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Publication numberUS20040009946 A1
Publication typeApplication
Application numberUS 10/444,925
Publication dateJan 15, 2004
Filing dateMay 23, 2003
Priority dateMay 23, 2002
Also published asCA2525976A1, EP1546379A2, EP1546379A4, US7399586, US20040077574, US20040121353, WO2003099227A2, WO2003099227A3, WO2004016735A2
Publication number10444925, 444925, US 2004/0009946 A1, US 2004/009946 A1, US 20040009946 A1, US 20040009946A1, US 2004009946 A1, US 2004009946A1, US-A1-20040009946, US-A1-2004009946, US2004/0009946A1, US2004/009946A1, US20040009946 A1, US20040009946A1, US2004009946 A1, US2004009946A1
InventorsStephen Lewis, Richard Klinghoffer, Linda Wilson
Original AssigneeCeptyr, Inc.
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Modulation of PTP1B expression and signal transduction by RNA interference
US 20040009946 A1
Abstract
Compositions and methods relating to small interfering RNA (siRNA) polynucleotides are provided as pertains to modulation of biological signal transduction. Shown are siRNA polynucleotides that interfere with expression of PTP1B, a member of the protein tyrosine phosphatase (PTP) class of enzymes that mediate signal transduction. In certain preferred embodiments siRNA modulate signal transduction pathways comprising human or murine PTP-1B and, in certain further embodiments, insulin receptor and/or Jak2. Modulation of PTP1B-mediated biological signal transduction has uses in diseases associated with defects in cell proliferation, cell differentiation and/or cell survival, such as metabolic disorders (including diabetes and obesity), cancer, autoimmune disease, infectious and inflammatory disorders and other conditions.
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Claims(41)
What is claimed is:
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, NM002827, 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, NM002827, 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, NM002827, NM011201, 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, NM002827, 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, NM002827, 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, NM002827, NM011201, 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, NM002827, NM011201, 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.
Description
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.

BACKGROUND OF THE INVENTION

[0002] 1. Technical Field

[0003] The present invention relates generally to compositions and methods useful for treating conditions associated with defects in cell proliferation, cell differentiation, and cell survival. The invention is more particularly related to double-stranded RNA polynucleotides that interfere with expression of a protein tyrosine phosphatase, PTP1B, and polypeptide variants thereof. The present invention is also related to the use of such RNA polynucleotides to alter activation of signal transduction pathway components or to alter cellular metabolic processes that lead to proliferative responses, cell differentiation and development, and cell survival.

[0004] 2. Description of the Related Art

[0005] Reversible protein tyrosine phosphorylation, coordinated by the action of protein tyrosine kinases (PTKs) that phosphorylate certain tyrosine residues in polypeptides, and protein tyrosine phosphatases (PTPs) that dephosphorylate certain phosphotyrosine residues, is a key mechanism in regulating many cellular activities. It is becoming apparent that the diversity and complexity of the PTPs and PTKs are comparable, and that PTPs are equally important in delivering both positive and negative signals for proper function of cellular machinery. Regulated tyrosine phosphorylation contributes to specific pathways for biological signal transduction, including those associated with cell division, cell survival, apoptosis, proliferation and differentiation. Defects and/or malfunctions in these pathways may underlie certain disease conditions for which effective means for intervention remain elusive, including for example, malignancy, autoimmune disorders, diabetes, obesity, and infection.

[0006] The protein tyrosine phosphatase (PTP) family of enzymes consists of more than 100 structurally diverse proteins in vertebrates, including almost 40 human PTPs that have in common the conserved 250 amino acid PTP catalytic domain, but which display considerable variation in their non-catalytic segments (Charbonneau and Tonks, 1992 Annu. Rev. Cell Biol. 8:463-493; Tonks, 1993 Semin. Cell Biol. 4:373-453; Andersen et al., Mol Cell Biol. 21:7117-36 (2001)). This structural diversity presumably reflects the diversity of physiological roles of individual PTP family members, which in certain cases have been demonstrated to have specific functions in growth, development and differentiation (Desai et al., 1996 Cell 84:599-609; Kishihara et al., 1993 Cell 74:143-156; Perkins et al., 1992 Cell 70:225-236; Pingel and Thomas, 1989 Cell 58:1055-1065; Schultz et al., 1993 Cell 73:1445-1454). The PTP family includes receptor-like and non-transmembrane enzymes that exhibit exquisite substrate specificity in vivo and that are involved in regulating a wide variety of cellular signaling pathways (Andersen et al., Mol. Cell. Biol. 21:7117 (2001); Tonks and Neel, Curr. Opin. Cell Biol. 13:182 (2001)). PTPs thus participate in a variety of physiologic functions, providing a number of opportunities for therapeutic intervention in physiologic processes through alteration (i.e., a statistically significant increase or decrease) or modulation (e.g., up-regulation or down-regulation) of PTP activity.

[0007] Although recent studies have also generated considerable information regarding the structure, expression and regulation of PTPs, the nature of many tyrosine phosphorylated substrates through which the PTPs exert their effects remains to be determined. Studies with a limited number of synthetic phosphopeptide substrates have demonstrated some differences in the substrate selectivities of different PTPs (Cho et al., 1993 Protein Sci. 2: 977-984; Dechert et al., 1995 Eur. J. Biochem. 231:673-681). Analyses of PTP-mediated dephosphorylation of PTP substrates suggest that catalytic activity may be favored by the presence of certain amino acid residues at specific positions in the substrate polypeptide relative to the phosphorylated tyrosine residue (Salmeen et al., 2000 Molecular Cell 6:1401; Myers et al., 2001 J. Biol. Chem. 276:47771; Myers et al., 1997 Proc. Natl. Acad. Sci. USA 94:9052; Ruzzene et al., 1993 Eur. J. Biochem. 211:289-295; Zhang et al., 1994 Biochemistry 33:2285-2290). Thus, although the physiological relevance of the substrates used in these studies is unclear, PTPs display a certain level of substrate selectivity in vitro.

[0008] The PTP family of enzymes contains a common evolutionarily conserved segment of approximately 250 amino acids known as the PTP catalytic domain. Within this conserved domain is a unique signature sequence motif, CX5R (SEQ ID NO: __, that is invariant among all PTPs. In a majority of PTPs, an 11 amino acid conserved sequence ([I/V]HCXAGXXR[S/T)G (SEQ ID NO: 1)) containing the signature sequence motif is found. The cysteine residue in this motif is invariant in members of the family and is essential for catalysis of the phosphotyrosine dephosphorylation reaction. It functions as a nucleophile to attack the phosphate moiety present on a phosphotyrosine residue of the incoming substrate. If the cysteine residue is altered by site-directed mutagenesis to serine (e.g., in cysteine-to-serine or “CS” mutants) or alanine (e.g., cysteine-to-alanine or “CA” mutants), the resulting PTP is catalytically deficient but retains the ability to complex with, or bind, its substrate, at least in vitro.

[0009] CS mutants of certain PTP family members, for example, MKP-1 (Sun et al., 1993 Cell 75:487), may effectively bind phosphotyrosyl polypeptide substrates in vitro to form stable enzyme-substrate complexes, thereby functioning as “substrate trapping” mutant PTPs. Such complexes can be isolated from cells in which both the mutant PTP and the phosphotyrosyl polypeptide substrates are present. According to non-limiting theory, expression of such a CS mutant PTP can thus antagonize the normal function of the corresponding wildtype PTP (and potentially other PTPs and/or other components of a PTP signaling pathway) via a mechanism whereby the CS mutant binds to and sequesters the substrate, precluding substrate interaction with catalytically active, wildtype enzyme (e.g., Sun et al., 1993).

[0010] CS mutants of certain other PTP family members, however, may bind phosphotyrosyl polypeptide substrates and form complexes that exist transiently and are not stable when the CS mutant is expressed in cells, i.e., in vivo. The CS mutant of one PTP, PTP1B (PTP-1B), is an example of such a PTP. Catalytically deficient mutants of such enzymes that are capable of forming stable complexes with phosphotyrosyl polypeptide substrates may be derived by mutating a wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue and replacing it with an amino acid that does not cause significant alteration of the Km of the enzyme but that results in a reduction in Kcat, as disclosed, for example, in U.S. Pat. Nos. 5,912,138 and 5,951,979, in U.S. application Ser. No. 09/323,426 and in PCT/US97/13016 and PCT/US00/14211. For instance, mutation of Asp 181 in PTP1B to alanine to create the aspartate-to-alanine (D to A or DA) mutant PTP1B-D181A results in a PTP1B “substrate trapping” mutant enzyme that forms a stable complex with its phosphotyrosyl polypeptide substrate (e.g., Flint et al., 1997 Proc. Natl. Acad. Sci. 94:1680). Substrates of other PTPs can be identified using a similar substrate trapping approach, for example substrates of the PTP family members PTP-PEST (Garton et al., 1996 J. Mol. Cell. Biol. 16:6408), TCPTP (Tiganis et al., 1998 Mol. Cell Biol. 18:1622), PTP-HSCF (Spencer et al., 1997 J. Cell Biol. 138:845), and PTP-H1 (Zhang et al., 1999 J. Biol. Chem. 274:17806).

[0011] One non-transmembrane PTP, PTP1B, recognizes several tyrosine-phosphorylated proteins as substrates, many of which are involved in human disease. For example, therapeutic inhibition of PTP1B in the insulin signaling pathway may serve to augment insulin action, thereby ameliorating the state of insulin resistance common in Type II diabetes patients. PTP1B acts as a negative regulator of signaling that is initiated by several growth factor/hormone receptor PTKs, including p210 Bcr-Abl (LaMontagne et al., Mol. Cell. Biol. 18:2965-75 (1998); LaMontagne et al., Proc. Natl. Acad. Sci. USA 95:14094-99 (1998)), receptor tyrosine kinases, such as EGF receptor, PDGF receptor, and insulin receptor (IR) (Tonks et al., Curr. Opin. Cell Biol. 13:182-95 (2001)), and JAK family members such as Jak2 and others (Myers et al., J. Biol. Chem. 276:47771-74 (2001)), as well as signaling events induced by cytokines (Tonks and Neel, 2001). Activity of PTP1B is regulated by modifications of several amino acid residues, such as phosphorylation of Ser residues (Brautigan and Pinault, 1993; Dadke et al., 2001; Flint et al., 1993), and oxidation of the active Cys residue in its catalytic motif (Lee et al., 1998; Meng et al., 2002) which is evolutionary conserved among protein tyrosine phosphatases and dual phosphatase family members (Andersen et al., 2001). In addition, changes in the expression levels of PTP1B have been noted in several human diseases, particularly those associated with disruption of the normal patterns of tyrosine phosphorylation. For example, therapeutic inhibition of PTPs such as PTP1B in the insulin signaling pathway may serve to augment insulin action, thereby ameliorating the state of insulin resistance common in patients with type 2 diabetes.

[0012] Diabetes mellitus is a common, degenerative disease affecting 5-10% of the human population in developed countries, and in many countries, it may be one of the five leading causes of death. Approximately 2% of the world's population has diabetes, the overwhelming majority of cases (>97%) being type 2 diabetes and the remainder being type 1. In type 1 diabetes, which is frequently diagnosed in children or young adults, insulin production by pancreatic islet beta cells is destroyed. Type 2 diabetes, or “late onset” or “adult onset” diabetes, is a complex metabolic disorder in which cells and tissues cannot effectively use available insulin; in some cases insulin production is also inadequate. At the cellular level, the degenerative phenotype that may be characteristic of late onset diabetes mellitus includes, for example, impaired insulin secretion and decreased insulin sensitivity, i.e., an impaired response to insulin.

[0013] Studies have shown that diabetes mellitus may be preceded by or is associated with certain related disorders. For example, an estimated forty million individuals in the U.S. suffer from late onset impaired glucose tolerance (IGT). IGT patients fail to respond to glucose with increased insulin secretion. Each year a small percentage (5-10%) of IGT individuals progress to insulin deficient non-insulin dependent diabetes (NIDDM). Some of these individuals further progress to insulin dependent diabetes mellitus (IDDM). NIDDM and IDDM are associated with decreased release of insulin by pancreatic beta cells and/or a decreased response to insulin by cells and tissues that normally exhibit insulin sensitivity. Other symptoms of diabetes mellitus and conditions that precede or are associated with diabetes mellitus include obesity, vascular pathologies, and various neuropathies, including blindness and deafness.

[0014] Type 1 diabetes is treated with lifelong insulin therapy, which is often associated with undesirable side effects such as weight gain and an increased risk of hypoglycemia. Current therapies for type 2 diabetes (NIDDM) include altered diet, exercise therapy, and pharmacological intervention with injected insulin or oral agents that are designed to lower blood glucose levels. Examples of such presently available oral agents include sulfonylureas, biguanides, thiazolidinediones, repaglinide, and acarbose, each of which alters insulin and/or glucose levels. None of the current pharmacological therapies, however, controls the disease over its full course, nor do any of the current therapies correct all of the physiological abnormalities in type 2 NIDDM, such as impaired insulin secretion, insulin resistance, and excessive hepatic glucose output. In addition, treatment failures are common with these agents, such that multi-drug therapy is frequently necessary.

[0015] In certain metabolic diseases or disorders, one or more biochemical processes, which may be either anabolic or catabolic (e.g., build-up or breakdown of substances, respectively), are altered (e.g., increased or decreased in a statistically significant manner) or modulated (e.g., up- or down-regulated to a statistically significant degree) relative to the levels at which they occur in a disease-free or normal subject such as an appropriate control individual. The alteration may result from an increase or decrease in a substrate, enzyme, cofactor, or any other component in any biochemical reaction involved in a particular process. Altered (i.e., increased or decreased in a statistically significant manner relative to a normal state) PTP activity can underlie certain disorders and suggests a PTP role in certain metabolic diseases.

[0016] For example, disruption of the murine PTP1B gene homolog in a knock-out mouse model results in PTP1B−/− mice exhibiting enhanced insulin sensitivity, decreased levels of circulating insulin and glucose, and resistance to weight gain even on a high-fat diet, relative to control animals having at least one functional PTP1B gene (Elchebly et al., Science 283:1544 (1999)). Insulin receptor hyperphosphorylation has also been detected in certain tissues of PTP1B deficient mice, consistent with a PTP1B contribution to the physiologic regulation of insulin and glucose metabolism (Id.). PTP-1B-deficient mice exhibit decreased adiposity (reduced fat cell mass but not fat cell number), increased basal metabolic rate and energy expenditure, and enhanced insulin-stimulated glucose utilization (Klaman et al., 2000 Mol. Cell. Biol. 20:5479). Additionally, altered PTP activity has been correlated with impaired glucose metabolism in other biological systems (e.g., McGuire et al., Diabetes 40:939 (1991); Myerovitch et al., J. Clin. Invest. 84:976 (1989); Sredy et al., Metabolism 44:1074 (1995)), including PTP involvement in biological signal transduction via the insulin receptor (see, e.g., WO 99/46268 and references cited therein).

[0017] An integration of crystallographic, kinetic, and PTP1B-peptide binding assays illustrated the interaction of PTP1B and insulin receptor (IR) (Salmeen et al., Mol. Cell 6:1401-12 (2000)). The insulin receptor (IR) comprises two extracellular a subunits and two transmembrane β subunits. Activation of the receptor results in autophosphorylation of tyrosine residues in both β subunits, each of which contains a protein kinase domain. Extensive interactions that form between PTP1B and insulin receptor kinase (IRK) encompass tandem pTyr residues at 1162 and 1163 of IRK, such that pTyr-1 162 is located in the active site of PTP1B (id.). The Asp/Glu-pTyr-pTyr-Arg/Lys motif has been implicated for optimal recognition by PTP1B for IRK. This motif is also present in other receptor PTKs, including Trk, FGFR, and Axl. In addition, this motif is found in the JAK family of PTKs, members of which transmit signals from cytokine receptors, including a classic cytokine receptor that is recognized by the satiety hormone leptin (Touw et al., Mol. Cell. Endocrinol. 160:1-9 (2000)).

[0018] Changes in the expression levels of PTP1B have been observed in several human diseases, particularly in diseases associated with disruption of the normal patterns of tyrosine phosphorylation. For example, the expression of PTP1B is induced specifically by the p210 Bcr-Abl oncoprotein, a PTK that is directly responsible for the initial manifestations of chronic myelogenous leukemia (CML) (LaMontagne et al., Mol. Cell. Biol. 18:2965-75 (1998); LaMontagne et al., Proc. Natl. Acad. Sci. USA 95:14094-99 (1998)). Expression of PTPB1 in response to this oncoprotein is regulated, in part, by transcription factors Sp1, Sp3, and Egr-1 (Fukada et al., J. Biol. Chem. 276:25512-19 (2001)). These transcription factors have been shown to bind to a p210 Bcr-Abl responsive sequence (PRS) in the human PTP1B promoter, located between −49 to −37 base pairs from the transcription start site, but do not appear to mediate certain additional, independent PTP1B transcriptional events, for which neither transcription factor(s) nor transcription factor recognition element(s) have been defined (id.).

[0019] RNA interference (RNAi) is a polynucleotide sequence-specific, post-transcriptional gene silencing mechanism effected by double-stranded RNA that results in degradation of a specific messenger RNA (mRNA), thereby reducing the expression of a desired target polypeptide encoded by the mRNA (see, e.g., WO 99/32619; WO 01/75164; U.S. Pat. No. 6,506,559; Fire et al., Nature 391:806-11 (1998); Sharp, Genes Dev. 13:139-41 (1999); Elbashir et al. Nature 411:494-98 (2001); Harborth et al., J. Cell Sci. 114:4557-65 (2001)). RNAi is mediated by double-stranded polynucleotides as also described hereinbelow, for example, double-stranded RNA (dsRNA), having sequences that correspond to exonic sequences encoding portions of the polypeptides for which expression is compromised. RNAi reportedly is not effected by double-stranded RNA polynucleotides that share sequence identity with intronic or promoter sequences (Elbashir et al., 2001). RNAi pathways have been best characterized in Drosophila and Caenorhabditis elegans, but “small interfering RNA” (siRNA) polynucleotides that interfere with expression of specific polypeptides in higher eukaryotes such as mammals (including humans) have also been considered (e.g., Tuschl, 2001 Chembiochem. 2:239-245; Sharp, 2001 Genes Dev. 15:485; Bernstein et al., 2001 RNA 7:1509; Zamore, 2002 Science 296:1265; Plasterk, 2002 Science 296:1263; Zamore 2001 Nat. Struct. Biol. 8:746; Matzke et al., 2001 Science 293:1080; Scadden et al., 2001 EMBO Rep. 2:1107).

[0020] According to a current non-limiting model, the RNAi pathway is initiated by ATP-dependent, processive cleavage of long dsRNA into double-stranded fragments of about 18-27 (e.g., 19, 20, 21, 22, 23, 24, 25, 26, etc.) nucleotide base pairs in length, called small interfering RNAs (siRNAs) (see review by Hutvagner et al., Curr. Opin. Gen. Dev. 12:225-32 (2002); Elbashir et al., 2001; Nykänen et al., Cell 107:309-21 (2001); Bass, Cell 101:235-38 (2000)); Zamore et al., Cell 101:25-33 (2000)). In Drosophila, an enzyme known as “Dicer” cleaves the longer double-stranded RNA into siRNAs; Dicer belongs to the RNase III family of dsRNA-specific endonucleases (WO 01/68836; Bernstein et al., Nature 409:363-66 (2001)). Further according to this non-limiting model, the siRNA duplexes are incorporated into a protein complex, followed by ATP-dependent unwinding of the siRNA, which then generates an active RNA-induced silencing complex (RISC) (WO 01/68836). The complex recognizes and cleaves a target RNA that is complementary to the guide strand of the siRNA, thus interfering with expression of a specific protein (Hutvagner et al., supra).

[0021] In C. elegans and Drosophila, RNAi may be mediated by long double-stranded RNA polynucleotides (WO 99/32619; WO 01/75164; Fire et al., 1998; Clemens et al., Proc. Natl. Acad. Sci. USA 97:6499-6503 (2000); Kisielow et al., Biochem. J. 363:1-5 (2002); see also WO 01/92513 (RNAi-mediated silencing in yeast)). In mammalian cells, however, transfection with long dsRNA polynucleotides (i.e., greater than 30 base pairs) leads to activation of a non-specific sequence response that globally blocks the initiation of protein synthesis and causes mRNA degradation (Bass, Nature 411:428-29 (2001)). Transfection of human and other mammalian cells with double-stranded RNAs of about 18-27 nucleotide base pairs in length interferes in a sequence-specific manner with expression of particular polypeptides encoded by messenger RNAs (mRNA) containing corresponding nucleotide sequences (WO 01/75164; Elbashir et al., 2001; Elbashir et al., Genes Dev. 15:188-200 (2001)); Harborth et al., J. Cell Sci. 114:4557-65 (2001); Carthew et al., Curr. Opin. Cell Biol. 13:244-48 (2001); Mailand et al., Nature Cell Biol. Advance Online Publication (Mar. 18, 2002); Mailand et al. 2002 Nature Cell Biol. 4:317).

[0022] siRNA polynucleotides may offer certain advantages over other polynucleotides known to the art for use in sequence-specific alteration or modulation of gene expression to yield altered levels of an encoded polypeptide product. These advantages include lower effective siRNA polynucleotide concentrations, enhanced siRNA polynucleotide stability, and shorter siRNA polynucleotide oligonucleotide lengths relative to such other polynucleotides (e.g., antisense, ribozyme or triplex polynucleotides). By way of a brief background, “antisense” polynucleotides bind in a sequence-specific manner to target nucleic acids, such as mRNA or DNA, to prevent transcription of DNA or translation of the mRNA (see, e.g., U.S. Pat. No. 5,168,053; U.S. Pat. No. 5,190,931; U.S. Pat. No. 5,135,917; U.S. Pat. No. 5,087,617; see also, e.g., Clusel et al., 1993 Nucl. Acids Res. 21:3405-11, describing “dumbbell” antisense oligonucleotides). “Ribozyme” polynucleotides can be targeted to any RNA transcript and are capable of catalytically cleaving such transcripts, thus impairing translation of mRNA (see, e.g., U.S. Pat. No. 5,272,262; U.S. Pat. No. 5,144,019; and U.S. Pat. Nos. 5,168,053, 5,180,818, 5,116,742 and 5,093,246; U.S. 2002/193579). “Triplex” DNA molecules refers to single DNA strands that bind duplex DNA to form a colinear triplex molecule, thereby preventing transcription (see, e.g., U.S. Pat. No. 5,176,996, describing methods for making synthetic oligonucleotides that bind to target sites on duplex DNA). Such triple-stranded structures are unstable and form only transiently under physiological conditions. Because single-stranded polynucleotides do not readily diffuse into cells and are therefore susceptible to nuclease digestion, development of single-stranded DNA for antisense or triplex technologies often requires chemically modified nucleotides to improve stability and absorption by cells. siRNAs, by contrast, are readily taken up by intact cells, are effective at interfering with the expression of specific polypeptides at concentrations that are several orders of magnitude lower than those required for either antisense or ribozyme polynucleotides, and do not require the use of chemically modified nucleotides.

