construct of claim 25 comprising at least one transcriptional terminator that is operably linked to the DNA polynucleotide segment.
- 30. A host cell transformed or transfected with the recombinant nucleic acid construct of any one of claims 20, 21, 23 and 25-29.
- 31. A pharmaceutical composition comprising an siRNA polynucleotide and a physiologically acceptable carrier, wherein the siRNA polynucleotide is selected from the group consisting of:
(i) an RNA polynucleotide which comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, 104-105, and 108-109, (ii) an RNA polynucleotide that comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 83-86, 88-91, 93-96, 98-101 and the complementary polynucleotide thereto, (iii) an RNA polynucleotide according to (i) or (ii) wherein the nucleotide sequence of the siRNA polynucleotide differs by one, two or three nucleotides at any of positions 1-19 of a sequence selected from the group consisting of the sequences set forth in SEQ ID NOS: 83-86, 88-91, 93-96, and 98-101, or at any position of a sequence selected from the group consisting of the sequences set forth in SEQ ID NOS: 104, 105, 108, and 109, and (iv) an RNA polynucleotide according to (i) or (ii) wherein the nucleotide sequence of the siRNA polynucleotide differs by two, three or four nucleotides at any of positions 1-19 of a sequence selected from the group consisting of the sequences set forth in SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, or at any position of a sequence selected from the group consisting of the sequences set forth in SEQ ID NOS: 104, 105, 108, and 109.
- 32. The pharmaceutical composition of claim 31 wherein the carrier comprises a liposome.
- 33. A method for interfering with expression of a PTP1B polypeptide, or variant thereof, comprising contacting a subject that comprises at least one cell which is capable of expressing a PTP1B polypeptide with a siRNA polynucleotide for a time and under conditions sufficient to interfere with PTP1B polypeptide expression, wherein:
(a) the PTP1B polypeptide comprises an amino acid sequence as set forth in a sequence selected from the group consisting of GenBank Acc. Nos. M31724, NM—002827, NM—011201, and M33689, (b) the siRNA polynucleotide is selected from the group consisting of
(i) an RNA polynucleotide which comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, 104-105, and 108-109, (ii) an RNA polynucleotide that comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, and the complementary polynucleotide thereto, (iii) an RNA polynucleotide according to (i) or (ii) wherein the nucleotide sequence of the siRNA polynucleotide differs by one, two or three nucleotides at any of positions 1-19 of a sequence selected from the group consisting of the sequences set forth in SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, or at any position of a sequence selected from the group consisting of the sequences set forth in SEQ ID NOS: 104, 105, 108, and 109, and (iv) an RNA polynucleotide according to (i) or (ii) wherein the nucleotide sequence of the siRNA polynucleotide differs by two, three or four nucleotides at any of positions 1-19 of a sequence selected from the group consisting of the sequences set forth in SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, or at any position of a sequence selected from the group consisting of the sequences set forth in SEQ ID NOS: 104, 105, 108, and 109.
- 34. A method for interfering with expression of a PTP1B polypeptide that comprises an amino acid sequence as set forth in a sequence selected from the group consisting of GenBank Ace. Nos. M31724, NM—002827, and M33689, or a variant of said PTP1B polypeptide, said method comprising contacting, under conditions and for a time sufficient to interfere with PTP1B polypeptide expression, (i) a subject that comprises at least one cell that is capable of expressing the PTP1B polypeptide, and (ii) a recombinant nucleic acid construct according to either claim 18 or claim 22.
- 35. A method for identifying a component of a PTP1B signal transduction pathway comprising:
A. contacting a siRNA polynucleotide and a first biological sample comprising at least one cell that is capable of expressing a PTP1B polypeptide, or a variant of said PTP1B polypeptide, under conditions and for a time sufficient for PTP1B expression when the siRNA polynucleotide is not present, wherein
(1) the PTP1B polypeptide comprises an amino acid sequence as set forth in a sequence selected from the group consisting of GenBank Acc. Nos. M31724, NM—002827, and M33689, (2) the siRNA polynucleotide is selected from the group consisting of
(i) an RNA polynucleotide which comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, 104-105, and 108-109, (ii) an RNA polynucleotide that comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 83-86, 88-91, 93-96, 98-101 and the complementary polynucleotide thereto, (iii) an RNA polynucleotide according to (i) or (ii) wherein the nucleotide sequence of the siRNA polynucleotide differs by one, two or three nucleotides at any of positions 1-19 of a sequence selected from the group consisting of the sequences set forth in SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, or at any position of a sequence selected from the group consisting of the sequences set forth in SEQ ID NOS: 104, 105, 108, and 109, and (iv) an RNA polynucleotide according to (i) or (ii) wherein the nucleotide sequence of the siRNA polynucleotide differs by two, three or four nucleotides at any of positions 1-19 of a sequence selected from the group consisting of the sequences set forth in SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, or at any position of a sequence selected from the group consisting of the sequences set forth in SEQ ID NOS: 104, 105, 108, and 109; and B. comparing a level of phosphorylation of at least one protein that is capable of being phosphorylated in the cell with a level of phosphorylation of the protein in a control sample that has not been contacted with the siRNA polynucleotide,
wherein an altered level of phosphorylation of the protein in the presence of the siRNA polynucleotide relative to the level of phosphorylation of the protein in an absence of the siRNA polynucleotide indicates that the protein is a component of the PTP1B signal transduction pathway.