[0023] Importantly, despite a number of attempts to devise selection criteria for identifying oligonucleotide sequences that will be effective in siRNA based on features of the desired target mRNA sequence (e.g., percent GC content, position from the translation start codon, or sequence similarities based on an in silico sequence database search for homologues of the proposed siRNA) it is presently not possible to predict with any degree of confidence which of myriad possible candidate siRNA sequences that can be generated as nucleotide sequences that correspond to a desired target mRNA (e.g., dsRNA of about 18-27 nucleotide base pairs) will in fact exhibit siRNA activity (i.e., interference with expression of the polypeptide encoded by the mRNA). Instead, individual specific candidate siRNA polynucleotide or oligonucleotide sequences must be generated and tested to determine whether interference with expression of a desired polypeptide target can be effected. Accordingly, no routine method exists in the art for designing a siRNA polynucleotide that is, with certainty, capable of specifically altering the expression of a given PTP polypeptide, and thus for the overwhelming majority of PTPs no effective siRNA polynucleotide sequences are presently known.

[0024] Currently, therefore, desirable goals for therapeutic regulation of biological signal transduction include modulation of PTP1B-mediated cellular events include, inter alia, inhibition or potentiation of interactions among PTP1B-binding molecules, substrates and binding partners, or of other agents that regulate PTP1B activities. Accordingly, a need exists in the art for an improved ability to intervene in the regulation of phosphotyrosine signaling, including regulating PTP1B by altering PTP1B catalytic activity, PTP1B binding to PTP1B substrate molecules, and/or PTP1B -encoding gene expression. An increased ability to so regulate PTP1B may facilitate the development of methods for modulating the activity of proteins involved in phosphotyrosine signaling pathways and for treating conditions associated with such pathways. The present invention fulfills these needs and further provides other related advantages.

SUMMARY OF THE INVENTION

[0025] The present invention relates to compositions and methods, including specific siRNA polynucleotides comprising nucleotide sequences disclosed herein, for modulating PTP1B. It is therefore an aspect of the invention to provide an isolated small interfering RNA (siRNA) polynucleotide, comprising in certain embodiments at least one nucleotide sequence selected from SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, 104-105, and 108-109, and in certain further embodiments at least one nucleotide sequence selected from SEQ ID NOS: 83-86, 88-91, 93-96, 98-101 and the complementary polynucleotide thereto. In another embodiment the small interfering RNA polynucleotide of either is capable of interfering with expression of a PTP1B polypeptide, wherein the PTP-1B polypeptide comprises an amino acid sequence as set forth in GenBank Ace. Nos. M31724, NM002827, or M33689. In another embodiment 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 SEQ ID NOS: 83-86, 88-91, 93-96, and 98-101, or at any position of a sequence selected from SEQ ID NOS: 104, 105, 108, and 109. In other embodiments 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 SEQ ID NOS: 83-86, 88-91, 93-96, and 98-101, or at any position of a sequence selected from SEQ ID NOS: 104, 105, 108, and 109.

[0026] In certain preferred embodiments the invention provides an isolated siRNA polynucleotide comprising a nucleotide sequence according to SEQ ID NO: 83, or the complement thereof, or an isolated siRNA polynucleotide comprising a nucleotide sequence according to SEQ ID NO: 88, or the complement thereof, or an isolated siRNA polynucleotide comprising a nucleotide sequence according to SEQ ID NO: 93, or the complement thereof, or an isolated siRNA polynucleotide comprising a nucleotide sequence according to SEQ ID NO: 98, or the complement thereof, or an isolated siRNA polynucleotide comprising a nucleotide sequence according to SEQ ID NO: 104 or 105, or an isolated siRNA polynucleotide comprising a nucleotide sequence according to SEQ ID NO: 108 or 109.

[0027] According to certain further embodiments of the above described invention, the polynucleotide comprises at least one synthetic nucleotide analogue of a naturally occurring nucleotide. In another embodiment the polynucleotide is linked to a detectable label, which in certain further embodiments is a reporter molecule that may in certain still further embodiments be selected from a dye, a radionuclide, a luminescent group, a fluorescent group, and biotin, wherein in a still further embodiment the fluorescent group is fluorescein isothiocyanate. In another embodiment the detectable label is a magnetic particle. According to related embodiments there is provided a pharmaceutical composition comprising any of the above described siRNA polynucleotides and a physiologically acceptable carrier, which in certain further embodiments comprises a liposome.

[0028] The invention also provides 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 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. In another embodiment there is provided 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 SEQ ID NOs: 102, 103, 106, and 107. In a further embodiment the recombinant nucleic acid construct comprises 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. In certain embodiments the recombinant nucleic acid construct comprises 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. In certain other embodiments 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, NM002827, and M33689. In another embodiment the invention provides 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 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. In certain further embodiments the siRNA comprises an overhang of at least one and no more than four nucleotides, the overhang being located immediately 3′ to (iii). In a further embodiment the spacer sequence comprises at least 9 nucleotides. In other further embodiments the spacer sequence comprises two uridine nucleotides that are contiguous with (iii). In another embodiment the recombinant nucleic acid construct comprises at least one transcriptional terminator that is operably linked to the DNA polynucleotide segment. According to related embodiments, the invention provides a host cell transformed or transfected with the above described recombinant nucleic acid constructs.

[0029] Certain embodiments of the invention provide a pharmaceutical composition comprising an siRNA polynucleotide and a physiologically acceptable carrier, wherein the siRNA polynucleotide is selected from (i) an RNA polynucleotide which comprises at least one nucleotide sequence selected from 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 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 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 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 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 sequences set forth in SEQ ID NOS: 104, 105, 108, and 109. In a further embodiment the carrier comprises a liposome.

[0030] Turning to another aspect of the invention, there is provided 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 GenBank Ace. Nos. M31724, NM002827, NM011201, and M33689, (b) the siRNA polynucleotide is selected from (i) an RNA polynucleotide which comprises at least one nucleotide sequence selected from 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 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 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 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 sequences set forth in SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, or at any position of a sequence selected from sequences set forth in SEQ ID NOS: 104, 105, 108, and 109.

[0031] The invention also provides a method for interfering with expression of a PTP1B polypeptide that comprises an amino acid sequence as set forth in a sequence selected from GenBank Acc. Nos. M31724, NM002827, 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 as described above. In another embodiment there is provided 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 GenBank Ace. Nos. M31724, NM002827, and M33689, (2) the siRNA polynucleotide is selected from (i) an RNA polynucleotide which comprises at least one nucleotide sequence selected from 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 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 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 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 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 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. In certain further embodiments the signal transduction pathway comprises a Jak2 kinase.

[0032] In another aspect the present invention provides 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 f GenBank Acc. Nos. M31724, NM002827, NM011201, and M33689, (b) the siRNA polynucleotide is selected from (i) an RNA polynucleotide which comprises at least one nucleotide sequence selected from 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 f 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 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 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 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 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.

[0033] In another embodiment there is provided 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 GenBank Acc. Nos. M31724, NM002827, NM011201, and M33689, (b) the siRNA polynucleotide is selected from (i) an RNA polynucleotide which comprises at least one nucleotide sequence selected from 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 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 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 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 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 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. Another embodiment of the invention provides a method for treating a Jak2-associated disorder comprising administering to a subject in need thereof a pharmaceutical composition as described above, wherein the siRNA polynucleotide inhibits expression of a PTP1B polypeptide, or a variant thereof. In certain embodiments the Jak2-associated disorder is diabetes, obesity, hyperglycemia-induced apoptosis, inflammation, or a neurodegenerative disorder. In another aspect the invention provides a small interfering RNA (siRNA) polynucleotide, comprising in certain embodiments at least one nucleotide sequence selected from SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, 104-105, and 108-109, and in certain further embodiments at least one nucleotide sequence selected from SEQ ID NOS: 83-86, 88-91, 93-96, 98-101 and the complementary polynucleotide thereto. The invention also provides a small interfering RNA (siRNA) polynucleotide, comprising an RNA polynucleotide which comprises at least one nucleotide sequence selected from SEQ ID NOS: 18-21, 33-36, 43-46, 53-56, and 58-61. Certain further embodiments relate to isolated siRNA polynucleotides that comprise nucleotide sequences having the above recited SEQ ID NOS, including compositions and methods for producing and therapeutically using such siRNA.

[0034] These and other embodiments of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entireties as if each was incorporated individually. Also incorporated by reference are co-pending applications, Ser. No. ______ and Ser. No. ______ (attorney docket numbers 200125.441D1 and 200125.448, respectively), which have been filed concurrently.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035]FIG. 1 depicts an immunoblot of the effect on endogenous expression of murine PTP1B by siRNAs specific for the murine PTP1B or the human PTP1B polynucleotide sequences. Expression was detected using a murine anti-PTP1B monoclonal antibody. Data are presented for two different clones of C57B16 #3 murine cells. Both clones were transfected with mPTP1B1.1 siRNA (lanes 3 and 8); mPTP1B1.2 (lanes 4 and 9); mPTP1B1.3 (lanes 5 and 10). One clone, C57B16 #3 clone 3, was transfected with hPTP1B1.1 (lane 6). Lane 2: untransfected C57B16 #3, clone 3 (NT); lane 7: untransfected C57B16 #3, clone 10.

[0036]FIG. 2 depicts an immunoblot analysis of the expression of human PTP-1B co-transfected into 1 BKO+HIR murine fibroblasts with human PTP-1B siRNA hairpin vectors. Expression was detected with an anti-human PTP1B antibody (h1B) (lower portion of immunoblot). As a protein expression control, cell lysates were probed with an anti-human insulin receptor (IR) antibody (upper portion of immunoblot).

[0037]FIG. 3 presents the results of an ELISA in which the level of insulin receptor (IR) phosphorylated tyrosine was measured in 293-HEK HIR cells transfected with 0, 0.5, 3, or 10 nM hPTP1B1.3 (H1.3, SEQ ID NO: __) (FIG. 3A) or mPTP1B1.1b (M1.1, SEQ ID NO: __) (FIG. 3B) siRNAs. The level of expression of human PTP1B in the cells was compared by immunoblot (see tables to right of each figure).

[0038]FIG. 4 depicts the results of an ELISA in which the level of insulin receptor (IR) phosphorylated tyrosine was measured in 293-HEK HIR cells transfected with 0, 0.5, 3, or 10 nM siRNAs. The siRNA polynucleotides transfected into the cells included mPTP1B1.1b (M1.1, SEQ ID NO: __) (FIG. 4A); hPTP1B1.2 (H1.2, SEQ ID NO: __) (FIG. 4B); hPTP1B1.3 (H1.3, SEQ ID NO: __) (FIG. 4C); and rPTP1B1.2 (R1.2, SEQ ID NO: __) (FIG. 4D). Seventy-two hours after transfection, cells were exposed to insulin for 7 minutes at the designated concentrations. Cell lysates were prepared and coated onto 96-well plates and probed with an anti-pY-IR-β antibody.

[0039]FIG. 5 represents ELISA data from three separate experiments (Exp. 1, 2, 3) that represent the level of insulin receptor phosphorylation in cells transfected with hPTP1B1.3 and stimulated with 50 nM insulin (Ins). Each data point represents the average optical density measured in duplicate wells.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention is directed in part to the unexpected discovery of short RNA polynucleotide sequences that are capable of specifically modulating expression of a desired PTP1B polypeptide, such as a human or murine PTP1B polypeptide (e.g., GenBank Acc. Nos. M31724, NM002827, NM011201, M33689, NM012637, NM012637, M33962; SEQ ID NOS: __-__; Andersen et al., 2001 Mol. Cell. Biol. 21:7117), or variant thereof. Without wishing to be bound by theory, the RNA polynucleotides of the present invention specifically reduce expression of a desired target polypeptide through recruitment of small interfering RNA (siRNA) mechanisms. In particular, and as described in greater detail herein, according to the present invention there are provided compositions and methods that relate to the surprising identification of certain specific RNAi oligonucleotide sequences of 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides that can be derived from corresponding polynucleotide sequences encoding the desired PTP1B target polypeptide. These sequences cannot be predicted through any algorithm, sequence alignment routine, or other systematic paradigm, but must instead be obtained through generation and functional testing for RNAi activity of actual candidate oligonucleotides, such as those disclosed for the first time herein.

[0041] In preferred embodiments of the invention, the siRNA polynucleotide interferes with expression of a PTP1B target polypeptide or a variant thereof, and comprises a RNA oligonucleotide or RNA polynucleotide uniquely corresponding in its nucleotide base sequence to the sequence of a portion of a target polynucleotide encoding the target polypeptide, for instance, a target mRNA sequence or an exonic sequence encoding such mRNA. The invention relates in preferred embodiments to siRNA polynucleotides that interfere with expression of specific polypeptides in mammals, which in certain particularly preferred embodiments are humans and in certain other particularly preferred embodiments are non-human mammals. Hence, according to non-limiting theory, the siRNA polynucleotides of the present invention direct sequence-specific degradation of mRNA encoding a desired PTP1B.

[0042] SiRNA Polynucleotides

[0043] As used herein, the term “siRNA” means either: (i) a double stranded RNA oligonucleotide, or polynucleotide, that is 18 base pairs, 19 base pairs, 20 base pairs, 21 base pairs, 22 base pairs, 23 base pairs, 24 base pairs, 25 base pairs, 26 base pairs, 27 base pairs, 28 base pairs, 29 base pairs or 30 base pairs in length and that is capable of interfering with expression and activity of a PTP-1B polypeptide, or a variant of the PTP-1B polypeptide, wherein a single strand of the siRNA comprises a portion of a RNA polynucleotide sequence that encodes the PTP-1B polypeptide, its variant, or a complementary sequence thereto; (ii) a single stranded oligonucleotide, or polynucleotide of 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides or 30 nucleotides in length and that is either capable of interfering with expression and/or activity of a target PTP-1B polypeptide, or a variant of the PTP-1B polypeptide, or that anneals to a complementary sequence to result in a dsRNA that is capable of interfering with target polypeptide expression, wherein such single stranded oligonucleotide comprises a portion of a RNA polynucleotide sequence that encodes the PTP-1B polypeptide, its variant, or a complementary sequence thereto; or (iii) an oligonucleotide, or polynucleotide, of either (i) or (ii) above wherein such oligonucleotide, or polynucleotide, has one, two, three or four nucleic acid alterations or substitutions therein. Certain RNAi oligonucleotide sequences described below are complementary to the 3′ non-coding region of target mRNA that encodes the PTP1B polypeptide.

[0044] A siRNA polynucleotide is a RNA nucleic acid molecule that mediates the effect of RNA interference, a post-transcriptional gene silencing mechanism. A siRNA polynucleotide preferably comprises a double-stranded RNA (dsRNA) but is not intended to be so limited and may comprise a single-stranded RNA (see, e.g., Martinez et al. Cell 110:563-74 (2002)). A siRNA polynucleotide may comprise other naturally occurring, recombinant, or synthetic single-stranded or double-stranded polymers of nucleotides (ribonucleotides or deoxyribonucleotides or a combination of both) and/or nucleotide analogues as provided herein (e.g., an oligonucleotide or polynucleotide or the like, typically in 5′ to 3′ phosphodiester linkage). Accordingly it will be appreciated that certain exemplary sequences disclosed herein as DNA sequences capable of directing the transcription of the subject invention siRNA polynucleotides are also intended to describe the corresponding RNA sequences and their complements, given the well established principles of complementary nucleotide base-pairing. A siRNA may be transcribed using as a template a DNA (genomic, cDNA, or synthetic) that contains a RNA polymerase promoter, for example, a U6 promoter or the H1 RNA polymerase III promoter, or the siRNA may be a synthetically derived RNA molecule. In certain embodiments the subject invention siRNA polynucleotide may have blunt ends, that is, each nucleotide in one strand of the duplex is perfectly complementary (e.g., by Watson-Crick base-pairing) with a nucleotide of the opposite strand. In certain other embodiments, at least one strand of the subject invention siRNA polynucleotide has at least one, and preferably two nucleotides that “overhang” (i.e., that do not base pair with a complementary base in the opposing strand) at the 3′ end of either strand, or preferably both strands, of the siRNA polynucleotide. In a preferred embodiment of the invention, each strand of the siRNA polynucleotide duplex has a two-nucleotide overhang at the 3′ end. The two-nucleotide overhang is preferably a thymidine dinucleotide (TT) but may also comprise other bases, for example, a TC dinucleotide or a TG dinucleotide, or any other dinucleotide. For a discussion of 3′ ends of siRNA polynucleotides see, e.g., WO 01/75164.

[0045] Preferred siRNA polynucleotides comprise double-stranded oligomeric nucleotides of about 18-30 nucleotide base pairs, preferably about 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 base pairs, and in other preferred embodiments about 19, 20, 21, 22 or 23 base pairs, or about 27 base pairs, whereby the use of “about” indicates, as described above, that in certain embodiments and under certain conditions the processive cleavage steps that may give rise to functional siRNA polynucleotides that are capable of interfering with expression of a selected polypeptide may not be absolutely efficient. Hence, siRNA polynucleotides, for instance, of “about” 18, 19, 20, 21, 22, 23, 24, or 25 base pairs may include one or more siRNA polynucleotide molecules that may differ (e.g., by nucleotide insertion or deletion) in length by one, two, three or four base pairs, by way of non-limiting theory as a consequence of variability in processing, in biosynthesis, or in artificial synthesis. The contemplated siRNA polynucleotides of the present invention may also comprise a polynucleotide sequence that exhibits variability by differing (e.g., by nucleotide substitution, including transition or transversion) at one, two, three or four nucleotides from a particular sequence, the differences occurring at any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of a particular siRNA polynucleotide sequence, or at positions 20, 21, 22, 23, 24, 25, 26, or 27 of siRNA polynucleotides depending on the length of the molecule, whether situated in a sense or in an antisense strand of the double-stranded polynucleotide. The nucleotide substitution may be found only in one strand, by way of example in the antisense strand, of a double-stranded polynucleotide, and the complementary nucleotide with which the substitute nucleotide would typically form hydrogen bond base pairing may not necessarily be correspondingly substituted in the sense strand. In preferred embodiments, the siRNA polynucleotides are homogeneous with respect to a specific nucleotide sequence. As described herein, preferred siRNA polynucleotides interfere with expression of a PTP-1B polypeptide. These polynucleotides may also find uses as probes or primers.

[0046] Polynucleotides that are siRNA polynucleotides of the present invention may in certain embodiments be derived from a single-stranded polynucleotide that comprises a single-stranded oligonucleotide fragment (e.g., of about 18-30 nucleotides, which should be understood to include any whole integer of nucleotides including and between 18 and 30) and its reverse complement, typically separated by a spacer sequence. According to certain such embodiments, cleavage of the spacer provides the single-stranded oligonucleotide fragment and its reverse complement, such that they may anneal to form (optionally with additional processing steps that may result in addition or removal of one, two, three or more nucleotides from the 3′ end and/or the 5′ end of either or both strands) the double-stranded siRNA polynucleotide of the present invention. In certain embodiments the spacer is of a length that permits the fragment and its reverse complement to anneal and form a double-stranded structure (e.g., like a hairpin polynucleotide) prior to cleavage of the spacer (and, optionally, subsequent processing steps that may result in addition or removal of one, two, three, four, or more nucleotides from the 3′ end and/or the 5′ end of either or both strands). A spacer sequence may therefore be any polynucleotide sequence as provided herein that is situated between two complementary polynucleotide sequence regions which, when annealed into a double-stranded nucleic acid, comprise a siRNA polynucleotide. Preferably a spacer sequence comprises at least 4 nucleotides, although in certain embodiments the spacer may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21-25, 26-30, 31-40, 41-50, 51-70, 71-90, 91-110, 111-150, 151-200 or more nucleotides. Examples of siRNA polynucleotides derived from a single nucleotide strand comprising two complementary nucleotide sequences separated by a spacer have been described (e.g., Brummelkamp et al., 2002 Science 296:550; Paddison et al., 2002 Genes Develop. 16:948; Paul et al. Nat. Biotechnol. 20:505-508 (2002); Grabarek et al., BioTechniques 34:734-44 (2003)).

[0047] Polynucleotide variants may contain one or more substitutions, additions, deletions, and/or insertions such that the activity of the siRNA polynucleotide is not substantially diminished, as described above. The effect on the activity of the siRNA polynucleotide may generally be assessed as described herein or using conventional methods. Variants preferably exhibit at least about 75%, 78%, 80%, 85%, 87%, 88% or 89% identity and more preferably at least about 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity to a portion of a polynucleotide sequence that encodes a native PTP1B. The percent identity may be readily determined by comparing sequences of the polynucleotides to the corresponding portion of a full-length PTP1B polynucleotide such as those known to the art and cited herein, using any method including using computer algorithms well known to those having ordinary skill in the art, such as Align or the BLAST algorithm (Altschul, J. Mol. Biol. 219:555-565, 1991; Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992), which is available at the NCBI website (see [online] Internet:<URL: http://www/ncbi.nlm.nih.gov/cgi-bin/BLAST). Default parameters may be used.

[0048] Certain siRNA polynucleotide variants are substantially homologous to a portion of a native PTP1B gene. Single-stranded nucleic acids derived (e.g., by thermal denaturation) from such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA or RNA sequence encoding a native PTP1B polypeptide (or a complementary sequence). A polynucleotide that detectably hybridizes under moderately stringent conditions may have a nucleotide sequence that includes at least 10 consecutive nucleotides, more preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 consecutive nucleotides complementary to a particular polynucleotide. In certain preferred embodiments such a sequence (or its complement) will be unique to a PTP1B polypeptide for which interference with expression is desired, and in certain other embodiments the sequence (or its complement) may be shared by PTP1B and one or more PTPs for which interference with polypeptide expression is desired.

[0049] Suitable moderately stringent conditions include, for example, pre-washing in a solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50° C.-70° C., 5×SSC for 1-16 hours (e.g., overnight); followed by washing once or twice at 22-65° C. for 20-40 minutes with one or more each of 2×, 0.5× and 0.2×SSC containing 0.05-0.1% SDS. For additional stringency, conditions may include a wash in 0.1×SSC and 0.1% SDS at 50-60° C. for 15-40 minutes. As known to those having ordinary skill in the art, variations in stringency of hybridization conditions may be achieved by altering the time, temperature, and/or concentration of the solutions used for pre-hybridization, hybridization, and wash steps. Suitable conditions may also depend in part on the particular nucleotide sequences of the probe used, and of the blotted, proband nucleic acid sample. Accordingly, it will be appreciated that suitably stringent conditions can be readily selected without undue experimentation when a desired selectivity of the probe is identified, based on its ability to hybridize to one or more certain proband sequences while not hybridizing to certain other proband sequences.

[0050] Sequence specific siRNA polynucleotides of the present invention may be designed using one or more of several criteria. For example, to design a siRNA polynucleotide that has 19 consecutive nucleotides identical to a sequence encoding a polypeptide of interest (e.g., PTP1B and other polypeptides described herein), the open reading frame of the polynucleotide sequence may be scanned for 21-base sequences that have one or more of the following characteristics: (1) an A+T/G+C ratio of approximately 1:1 but no greater than 2:1 or 1:2; (2) an AA dinucleotide or a CA dinucleotide at the 5′ end; (3) an internal hairpin loop melting temperature less than 55° C.; (4) a homodimer melting temperature of less than 37° C. (melting temperature calculations as described in (3) and (4) can be determined using computer software known to those skilled in the art); (5) a sequence of at least 16 consecutive nucleotides not identified as being present in any other known polynucleotide sequence (such an evaluation can be readily determined using computer programs available to a skilled artisan such as BLAST to search publicly available databases). Alternatively, an siRNA polynculeotide sequence may be designed and chosen using a computer software available commercially from various vendors (e.g., OligoEngine™ (Seattle, Wash.); Dharmacon, Inc. (Lafayette, Colo.); Ambion Inc. (Austin, Tex.); and QIAGEN, Inc. (Valencia, Calif.)). (See also Elbashir et al., Genes & Development 15:188-200 (2000); Elbashir et al., Nature 411:494-98 (2001); and [online] Internet:URL<http://www.mpibpc.gwdg.de/abteilungen/100/105/Tuschl_MIV2(3)2002.p df.) The siRNA polynucleotides may then be tested for their ability to interfere with the expression of the target polypeptide according to methods known in the art and described herein. The determination of the effectiveness of an siRNA polynucleotide includes not only consideration of its ability to interfere with polypeptide expression but also includes consideration of whether the siRNA polynucleotide manifests undesirably toxic effects, for example, apoptosis of a cell for which cell death is not a desired effect of RNA interference (e.g., interference of PTP1B expression in a cell).

[0051] It should be appreciated that not all siRNAs designed using the above methods will be effective at silencing or interfering with expression of a PTP1B target polypeptide. And further, that the siRNAs will effect silencing to different degrees. Such siRNAs must be tested for their effectiveness, and selections made therefrom based on the ability of a given siRNA to interfere with or modulate (e.g., decrease in a statistically significant manner) the expression of PTP1B. Accordingly, identification of specific siRNA polynucleotide sequences that are capable of interfering with expression of a PTP1B polypeptide requires production and testing of each siRNA, as demonstrated in greater detail below (see Examples).

[0052] Furthermore, not all siRNAs that interfere with protein expression will have a physiologically important effect. The inventors here have designed, and describe herein, physiologically relevant assays for measuring the influence of modulated target polypeptide expression, for instance, cellular proliferation, induction of apoptosis, and/or altered levels of protein tyrosine phosphorylation (e.g., insulin receptor phosphorylation), to determine if the levels of interference with target protein expression that were observed using the siRNAs of the invention have clinically relevant significance. Additionally, and according to non-limiting theory, the invention contemplates altered (e.g., decreased or increased in a statistically significant manner) expression levels of one or more polypeptides of interest, and/or altered (i.e., increased or decreased) phosphorylation levels of one or more phosphoproteins of interest, which altered levels may result from impairment of PTP1B protein expression and/or cellular compensatory mechanisms that are induced in response to RNAi-mediated inhibition of a specific target polypeptide expression.

[0053] Persons having ordinary skill in the art will also readily appreciate that as a result of the degeneracy of the genetic code, many nucleotide sequences may encode a polypeptide as described herein. That is, an amino acid may be encoded by one of several different codons and a person skilled in the art can readily determine that while one particular nucleotide sequence may differ from another (which may be determined by alignment methods disclosed herein and known in the art), the sequences may encode polypeptides with identical amino acid sequences. By way of example, the amino acid leucine in a polypeptide may be encoded by one of six different codons (TTA, TTG, CTT, CTC, CTA, and CTG) as can serine (TCT, TCC, TCA, TCG, AGT, and AGC). Other amino acids, such as proline, alanine, and valine, for example, may be encoded by any one of four different codons (CCT, CCC, CCA, CCG for proline; GCT, GCC, GCA, GCG for alanine; and GTT, GTC, GTA, GTG for valine). Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention.

[0054] Polynucleotides, including target polynucleotides (e.g., polynucleotides capable of encoding a target polypeptide of interest), may be prepared using any of a variety of techniques, which will be useful for the preparation of specifically desired siRNA polynucleotides and for the identification and selection of desirable sequences to be used in siRNA polynucleotides. For example, a polynucleotide may be amplified from cDNA prepared from a suitable cell or tissue type. Such polynucleotides may be amplified via polymerase chain reaction (PCR). For this approach, sequence-specific primers may be designed based on the sequences provided herein and may be purchased or synthesized. An amplified portion may be used to isolate a full-length gene, or a desired portion thereof, from a suitable library (e.g., human skeletal muscle cDNA) using well known techniques. Within such techniques, a library (cDNA or genomic) is screened using one or more polynucleotide probes or primers suitable for amplification. Preferably, a library is size-selected to include larger molecules. Random primed libraries may also be preferred for identifying 5′ and upstream regions of genes. Genomic libraries are preferred for obtaining introns and extending 5′ sequences. Suitable sequences for a siRNA polynucleotide contemplated by the present invention may also be selected from a library of siRNA polynucleotide sequences.

[0055] For hybridization techniques, a partial sequence may be labeled (e.g., by nick-translation or end-labeling with 32P) using well known techniques. A bacterial or bacteriophage library may then be screened by hybridizing filters containing denatured bacterial colonies (or lawns containing phage plaques) with the labeled probe (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 2001). Hybridizing colonies or plaques are selected and expanded, and the DNA is isolated for further analysis. Clones may be analyzed to determine the amount of additional sequence by, for example, PCR using a primer from the partial sequence and a primer from the vector. Restriction maps and partial sequences may be generated to identify one or more overlapping clones. A full-length cDNA molecule can be generated by ligating suitable fragments, using well known techniques.

[0056] Alternatively, numerous amplification techniques are known in the art for obtaining a full-length coding sequence from a partial cDNA sequence. Within such techniques, amplification is generally performed via PCR. One such technique is known as “rapid amplification of cDNA ends” or RACE. This technique involves the use of an internal primer and an external primer, which hybridizes to a polyA region or vector sequence, to identify sequences that are 5′ and 3′ of a known sequence. Any of a variety of commercially available kits may be used to perform the amplification step. Primers may be designed using, for example, software well known in the art. Primers (or oligonucleotides for other uses contemplated herein, including, for example, probes and antisense oligonucleotides) are preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 nucleotides in length, have a GC content of at least 40% and anneal to the target sequence at temperatures of about 54° C. to 72° C. The amplified region may be sequenced as described above, and overlapping sequences assembled into a contiguous sequence. Certain oligonucleotides contemplated by the present invention may, for some preferred embodiments, have lengths of 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33-35, 35-40, 41-45, 46-50, 56-60, 61-70, 71-80, 81-90 or more nucleotides.

[0057] A number of specific siRNA polynucleotide sequences useful for interfering with PTP1B polypeptide expression are presented in the Examples, the Drawings, and the Sequence Listing. SiRNA polynucleotides may generally be prepared by any method known in the art, including, for example, solid phase chemical synthesis. Modifications in a polynucleotide sequence may also be introduced using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis. Further, siRNAs may be chemically modified or conjugated to improve their serum stability and/or delivery properties. Included as an aspect of the invention are the siRNAs described herein wherein the ribose has been removed therefrom. Alternatively, siRNA polynucleotide molecules may be generated by in vitro or in vivo transcription of suitable DNA sequences (e.g., polynucleotide sequences encoding a PTP, or a desired portion thereof), provided that the DNA is incorporated into a vector with a suitable RNA polymerase promoter (such as T7, U6, H1, or SP6). In addition, a siRNA polynucleotide may be administered to a patient, as may be a DNA sequence (e.g, a recombinant nucleic acid construct as provided herein) that supports transcription (and optionally appropriate processing steps) such that a desired siRNA is generated in vivo.

[0058] Accordingly, a siRNA polynucleotide that is complementary to at least a portion of a PTP1B coding sequence may be used to modulate gene expression, or as a probe or primer. Identification of siRNA polynucleotide sequences and DNA encoding genes for their targeted delivery involves techniques described herein with regard to PTP1B. Identification of such siRNA polynucleotide sequences and DNA encoding genes for their targeted delivery involves techniques that are also described herein. As discussed above, siRNA polynucleotides exhibit desirable stability characteristics and may, but need not, be further designed to resist degradation by endogenous nucleolytic enzymes by using such linkages as phosphorothioate, methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphate esters, and other such linkages (see, e.g., Agrwal et al., Tetrahedron Lett. 28:3539-3542 (1987); Miller et al., J. Am. Chem. Soc. 93:6657-6665 (1971); Stec et al., Tetrahedron Lett. 26:2191-2194 (1985); Moody et al., Nucleic Acids Res. 12:4769-4782 (1989); Uznanski et al., Nucleic Acids Res. (1989); Letsinger et al., Tetrahedron 40:137-143 (1984); Eckstein, Annu. Rev. Biochem. 54:367-402 (1985); Eckstein, Trends Biol. Sci. 14:97-100 (1989); Stein, In: Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, Cohen, ed., Macmillan Press, London, pp. 97-117 (1989); Jager et al., Biochemistry 27:7237-7246 (1988)).

[0059] Any polynucleotide of the invention may be further modified to increase stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5′ and/or 3′ ends; the use of phosphorothioate or 2′ O-methyl rather than phosphodiester linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine, and wybutosine and the like, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine, and uridine.

[0060] Nucleotide sequences as described herein may be joined to a variety of other nucleotide sequences using established recombinant DNA techniques. For example, a polynucleotide may be cloned into any of a variety of cloning vectors, including plasmids, phagemids, lambda phage derivatives, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. In general, a suitable vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and one or more selectable markers. (See, e.g., WO 01/96584; WO 01/29058; U.S. Pat. No. 6,326,193; U.S. 2002/0007051). Other elements will depend upon the desired use, and will be apparent to those having ordinary skill in the art. For example, the invention contemplates the use of siRNA polynucleotide sequences in the preparation of recombinant nucleic acid constructs including vectors for interfering with the expression of a desired target polypeptide such as a PTP1B polypeptide in vivo; the invention also contemplates the generation of siRNA transgenic or “knock-out” animals and cells (e.g., cells, cell clones, lines or lineages, or organisms in which expression of one or more desired polypeptides (e.g., a target polypeptide) is fully or partially compromised). An siRNA polynucleotide that is capable of interfering with expression of a desired polypeptide (e.g., a target polypeptide) as provided herein thus includes any siRNA polynucleotide that, when contacted with a subject or biological source as provided herein under conditions and for a time sufficient for target polypeptide expression to take place in the absence of the siRNA polynucleotide, results in a statistically significant decrease (alternatively referred to as “knockdown” of expression) in the level of target polypeptide expression that can be detected. Preferably the decrease is greater than 10%, more preferably greater than 20%, more preferably greater than 30%, more preferably greater than 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 98% relative to the expression level of the polypeptide detected in the absence of the siRNA, using conventional methods for determining polypeptide expression as known to the art and provided herein. Preferably, the presence of the siRNA polynucleotide in a cell does not result in or cause any undesired toxic effects, for example, apoptosis or death of a cell in which apoptosis is not a desired effect of RNA interference.

[0061] Within certain embodiments, siRNA polynucleotides may be formulated so as to permit entry into a cell of a mammal, and expression therein. Such formulations are particularly useful for therapeutic purposes, as described below. Those having ordinary skill in the art will appreciate that there are many ways to achieve expression of a polynucleotide in a target cell, and any suitable method may be employed. For example, a polynucleotide may be incorporated into a viral vector using well known techniques (see also, e.g., U.S. 2003/0068821). A viral vector may additionally transfer or incorporate a gene for a selectable marker (to aid in the identification or selection of transduced cells) and/or a targeting moiety, such as a gene that encodes a ligand for a receptor on a specific target cell, to render the vector target specific. Targeting may also be accomplished using an antibody, by methods known to those having ordinary skill in the art.

[0062] Other formulations for therapeutic purposes include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e., an artificial membrane vesicle). The preparation and use of such systems is well known in the art.

[0063] Within other embodiments, one or more promoters may be identified, isolated and/or incorporated into recombinant nucleic acid constructs of the present invention, using standard techniques. The present invention provides nucleic acid molecules comprising such a promoter sequence or one or more cis- or trans-acting regulatory elements thereof. Such regulatory elements may enhance or suppress expression of a siRNA. A 5′ flanking region may be generated using standard techniques, based on the genomic sequence provided herein. If necessary, additional 5′ sequences may be generated using PCR-based or other standard methods. The 5′ region may be subcloned and sequenced using standard methods. Primer extension and/or RNase protection analyses may be used to verify the transcriptional start site deduced from the cDNA.

[0064] To define the boundary of the promoter region, putative promoter inserts of varying sizes may be subcloned into a heterologous expression system containing a suitable reporter gene without a promoter or enhancer. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the Green Fluorescent Protein gene (see, e.g., Ui-Tei et al., FEBS Lett. 479:79-82 (2000). Suitable expression systems are well known and may be prepared using well known techniques or obtained commercially. Internal deletion constructs may be generated using unique internal restriction sites or by partial digestion of non-unique restriction sites. Constructs may then be transfected into cells that display high levels of siRNA polynucleotide and/or polypeptide expression. In general, the construct with the minimal 5′ flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

[0065] Once a functional promoter is identified, cis- and trans-acting elements may be located. Cis-acting sequences may generally be identified based on homology to previously characterized transcriptional motifs. Point mutations may then be generated within the identified sequences to evaluate the regulatory role of such sequences. Such mutations may be generated using site-specific mutagenesis techniques or a PCR-based strategy. The altered promoter is then cloned into a reporter gene expression vector, as described above, and the effect of the mutation on reporter gene expression is evaluated.

[0066] In general, polypeptides and polynucleotides as described herein are isolated. An “isolated” polypeptide or polynucleotide is one that is removed from its original environment. For example, a naturally occurring protein is isolated if it is separated from some or all of the coexisting materials in the natural system. Preferably, such polypeptides are at least about 90% pure, more preferably at least about 95% pure and most preferably at least about 99% pure. A polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of the natural environment. A “gene” includes the segment of DNA involved in producing a polypeptide chain; it further includes regions preceding and following the coding region “leader and trailer,” for example promoter and/or enhancer and/or other regulatory sequences and the like, as well as intervening sequences (introns) between individual coding segments (exons).

[0067] The effect of siRNA interference with expression of a component in the signal transduction pathway induced by insulin, for example, may be evaluated by determining the level of tyrosine phosphorylation of insulin receptor beta (IR-β) and/or of the downstream signaling molecule PKB/Akt and/or of any other downstream polypeptide that may be a component of a particular signal transduction pathway as provided herein.

[0068] As noted above, regulated tyrosine phosphorylation contributes to specific pathways for biological signal transduction, including those associated with cell division, cell survival, apoptosis, proliferation and differentiation, and “biological signal transduction pathways,” or “inducible signaling pathways” in the context of the present invention include transient or stable associations or interactions among molecular components involved in the control of these and similar processes in cells. Depending on the particular pathway of interest, an appropriate parameter for determining induction of such pathway may be selected. For example, for signaling pathways associated with cell proliferation, a variety of well known methodologies are available for quantifying proliferation, including, for example, incorporation of tritiated thymidine into cellular DNA, monitoring of detectable (e.g., fluorimetric or calorimetric) indicators of cellular respiratory activity (for example, conversion of the tetrazolium salts (yellow) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) to formazan dyes (purple) in metabolically active cells), or cell counting, or the like. Similarly, in the cell biology arts, multiple techniques are known for assessing cell survival (e.g., vital dyes, metabolic indicators, etc.) and for determining apoptosis (for example, annexin V binding, DNA fragmentation assays, caspase activation, marker analysis, e.g., poly(ADP-ribose) polymerase (PARP), etc.). Other signaling pathways will be associated with particular cellular phenotypes, for example specific induction of gene expression (e.g., detectable as transcription or translation products, or by bioassays of such products, or as nuclear localization of cytoplasmic factors), altered (e.g., statistically significant increases or decreases) levels of intracellular mediators (e.g., activated kinases or phosphatases, altered levels of cyclic nucleotides or of physiologically active ionic species, etc.), altered cell cycle profiles, or altered cellular morphology, and the like, such that cellular responsiveness to a particular stimulus as provided herein can be readily identified to determine whether a particular cell comprises an inducible signaling pathway.

[0069] PTP1B

[0070] The sequence of PTP-1B as used herein, means any sequence, as the context requires, selected from the following group (e.g., GenBank Accession Nos. M31724 (SEQ ID NOS: __-__); NM002827 (SEQ ID NOS: __-__); NM011201 (SEQ ID NOS: __-__; M31724 (SEQ ID NOS: __-__); M33689 (SEQ ID NOS: __-__); M33962 (SEQ ID NOS: __-__) The invention also includes variants or mutated forms of PTP1B that contain single nucleotide polymorphisms (SNPs), or allelic forms.

[0071] Specific substitutions of individual amino acids through introduction of site-directed mutations are well-known and may be made according to methodologies with which those having ordinary skill in the art will be familiar. The effects on catalytic activity of the resulting mutant PTP may be determined empirically by testing the resulting modified protein for the preservation of the Km and reduction of Kcat to less than 1 per minute as provided herein and as previously disclosed (e.g., WO98/04712; Flint et al., 1997 Proc. Nat. Acad. Sci. USA 94:1680). In addition, the effect on the ability of the resulting mutant PTP molecule to phosphorylate one or more tyrosine residues can also be determined empirically merely by testing such a mutant for the presence of phosphotyrosine, as also provided herein, for example, following exposure of the mutant to conditions in vitro or in vivo where it may act as a phosphate acceptor for a protein tyrosine kinase.

[0072] In particular, portions of two PTP1B polypeptide sequences are regarded as “corresponding” amino acid sequences, regions, fragments or the like, based on a convention of numbering one PTP1B sequence according to amino acid position number, and then aligning the sequence to be compared in a manner that maximizes the number of amino acids that match or that are conserved residues, for example, that remain polar (e.g., D, E, K, R, H, S, T, N, Q), hydrophobic (e.g, A, P, V, L, I, M, F, W, Y) or neutral (e.g, C, G) residues at each position. Similarly, a DNA sequence encoding a candidate PTP that is to be mutated as provided herein, or a portion, region, fragment or the like, may correspond to a known wildtype PTP1B-encoding DNA sequence according to a convention for numbering nucleic acid sequence positions in the known wildtype PTP1B DNA sequence, whereby the candidate PTP DNA sequence is aligned with the known PTP1B DNA such that at least 70%, preferably at least 80% and more preferably at least 90% of the nucleotides in a given sequence of at least 20 consecutive nucleotides of a sequence are identical. In certain preferred embodiments, a candidate PTP DNA sequence is greater than 95% identical to a corresponding known PTP1B DNA sequence. In certain particularly preferred embodiments, a portion, region or fragment of a candidate PTP DNA sequence is identical to a corresponding known PTP1B DNA sequence. As is well known in the art, an individual whose DNA contains no irregularities (e.g, a common or prevalent form) in a particular gene responsible for a given trait may be said to possess a wildtype genetic complement (genotype) for that gene, while the presence of irregularities known as mutations in the DNA for the gene, for example, substitutions, insertions or deletions of one or more nucleotides, indicates a mutated or mutant genotype.

[0073] Modification of DNA may be performed by a variety of methods, including site-specific or site-directed mutagenesis of DNA encoding the polypeptide of interest (e.g., a siRNA target polypeptide) and the use of DNA amplification methods using primers to introduce and amplify alterations in the DNA template, such as PCR splicing by overlap extension (SOE). Site-directed mutagenesis is typically effected using a phage vector that has single- and double-stranded forms, such as M13 phage vectors, which are well-known and commercially available. Other suitable vectors that contain a single-stranded phage origin of replication may be used (see, e.g., Veira et al., Meth. Enzymol. 15:3, 1987). In general, site-directed mutagenesis is performed by preparing a single-stranded vector that encodes the protein of interest (e.g., PTP1B). An oligonucleotide primer that contains the desired mutation within a region of homology to the DNA in the single-stranded vector is annealed to the vector followed by addition of a DNA polymerase, such as E. coli DNA polymerase I (Klenow fragment), which uses the double stranded region as a primer to produce a heteroduplex in which one strand encodes the altered sequence and the other the original sequence. Additional disclosure relating to site-directed mutagenesis may be found, for example, in Kunkel et al. (Methods in Enzymol. 154:367, 1987) and in U.S. Pat. Nos. 4,518,584 and 4,737,462. The heteroduplex is introduced into appropriate bacterial cells, and clones that include the desired mutation are selected. The resulting altered DNA molecules may be expressed recombinantly in appropriate host cells to produce the modified protein.

[0074] SiRNAs of the invention may be fused to other nucleotide molecules, or to polypeptides, in order to direct their delivery or to accomplish other functions. Thus, for example, fusion proteins comprising a siRNA oligonucleotide that is capable of specifically interfering with expression of PTP1B may comprise affinity tag polypeptide sequences, which refers to polypeptides or peptides that facilitate detection and isolation of the such polypeptide via a specific affinity interaction with a ligand. The ligand may be any molecule, receptor, counterreceptor, antibody or the like with which the affinity tag may interact through a specific binding interaction as provided herein. Such peptides include, for example, poly-His or “FLAG®” or the like, e.g., the antigenic identification peptides described in U.S. Pat. No. 5,011,912 and in Hopp et al., (1988 Bio/Technology 6:1204), or the XPRESS™ epitope tag (Invitrogen, Carlsbad, Calif.). The affinity sequence may be a hexa-histidine tag as supplied, for example, by a pBAD/His (Invitrogen) or a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the affinity sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g., COS-7 cells, is used. The HA tag corresponds to an antibody defined epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984 Cell 37:767).

[0075] The present invention also relates to vectors and to constructs that include or encode siRNA polynucleotides of the present invention, and in particular to “recombinant nucleic acid constructs” that include any nucleic acid such as a DNA polynculeotide segment that may be transcribed to yield PTP1B polynucleotide-specific siRNA polynucleotides according to the invention as provided above; to host cells which are genetically engineered with vectors and/or constructs of the invention and to the production of siRNA polynucleotides, polypeptides, and/or fusion proteins of the invention, or fragments or variants thereof, by recombinant techniques. SiRNA sequences disclosed herein as RNA polynucleotides may be engineered to produce corresponding DNA sequences using well-established methodologies such as those described herein. Thus, for example, a DNA polynucleotide may be generated from any siRNA sequence described herein (including in the Sequence Listing), such that the present siRNA sequences will be recognized as also providing corresponding DNA polynucleotides (and their complements). These DNA polynucleotides are therefore encompassed within the contemplated invention, for example, to be incorporated into the subject invention recombinant nucleic acid constructs from which siRNA may be transcribed.

[0076] According to the present invention, a vector may comprise a recombinant nucleic acid construct containing one or more promoters for transcription of an RNA molecule, for example, the human U6 snRNA promoter (see, e.g., Miyagishi et al, Nat. Biotechnol. 20:497-500 (2002); Lee et al., Nat. Biotechnol. 20:500-505 (2002); Paul et al., Nat. Biotechnol. 20:505-508 (2002); Grabarek et al., BioTechniques 34:73544 (2003); see also Sui et al., Proc. Natl. Acad. Sci. USA 99:5515-20 (2002)). Each strand of a siRNA polynucleotide may be transcribed separately each under the direction of a separate promoter and then may hybridize within the cell to form the siRNA polynucleotide duplex. Each strand may also be transcribed from separate vectors (see Lee et al., supra). Alternatively, the sense and antisense sequences specific for a PTP1B sequence may be transcribed under the control of a single promoter such that the siRNA polynucleotide forms a hairpin molecule (Paul et al., supra). In such an instance, the complementary strands of the siRNA specific sequences are separated by a spacer that comprises at least four nucleotides, but may comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 94 18 nucleotides or more nucleotides as described herein. In addition, siRNAs transcribed under the control of a U6 promoter that form a hairpin may have a stretch of about four uridines at the 3′ end that act as the transcription termination signal (Miyagishi et al., supra; Paul et al., supra). By way of illustration, if the target sequence is 19 nucleotides, the siRNA hairpin polynucleotide (beginning at the 5′ end) has a 19-nucleotide sense sequence followed by a spacer (which as two uridine nucleotides adjacent to the 3′ end of the 19-nucleotide sense sequence), and the spacer is linked to a 19 nucleotide antisense sequence followed by a 4-uridine terminator sequence, which results in an overhang. SiRNA polynucleotides with such overhangs effectively interfere with expression of the target polypeptide (see id.). A recombinant construct may also be prepared using another RNA polymerase III promoter, the HI RNA promoter, that may be operatively linked to siRNA polynucleotide specific sequences, which may be used for transcription of hairpin structures comprising the siRNA specific sequences or separate transcription of each strand of a siRNA duplex polynucleotide (see, e.g., Brummelkamp et al., Science 296:550-53 (2002); Paddison et al., supra). DNA vectors useful for insertion of sequences for transcription of an siRNA polynucleotide include pSUPER vector (see, e.g., Brummelkamp et al., supra); pAV vectors derived from pCWRSVN (see, e.g., Paul et al., supra); and pIND (see, e.g., Lee et al., supra), or the like.