- 36. The method of claim 35 wherein the signal transduction pathway comprises a Jak2 kinase.
- 37. A method for modulating an insulin receptor protein phosphorylation state in a cell, comprising contacting the cell with a siRNA polynucleotide under conditions and for a time sufficient to interfere with expression of a PTP1B polypeptide, wherein
(a) the PTP1B polypeptide comprises an amino acid sequence as set forth in a sequence selected from the group consisting of GenBank Acc. Nos. M31724, NM—002827, NM—011201, and M33689, (b) the siRNA polynucleotide is selected from the group consisting of
(i) an RNA polynucleotide which comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 83-86, 88-91, 93-96, 98-101 or the complements thereof and SEQ ID NOS: 104, 105, 108, and 109, (ii) an RNA polynucleotide that comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 83-86, 88-91, 93-96, 98-101 and the complementary polynucleotide thereto, (iii) an RNA polynucleotide according to (i) or (ii) wherein the nucleotide sequence of the siRNA polynucleotide differs by one, two or three nucleotides at any of positions 1-19 of a sequence selected from the group consisting of the sequences set forth in SEQ ID NOS: 83-86, 88-91, 93-96, and 98-101, or at any position of a sequence selected from the group consisting of the sequences set forth in SEQ ID NOS: 104, 105, 108, and 109, and (iv) an RNA polynucleotide according to (i) or (ii) wherein the nucleotide sequence of the siRNA polynucleotide differs by two, three or four nucleotides at any of positions 1-19 of a sequence selected from the group consisting of the sequences set forth in SEQ ID NOS: 83-86, 88-91, 93-96, and 98-101, or at any position of a sequence selected from the group consisting of the sequences set forth in SEQ ID NOS: 104, 105, 108, and 109; and (c) the insulin receptor protein comprises a polypeptide which comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: __-__ or a variant thereof.
- 38. A method for altering Jak2 protein phosphorylation state in a cell, comprising contacting the cell with a siRNA polynucleotide under conditions and for a time sufficient to interfere with expression of a PTP1B polypeptide, wherein
(a) the PTP1B polypeptide comprises an amino acid sequence as set forth in a sequence selected from the group consisting of GenBank Ace. Nos. M31724, NM—002827, NM—011201, and M33689, (b) the siRNA polynucleotide is selected from the group consisting of
(i) an RNA polynucleotide which comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 83-86, 88-91, 93-96, and 98-101 or the complements thereof, and SEQ ID NOS: 104, 105, 108, and 109, (ii) an RNA polynucleotide that comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 83-86, 88-91, 93-96, and 98-101 and the complementary polynucleotide thereto, (iii) an RNA polynucleotide according to (i) or (ii) wherein the nucleotide sequence of the siRNA polynucleotide differs by one, two or three nucleotides at any of positions 1-19 of a sequence selected from the group consisting of the sequences set forth in SEQ ID NOS: 83-86, 88-91, 93-96, and 98-101, or at any position of a sequence selected from the group consisting of the sequences set forth in SEQ ID NOS: 104, 105, 108, and 109, and (iv) an RNA polynucleotide according to (i) or (ii) wherein the nucleotide sequence of the siRNA polynucleotide differs by two, three or four nucleotides at any of positions 1-19 of a sequence selected from the group consisting of the sequences set forth in SEQ ID NOS: 83-86, 88-91, 93-96, and 98-101, or at any position of a sequence selected from the group consisting of the sequences set forth in SEQ ID NOS: 104, 105, 108, and 109; and (c) the Jak2 protein comprises a polypeptide which comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: __-__, or a variant thereof.
- 39. A method for treating a Jak2-associated disorder comprising administering to a subject in need thereof a pharmaceutical composition according to claim 31, wherein the siRNA polynucleotide inhibits expression of a PTP1B polypeptide, or a variant thereof.
- 40. The method of claim 39 wherein the Jak2-associated disorder is selected from the group consisting of diabetes, obesity, hyperglycemia-induced apoptosis, inflammation, and a neurodegenerative disorder.
- 41. An isolated small interfering RNA (siRNA) polynucleotide, comprising an RNA polynucleotide which comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 18-21, 33-36, 43-46, 53-56, and 58-61.
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional Patent Application No. 60/383,249 filed May 23, 2002, and U.S. Provisional Patent Application No. 60/462,942 filed Apr. 14, 2003, which are incorporated herein by reference in their entirety.
Provisional Applications (2)
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Number |
Date |
Country |
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60462942 |
Apr 2003 |
US |
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60383249 |
May 2002 |
US |