[0077] PTP1B polypeptides can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters, providing ready systems for determination of siRNA polynucleotides that are capable of interfering with polypeptide expression as provided herein. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described, for example, by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor, N.Y., (2001).

[0078] Generally, recombinant expression vectors for use in the preparation of recombinant nucleic acid constructs or vectors of the invention will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence (e.g., a siRNA polynucleotide sequence). Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heat shock proteins, among others. For PTP polypeptide expression (including PTP fusion proteins and substrate trapping mutant PTPs), and for other expression of other polypeptides of interest, the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.

[0079] Useful expression constructs for bacterial use are constructed by inserting into an expression vector a structural DNA sequence encoding a desired siRNA polynucleotide, together with suitable transcription initiation and termination signals in operable linkage, for example, with a functional promoter. The construct may comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector construct and, if desirable, to provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice. Any other plasmid or vector may be used as long as they are replicable and viable in the host.

[0080] As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., USA). These pBR322 “backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed.

[0081] Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter, if it is a regulated promoter as provided herein, is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents; such methods are well know to those skilled in the art.

[0082] Thus, for example, the nucleic acids of the invention as described herein (e.g., DNA sequences from which siRNA may be transcribed) may be included in any one of a variety of expression vector constructs as a recombinant nucleic acid construct for expressing a PTP1B polynucleotide-specific siRNA polynucleotide as provided herein. Such vectors and constructs include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA, such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used for preparation of a recombinant nucleic acid construct as long as it is replicable and viable in the host.

[0083] The appropriate DNA sequence(s) may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described, for example, in Ausubel et al. (1993 Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., Boston, Mass.); Sambrook et al. (2001 Molecular Cloning, Third Ed., Cold Spring Harbor Laboratory, Plainview, N.Y.); Maniatis et al. (1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.); and elsewhere.

[0084] The DNA sequence in the expression vector is operatively linked to at least one appropriate expression control sequences (e.g., a promoter or a regulated promoter) to direct mRNA synthesis. Representative examples of such expression control sequences include LTR or SV40 promoter, the E. coli lac or trp, the phage lambda PL promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lacd, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art, and preparation of certain particularly preferred recombinant expression constructs comprising at least one promoter or regulated promoter operably linked to a nucleic acid encoding a PTP1B polypeptide is described herein.

[0085] As noted above, in certain embodiments the vector may be a viral vector such as a retroviral vector. For example, retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.

[0086] The viral vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques 7:980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and β-actin promoters). Other viral promoters that may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein, and may be from among either regulated promoters or promoters as described above.

[0087] The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller, Human Gene Therapy, 1:5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and calcium phosphate precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.

[0088] The producer cell line generates infectious retroviral vector particles that include the nucleic acid sequence(s) encoding the PTP1B polypeptide and variants and fusion proteins thereof. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the siRNA polynucleotide that is capable of specifically interfering with expression of a polypeptide or fusion protein. Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, bronchial epithelial cells and various other culture-adapted cell lines.

[0089] In another aspect, the present invention relates to host cells containing the above described recombinant PTP1B expression constructs. Host cells are genetically engineered (transduced, transformed or transfected) with the vectors and/or expression constructs of this invention that may be, for example, a cloning vector, a shuttle vector, or an expression construct. The vector or construct may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying particular genes such as genes encoding siRNA polynucleotides or fusion proteins thereof. The culture conditions for particular host cells selected for expression, such as temperature, pH and the like, will be readily apparent to the ordinarily skilled artisan.

[0090] The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Representative examples of appropriate host cells according to the present invention include, but need not be limited to, bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells, such as Drosophila S2 and Spodoptera S19; animal cells, such as CHO, COS or 293 cells; adenoviruses; plant cells, or any suitable cell already adapted to in vitro propagation or so established de novo. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

[0091] Various mammalian cell culture systems can also be employed to produce siRNA polynucleotides from recombinant nucleic acid constructs of the present invention. The invention is therefore directed in part to a method of producing a siRNA polynucleotide, by culturing a host cell comprising a recombinant nucleic acid construct that comprises at least one promoter operably linked to a nucleic acid sequence encoding a siRNA polynucleotide specific for a PTP1B polypeptide. In certain embodiments, the promoter may be a regulated promoter as provided herein, for example a tetracylcine-repressible promoter. In certain embodiments the recombinant expression construct is a recombinant viral expression construct as provided herein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa, HEK, and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences, for example as described herein regarding the preparation of recombinant siRNA polynucleotide constructs. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements. Introduction of the construct into the host cell can be effected by a variety of methods with which those skilled in the art will be familiar, including but not limited to, for example, liposomes including cationic liposomes, calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis et al., 1986 Basic Methods in Molecular Biology), or other suitable technique.

[0092] The expressed recombinant siRNA polynucleotides may be useful in intact host cells; in intact organelles such as cell membranes, intracellular vesicles or other cellular organelles; or in disrupted cell preparations including but not limited to cell homogenates or lysates, microsomes, uni- and multilamellar membrane vesicles or other preparations. Alternatively, expressed recombinant siRNA polynucleotides can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.

[0093] Samples

[0094] According to the present invention, a method is provided for interfering with expression of a PTP1B polypeptide as provided herein. A method is also provided for interfering with expression of a PTP1B polypeptide, comprising contacting a siRNA polynucleotide with a cell that is capable of expressing PTP1B, typically in a biological sample or in a subject or biological source. A “sample” as used herein refers to a biological sample containing PTP1B, and may be provided by obtaining a blood sample, biopsy specimen, tissue explant, organ culture or any other tissue or cell preparation from a subject or a biological source. A sample may further refer to a tissue or cell preparation in which the morphological integrity or physical state has been disrupted, for example, by dissection, dissociation, solubilization, fractionation, homogenization, biochemical or chemical extraction, pulverization, lyophilization, sonication or any other means for processing a sample derived from a subject or biological source. In certain preferred embodiments, the sample is a cell that comprises at least one PTP1B polypeptide, and in certain particularly preferred embodiments the cell comprises an inducible biological signaling pathway, at least one component of which is PTP1B. In particularly preferred embodiments the cell is a mammalian cell, for example, Rat-1 fibroblasts, COS cells, CHO cells, HEK-293 cells, HepG2, HII4E-C3, L6, and 3T3-L1, or other well known model cell lines, which are available from the American Type Culture Collection (ATCC, Manassas, Va.). In other preferred embodiments, the cell line is derived from PTP-1B knockout animals and which may be transfected with human insulin receptor (HIR), for example, 1BKO mouse embryo fibroblasts.

[0095] The subject or biological source may be a human or non-human animal, a primary cell culture or culture adapted cell line including but not limited to genetically engineered cell lines that may contain chromosomally integrated or episomal recombinant nucleic acid sequences, immortalized or immortalizable cell lines, somatic cell hybrid cell lines, differentiated or differentiatable cell lines, transformed cell lines and the like. Optionally, in certain situations it may be desirable to treat cells in a biological sample with hydrogen peroxide and/or with another agent that directly or indirectly promotes reactive oxygen species (ROS) generation, including biological stimuli as described herein; in certain other situations it may be desirable to treat cells in a biological sample with a ROS scavenger, such as N-acetyl cysteine (MAC) or superoxide dismutase (SOD) or other ROS scavengers known in the art; in other situations cellular glutathione (GSH) may be depleted by treating cells with L-buthionine-SR-sulfoximine (Bso); and in other circumstances cells may be treated with pervanadate to enrich the sample in tyrosine phosphorylated proteins. Other means may also be employed to effect an increase in the population of tyrosine phosphorylated proteins present in the sample, including the use of a subject or biological source that is a cell line that has been transfected with at least one gene encoding a protein tyrosine kinase.

[0096] Additionally or alternatively, a biological signaling pathway may be induced in subject or biological source cells by contacting such cells with an appropriate stimulus, which may vary depending upon the signaling pathway under investigation, whether known or unknown. For example, a signaling pathway that, when induced, results in protein tyrosine phosphorylation and/or protein tyrosine dephosphorylation may be stimulated in subject or biological source cells using any one or more of a variety of well known methods and compositions known in the art to stimulate protein tyrosine kinase (PTK) and/or PTP activity. These stimuli may include, without limitation, exposure of cells to cytokines, growth factors, hormones, peptides, small molecule mediators, cell stressors (e.g., ultraviolet light; temperature shifts; osmotic shock; ROS or a source thereof, such as hydrogen peroxide, superoxide, ozone, etc. or any agent that induces or promotes ROS production (see, e.g., Halliwell and Gutteridge, Free Radicals in Biology and Medicine (3rd Ed.) 1999 Oxford University Press, Oxford, UK); heavy metals; alcohol) or other agents that induce PTK-mediated protein tyrosine phosphorylation and/or PTP-mediated phosphoprotein tyrosine dephosphorylation. Such agents may include, for example, interleukins (e.g., IL-1, IL-3), interferons (e.g., IFN-γ), human growth hormone, insulin, epidermal growth factor (EGF), platelet derived growth factor (PDGF), granulocyte colony stimulating factor (G-CSF), granulocyte-megakaryocyte colony stimulating factor (GM-CSF), transforming growth factor (e.g., TGF-β1), tumor necrosis factor (e.g., TNF-α) and fibroblast growth factor (FGF; e.g., basic FGF (bFGF)), any agent or combination of agents capable of triggering T lymphocyte activation via the T cell receptor for antigen (TCR; TCR-inducing agents may include superantigens, specifically recognized antigens and/or MHC-derived peptides, MHC peptide tetramers (e.g., Altman et al., 1996 Science 274:94-96); TCR-specific antibodies or fragments or derivatives thereof), lectins (e.g., PHA, PWM, ConA, etc.), mitogens, G-protein coupled receptor agonists such as angiotensin-2, thrombin, thyrotropin, parathyroid hormone, lysophosphatidic acid (LPA), sphingosine-1-phosphate, serotonin, endothelin, acetylcholine, platelet activating factor (PAF) or bradykinin, as well as other agents with which those having ordinary skill in the art will be familiar (see, e.g., Rhee et al., [online] Oct. 10, 2000 Science's stke, Internet:URL<www.stke.org/cgl/content/full/OC_sigtrans;2000/53/pe1>), and references cited therein).

[0097] As noted above, regulated tyrosine phosphorylation contributes to specific pathways for biological signal transduction, including those associated with cell division, cell survival, apoptosis, proliferation and differentiation, and “inducible signaling pathways” in the context of the present invention include transient or stable associations or interactions among molecular components involved in the control of these and similar processes in cells. Depending on the particular pathway of interest, an appropriate parameter for determining induction of such pathway may be selected. For example, for signaling pathways associated with cell proliferation, a variety of well known methodologies are available for quantifying proliferation, including, for example, incorporation of tritiated thymidine into cellular DNA, monitoring of detectable (e.g., fluorimetric or calorimetric) indicators of cellular respiratory activity, (e.g., MTT assay) or cell counting, or the like. Similarly, in the cell biology arts there are known multiple techniques for assessing cell survival (e.g., vital dyes, metabolic indicators, etc.) and for determining apoptosis (e.g., annexin V binding, DNA fragmentation assays, caspase activation, PARP cleavage, etc.). Other signaling pathways will be associated with particular cellular phenotypes, for example specific induction of gene expression (e.g., detectable as transcription or translation products, or by bioassays of such products, or as nuclear localization of cytoplasmic factors), altered (e.g., statistically significant increases or decreases) levels of intracellular mediators (e.g., activated kinases or phosphatases, altered levels of cyclic nucleotides or of physiologically active ionic species, etc.), altered cell cycle profiles, or altered cellular morphology, and the like, such that cellular responsiveness to a particular stimulus as provided herein can be readily identified to determine whether a particular cell comprises an inducible signaling pathway.

[0098] In preferred embodiments, a PTP1B substrate may be any naturally or non-naturally occurring phosphorylated peptide, polypeptide or protein that can specifically bind to and/or be dephosphorylated by PTP1B.

[0099] Identification and selection of PTP1B substrates as provided herein, for use in the present invention, may be performed according to procedures with which those having ordinary skill in the art will be familiar, or may, for example, be conducted according to the disclosures of WO 00/75339, U.S. application Ser. No. 09/334,575, or U.S. application Ser. No. 10/366,547, and references cited therein. The phosphorylated protein/PTP complex may be isolated, for example, by conventional isolation techniques as described in U.S. Pat. No. 5,352,660, including salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, combinations thereof or other strategies. PTP1B substrates that are known may also be prepared according to well known procedures that employ principles of molecular biology and/or peptide synthesis (e.g., Ausubel et al., Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., Boston, Mass. (1993); Sambrook et al., Molecular Cloning, Third Ed., Cold Spring Harbor Laboratory, Plainview, N.Y. (2001); Fox, Molec. Biotechnol. 3:249 (1995); Maeji et al., Pept. Res. 8:33 (1995)).

[0100] The PTP1B substrate peptides of the present invention may therefore be derived from PTP1B substrate proteins, polypeptides and peptides as provided herein having amino acid sequences that are identical or similar to tyrosine phosphorylated PTP1B substrate sequences known in the art. For example by way of illustration and not limitation, peptide sequences derived from the known PTP1B substrate proteins referred to above are contemplated for use according to the instant invention, as are peptides having at least 70% similarity (preferably 70% identity), more preferably 80% similarity (more preferably 80% identity), more preferably 90% similarity (more preferably 90% identity) and still more preferably 95% similarity (still more preferably 95% identity) to the polypeptides described in references cited herein and in the Examples and to portions of such polypeptides as disclosed herein. As known in the art “similarity” between two polypeptides is determined by comparing the amino acid sequence and conserved amino acid substitutes thereto of the polypeptide to the sequence of a second polypeptide (e.g., using GENEWORKS, Align or the BLAST algorithm, or another algorithm, as described above).

[0101] In certain preferred embodiments of the present invention, the siRNA polynucleotide and/or the PTP1B substrate is detectably labeled, and in particularly preferred embodiments the siRNA polynucleotide and/or PTP substrate is capable of generating a radioactive or a fluorescent signal. The siRNA polynucleotide and/or PTP substrate can be detectably labeled by covalently or non-covalently attaching a suitable reporter molecule or moiety, for example a radionuclide such as 32P (e.g., Pestka et al., 1999 Protein Expr. Purif. 17:203-14), a radiohalogen such as iodine [125I or 131I] (e.g., Wilbur, 1992 Bioconjug. Chem. 3:433-70), or tritium [3H]; an enzyme; or any of various luminescent (e.g., chemiluminescent) or fluorescent materials (e.g., a fluorophore) selected according to the particular fluorescence detection technique to be employed, as known in the art and based upon the present disclosure. Fluorescent reporter moieties and methods for labeling siRNA polynucleotides and/or PTP substrates as provided herein can be found, for example in Haugland (1996 Handbook of Fluorescent Probes and Research Chemicals—Sixth Ed., Molecular Probes, Eugene, Oreg.; 1999 Handbook of Fluorescent Probes and Research Chemicals—Seventh Ed., Molecular Probes, Eugene, Oreg., [Internet]<: http://www.probes.com/lit/>) and in references cited therein. Particularly preferred for use as such a fluorophore in the subject invention methods are fluorescein, rhodamine, Texas Red, AlexaFluor-594, AlexaFluor-488, Oregon Green, BODIPY-FL, umbelliferone, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin or Cy-5. Examples of suitable enzymes include, but are not limited to, horseradish peroxidase, biotin, alkaline phosphatase, β-galactosidase and acetylcholinesterase. Appropriate luminescent materials include luminol, and suitable radioactive materials include radioactive phosphorus [32P]. In certain other preferred embodiments of the present invention, a detectably labeled siRNA polynucleotide comprises a magnetic particle, for example a paramagnetic or a diamagnetic particle or other magnetic particle or the like (preferably a microparticle) known to the art and suitable for the intended use. Without wishing to be limited by theory, according to certain such embodiments there is provided a method for selecting a cell that has bound, adsorbed, absorbed, internalized or otherwise become associated with a siRNA polynucleotide that comprises a magnetic particle. For example, selective isolation of a population or subpopulation of cells containing one or more PTP1B-specific siRNA polynucleotide-magnetic particle conjugates may offer certain advantages in the further characterization or regulation of PTP signaling pathways.

[0102] In certain embodiments of the present invention, particular PTP1B-specific siRNA polynucleotides of interest may be identified by contacting a candidate siRNA polynucleotide with a sample comprising a cell that comprises a PTP1B gene and that is capable of PTP1B gene transcription or expression (e.g., translation), under conditions and for a time sufficient to detect PTP1B gene transcription or expression, and comparing PTP1B transcription levels, PTP1B polypeptide expression and/or PTP1B functional expression (e.g., PTP1B catalytic activity) in the absence and presence of the candidate siRNA polynucleotide. Preferably PTP1B transcription or expression is decreased in the presence of the siRNA polynucleotide, thereby providing an alternative to PTP active site directed approaches to modulating PTP1B activity. (The invention need not be so limited, however, and contemplates other embodiments wherein transcription and/or expression levels of a signal transduction component other than that which is specifically targeted by the siRNA may be increased in the presence of a certain PTP1B-specific siRNA polynucleotide. By way of non-limiting theory, such an increase may result from a cellular compensatory mechanism that is induced as a result of the siRNA.)

[0103] For a cell that expresses PTP1B and comprises an insulin receptor, such as IR-β, and the siRNA polynucleotide effects an increase in insulin receptor phosphorylation, presumably (and according to non-binding theory) by decreasing PTP1B levels through interference with PTP-1B expression. Methods for determining insulin receptor phosphorylation are known in the art (e.g., Cheatham et al., 1995 Endocr. Rev. 16:117-142) and are described in greater detail below. In certain other further embodiments wherein the cell comprises an insulin receptor, any of a variety of cellular insulin responses may be monitored according to art-established methodologies, including but not limited to glucose uptake (e.g., Elchebly et al., 1999 Science 283:1544; McGuire et al., 1991 Diabetes 40:939; Myerovitch et al., 1989 J. Clin. Invest. 84:976; Sredy et al. 1995 Metabolism 44:1074; WO 99/46268); glycogen synthesis (e.g., Berger et al., 1998 Anal. Biochem. 261:159), Glut4 recruitment to a plasma membrane (Robinson et al., 1992 J. Cell Biol. 117:1181); liver transcription events, or amino acid import (Hyde et al., 2002 J. Biol. Chem. 277:13628-34 (2002)). In certain other further embodiments wherein the cell comprises an insulin receptor, cellular insulin responses that may be monitored include MAP kinase phosphorylation, AKT phosphorylation, and other insulin-stimulated phosphorylation events downstream of the insulin receptor, such as P13 kinase, perk, pSTAT5, and IRS1, and inhibition of phosphoenolpyruvate carboxykinase transcription (Forest et al., 1990 Molec. Endocrinol. 4:1302), phosphatidylinositoltriphosphate kinase activation (Endeman et al., 1990 J. Biol. Chem. 265:396), lipogenesis (Moody et al., 1974 Horm. Metab. Res. 6:12), lipolysis (Hess et al., 1991 J. Cell. Biochem. 45:374), TYK2 dephosphorylation and JAK2 (see GenBank Nos. NM004972, AF058925, AF005216, NM031514, and NM008261) dephosphorylation (Myers et al., 2001 J. Biol. Chem. 276:47771), interferon-stimulated pSTAT1 and pSTAT3, and EGF or PDGRF phosphorylation (Ullrich et al., 1990 Cell 61:203). In addition, phosphorylation of the insulin receptor, such as at positions tyr1162/tyr1163 and at position tyr972, may be detected with anti-phosphotyrosine antibodies that are site-specific for tyr1162/tyr1163 or tyr972.

[0104] PTP1B activity may also be measured in whole cells transfected with a reporter gene whose expression is dependent upon the activation of an appropriate substrate. For example, appropriate cells (i.e., cells that are capable of expressing PTP1B and that have been transfected with a PTP1B-specific siRNA polynucleotide that is either known or suspected of being capable of interfering with PTP-1B polypeptide expression) may be transfected with a substrate-dependent promoter linked to a reporter gene. In such a system, expression of the reporter gene (which may be readily detected using methods well known to those of ordinary skill in the art) depends upon activation of substrate. Dephosphorylation of substrate may be detected based on a decrease in reporter activity. Candidate siRNA polynucleotides specific for PTP1B may be added to such a system, as described above, to evaluate their effect on PTP1B activity.

[0105] Within other aspects, the present invention provides animal models in which an animal, by virtue of introduction of an appropriate PTP1B-specific siRNA polynucleotide, for example, as a transgene, does not express (or expresses a significantly reduced amount of) a functional PTP1B. Such animals may be generated, for example, using standard homologous recombination strategies, or alternatively, for instance, by oocyte microinjection with a plasmid comprising the siRNA-encoding sequence that is regulated by a suitable promoter (e.g., ubiquitous or tissue-specific) followed by implantation in a surrogate mother. Animal models generated in this manner may be used to study activities of PTP signaling pathway components and modulating agents in vivo.

[0106] Therapeutic Methods

[0107] One or more siRNA polynucleotides capable of interfering with PTP1B polypeptide expression and identified according to the above-described methods may also be used to modulate (e.g., inhibit or potentiate) PTP1B activity in a patient. As used herein, a “patient” may be any mammal, including a human, and may be afflicted with a condition associated with undesired PTP1B activity or may be free of detectable disease. Accordingly, the treatment may be of an existing disease or may be prophylactic. Conditions associated with signal transduction and/or PTP1B activity include any disorder associated with cell proliferation, including cancer, graft-versus-host disease (GVHD), autoimmune diseases, allergy or other conditions in which unregulated PTP1B activity may be involved.

[0108] For administration to a patient, one or more specific siRNA polynucleotides, either alone, with or without chemical modification or removal of ribose, or comprised in an appropriate vector as described herein (e.g., including a vector which comprises a DNA sequence from which a specific siRNA can be transcribed) are generally formulated as a pharmaceutical composition. A pharmaceutical composition may be a sterile aqueous or non-aqueous solution, suspension or emulsion, which additionally comprises a physiologically acceptable carrier (i.e., a non-toxic material that does not interfere with the activity of the active ingredient). Such compositions may be in the form of a solid, liquid or gas (aerosol). Alternatively, compositions of the present invention may be formulated as a lyophilizate or compounds may be encapsulated within liposomes using well known technology. Pharmaceutical compositions within the scope of the present invention may also contain other components, which may be biologically active or inactive. Such components include, but are not limited to, buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, stabilizers, dyes, flavoring agents, and suspending agents and/or preservatives.

[0109] Any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of the present invention. Carriers for therapeutic use are well known, and are described, for example, in Remingtons Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro ed. 1985). In general, the type of carrier is selected based on the mode of administration. Pharmaceutical compositions may be formulated for any appropriate manner of administration, including, for example, topical, oral, nasal, intrathecal, rectal, vaginal, sublingual or parenteral administration, including subcutaneous, intravenous, intramuscular, intrasternal, intracavernous, intrameatal or intraurethral injection or infusion. For parenteral administration, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, kaolin, glycerin, starch dextrins, sodium alginate, carboxymethylcellulose, ethyl cellulose, glucose, sucrose and/or magnesium carbonate, may be employed.

[0110] A pharmaceutical composition (e.g., for oral administration or delivery by injection) may be in the form of a liquid (e.g., an elixir, syrup, solution, emulsion or suspension). A liquid pharmaceutical composition may include, for example, one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. A parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. The use of physiological saline is preferred, and an injectable pharmaceutical composition is preferably sterile.

[0111] The compositions described herein may be formulated for sustained release (i.e., a formulation such as a capsule or sponge that effects a slow release of compound following administration). Such compositions may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained-release formulations may contain an agent dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane. Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release. The amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.

[0112] Within a pharmaceutical composition, a therapeutic agent comprising a polypeptide-directed siRNA polynucleotide as described herein (or, e.g., a recombinant nucleic acid construct encoding a siRNA polynucleotide) may be linked to any of a variety of compounds. For example, such an agent may be linked to a targeting moiety (e.g., a monoclonal or polyclonal antibody, a protein or a liposome) that facilitates the delivery of the agent to the target site. As used herein, a “targeting moiety” may be any substance (such as a compound or cell) that, when linked to an agent enhances the transport of the agent to a target cell or tissue, thereby increasing the local concentration of the agent. Targeting moieties include antibodies or fragments thereof, receptors, ligands and other molecules that bind to cells of, or in the vicinity of, the target tissue. An antibody targeting agent may be an intact (whole) molecule, a fragment thereof, or a functional equivalent thereof. Examples of antibody fragments are F(ab′)2, Fab′, Fab and F[v] fragments, which may be produced by conventional methods or by genetic or protein engineering. Linkage is generally covalent and may be achieved by, for example, direct condensation or other reactions, or by way of bi- or multi-functional linkers. Targeting moieties may be selected based on the cell(s) or tissue(s) toward which the agent is expected to exert a therapeutic benefit.

[0113] Pharmaceutical compositions may be administered in a manner appropriate to the disease to be treated (or prevented). An appropriate dosage and a suitable duration and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the patient's disease, the particular form of the active ingredient and the method of administration. In general, an appropriate dosage and treatment regimen provides the agent(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (e.g., an improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free and/or overall survival, or a lessening of symptom severity). For prophylactic use, a dose should be sufficient to prevent, delay the onset of or diminish the severity of a disease associated with cell proliferation.

[0114] Optimal dosages may generally be determined using experimental models and/or clinical trials. In general, the amount of siRNA polynucleotide present in a dose, or produced in situ by DNA present in a dose (e.g, from a recombinant nucleic acid construct comprising a siRNA polynucleotide), ranges from about 0.01 μg to about 100 μg per kg of host, typically from about 0.1 μg to about 10 μg. The use of the minimum dosage that is sufficient to provide effective therapy is usually preferred. Patients may generally be monitored for therapeutic or prophylactic effectiveness using assays suitable for the condition being treated or prevented, which will be familiar to those having ordinary skill in the art. Suitable dose sizes will vary with the size of the patient, but will typically range from about 10 mL to about 500 mL for 10-60 kg animal.

[0115] The following Examples are offered by way of illustration and not by way of limitation.

EXAMPLE 1

[0116] Interferences of PTP1B Expression by Specific siRNA

[0117] This Example describes the effect on expression of PTP-1B expression in cells transfected with sequence-specific siRNA polynucleotides.

[0118] Interference of Endogenous Expression of Murine PTP1B in Mouse Fibroblasts by Sequence Specific siRNA Polynucleotides

[0119] Three siRNA sequences that were specific for murine PTP1B polynucleotide (GenBank Ace. No. NM011201, SEQ ID NO: __) encoding a murine PTP1B polypeptide (GenBank Ace. No. NM011201, SEQ ID NO: __) and one siRNA sequence specific for human PTP1B polynucleotide (GenBank Ace. No. NM002827, SEQ ID NO: __) encoding a human PTP1B polypeptide (GenBank Ace. No. NM002827, SEQ ID NO: __) were designed as follows. The siRNA nucleotide sequences specific for each PTP1B were chosen by first scanning the open reading frame of the target cDNA for 21-base sequences that were flanked on the 5′ end by two adenine bases (AA) and that had A+T/G+C ratios that were nearly 1:1. Twenty-one-base sequences with an A+T/G+C ratio greater than 2:1 or 1:2 were excluded. If no 21-base sequences were identified that met this criteria, the polynucleotide sequence encoding the PTP1B was searched for a 21-base sequence having the bases CA at the 5′ end. The specificity of each 21-mer was determined by performing a BLAST search of public databases. Sequences that contained at least 16 of 21 consecutive nucleotides with 100% identity with a polynucleotide sequence other than the target sequence were not used in the experiments.

[0120] Sense and antisense oligonucleotides for TCPTP analysis were synthesized according to the standard protocol of the vendor (Dharmacon Research, Inc., Lafayette, Colo.). For some experiments described in this and other examples, the vendor gel-purified the double-stranded siRNA polynucleotide, which was then used. In the instances when the vendor did not prepare double-stranded siRNA, just before transfection, double-stranded siRNAs were prepared by annealing the sense and anti-sense oligonucleotides in annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 minute at 90° C., followed by a 60-minute incubation at 37° C.

[0121] In each of the examples, each siRNA sequence represents the sense strand of the siRNA polynucleotide and its corresponding sequence identifier. Unless otherwise stated, it is to be understood that the siRNA transfected into a cell is composed of the sense strand and its complementary antisense strand, which form a duplex siRNA polynucleotide.

[0122] Mouse C57B16 #3 cells, clones 3 and 10, were maintained in cell culture according to standard cell culture methods. Each C57B16 #3 clone was transfected with 200 nM of the following siRNAs: mPTP1B.1 (SEQ ID NO: __), mPTP1B.2 (SEQ ID NO: __, mPTP1B.3 (SEQ ID NO: __), and hPTP1B.1 (SEQ ID NO: __). Each siRNA was diluted in 50 μl OPTIMEM® to provide a final concentration of 200 nM per well. In a separate tube, 3 μl of Lipofectamine™ was combined with 10 μl OPTIMEM®. Each solution was incubated for 7 minutes. The two solutions were then mixed and incubated at room temperature for 22 minutes. The final volume of the mixed solution was adjusted to 100 μl and then the C57B16 #3 cells were added. Cells were transfected with the specific siRNAs, the human PTP1B siRNA, or annealing buffer alone. The transfected cells were incubated with siRNAs for six days.

[0123] Cell lysates were prepared by extracting the cells in ELISA extraction buffer (50 mM Tris-HCl, pH 7.5 (room temperature); 2 mM EDTA, pH 7-8; 1 mM phosphate (polyphosphate); 1 mM NaVO4 (monomeric), pH 10; 0.1% Triton X-100; Protease Inhibitor Cocktail set III, (Calbiochem, San Diego, Calif., catalog #539134)). The lysates were separated by SDS-PAGE gel and analyzed by immunoblot. The lysates were centrifuged and aliquots of supernatant (10 μl) from each transfected cell culture sample were combined with 10 μl of SDS-PAGE reducing sample buffer. The samples were heated at 95° C. for five minutes, and then applied to a 14% Tris-glycine SDS-PAGE gel (NOVEX® from Invitrogen Life Technologies, Carlsbad, Calif.). After electrophoresis, the separated proteins were electrophoretically transferred from the gel onto an Immobilon-P polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, Mass.). The PVDF membrane was blocked in 5% milk in TBST (20 mM Tris pH 7.5, 150 mM NaCl, 0.05% Tween-20); incubated with an anti-murine PTP1B monoclonal antibody (Dr. Ben Neel, Harvard University, Cambridge, Mass.) for 2-16 hours at room temperature; washed 3×10 minutes with TBST; and then incubated with an appropriate horseradish peroxidase (HRP) conjugate IgG (1:10,000) (Amersham Biosciences, Piscataway, N.J.) for 30 minutes at room temperature. Binding was detected with the ECL chemiluminescent reagent used according to the vendor's instructions (Amersham Biosciences, Piscataway, N.J.). As shown in FIG. 1, the levels of expression of endogenous PTP1B were decreased only in C57B16 cells transfected with the murine PTP1B sequence specific siRNA polynucleotides.

[0124] The effect of RNAi on endogenous expression of murine PTP1B in a second murine cell line was examined. Mouse PTP1B:3T31R fibroblasts were transfected with 20 nM mPTP1B1.1 (SEQ ID NO: __); mPTP1B1.6 (SEQ ID NO: __); and mPTP1B1.8 (SEQ ID NO: __) according to the method described above. The level of murine PTP1B expression in the cells transfected with mPTP1B1.1 decreased approximately 80% compared with cells transfected with a non-specific siRNA (hPTP1B1.3 (SEQ ID NO: __); cells transfected with mPTP1B1.6 decreased approximately 40%; and cells transfected with mPTP1B1.8 decreased approximately 60%.

[0125] Interference with Murine PTP1B Expression by siRNA in Co-Transfection Assays

[0126] A recombinant expression construct was prepared that encodes wild-type murine PTP1B (mPTP1B) (GenBank Acc. No. NM011201, SEQ ID NOs: __ and __). The following oligonucleotide primers were used for the wild-type construct. The sequences of the BamHI and EcoRI restriction sites are underlined.

mPTP1B-sense (mPTP1B 5′ BamHI) 5′-GGGGGGGATCCATGGAGATGGAGAAGGAGTTCGAGG-3′ (SEQ ID NO:_)
mPTP1B anti sense (mPTP1B 3′ EcoRI) 5′-GGGGGAATTCTCAGTGAAAACACACCCGGTAGCAC-3′ (SEQ ID NO:_)

[0127] Vector pCMVTag2B (Stratagene, La Jolla, Calif.) was digested with restriction endonuclease BamHI (New England Biolabs, Beverly, Mass.) for 3 hours at 37° C. The digested vector was then incubated with Klenow polymerase (New England Biolabs) for 15 minutes at 25° C. to fill in the recessed 3′ termini, followed by an incubation of 30 minutes at 37° C. with calf intestinal phosphatase (New England Biolabs). The GATEWAY™ Reading Frame Cassette B (Invitrogen Life Technologies, Carlsbad, Calif.) was inserted into the pCMVTag2B vector by ligation with T4 DNA ligase (Invitrogen Life Technologies) overnight at 16° C. according to the supplier's instructions. DB3.1™ competent E. coli cells were transformed with the ligated vector (GWpCMVTag2) and DNA was isolated by standard molecular biology methods.

[0128] Vectors for expression of mPTP1B wild type were prepared as follows. The mPTP1B construct was subcloned into a GATEWAY™ entry vector pENTR3C™ (Invitrogen Life Technologies) by digesting 20 μl of the mPTP1B cDNA or 20 μl of the pENTR3C™ vector with 1 μl of BamHI (New England Biolabs); 1 μl of EcoRI (New England Biolabs); 5 μl 10× EcoRi buffer (New England Biolabs); 5 μl 10×BSA (New England Biolabs); and 18 μl distilled water for 3 hours at 37° C. Digested DNA was run on a 1% agarose gel, digested bands were excised, and the DNA was gel-purified using a QIAGEN Gel Extraction kit (QIAGEN, Inc., Valencia, Calif.). Four microliters of the mPTP1B cDNA was ligated into 2 μl of the pENTR3C™ vector overnight at 16° C. with 1 μl 10× Ligation Buffer (Invitrogen Life Technologies), 1 μl T4 DNA Ligase (4U/μl) (Invitrogen, Carlsbad, Calif.), and 2 μl distilled water. The construct was transformed into LIBRARY EFFICIENCY® DH5α™ cells. The FLAG® epitope-tagged mPTP1B construct was prepared by cloning the pENTR3 C™ mPTP1B WT construct into the GWpCMVTag2 vector. The pENTR3C™ construct containing the mPTP1B polynucleotide was linearized by digesting the construct with Vsp I (Promega Corp., Madison, Wis.) at 37° C. for 2 hours. The DNA was purified using a QIAGEN PCR Purification kit (QIAGEN, Inc.). Three microliters (100 ng/μl) of the GWpCMVTag2 vector were combined in a GATEWAY™ LR reaction with 6 μl linearized pENTR3C™ mPTP1B WT, 3 μl TE buffer, 4 μl Clonase™ Enzyme, and 4 μl LR reaction buffer (Invitrogen Life Technologies) for 1 hour at room temperature. After addition of Proteinase K (Invitrogen Life Technologies) to the reaction for 10 minutes, LIBRARY EFFICIENCY® DH5α™ cells were transformed with the expression construct.

[0129] The murine PTP1B expression vector (0.5 μg) was co-transfected with 20 nM murine PTP1B sequence-specific siRNA polynucleotides into PTP1B knockout mouse fibroblasts (PTP1B KO mouse embryonic fibroblasts were prepared from 13-day embryos from PTP1B knock out mice to establish the cell line, which was then transfected with human insulin receptor (1BKO+HIR) (HIR, Julie Moyers, Eli Lilly and Company, Indianapolis, Ind.)). Cells were transfected with siRNAs or annealing buffer alone. Each siRNA was diluted in 250 μl OPTIMEM® low serum medium (Gibco, Inc.) to a final concentration of 20 nM. In a separate tube, 10 μl of Lipofectamine™ 2000 (Invitrogen Life Technologies, Carlsbad, Calif.) was combined with 250 μl OPTIMEM®. Each solution was incubated for 7 minutes. The two solutions were then mixed and incubated at room temperature for 22 minutes. The final volume of the mixed solution was adjusted to 100 μl and then the cells were added. After incubating the transfected cells for 18 hours at 37° C., cell lysates were prepared, separated by 4-12% SDS-PAGE, and immunoblotted using the anti-PTP1B murine monoclonal antibody (see above). The results are summarized in Table 1, and it is noted that each 21-mer sequence below contains a dinucleotide “overhang” at the 3′ end, and that certain preferred embodiments of the invention described herein should be considered to include the 19-mer polynucleotide sequences beginning at the 5′ end therein as well as the 21-mer polynucleotide shown in the Table.

TABLE 1
siRNA INTERFERENCE WITH MURINE PIP-1B EXPRESSION
IN CO-TRANSFECTION ASSAYS
SEQ ID Decrease in
Target siRNA Sequence siRNA Name NO: Expression
Murine PTP1B 5′-gaagcccagaggagcuauatt-3′ mPTP1B1.1 95%
5′-cuacaccacauggccugactt-3′ mPTP1B1.2 Not
analyzed
5′-gacugccgaccagcugcgctt-3′ mPTP1B1.3 Not
analyzed
5′-gguaccgagaugucagccctt-3′ mPTP1B1.4 25%
5′-ugacuauaucaaugccagctt-3′ mPTP1B1.5 Not
analyzed
5′-agaagaaaaggagaugguctt-3′ mPTP1B1.6 80%
5′-cgggaagugcaaggagcuctt-3′ mPTP1B1.7 Not
analyzed
5′-ggaucaguggaaggagcuctc-3′ mPTP1B1.8 80%

[0130] Interference with Rat PTP1B Expression by siRNA in Co-Transfection Assays

[0131] A co-transfection assay was performed as described above in which 1BKO+HIR mouse fibroblasts were co-transfected with an expression vector containing the sequence encoding a rat PTP1B polypeptide (SEQ ID NO: __) (GenBank Accession No. NM012637) and a sequence specific siRNA, rPTP1B1.1 (5′-agaagaaaaagagaugguctt-3′ (SEQ ID NO: __) (20 nM). Additional rat PTP1B specific siRNA polynucleotides examined in the co-transfection assay included rPTP1B1.2 (5′-cggaugguggguggagguctt-3′ (SEQ ID NO: __); rPTP1B1.3 (5′-uggcaagugcaaggagcuctt-3′ (SEQ ID NO: __); and rPTP]B1.4 (5′-cuacaccaccuggccugactt-3′ (SEQ ID NO: __). The level of expression of the rat PTP1B polypeptide was determined by immunoblotting cell lysates with an anti-human PTP1B antibody that also specifically binds to rat PTP1B (PHO2, Oncogene Research Products™ Inc. San Diego, Calif.). Expression of rat PTP1B decreased approximately 50% in cells transfected with rPTP1B1.1.

[0132] Interference with Human PTP-1B Expression by siRNA in Co-Transfection Assays

[0133] Human PTP1B encoding sequence was cloned into a Pmt vector according to standard molecular biology procedures (see Flint et al., EMBO J. 12:1937-46 (1993)). 1BKO+HIR cells were co-transfected with the human PTP-1B expression vector and siRNA polynucleotides (20 nM) specific for human PTP-1B sequences overnight using Lipofectamine 2000. Cells were lysed as described above, and the lysates were separated by 4-12% SDS-PAGE and transferred onto a PDVF membrane. The level of expression of human PTP-1 B was determined by immunoblotting with an anti-human PTP-1B antibody (PHO2, Oncogene Research Products™, Inc. San Diego, Calif.). Interference with expression of human PTP-1B was observed with four siRNA polynucleotides as indicated in Table 2, and it is noted that each 21-mer sequence below contains a dinucleotide “overhang” at the 3′ end, and that the invention herein should be considered to include the 19-mer polynucleotide sequences beginning at the 5′ end therein as well as the 21-mer polynucleotide shown in the Table.

TABLE 2
sIRNA INTERFERENCE WITH HUMAN PTP-1B EXPRESSION
IN CO-TRANSFECTION ASSAYS
siRNA SEQ ID Decrease in
Target siRNA Sequence Name NO: Expression
Human PTP1B 5′-cuauaccacauggccugactt-3′ hPTP1B1.1 Not analyzed
5′-gcccaaaggaguuacauuctt-3′ hPTP1B1.2 >95%
5′-ggaagaaaaaggaagcccctt-3′ hPTP1B1.3 >95%
5′-caaugggaaaugcagggagtt-3′ hPTP1B1.4 >95%
5′-ggaucaguggaaggagcuutc-3′ hPTP1B1.5 >95%

[0134] Interference with Endogenous Expression of Human PTP1B by siRNA

[0135] The effect of sequence specific siRNA on endogenous expression of human PTP1B was examined in two different cell lines. HeLa cells were transfected as described above with hPTP1B1.1, hPTP1B1.2, hPTP1B1.3, hPTP1B1.4, and hPTP1B1.5 at 20 nM using Lipofectamine 2000, and after three days, the level of expression of PTP1B was analyzed by immunoblot. No significant decrease in expression of human PTP1B was observed in HeLa cells transfected with the siRNA hPTP1B1.1. In HeLa cells transfected with hPTP1B1.2 and hPTP1B1.4, the level of expression of human PTP1B decreased 80%, and in cells transfected with hPTP1B1.3, the level of expression decreased 90%. Endogenous expression of human PTP1B in the second cell line, 293-HEK-HIR, (gift from Julie Moyers, Eli Lilly and Company) transfected with sequence specific siRNAs hPTP1B1.2, hPTPB1.3, hPTP1B1.4, hPTP1B1.5 (20 nM) was reduced by 90%.

[0136] Transient Transfection of Human PTP1B and Sequence Specific Hairpin Vectors

[0137] Effectiveness of a human PTP1B sequence-specific siRNA in the form of a hairpin insert was examined in a transient co-transfection assay. Cells (1BKO+HIR mouse fibroblasts) were transfected with a human PTP1B expression vector (see above) and co-transfected with hPTP1B hairpin vectors (1, 0.5, and 0.25 μg) according to the transfection method described above. The human PTP1B specific sequences were inserted in frame with a human U6 small nuclear RNA promoter into a vector, which was a gift from David Engelke (University of Michigan, Ann Arbor, Mich.) (see also Paul et al., Nat. Biotechnol. 20:446-48 (2002)). The sequences of each strand inserted into the hairpin vectors are as follows.

hPTP1B H1.2-HP4
5′-tttGCCCAAAGGAGTTACATTCGTAAGAATGTAACTCCTTTGGGCttttt-3′ (SEQ ID NO:_)
3′-GGGTTTCCTCAATGTAAGCATTCTTACATTGAGGAAACCCGaaaaagatc-5′ (SEQ ID NO:_)
hPTP1B H1.2-HP9
5′-tttGCCCAAAGGAGTTACATTCCCTGGGTAAGAATGTAACTCCTTTGGGCttttt-3′ (SEQ ID NO:_)
3′-GGGTTTCCTCAATGTAAGGGACCCATTCTTACATTGAGGAAACCCGaaaaagatc-5′ (SEQ ID NO:_)

[0138] Twenty-four hours after the cells were transfected, cell lysates were prepared and expression of human PTP1B was determined by immunoblotting with an anti-human PTP1B antibody (see above). Cell lysates were also immunoblotted with an antibody specific for human insulin receptor beta chain (IRβ) (C-19, Cat. No. SC-711, Santa Cruz Biotechnology). The results are presented in FIG. 2.

[0139] Hairpin vectors are also prepared that contain sequences specific for murine PTP1B. The following sequences of each strand are inserted into a hairpin vector.

mPTP1B M1.1-HP4
5′-tttGAAGCCCAGAGGAGCTATAAGAATATAGCTCCTCTGGGCTTCttttt-3′ (SEQ ID NO:_)
3′-TTCGGGTCTCCTCGATA1TCTTATATCGAGGAGACCCGAAGaaaaagatc-5′ (SEQ ID NO:_)
mPTP1B M1.1-HP9
5′-tttGAAGCCCAGAGGAGCTATAGGGTGAGAATATAGCTCCTCTGGGCUCttttt-3′ (SEQ ID NO:_)
3′-TTCGGGTCTCCTCGATATCCCACTCTTATATCGAGGAGACCCGAAGaaaaagatc-5′ (SEQ ID NO:_)

EXAMPLE 2

[0140] Effect of siRNAs Specific for PTP1B on Insulin Receptor Tyrosine Phosphorylation

[0141] This example illustrates the effect of RNAi on the function of components in a cell signaling pathway. The role of PTP1B in the down regulation of insulin signaling has been illustrated by data derived from a variety of approaches (Cheng et al., Eur. J. Biochem. 269:1050-59 (2002)), including the phenotype of the PTP1B knockout mouse (Elchebly et al., Science 283:1544-48 (1999); Klaman et al., Mol. Cell Biol. 20:5479-89 (2000); see also U.S. patent application Ser. No. 10/366,547).

[0142] The effect of human PTP1B siRNA on the level of phosphorylation of IR-β was evaluated by ELISA. 292-HEK HIR cells were transfected with 0, 1, 5, and 10 nM hPTP1B1.3 (SEQ ID NO: __) or mPTP1B1.1 (SEQ ID NO: __). Seventy-two hours after transfection, cells were exposed to insulin for 7 minutes at concentrations of 0, 20, 50, and 100 nM. Cell lysates were prepared as described in Example 1, and total cell protein was quantified by the Bio-Rad Protein Assay performed according to the manufacturer's instructions (Bio-Rad, Hercules, Calif.). An ELISA was performed as follows. Dynex Immulon HB4× plates were coated with anti-insulin receptor antibody Ab-1 (1 mg/ml; NeoMarkers, Inc., Fremont, Calif.) that was diluted 1:1000 in CMF (calcium magnesium free)-PBS containing 5 μg/ml fatty acid free BSA (faf-BSA). The plates were incubated at 4° C. for at least four hours. The antibody solution was removed by aspiration, followed by the addition of 300 μl of 3% faf-BSA+CMF-PBS. The plates were incubated for 1 hr with agitation on a vortex platform shaker (setting #5) at room temperature. After aspirating the 3% faf-BSA+CMF-PBS solution, approximately 10-20 μg of lysate were added to the wells and incubated at room temperature for one hour. Plates were washed three times with TBST (20 mM Tris, -HCl, pH 7.5 150 mM NaCl; 0.05% Tween 20). An anti-insulin receptor phosphotyrosine specific antibody (pTyr 1162/63, Biosource International, Camarillo, Calif., Catalog #44-804) was diluted 1:2000 in TBST and added to the plates for one hour at room temperature. The plates were washed three times with TBST. HRP-conjugated anti-rabbit antibody (Amersham Biosciences, catalog #NA934V) (1:2000 in TBST) was then added to the wells and incubated at room temperature for one hour. The plates were washed three times with TBST and once with deionized, sterile water. TMB solution (Sigma Aldrich) (100 μl per well) was added and developed until a modest color change (10-30 minutes depending on cell type and insulin response). The reaction was stopped with 100 μl of 1.8 N H2S04 and then mixed. The optical density of each well was measured at 450 nM in a Spectramax plate reader (Molecular Devices Corp., Sunnyvale, Calif.). The data are presented in FIG. 3. The level of expression of PTP1B in each cell lysates was determined by immunoblot as described above. PTP1B polypeptide was detected using an anti-human PTP-1B antibody (PHO2, Oncogene Research Products™, Inc.). The amount of PTP1B expressed in cells transfected with varying concentrations of either siRNA was quantified by densitometric analysis of the immunoblot. The level of expression of human PTP1B is presented as a percent of the level of expression in cells that were not transfected with hPTP1B1.3 siRNA (i.e., the level of expression in untransfected cells equals 100%) (see tables in FIG. 3).

[0143] In a second experiment, 292-HEK HIR cells were transfected with 0, 0.5, 3, or 10 nM siRNAs. The siRNA polynucleotides transfected into the cells included hPTP1B1.2 (SEQ ID NO: __), hPTP1B1.3 (SEQ ID NO: __), mPTP1B1.1 (SEQ ID NO: __), and rPTP1B1.2 (SEQ ID NO: __). Seventy-two hours after transfection, cells were exposed to insulin for 7 minutes at concentrations of 0, 1, 5, 10, 50, and 100 nM. Cell lysates were prepared and total cell protein was quantified as described above. An ELISA was performed as described above. Cell lysates were coated onto 96-well plates, blocked, and probed with an anti-pYpY1162/1163-IR-β antibody. Binding was detected using an enzyme conjugated secondary reagent. As shown in FIG. 4, increased phosphorylation of the insulin receptor was observed in cells transfected with hPTP1B1.3.

[0144] The percent decrease in the level of PTP1B expression was compared with the level of phosphorylation of the insulin receptor. In three separate experements, 292-HEK HIR cells were transfected with 0, 0.5, 3, or 10 nM hPTP1B1.3 siRNA and then exposed to insulin for 7 minutes at concentrations of 0, 5, 10, 20, 50, and 100 nM. An ELISA and immunoblot of cell lysates were performed as described above. The effect of hPTP1B1.3 siRNA on the phosphorylation state of the insulin receptor is summarized in FIG. 5. Each data point represents the average optical density measured in duplicate wells.

EXAMPLE 3

[0145] Human and Mouse PTP1B Specific siRNA Polynucleotides

[0146] The level of expression of human PTP1B in cells that are capable of expressing human PTP1B and that are transfected with any one of the following siRNA polynucleotides is determined according to methods and procedures described in Example 1. The effect of the siRNA specific for human PTP1B on insulin receptor tyrosine phosphorylation is determined according to the method described in Example 2. The siRNA sequences that are incorporated into a vector from which a hairpin vector is transcribed and/or that are transfected via liposomes according to methods described in Example 1 are presented in Tables 3-5. The human PTP1B target sequences were derived from the human PTP1B nucleotide sequence set forth in GenBank Accession No. NM002827 (SEQ ID NO: __). Table 3 presents 19-base pair human PTP1B target sequences that are preceded by a AA dinucleotide leader sequence. Table 4 presents 19-base pair human PTP1B target sequences that are preceded by a CA dinucleotide leader sequence. The leader sequence refers to the two nucleotides that are 5′ to the 19 base pair target mRNA sequence and the “ending sequence” refers to the two nucleotides that are just 3′ to the mRNA sequence. The position number connotes the nucleotide position at or about the first nucleotide of the 19-nucleotide target sequence. The regions of the mRNA are referred to as the coding region (CR) or open reading frame/coding region (ORF/CF) and untranslated region (UTR). The stop codon (UAG) present in any sequence is underlined and bolded. Table 5 presents human PTP1B siRNA polynucleotide sequences that were selected using the Dharmacon siDESIGN system. These sequences were generated using the following parameters: (1) leader sequences included dinucleotides AA, CA, TA, and GA; (2) 5′ UTR, coding region, and 3′ UTR were scanned; (4) the G+C content varied from approximately 31-63%; (5) overlaps of sequences within different 19 nucleotide sequences were permitted. These sequences were then compared to known human genome sequences using the BLAST program. Potential target sequences were eliminated if 16 or more consecutive nucleotides within the 19-nucleotide target sequence were identified in another human polynucleotide sequence. The remaining 19-nucleotide siRNA sequences are presented in Table 5. The sequences in shaded rows were identified by other methods as well.

[0147] Similarly, the level of expression of mouse or rat PTP1B in cells that are transfected with sequence specific siRNA polynucleotides is determined according to the methods and procedures described in Example 1. The effect of the siRNA specific for mouse or rat PTP1B on insulin receptor tyrosine phosphorylation is determined according to the method described in Example 2. Tables 6 and 7 present 19-nucleotide siRNA sequences specific to mouse PTP1B (GenBank Accession No. NM011201, SEQ ID NO: __) that have a AA dinucleotide leader sequence and a CA dinucleotide leader sequence, respectively. Table 8 presents 1 9-nucleotide siRNA sequences that were selected using Dharmacon siDESIGN and the BLAST program as described above except that the sequences were compared with known mouse genome sequences. Table 9 presents 19-nucleotide siRNA sequences that were selected using Dharmacon siDESIGN and the BLAST program as described above except that the sequences were compared with known rat genome sequences.

[0148] Each siRNA sequence represented in Tables 3-9 lists the sequence of the sense strand of the siRNA and its corresponding sequence identifier. An siRNA polynucleotide as described herein is understood to be composed of the 19 nucleotide sense strand and its complementary (or antisense) strand. In addition, a siRNA polynucleotide of the present invention typically has a dinucleotide overhang at the 3′ end of each strand, which may be any two nucleotides.

TABLE 3
HUMAN PTP1B sIRNA POLYNUCLEOTIDE SEQUENCES
Ending Identified
Leader 19 nucleotide target (mRNA) sequence Sequence
Sequence (SEQ ID NO) (mRNA) Position Region Name
AA AAGGAGUUCGAGCAGAUCG (_) AC 187 CR
AA AGGAGUUCGAGCAGAUCGA CA 188 CR
AA GGAGUUCGAGCAGAUCGAC AA 189 CR
AA GCCAGUGACUUCCCAUGUA GA 253 CR
AA CCGAAAUAGGUACAGAGAC GU 300 CR
AA AUAGGUACAGAGACGUCAG UC 305 CR
AA UAGGUACAGAGACGUCAGU CC 306 CR
AA AAUGGAAGAAGCCCAAAGG AG 393 CR
AA AUGGAAGAAGCCCAAAGGA GU 394 CR
AA UGGAAGAAGCCCAAAGGAG UU 395 CR
AA GAAGCCCAAAGGAGUUACA UU 400 CR
AA GCCCAAAGGAGUUACAUUC UU 403 CR hPTP1B 1.2
AA CACAUGCGGUCACUUUUGG GA 444 CR
AA CAGAGUGAUGGAGAAAGGU UC 507 CR
AA GGUUCGUUAAAAUGCGCAC AA 527 CR
AA AAUGCGCACAAUACUGGCC AC 533 CR
AA AUGCGCACAAUACUGGCCA CA 534 CR
AA UGCGCACAAUACUGGCCAC AA 535 CR
AA CCCAAGAAACUCGAGAGAU CU 668 CR
AA UCACCAGCCUCAUUCUUGA AC 773 CR
AA CCUUCUGUCUGGCUGAUAC CU 845 CR
AA GAGGAAAGACCCUUCUUCC GU 885 CR
AA AGACCCUUCUUCCGUUGAU AU 891 CR
AA GACCCUUCUUCCGUUGAUA UC 892 CR
AA AUGAGGAAGUUUCGGAUGG CC 931 CR
AA GCUGCCAAAUUCAUCAUGC GG 1003 CR
AA GGAGCUUUCCCACGAGGAC CU 1050 CR
AA ACGAAUCCUGGAGCCACAC AA 1116 CR
AA CGAAUCCUGGAGCCACACA AU 1117 CR
AA UCCUGGAGCCACACAAUCC GA 1121 CR
AA UGGGAAAUGCAGGGAGUUC UU 1137 CR
AA GCAACAGACCCAGCAGGAU AA 1179 CR
AA GAGACCCACCAGGAUAAAG AC 1183 CR
AA AGACUGCCCCAUCAAGGAA GA 1200 CR
AA CACUGCCCCAUCAAGGAAG AA 1201 CR
AA GGAAGAAAAAGGAAGCCCC UU 1215 CR hPTP1B 1.3
AA AAGCAAGCCCCUUAAAUGC CG 1223 CR
AA AGGAAGCCCCUUAAAUGCC CC 1224 CR
AA GGAACCCCCUUAAAUGCCG CA 1225 CR
AA GCCCCUUAAAUGCCGCACC CU 1229 CR
AA AUGCCCCACCCUACCGCAU CG 1238 CR
AA AGCAUGAGUCAAGACACUG AA 1261 CR
AA CCAUCAGUCAACACACUGA AG 1262 CR
AA GGACGAGGACCAUGCACUG AG 1365 CR
AA GCCCUUCCUGGUCAACAUG UC 1395 CR
AA CAUGUGCGUCCCUACGGUC CU 1410 CR
AA CACCAACACA UAG CCUGAC CC 1470 CR & 3′UTR
AA CACA UAG CCUGACCCUCCU CC 1476 CR & 3′UTR
AA AACCCAUCUUCCCCGGAUG UC 1627 3′UTR
AA ACCCAUCUUCCCCGGAUGU CU 1628 3′UTR
AA CCCAUCUUCCCCGGAUGUG UC 1629 3′UTR
AA ACAGAGUACCAUGCUGGCC CC 1729 3′UTR
AA GAGAGUACCAUGCUGGCGG CC 1730 3′UTR
AA CAGCCCCCCCCUUGAAUCU CC 1835 3′UTR
AA AGGCAUCCAUACUGCACUA CC 1904 3′UTR
AA GGCAUCCAUAGUGCACUAG CA 1905 3′UTR
AA GGAGGACGGUUGUAAGCAG UU 2072 3′UTR
AA UCACUGCUCCCCCGUGUGU AU 2241 3′UTR
AA GGUCUUCUUGUGUCCUGAU GA 2276 3′UTR
AA UGUGCCCCAUGUCCAAGUC CA 2341 3′UTR
AA GUCCAACCUGCCUGUGCAU GA 2357 3′UTR
AA CCUGCCUGUGCAUGACCUG AU 2363 3′UTR
AA GCCUGUUGCUGAAGUCAUU GU 2407 3′UTR
AA GUCAUUGUCGCUCAGCAAU AG 2420 3′UTR
AA UUCCUGGCAUGACACUCUA GU 2474 3′UTR
AA GCCAUAUUCACACCUCACG CU 2571 3′UTR
AA GUCAACACUCUUCUUGAGC AG 2662 3′UTR
AA CACUCUUCUUGAGCAGACC GU 2667 3′UTR
AA GAGAGGCACCUGCUGGAAA CC 2697 3′UTR
AA CCACACUUCUUGAAACAGC CU 2716 3′UTR
AA GACCUCCACAUUAAGUGGC UU 2870 3′UTR
AA CAUGAAAAACACGGCAGCU GU 2896 3′UTR
AA AAACACGGCAGCUGUAGCU CC 2902 3′UTR
AA AACACGGCAGCUGUAGCUC CC 2903 3′UTR
AA ACACGGCAGCUGUAGCUCC CG 2904 3′UTR
AA CAUUCGAGGUGUCACCCUG CA 3003 3′UTR
AA GGCUUAGGUGCCAGGCUGU AA 3047 3′UTR
AA UGGACGUACUGGUUUAACC UC 3151 3′UTR
AA CCUCCUAUCCUUGGAGAGC AG 3168 3′UTR

[0149]

TABLE 4
HUMAN PTP1B siRNA POLYNUCLEOTIDE SEQUENCES (CA LEADER)
Ending Identified
Leader 19 nucleotide target (mRNA) sequence Sequence
Sequence (SEQ ID NO) (mRNA) Position Region Name
CA UGAAGAAGCAGCAGCGGCU AG 31 5′UTR
CA GGAUAUCCGACAUGAAGCC AG 237 CR
CA UGAAGCCAGUGACUUCCCA UG 249 CR
CA GUGACUUCCCAUGUAGAGU GG 257 CR
CA UGUAGUGUGGCCAAGCUUC CU 268 CR
CA GAGACGUCAGUCCCUUUGA CC 314 CR
CA GUCCCUUUGACCAUAGUCG GA 323 CR
CA UGCUCAACAGAGUGAUGGA GA 500 CR
CA ACAGAGUGAUGGAGAAAGG UU 506 CR
CA GAGUGAUGGAGAAAGGUUC GU 509 CR
CA GUGCGACAGCUAGAAUUGG AA 637 CR
CA ACCCAAGAAACUCGAGAGA UC 667 CR
CA CUAUACCACAUGGCCUGAC UU 699 CR hPTPLB 1.1
CA CAUGGCCUGACUUUGGAGU CC 707 CR
CA UGGCCUGACUUUGGAGUCC CU 709 CR
CA CCAGCCUCAUUCUUGAACU UU 736 CR
CA GGCAUCGGCAGGUCUGGAA CC 826 CR
CA UCGGCAGGUCUGGAACCUU CU 830 CR
CA GGUCUGGAACCUUCUGUCU GG 836 CR
CA AGAGGAAAGACCCUUCUUC CG 884 CR
CA GCUGCGCUUCUCCUACCUG GC 972 CR
CA GGAUCAGUGGAAGGAGCUU UC 1038 CR hPTP1B 1.5
CA GUGGAAGGAGCUUUCCCAC GA 1044 CR
CA CCCAAACGAAUCCUGGAGC CA 1111 CR
CA AACGAAUCCUGGAGCCACA CA 1115 CR
CA CACAAUGGGAAAUGCAGGG AG 1132 CR
CA CAAUGGGAAAUGCAGGGAG UU 1134 CR hPTP1B 1.4
CA AUGGGAAAUGCAGGGAGUU CU 1136 CR
CA GGAGGAUAAAGACUGCCCC AU 1191 CR
CA AGGAAGAAAAAGGAAGCCC CU 1214 CR
CA CCCUACGGCAUCGAAAGCA UG 1246 CR
CA UGCACUGAGUUACUGGAAG CC 1377 CR
CA CUGAGUUACUGGAAGCCCU UC 1381 CR
CA ACAUGUGCGUGGCUACGGU CC 1409 CR
CA UGUGCGUGGCUACGGUCCU CA 1412 CR
CA GGUUCCUGUUCAACAGCAA CA 1457 CR
CA ACAGCAACACA UAG CCUGA CC 1469 CR & 3′UTR
CA GCAACACA UAG CCUGACCC UC 1472 CR & 3′UTR
CA ACACA UAG CCUGACCCUCC UC 1475 CR & 3′UTR
CA CA UAG CCUGACCCUCCUCC AC 1478 CR & 3′UTR
CA UAG CCUGACCCUCCUCCAC UC 1480 CR & 3′UTR
CA CUCCACCUCCACCCACUGU CC 1498 3′UTR
CA GGCAUGCCGCGGUAGGUAA GG 1552 3′UTR
CA CUAAAACCCAUCUUCCCCG GA 1623 3′UTR
CA UCUUCCCCGGAUGUGUGUC UC 1633 3′UTR
CA ACAGCCCCCCCCUUGAAUC UG 1834 3′UTR
CA AAGGCAUCCAUAGUGCACU AG 1903 3′UTR
CA AUCACUGCUCCCCCGUGUG UA 2240 3′UTR
CA CUGCUCCCCCGUGUGUAUU UG 2244 3′UTR
CA UGUCCAAGUCCAACCUGCC UG 2350 3′UTR
CA AGUCCAACCUGCCUGUGCA UG 2356 3′UTR
CA ACCUGCCUGUGCAUGACCU GA 2362 3′UTR
CA UUACAUGGCUGUGGUUCCU AA 2386 3′UTR
CA UGGCUGUGGUUCCUAAGCC UG 2391 3′UTR
CA UGACACUCUAGUGACUUCC UG 2483 3′UTR
CA CUCUAGUGACUUCCUGGUG AG 2488 3′UTR
CA GCCUGUCCUGGUACAGCAG GG 2514 3′UTR
CA UAUUCACACCUCACGCUCU GG 2575 3′UTR
CA CACCUCACGCUCUGGACAU GA 2581 3′UTR
CA CCUCACGCUCUGGACAUGA UU 2583 3′UTR
CA CGCUCUGGAGAUGAUUUAG GG 2588 3′UTR
CA GCCUCCGCCAUUCCAAGUC AA 2646 3′UTR
CA ACACUCUUCUUGAGCAGAC CG 2666 3′UTR
CA CUCUUCUUGAGCAGACCGU GA 2669 3′UTR
CA GACCGUGAUUUGGAAGAGA GG 2682 3′UTR
CA CCUGCUGGAAACCACACUU CU 2705 3′UTR
CA CACUUCUUGAAACAGCCUG GG 2719 3′UTR
CA UGAAAAACACGGCAGCUGU AG 2898 3′UTR
CA GCUGUAGCUCCCGAGCUAC UC 2912 3′UTR
CA CAUUUUGCCUUUCUCGUGG UA 2950 3′UTR
CA UUCGAGGUGUCACCCUGCA GA 3005 3′UTR
CA CCCUGCAGAGCUAUGGUGA GG 3017 3′UTR
CA GAGCUAUGGUGAGGUGUGG AU 3024 3′UTR
CA GGCUGUAAGCAUUCUGAGC UG 3060 3′UTR
CA GCUGGCUCUCCACCUUGUU AC 3188 3′UTR

[0150]

TABLE 6
MOUSE PTP1B siRNA POLYNUCLEOTIDE SEQUENCES (AA LEADER)
Ending Identified
Leader 19 nucleotide target (mRNA) sequence Sequence
Sequence (SEQ ID NO) (mRNA) Position Region Name
AA CCAAACGGACAACCCAUAG UA 79 5′UTR
AA ACGGACAACCCAUAGUACC CG 83 5′UTR
AA CGGACAACCCAUAGUACCC GA 84 5′UTR
AA CCCAUAGUACCCGAAGACA GG 91 5′UTR
AA CCAGACAAUCGUAAGCUUG AU 117 5′UTR
AA GCUUGAUGGUGUUUUCCCU GA 131 5′UTR
AA GCAUCUCAUGAAUGUCAGC CA 165 5′UTR
AA UGUCAGCCAAAUUCCGUAC AG 177 5′UTR
AA AUUCCGUACAGUUCGGUGC GG 187 5′UTR
AA UUCCGUACAGUUCGGUGCG GA 188 5′UTR
AA CGAAACACCUCCUGUACCA GG 215 5′UTR
AA ACACCUCCUGUACCAGGUU CC 219 5′UTR
AA CACCUCCUGUACCAGGUUC CC 220 5′UTR
AA CUUCAGAAUCAUCCAGGCU UC 354 5′UTR
AA UCAUCCAGGCUUCAUCAUG UU 362 5′UTR
AA GGUGAGAGCCACCACAGAG GA 423 5′UTR
AA CUGGUAGGCUGAACCCAUG CU 492 5′UTR
AA CCCAUGCUGAAGCUCCACC CG 505 5′UTR
AA CAUGCAGAAGCCGCUGCUG GG 583 5′UTR
AA GGAGUUCGAGGAGAUCGAC AA 724 5′UTR
AA GCCAGCGACUUCCCAUGCA AA 788 CR
AA AGUCGCGAAGCUUCCUAAG AA 808 CR
AA GUCGCGAAGCUUCCUAAGA AC 809 CR
AA GAACAAAAACCGGAACAGG UA 826 CR
AA CAAAAACCGGAACAGGUAC CG 829 CR
AA AAACCGGAACAGGUACCGA GA 832 CR
AA AACCGGAACAGGUACCGAG AU 833 CR
AA ACCGGAACAGGUACCGAGA UG 834 CR
AA CCGGAACAGGUACCGAGAU GU 835 CR
AA CAGGUACCGAGAUGUCAGC CC 841 CR
AA AAAUGGAAGAAGCCCAGAG GA 927 CR
AA AAUGGAAGAAGCCCAGAGG AG 928 CR
AA AUGGAAGAAGCCCAGAGGA GC 929 CR
AA UGGAAGAAGCCCAGAGGAG CU 930 CR
AA GAAGCCCAGAGGAGCUAUA UU 935 CR mPTPIB 1.1
AA GCCCAGAGGAGCUAUAUUC UC 938 CR
AA CCGCAUCAUGGAGAAAGGC UC 1042 CR
AA AGGCUCGUUAAAAUGUGCC CA 1057 CR AA GGCUCGUUAAAAUGUGCCC AG 1058 CR
AA AAUGUGCCCAGUAUUGGCC AC 1068 CR
AA AUGUGCCCAGUAUUGGCCA CA 1069 CR AA UGUGCCCAGUAUUGGCCAC AG 1070 CR
AA GGAGAUGGUCUUUGAUGAC AC 1102 CR
AA AACCUGACUACCAAGGAGA CU 1193 CR
AA ACCUGACUACCAAGGAGAC UC 1194 CR
AA CCUGACUACCAAGGAGACU CG 1195 CR
AA GGAGACUCGAGAGAUCCUG CA 1207 CR
AA AGUCCGAGAGUCAGGCUCA CU 1300 CR
AA GUCCGAGAGUCAGGCUCAC UC 1301 CR
AA GAGGAAAGACCCAUCUUCC GU 1420 CR
AA AGACCCAUCUUCCGUGGAC AU 1426 CR
AA GACCCAUCUUCCGUGGACA UC 1427 CR
AA GAAAGUACUGCUGGAGAUG CG 1450 CR
AA AGUACUGCUGGAGAUGCGC AG 1453 CR
AA GUACUGCUGGAGAUGCGCA GG 1454 CR
AA ACGCACACUGGAGCCUCAC AA 1651 CR
AA CGCACACUGGAGCCUCACA AC 1652 CR
AA GUGCAAGGAGCUCUUCUCC AG 1678 CR
AA GGAGCUCUUCUCCAGCCAC CA 1684 CR
AA GGCAGAGCCCAGUCAAGUG CC 1757 CR
AA GUGCCAUGCACAGCGUGAG CA 1773 CR
AA GUUAGGAGACGGAUGGUGG GU 1814 CR
AA AGUGCUCAGGCGUCUGUCC CC 1847 CR
AA GAGCUGUCCUCCACUGAGG AG 1877 CR
AA CACAAGGCACAUUGGCCAA GU 1901 CR
AA GGCACAUUGGCCAAGUCAC UG 1906 CR
AA GUCACUGGAAGCCCUUCCU GG 1920 CR
AA GCCCUUCCUGGUCAAUGUG UG 1930 CR
AA UGUGUGCAUGGCCACGCUC CU 1945 CR
AA CAACAACUCGCAAGCCUGC UC 2079 3′UTR
AA CAACUCGCAAGCCUGCUCU GG 2082 3′UTR
AA CUCGCAAGCCUGCUCUGGA AC 2085 3′UTR
AA GCCUGCUCUGGAACUGGAA GG 2092 3′UTR
AA CCUGUUCAGGAGAAGUAGA GG 2195 3′UTR
AA UACUCUUCUUGCUCUCACC UC 2226 3′UTR
AA CAUUUAUAAAGGCAGGCCC GA 2322 3′UTR
AA CGGGAAGUGCAAGGAGCUC UU 1674 CR mPTPIB 1.7
AA UGACUAUAUCAAUGCCAGC UU 903 CR mPTPIB 1.5

[0151]

TABLE 7
MOUSE PTP1B siRNA POLYNUCLEOTIDE SEQUENCES (CA LEADER)
Ending Identified
Leader 19 nucleotide target (mRNA) sequence Sequence
Sequence (SEQ ID NO) (mRNA) Position Region Name
CA CAUUCCUAGUUAGCAGUGC (_) AU 21 5′UTR
CA GUGCAUACUCAUCAGACUG (_) GA 36 5′UTR
CA AACGGACAACCCAUAGUAC (_) CC 82 5′UTR
CA ACCCAUAGUACCCGAAGAC AG 90 5′UTR
CA GCCAAAUUCCGUACAGUUC GG 182 5′UTR
CA AAUUCCGUACAGUUCGGUG CG 186 5′UTR
CA GUUCGGUGCGGAUCCGAAC GA 197 5′UTR
CA CCUCCUGUACCAGGUUCCC GU 222 5′UTR
CA GGUUCCCGUGUCGCUCUCA AU 234 5′UTR
CA GAAUCAUCCAGGCUUCAUC AU 359 5′UTR
CA GGCUUCAUCAUGUUUUCCC AC 369 5′UTR
CA UCAUGUUUUCCCACCUCCA GC 376 5′UTR
CA UGUUUUCCCACCUCCAGCA AG 379 5′UTR
CA CCUCCAGCAAGAACCGAGG GC 389 5′UTR
CA UGAAGGUGAGAGCCACCAC AG 419 5′UTR
CA CCACAGAGGAGACGCAUGG GA 434 5′UTR
CA CAGACGAUGACGAAGACGC GC 460 5′UTR
CA GACGAUGACGAAGACGCGC CA 462 5′UTR
CA CGUGUGGAACUGGUAGGCU GA 483 5′UTR
CA UGCUGAAGCUCCACCCGUA GU 509 5′UTR
CA GGCAUGGCGGAGGCUAGAU GC 546 5′UTR
CA UCCAGAACAUGCAGAAGCC GC 576 5′UTR
CA GAACAUGCAGAAGCCGCUG CU 580 5′UTR
CA UGGAGAUGGAGAAGGAGUU CG 711 CR
CA GGACAUUCGACAUGAAGCC AG 772 CR
CA UUCGACAUGAAGCCAGCGA CU 777 CR
CA UGAAGCCAGCGACUUCCCA UG 784 CR
CA GCGACUUCCCAUGCAAAGU CG 792 CR
CA UGCAAAGUCGCGAAGCUUC CU 803 CR
CA AAGUCGCGAAGCUUCCUAA GA 807 CR
CA AAAACCGGAACAGGUACCG AG 831 CR
CA GGUACCGAGAUGUCAGCCC UU 843 CR
CA GCCCUUUUGACCACAGUCG GA 858 CR
CA GAGGAGCUAUAUUCUCACC CA 943 CR
CA UGCUCAACCGCAUCAUGGA GA 1035 CR
CA ACCGCAUCAUGGAGAAAGG CU 1041 CR
CA UCAUGGAGAAAGGCUCGUU AA 1047 CR
CA GUAUUGGCCACAGCAAGAA GA 1078 CR
CA CAGUACGACAGUUGGAGUU GG 1170 CR
CA GUACGACAGUUGGAGUUGG AA 1172 CR
CA GUUGGAGUUGGAAAACCUG AC 1180 CR
CA AGGAGACUCGAGAGAUCCU GC 1206 CR
CA UUUCCACUACACCACAUGG CC 1228 CR
CA CUACACCACAUGGCCUGAC UU 1234 CR mPTPIB 1.2
CA CCACAUGGCCUGACUUUGG AG 1239 CR
CA CAUGGCCUGACUUUGGAGU CC 1242 CR
CA UGGCCUGACUUUGGAGUCC CC 1244 CR
CA CCGGCUUCUUUCCUCAAUU UC 1271 CR
CA AAGUCCGAGAGUCAGGCUC AC 1299 CR
CA UGGCCCCAUUGUGGUCCAC UG 1333 CR
CA UUGUGGUCCACUGCAGCGC CG 1341 CR
CA CCUGCCUCUUACUGAUGGA CA 1398 CR
CA AGAGGAAAGACCCAUCUUC CG 1419 CR
CA UCUUCCGUGGACAUCAAGA AA 1433 CR
CA UCCAGACUGCCGACCAGCU GC 1491 CR
CA GCUGCGCUUCUCCUACCUG GC 1507 CR
CA GUGCAGGAUCAGUGGAAGG AG 1568 CR
CA GGAUCAGUGGAAGGAGCUC UC 1573 CR mPTPIB 1.8
CA CCCAAACGCACACUGGAGC CU 1646 CR
CA AACGCACACUGGAGCCUCA CA 1650 CR
CA CACUGGAGCCUCACAACGG GA 1656 CR
CA AGGAGCUCUUCUCCAGCCA CC 1683 CR
CA GAGAGGAAGGCAGAGCCCA GU 1749 CR
CA GAGCCCAGUCAAGUGCCAU GC 1761 CR
CA GUCAAGUGCCAUGCACAGC GU 1768 CR
CA AGUGCCAUGCACAGCGUGA GC 1772 CR
CA UGCACAGCGUGAGCAGCAU GA 1779 CR
CA CAGCGUGAGCAGCAUGAGU CC 1783 CR
CA GCGUGAGCAGCAUGAGUCC AG 1785 CR
CA GCAUGAGUCCAGACACUGA AG 1794 CR
CA UGAGUCCAGACACUGAAGU UA 1797 CR
CA GACACUGAAGUUAGGAGAC GG 1805 CR
CA CUGAAGUUAGGAGACGGAU GG 1809 CR
CA AAGUGCUCAGGCGUCUGUC CC 1846 CR
CA CCGAGGAAGAGCUGUCCUC CA 1869 CR
CA CUGAGGAGGAACACAAGGC AC 1890 CR
CA CAAGGCACAUUGGCCAAGU CA 1903 CR
CA AGGCACAUUGGCCAAGUCA CU 1905 CR
CA CAUUGGCCAAGUCACUGGA AG 1910 CR
CA UUGGCCAAGUCACUGGAAG CC 1912 CR
CA AGUCACUGGAAGCCCUUCC UG 1919 CR
CA CUGGAAGCCCUUCCUGGUC AA 1924 CR
CA AUGUGUGCAUGGCCACGCU CC 1944 CR
CA CCGGCGCGUACUUGUGCUA CC 1971 CR
CA CUGCCACUGCCCAGCUUAG GA 2023 3′UTR
CA CUGCCCAGCUUAGGAUGCG GU 2029 3′UTR
CA GCUUAGGAUGCGGUCUGCG GC 2036 3′UTR
CA ACAACUCGCAAGCCUGCUC UG 2081 3′UTR
CA ACUCGCAAGCCUGCUCUGG AA 2084 3′UTR
CA AGCCUGCUCUGGAACUGGA AG 2091 3′UTR
CA GGAGAAGUAGAGGAAAUGC CA 2203 3′UTR
CA CCUCACUCCUCCCCUUUCU CU 2243 3′UTR
CA CUCCUCCCCUUUCUCUGAU UC 2248 3′UTR
CA UUUAUAAAGGCAGGCCCGA AU 2324 3′UTR
CA GGUACCGAGAUGUCAGCCC UU 846 CR mPTPIB 1.4
CA AGAAGAAAAGGAGAUGGUC UU 1095 CR mPTPIB 1.6
CA GACUGCCGACCAGCUGCGC UU 1497 CR mPTP1B 1.3

[0152]

TABLE 9
RAT PTP1B sIRNA POLYNUCLEOTIDE SEQUENCES (POST-
BLAST)
19 nucleotide target (mRNA) Position
(SEQ ID NO) Region Number
UCGAUAAGGCUGGGAACUG ORF/CR 148
GAAUAGCGAAACUUCCUAA ORF/CR 217
AUAGCGAAACUUCCUAAGA ORF/CR 219
CCACAGUCGGAUUAAAUUG ORF/CR 278
CAGUCGGAUUAAAUUGCAU ORF/CR 281
GUCGGAUUAAAUUGCAUCA ORF/CR 283
UUGCAUCAGGAAGAUAAUG ORF/CR 294
GCCCAGAGGAGCUAUAUCC ORF/CR 348
UCCUCACCCAGGGCCCUUU ORF/CR 364
ACCGCAUCAUGGAGAAAGG ORF/CR 451
UCAUGGAGAAAGGCUCGUU ORF/CR 457
UGGAGAAAGGCUCGUUAAA ORF/CR 460
GUAUUGGCCACAGAAAGAA ORF/CR 488
AGAGAUGGUCUUCGAUGAC ORF/CR 512
CACCAAUUUGAAGCUGACA ORF/CR 530
CCAAUUUGAAGCUGACACU ORF/CR 532
UUACACAGUACGGCAGUUG ORF/CR 575
CAGUACGGCAGUUGGAGUU ORF/CR 580
GGCUCGAGAGAUCCUGCAU ORF/CR 620
CUGAUGGACAAGAGGAAAG ORF/CR 819
CAUCAAGAAAGUGCUGUUG ORF/CR 854
GGGUGCAAAGUUCAUCAUG ORF/CR 947
UCAUGGGCGACUCGUCAGU ORF/CR 961
CUCGUCAGUGCAGGAUCAG ORF/CR 971
CGCACAUUGGAGCCUCACA ORF/CR 1062
AGUGCAAGGAGCUCUUCUC ORF/CR 1087
GCAUGAGCAGUAUGAGUCA ORF/CR 1195
GUAUGAGUCAAGACACUGA ORF/CR 1204
GUCAAGACACUGAAGUUAG ORF/CR 1210
UGGUGGGUGGAGGUCUUCA ORF/CR 1237
AAGGCACACAGGCCAGUUC ORF/CR 1314
AGGCACACAGGCCAGUUCA ORF/CR 1315
GGCACACAGGCCAGUUCAC ORF/CR 1316
AGCCCUUCCUGGUCAACGU ORF/CR 1339
UUUGGUCUGCGGCGUCUAA 3′UTR 1472
GAAGAAACAACAGCUUACA 3′UTR 1500
AGAAACAACAGCUUACAAG 3′UTR 1502
GUCUAAUCUCAGGGCCUUA 3′UTR 1604
AUGCCAAAUACUCUUCUUG 3′UTR 1647
UCAGAUUCACGAUUUACGU 3′UTR 1838
GCCACUCCACUGAGGUGUA 3′UTR 2197
CUCCACUGAGGUGUAAAGC 3′UTR 2201
GCCUUGGUGUCAUGGAAGU 3′UTR 2238
ACAACCUCUGAAACACUCA 3′UTR 2279
GUCUGGACUCAUGAAACAC 3′UTR 2381
AACACCGCCGAGCGCUUAC 3′UTR 2395
ACACCGCCGAGCGCUUACU 3′UTR 2396
GCCGCUCCACUGUUAUUUA 3′UTR 2764
UUCACUUUGCCCACAGACA 3′UTR 2783
CAGACAACAGUGGUGACAU 3′UTR 2975
GACAACAGUGGUGACAUGU 3′UTR 2977
ACAGUGGUGACAUGUAAAG 3′UTR 2981
CUGAUGACAUGUGUAGGAU 3′UTR 3012
CUCCCGGCAGGACUCUUCA 3′UTR 3231
CCUCAUUCCCUGGACACUU 3′UTR 3304
CCAGUCACCUUGCUCAGAA 3′UTR 3488
GUCACCUUGCUCAGAAGUG 3′UTR 3491
UAAGCGAAGGCAGCUGGAA 3′UTR 3602
AAGCUGCUGCAUGCCUUAA 3′UTR 3837
AGCUGCUGCAUGCCUUAAG 3′UTR 3838
CAACAAAGUGCUCUGGAAU 3′UTR 3977
CUGUCCGACUGCACCGUUU 3′UTR 4049
CUGCACCGUUUCCAACUUG 3′UTR 4057
ACUUGUGUCUCACUAAUGG 3′UTR 4071
ADDITIONAL REFERENCES

[0153] Agami et al., Cell 102:55-66 (2000)

[0154] Bass, Brenda L., Cell 101:235:238 (2000)

[0155] Brummelkamp et al., Science 296:550-53 (2002)

[0156] Carthew, Richard W., Current Opinion in Cell Biology 13:244-248 (2001)

[0157] Clemens et al., Proc. Natl. Acad. Sci. USA 97:6499-6503 (2000)

[0158] Elbashir et al., Genes & Development 15:188-200 (2001)

[0159] Elbashir, et al., Nature 411:494-498 (2001)

[0160] Fire et al., Nature 391:806-11 (1993)

[0161] Flint et al., Proc. Natl. Acad. Sci. USA 94:1680-1685 (1997)

[0162] Fukada et al., J. Biol. Chem. 276:25512-25519 (2001)

[0163] Harborth et al., J. Cell Sci. 114:4557-4565 (2001)

[0164] Hutvagner et al., Curr. Opin. Gen. & Dev. 12:225-232 (2002)

[0165] Kisielow et al., Biochem. J. 363:1-5 (2002)

[0166] Paddison et al., Genes & Development 16:948-958 (2002)

[0167] Salmeen et al., Moleular Cell 6:1401-1412 (2000)

[0168] Scadden et al., EMBO Reports 2:1107-1111 (2001)

[0169] Sharp, Phillip A., Genes & Development 13:139-141 (1999)

[0170] Sharp, Phillip A., Genes & Development 15:485-490 (2001)

[0171] Shen et al., Proc. Natl. Acad. Sci. USA 24:13613-13618 (2001)

[0172] Sui et al., Proc. Natl. Acad Sci. USA 99:5515-5520 (2002)

[0173] Tonks et al, Curr. Opin. Cell Biol. 13:182-195 (2001)

[0174] Tuschl, Thomas, Chembiochem. 2:239-245 (2001)

[0175] Ui-Tei et al., FEBS Letters 479:79-82 (2000)

[0176] Wen et al., Proc. Natl. Acad Sci. 98:4622-4627 (2001)

[0177] Zamore et al., Cell 101:25-33 (2000)

[0178] EPI 152 056

[0179] U.S. Pat. No. 2001/0029617

[0180] U.S. Pat. No. 2002/0007051

[0181] U.S. Pat. No. 6,326,193

[0182] U.S. Pat. No. 6,342,595

[0183] U.S. Pat. No. 6,506,559

[0184] WO 01/29058

[0185] WO 01/34815

[0186] WO 01/42443

[0187] WO 01/68836

[0188] WO 01/75164

[0189] WO 01/92513

[0190] WO 01/96584

[0191] WO 99/32619

[0192] From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for the purpose of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the present invention is not limited except as by the appended claims.

1 599 1 7 PRT Unknown Unique signature sequence motif contained within the conserved domain of the PTP family of enzymes. 1 Cys Xaa Xaa Xaa Xaa Xaa Arg 1 5 2 11 PRT Unknown An 11 amino acid conserved sequence containing the signature sequence motif in a majority of PTPs. 2 Xaa His Cys Xaa Ala Gly Xaa Xaa Arg Xaa Gly 1 5 10 3 3247 DNA Homo sapiens 3 gggcgggcct cggggctaag agcgcgacgc ctagagcggc agacggcgca gtgggccgag 60 aaggaggcgc agcagccgcc ctggcccgtc atggagatgg aaaaggagtt cgagcagatc 120 gacaagtccg ggagctgggc ggccatttac caggatatcc gacatgaagc cagtgacttc 180 ccatgtagag tggccaagct tcctaagaac aaaaaccgaa ataggtacag agacgtcagt 240 ccctttgacc atagtcggat taaactacat caagaagata atgactatat caacgctagt 300 ttgataaaaa tggaagaagc ccaaaggagt tacattctta cccagggccc tttgcctaac 360 acatgcggtc acttttggga gatggtgtgg gagcagaaaa gcaggggtgt cgtcatgctc 420 aacagagtga tggagaaagg ttcgttaaaa tgcgcacaat actggccaca aaaagaagaa 480 aaagagatga tctttgaaga cacaaatttg aaattaacat tgatctctga agatatcaag 540 tcatattata cagtgcgaca gctagaattg gaaaacctta caacccaaga aactcgagag 600 atcttacatt tccactatac cacatggcct gactttggag tccctgaatc accagcctca 660 ttcttgaact ttcttttcaa agtccgagag tcagggtcac tcagcccgga gcacgggccc 720 gttgtggtgc actgcagtgc aggcatcggc aggtctggaa ccttctgtct ggctgatacc 780 tgcctcctgc tgatggacaa gaggaaagac ccttcttccg ttgatatcaa gaaagtgctg 840 ttagaaatga ggaagtttcg gatggggttg atccagacag ccgaccagct gcgcttctcc 900 tacctggctg tgatcgaagg tgccaaattc atcatggggg actcttccgt gcaggatcag 960 tggaaggagc tttcccacga ggacctggag cccccacccg agcatatccc cccacctccc 1020 cggccaccca aacgaatcct ggagccacac aatgggaaat gcagggagtt cttcccaaat 1080 caccagtggg tgaaggaaga gacccaggag gataaagact gccccatcaa ggaagaaaaa 1140 ggaagcccct taaatgccgc accctacggc atcgaaagca tgagtcaaga cactgaagtt 1200 agaagtcggg tcgtgggggg aagtcttcga ggtgcccagg ctgcctcccc agccaaaggg 1260 gagccgtcac tgcccgagaa ggacgaggac catgcactga gttactggaa gcccttcctg 1320 gtcaacatgt gcgtggctac ggtcctcacg gccggcgctt acctctgcta caggttcctg 1380 ttcaacagca acacatagcc tgaccctcct ccactccacc tccacccact gtccgcctct 1440 gcccgcagag cccacgcccg actagcaggc atgccgcggt aggtaagggc cgccggaccg 1500 cgtagagagc cgggccccgg acggacgttg gttctgcact aaaacccatc ttccccggat 1560 gtgtgtctca cccctcatcc ttttactttt tgccccttcc actttgagta ccaaatccac 1620 aagccatttt ttgaggagag tgaaagagag taccatgctg gcggcgcaga gggaaggggc 1680 ctacacccgt cttggggctc gccccaccca gggctccctc ctggagcatc ccaggcggcg 1740 cacgccaaca gcccccccct tgaatctgca gggagcaact ctccactcca tatttattta 1800 aacaattttt tccccaaagg catccatagt gcactagcat tttcttgaac caataatgta 1860 ttaaaatttt ttgatgtcag ccttgcatca agggctttat caaaaagtac aataataaat 1920 cctcaggtag tactgggaat ggaaggcttt gccatgggcc tgctgcgtca gaccagtact 1980 gggaaggagg acggttgtaa gcagttgtta tttagtgata ttgtgggtaa cgtgagaaga 2040 tagaacaatg ctataatata taatgaacac gtgggtattt aataagaaac atgatgtgag 2100 attactttgt cccgcttatt ctcctccctg ttatctgcta gatctagttc tcaatcactg 2160 ctcccccgtg tgtattagaa tgcatgtaag gtcttcttgt gtcctgatga aaaatatgtg 2220 cttgaaatga gaaactttga tctctgctta ctaatgtgcc ccatgtccaa gtccaacctg 2280 cctgtgcatg acctgatcat tacatggctg tggttcctaa gcctgttgct gaagtcattg 2340 tcgctcagca atagggtgca gttttccagg aataggcatt tgctaattcc tggcatgaca 2400 ctctagtgac ttcctggtga ggcccagcct gtcctggtac agcagggtct tgctgtaact 2460 cagacattcc aagggtatgg gaagccatat tcacacctca cgctctggac atgatttagg 2520 gaagcaggga caccccccgc cccccacctt tgggatcagc ctccgccatt ccaagtcaac 2580 actcttcttg agcagaccgt gatttggaag agaggcacct gctggaaacc acacttcttg 2640 aaacagcctg ggtgacggtc ctttaggcag cctgccgccg tctctgtccc ggttcacctt 2700 gccgagagag gcgcgtctgc cccaccctca aaccctgtgg ggcctgatgg tgctcacgac 2760 tcttcctgca aagggaactg aagacctcca cattaagtgg ctttttaaca tgaaaaacac 2820 ggcagctgta gctcccgagc tactctcttg ccagcatttt cacattttgc ctttctcgtg 2880 gtagaagcca gtacagagaa attctgtggt gggaacattc gaggtgtcac cctgcagagc 2940 tatggtgagg tgtggataag gcttaggtgc caggctgtaa gcattctgag ctggcttgtt 3000 gtttttaagt cctgtatatg tatgtagtag tttgggtgtg tatatatagt agcatttcaa 3060 aatggacgta ctggtttaac ctcctatcct tggagagcag ctggctctcc accttgttac 3120 acattatgtt agagaggtag cgagctgctc tgctatatgc cttaagccaa tatttactca 3180 tcaggtcatt attttttaca atggccatgg aataaaccat ttttacaaaa ataaaaacaa 3240 aaaaagc 3247 4 435 PRT Homo sapiens 4 Met Glu Met Glu Lys Glu Phe Glu Gln Ile Asp Lys Ser Gly Ser Trp 1 5 10 15 Ala Ala Ile Tyr Gln Asp Ile Arg His Glu Ala Ser Asp Phe Pro Cys 20 25 30 Arg Val Ala Lys Leu Pro Lys Asn Lys Asn Arg Asn Arg Tyr Arg Asp 35 40 45 Val Ser Pro Phe Asp His Ser Arg Ile Lys Leu His Gln Glu Asp Asn 50 55 60 Asp Tyr Ile Asn Ala Ser Leu Ile Lys Met Glu Glu Ala Gln Arg Ser 65 70 75 80 Tyr Ile Leu Thr Gln Gly Pro Leu Pro Asn Thr Cys Gly His Phe Trp 85 90 95 Glu Met Val Trp Glu Gln Lys Ser Arg Gly Val Val Met Leu Asn Arg 100 105 110 Val Met Glu Lys Gly Ser Leu Lys Cys Ala Gln Tyr Trp Pro Gln Lys 115 120 125 Glu Glu Lys Glu Met Ile Phe Glu Asp Thr Asn Leu Lys Leu Thr Leu 130 135 140 Ile Ser Glu Asp Ile Lys Ser Tyr Tyr Thr Val Arg Gln Leu Glu Leu 145 150 155 160 Glu Asn Leu Thr Thr Gln Glu Thr Arg Glu Ile Leu His Phe His Tyr 165 170 175 Thr Thr Trp Pro Asp Phe Gly Val Pro Glu Ser Pro Ala Ser Phe Leu 180 185 190 Asn Phe Leu Phe Lys Val Arg Glu Ser Gly Ser Leu Ser Pro Glu His 195 200 205 Gly Pro Val Val Val His Cys Ser Ala Gly Ile Gly Arg Ser Gly Thr 210 215 220 Phe Cys Leu Ala Asp Thr Cys Leu Leu Leu Met Asp Lys Arg Lys Asp 225 230 235 240 Pro Ser Ser Val Asp Ile Lys Lys Val Leu Leu Glu Met Arg Lys Phe 245 250 255 Arg Met Gly Leu Ile Gln Thr Ala Asp Gln Leu Arg Phe Ser Tyr Leu 260 265 270 Ala Val Ile Glu Gly Ala Lys Phe Ile Met Gly Asp Ser Ser Val Gln 275 280 285 Asp Gln Trp Lys Glu Leu Ser His Glu Asp Leu Glu Pro Pro Pro Glu 290 295 300 His Ile Pro Pro Pro Pro Arg Pro Pro Lys Arg Ile Leu Glu Pro His 305 310 315 320 Asn Gly Lys Cys Arg Glu Phe Phe Pro Asn His Gln Trp Val Lys Glu 325 330 335 Glu Thr Gln Glu Asp Lys Asp Cys Pro Ile Lys Glu Glu Lys Gly Ser 340 345 350 Pro Leu Asn Ala Ala Pro Tyr Gly Ile Glu Ser Met Ser Gln Asp Thr 355 360 365 Glu Val Arg Ser Arg Val Val Gly Gly Ser Leu Arg Gly Ala Gln Ala 370 375 380 Ala Ser Pro Ala Lys Gly Glu Pro Ser Leu Pro Glu Lys Asp Glu Asp 385 390 395 400 His Ala Leu Ser Tyr Trp Lys Pro Phe Leu Val Asn Met Cys Val Ala 405 410 415 Thr Val Leu Thr Ala Gly Ala Tyr Leu Cys Tyr Arg Phe Leu Phe Asn 420 425 430 Ser Asn Thr 435 5 3318 DNA Homo sapiens 5 gtgatgcgta gttccggctg ccggttgaca tgaagaagca gcagcggcta gggcggcggt 60 agctgcaggg gtcggggatt gcagcgggcc tcggggctaa gagcgcgacg cggcctagag 120 cggcagacgg cgcagtgggc cgagaaggag gcgcagcagc cgccctggcc cgtcatggag 180 atggaaaagg agttcgagca gatcgacaag tccgggagct gggcggccat ttaccaggat 240 atccgacatg aagccagtga cttcccatgt agagtggcca agcttcctaa gaacaaaaac 300 cgaaataggt acagagacgt cagtcccttt gaccatagtc ggattaaact acatcaagaa 360 gataatgact atatcaacgc tagtttgata aaaatggaag aagcccaaag gagttacatt 420 cttacccagg gccctttgcc taacacatgc ggtcactttt gggagatggt gtgggagcag 480 aaaagcaggg gtgtcgtcat gctcaacaga gtgatggaga aaggttcgtt aaaatgcgca 540 caatactggc cacaaaaaga agaaaaagag atgatctttg aagacacaaa tttgaaatta 600 acattgatct ctgaagatat caagtcatat tatacagtgc gacagctaga attggaaaac 660 cttacaaccc aagaaactcg agagatctta catttccact ataccacatg gcctgacttt 720 ggagtccctg aatcaccagc ctcattcttg aactttcttt tcaaagtccg agagtcaggg 780 tcactcagcc cggagcacgg gcccgttgtg gtgcactgca gtgcaggcat cggcaggtct 840 ggaaccttct gtctggctga tacctgcctc ttgctgatgg acaagaggaa agacccttct 900 tccgttgata tcaagaaagt gctgttagaa atgaggaagt ttcggatggg gctgatccag 960 acagccgacc agctgcgctt ctcctacctg gctgtgatcg aaggtgccaa attcatcatg 1020 ggggactctt ccgtgcagga tcagtggaag gagctttccc acgaggacct ggagccccca 1080 cccgagcata tccccccacc tccccggcca cccaaacgaa tcctggagcc acacaatggg 1140 aaatgcaggg agttcttccc aaatcaccag tgggtgaagg aagagaccca ggaggataaa 1200 gactgcccca tcaaggaaga aaaaggaagc cccttaaatg ccgcacccta cggcatcgaa 1260 agcatgagtc aagacactga agttagaagt cgggtcgtgg ggggaagtct tcgaggtgcc 1320 caggctgcct ccccagccaa aggggagccg tcactgcccg agaaggacga ggaccatgca 1380 ctgagttact ggaagccctt cctggtcaac atgtgcgtgg ctacggtcct cacggccggc 1440 gcttacctct gctacaggtt cctgttcaac agcaacacat agcctgaccc tcctccactc 1500 cacctccacc cactgtccgc ctctgcccgc agagcccacg cccgactagc aggcatgccg 1560 cggtaggtaa gggccgccgg accgcgtaga gagccgggcc ccggacggac gttggttctg 1620 cactaaaacc catcttcccc ggatgtgtgt ctcacccctc atccttttac tttttgcccc 1680 ttccactttg agtaccaaat ccacaagcca ttttttgagg agagtgaaag agagtaccat 1740 gctggcggcg cagagggaag gggcctacac ccgtcttggg gctcgcccca cccagggctc 1800 cctcctggag catcccaggc gggcggcacg ccaacagccc cccccttgaa tctgcaggga 1860 gcaactctcc actccatatt tatttaaaca attttttccc caaaggcatc catagtgcac 1920 tagcattttc ttgaaccaat aatgtattaa aattttttga tgtcagcctt gcatcaaggg 1980 ctttatcaaa aagtacaata ataaatcctc aggtagtact gggaatggaa ggctttgcca 2040 tgggcctgct gcgtcagacc agtactggga aggaggacgg ttgtaagcag ttgttattta 2100 gtgatattgt gggtaacgtg agaagataga acaatgctat aatatataat gaacacgtgg 2160 gtatttaata agaaacatga tgtgagatta ctttgtcccg cttattctcc tccctgttat 2220 ctgctagatc tagttctcaa tcactgctcc cccgtgtgta ttagaatgca tgtaaggtct 2280 tcttgtgtcc tgatgaaaaa tatgtgcttg aaatgagaaa ctttgatctc tgcttactaa 2340 tgtgccccat gtccaagtcc aacctgcctg tgcatgacct gatcattaca tggctgtggt 2400 tcctaagcct gttgctgaag tcattgtcgc tcagcaatag ggtgcagttt tccaggaata 2460 ggcatttgcc taattcctgg catgacactc tagtgacttc ctggtgaggc ccagcctgtc 2520 ctggtacagc agggtcttgc tgtaactcag acattccaag ggtatgggaa gccatattca 2580 cacctcacgc tctggacatg atttagggaa gcagggacac cccccgcccc ccacctttgg 2640 gatcagcctc cgccattcca agtcaacact cttcttgagc agaccgtgat ttggaagaga 2700 ggcacctgct ggaaaccaca cttcttgaaa cagcctgggt gacggtcctt taggcagcct 2760 gccgccgtct ctgtcccggt tcaccttgcc gagagaggcg cgtctgcccc accctcaaac 2820 cctgtggggc ctgatggtgc tcacgactct tcctgcaaag ggaactgaag acctccacat 2880 taagtggctt tttaacatga aaaacacggc agctgtagct cccgagctac tctcttgcca 2940 gcattttcac attttgcctt tctcgtggta gaagccagta cagagaaatt ctgtggtggg 3000 aacattcgag gtgtcaccct gcagagctat ggtgaggtgt ggataaggct taggtgccag 3060 gctgtaagca ttctgagctg ggcttgttgt ttttaagtcc tgtatatgta tgtagtagtt 3120 tgggtgtgta tatatagtag catttcaaaa tggacgtact ggtttaacct cctatccttg 3180 gagagcagct ggctctccac cttgttacac attatgttag agaggtagcg agctgctctg 3240 ctatatgcct taagccaata tttactcatc aggtcattat tttttacaat ggccatggaa 3300 taaaccattt ttacaaaa 3318 6 435 PRT Homo sapiens 6 Met Glu Met Glu Lys Glu Phe Glu Gln Ile Asp Lys Ser Gly Ser Trp 1 5 10 15 Ala Ala Ile Tyr Gln Asp Ile Arg His Glu Ala Ser Asp Phe Pro Cys 20 25 30 Arg Val Ala Lys Leu Pro Lys Asn Lys Asn Arg Asn Arg Tyr Arg Asp 35 40 45 Val Ser Pro Phe Asp His Ser Arg Ile Lys Leu His Gln Glu Asp Asn 50 55 60 Asp Tyr Ile Asn Ala Ser Leu Ile Lys Met Glu Glu Ala Gln Arg Ser 65 70 75 80 Tyr Ile Leu Thr Gln Gly Pro Leu Pro Asn Thr Cys Gly His Phe Trp 85 90 95 Glu Met Val Trp Glu Gln Lys Ser Arg Gly Val Val Met Leu Asn Arg 100 105 110 Val Met Glu Lys Gly Ser Leu Lys Cys Ala Gln Tyr Trp Pro Gln Lys 115 120 125 Glu Glu Lys Glu Met Ile Phe Glu Asp Thr Asn Leu Lys Leu Thr Leu 130 135 140 Ile Ser Glu Asp Ile Lys Ser Tyr Tyr Thr Val Arg Gln Leu Glu Leu 145 150 155 160 Glu Asn Leu Thr Thr Gln Glu Thr Arg Glu Ile Leu His Phe His Tyr 165 170 175 Thr Thr Trp Pro Asp Phe Gly Val Pro Glu Ser Pro Ala Ser Phe Leu 180 185 190 Asn Phe Leu Phe Lys Val Arg Glu Ser Gly Ser Leu Ser Pro Glu His 195 200 205 Gly Pro Val Val Val His Cys Ser Ala Gly Ile Gly Arg Ser Gly Thr 210 215 220 Phe Cys Leu Ala Asp Thr Cys Leu Leu Leu Met Asp Lys Arg Lys Asp 225 230 235 240 Pro Ser Ser Val Asp Ile Lys Lys Val Leu Leu Glu Met Arg Lys Phe 245 250 255 Arg Met Gly Leu Ile Gln Thr Ala Asp Gln Leu Arg Phe Ser Tyr Leu 260 265 270 Ala Val Ile Glu Gly Ala Lys Phe Ile Met Gly Asp Ser Ser Val Gln 275 280 285 Asp Gln Trp Lys Glu Leu Ser His Glu Asp Leu Glu Pro Pro Pro Glu 290 295 300 His Ile Pro Pro Pro Pro Arg Pro Pro Lys Arg Ile Leu Glu Pro His 305 310 315 320 Asn Gly Lys Cys Arg Glu Phe Phe Pro Asn His Gln Trp Val Lys Glu 325 330 335 Glu Thr Gln Glu Asp Lys Asp Cys Pro Ile Lys Glu Glu Lys Gly Ser 340 345 350 Pro Leu Asn Ala Ala Pro Tyr Gly Ile Glu Ser Met Ser Gln Asp Thr 355 360 365 Glu Val Arg Ser Arg Val Val Gly Gly Ser Leu Arg Gly Ala Gln Ala 370 375 380 Ala Ser Pro Ala Lys Gly Glu Pro Ser Leu Pro Glu Lys Asp Glu Asp 385 390 395 400 His Ala Leu Ser Tyr Trp Lys Pro Phe Leu Val Asn Met Cys Val Ala 405 410 415 Thr Val Leu Thr Ala Gly Ala Tyr Leu Cys Tyr Arg Phe Leu Phe Asn 420 425 430 Ser Asn Thr 435 7 2346 DNA Mus musculus 7 gaattcggga tccttttgca cattcctagt tagcagtgca tactcatcag actggagatg 60 tttaatgaca tcagggaacc aaacggacaa cccatagtac ccgaagacag ggtgaaccag 120 acaatcgtaa gcttgatggt gttttccctg actgggtagt tgaagcatct catgaatgtc 180 agccaaattc cgtacagttc ggtgcggatc cgaacgaaac acctcctgta ccaggttccc 240 gtgtcgctct caatttcaat cagctcatct atttgtttgg gagtcttgat tttatttacc 300 gtgaagacct tctctggctg gccccgggct ctcatgttgg tgtcatgaat taacttcaga 360 atcatccagg cttcatcatg ttttcccacc tccagcaaga accgagggct ttctggcatg 420 aaggtgagag ccaccacaga ggagacgcat gggagcgcac agacgatgac gaagacgcgc 480 cacgtgtgga actggtaggc tgaacccatg ctgaagctcc acccgtagtg gggaatgatg 540 gcccaggcat ggcggaggct agatgccgcc aatcatccag aacatgcaga agccgctgct 600 ggggagcttg gggctgcggt ggtggcgggt gacgggcttc gggacgcgga gcgacgcggc 660 ctagcgcggc ggacggccgt gggaactcgg gcagccgacc cgtcccgcca tggagatgga 720 gaaggagttc gaggagatcg acaaggctgg gaactgggcg gctatttacc aggacattcg 780 acatgaagcc agcgacttcc catgcaaagt cgcgaagctt cctaagaaca aaaaccggaa 840 caggtaccga gatgtcagcc cttttgacca cagtcggatt aaattgcacc aggaagataa 900 tgactatatc aatgccagct tgataaaaat ggaagaagcc cagaggagct atattctcac 960 ccagggccct ttaccaaaca catgtgggca cttctgggag atggtgtggg agcagaagag 1020 caggggcgtg gtcatgctca accgcatcat ggagaaaggc tcgttaaaat gtgcccagta 1080 ttggccacag caagaagaaa aggagatggt ctttgatgac acaggtttga agttgacact 1140 aatctctgaa gatgtcaagt catattacac agtacgacag ttggagttgg aaaacctgac 1200 taccaaggag actcgagaga tcctgcattt ccactacacc acatggcctg actttggagt 1260 ccccgagtca ccggcttctt tcctcaattt ccttttcaaa gtccgagagt caggctcact 1320 cagcctggag catggcccca ttgtggtcca ctgcagcgcc ggcatcggga ggtcagggac 1380 cttctgtctg gctgacacct gcctcttact gatggacaag aggaaagacc catcttccgt 1440 ggacatcaag aaagtactgc tggagatgcg caggttccgc atggggctca tccagactgc 1500 cgaccagctg cgcttctcct acctggctgt catcgagggc gccaagttca tcatgggcga 1560 ctcgtcagtg caggatcagt ggaaggagct ctcccgggag gatctagacc ttccacccga 1620 gcacgtgccc ccacctcccc ggccacccaa acgcacactg gagcctcaca acgggaagtg 1680 caaggagctc ttctccagcc accagtgggt gagcgaggag acctgtgggg atgaagacag 1740 cctggccaga gaggaaggca gagcccagtc aagtgccatg cacagcgtga gcagcatgag 1800 tccagacact gaagttagga gacggatggt gggtggaggt cttcaaagtg ctcaggcgtc 1860 tgtccccacc gaggaagagc tgtcctccac tgaggaggaa cacaaggcac attggccaag 1920 tcactggaag cccttcctgg tcaatgtgtg catggccacg ctcctggcca ccggcgcgta 1980 cttgtgctac cgggtgtgtt ttcactgaca gactgggagg cactgccact gcccagctta 2040 ggatgcggtc tgcggcgtct gacctggtgt agagggaaca acaactcgca agcctgctct 2100 ggaactggaa gggcctgccc caggagggta ttagtgcact gggctttgaa ggagcccctg 2160 gtcccacgaa cagagtctaa tctcagggcc ttaacctgtt caggagaagt agaggaaatg 2220 ccaaatactc ttcttgctct cacctcactc ctcccctttc tctgattcat ttgtttttgg 2280 aaaaaaaaaa aaaaagaatt acaacacatt gttgttttta acatttataa aggcaggccc 2340 gaattc 2346 8 432 PRT Mus musculus 8 Met Glu Met Glu Lys Glu Phe Glu Glu Ile Asp Lys Ala Gly Asn Trp 1 5 10 15 Ala Ala Ile Tyr Gln Asp Ile Arg His Glu Ala Ser Asp Phe Pro Cys 20 25 30 Lys Val Ala Lys Leu Pro Lys Asn Lys Asn Arg Asn Arg Tyr Arg Asp 35 40 45 Val Ser Pro Phe Asp His Ser Arg Ile Lys Leu His Gln Glu Asp Asn 50 55 60 Asp Tyr Ile Asn Ala Ser Leu Ile Lys Met Glu Glu Ala Gln Arg Ser 65 70 75 80 Tyr Ile Leu Thr Gln Gly Pro Leu Pro Asn Thr Cys Gly His Phe Trp 85 90 95 Glu Met Val Trp Glu Gln Lys Ser Arg Gly Val Val Met Leu Asn Arg 100 105 110 Ile Met Glu Lys Gly Ser Leu Lys Cys Ala Gln Tyr Trp Pro Gln Gln 115 120 125 Glu Glu Lys Glu Met Val Phe Asp Asp Thr Gly Leu Lys Leu Thr Leu 130 135 140 Ile Ser Glu Asp Val Lys Ser Tyr Tyr Thr Val Arg Gln Leu Glu Leu 145 150 155 160 Glu Asn Leu Thr Thr Lys Glu Thr Arg Glu Ile Leu His Phe His Tyr 165 170 175 Thr Thr Trp Pro Asp Phe Gly Val Pro Glu Ser Pro Ala Ser Phe Leu 180 185 190 Asn Phe Leu Phe Lys Val Arg Glu Ser Gly Ser Leu Ser Leu Glu His 195 200 205 Gly Pro Ile Val Val His Cys Ser Ala Gly Ile Gly Arg Ser Gly Thr 210 215 220 Phe Cys Leu Ala Asp Thr Cys Leu Leu Leu Met Asp Lys Arg Lys Asp 225 230 235 240 Pro Ser Ser Val Asp Ile Lys Lys Val Leu Leu Glu Met Arg Arg Phe 245 250 255 Arg Met Gly Leu Ile Gln Thr Ala Asp Gln Leu Arg Phe Ser Tyr Leu 260 265 270 Ala Val Ile Glu Gly Ala Lys Phe Ile Met Gly Asp Ser Ser Val Gln 275 280 285 Asp Gln Trp Lys Glu Leu Ser Arg Glu Asp Leu Asp Leu Pro Pro Glu 290 295 300 His Val Pro Pro Pro Pro Arg Pro Pro Lys Arg Thr Leu Glu Pro His 305 310 315 320 Asn Gly Lys Cys Lys Glu Leu Phe Ser Ser His Gln Trp Val Ser Glu 325 330 335 Glu Thr Cys Gly Asp Glu Asp Ser Leu Ala Arg Glu Glu Gly Arg Ala 340 345 350 Gln Ser Ser Ala Met His Ser Val Ser Ser Met Ser Pro Asp Thr Glu 355 360 365 Val Arg Arg Arg Met Val Gly Gly Gly Leu Gln Ser Ala Gln Ala Ser 370 375 380 Val Pro Thr Glu Glu Glu Leu Ser Ser Thr Glu Glu Glu His Lys Ala 385 390 395 400 His Trp Pro Ser His Trp Lys Pro Phe Leu Val Asn Val Cys Met Ala 405 410 415 Thr Leu Leu Ala Thr Gly Ala Tyr Leu Cys Tyr Arg Val Cys Phe His 420 425 430 9 3215 DNA Homo sapiens 9 gcgcgacgcg gcctagagcg gcagacggcg cagtgggccg agaaggaggc gcagcagccg 60 ccctggcccg tcatggagat ggaaaaggag ttcgagcaga tcgacaagtc cgggagctgg 120 gcggccattt accaggatat ccgacatgaa gccagtgact tcccatgtag agtggccaag 180 cttcctaaga acaaaaaccg aaataggtac agagacgtca gtccctttga ccatagtcgg 240 attaaactac atcaagaaga taatgactat atcaacgcta gtttgataaa aatggaagaa 300 gcccaaagga gttacattct tacccagggc cctttgccta acacatgcgg tcacttttgg 360 gagatggtgt gggagcagaa aagcaggggt gtcgtcatgc tcaacagagt gatggagaaa 420 ggttcgttaa aatgcgcaca atactggcca caaaaagaag aaaaagagat gatctttgaa 480 gacacaaatt tgaaattaac attgatctct gaagatatca agtcatatta tacagtgcga 540 cagctagaat tggaaaacct tacaacccaa gaaactcgag agatcttaca tttccactat 600 accacatggc ctgactttgg agtccctgaa tcaccagcct cattcttgaa ctttcttttc 660 aaagtccgag agtcagggtc actcagcccg gagcacgggc ccgttgtggt gcactgcagt 720 gcaggcatcg gcaggtctgg aaccttctgt ctggctgata cctgcctctt gctgatggac 780 aagaggaaag acccttcttc cgttgatatc aagaaagtgc tgttagaaat gaggaagttt 840 cggatggggc tgatccagac agccgaccag ctgcgcttct cctacctggc tgtgatcgaa 900 ggtgccaaat tcatcatggg ggactcttcc gtgcaggatc agtggaagga gctttcccac 960 gaggacctgg agcccccacc cgagcatatc cccccacctc cccggccacc caaacgaatc 1020 ctggagccac acaatgggaa atgcagggag ttcttcccaa atcaccagtg ggtgaaggaa 1080 gagacccagg aggataaaga ctgccccatc aaggaagaaa aaggaagccc cttaaatgcc 1140 gcaccctacg gcatcgaaag catgagtcaa gacactgaag ttagaagtcg ggtcgtgggg 1200 ggaagtcttc gaggtgccca ggctgcctcc ccagccaaag gggagccgtc actgcccgag 1260 aaggacgagg accatgcact gagttactgg aagcccttcc tggtcaacat gtgcgtggct 1320 acggtcctca cggccggcgc ttacctctgc tacaggttcc tgttcaacag caacacatag 1380 cctgaccctc ctccactcca cctccaccca ctgtccgcct ctgcccgcag agcccacgcc 1440 cgactagcag gcatgccgcg gtaggtaagg gccgccggac cgcgtagaga gccgggcccc 1500 ggacggacgt tggttctgca ctaaaaccca tcttccccgg atgtgtgtct cacccctcat 1560 ccttttactt tttgcccctt ccactttgag taccaaatcc acaagccatt ttttgaggag 1620 agtgaaagag agtaccatgc tggcggcgca gagggaaggg gcctacaccc gtcttggggc 1680 tcgccccacc cagggctccc tcctggagca tcccaggcgg gcggcacgcc agacagcccc 1740 ccccttgaat ctgcagggag caactctcca ctccatattt atttaaacaa ttttttcccc 1800 aaaggcatcc atagtgcact agcattttct tgaaccaata atgtattaaa attttttgat 1860 gtcagccttg catcaagggc tttatcaaaa agtacaataa taaatcctca ggtagtactg 1920 ggaatggaag gctttgccat gggcctgctg cgtcagacca gtactgggaa ggaggacggt 1980 tgtaagcagt tgttatttag tgatattgtg ggtaacgtga gaagatagaa caatgctata 2040 atatataatg aacacgtggg tatttaataa gaaacatgat gtgagattac tttgtcccgc 2100 ttattctgct ccctgttatc tgctagatct agttctcaat cactgctccc ccgtgtgtat 2160 tagaatgcat gtaaggtctt cttgtgtcct gatgaaaaat atgtgcttga aatgagaaac 2220 tttgatctct gcttactaat gtgccccatg tccaagtcca acctgcctgt gcatgacctg 2280 atcattacat ggctgtggtt cctaagcctg ttgctgaagt cattgtcgct cagcaatagg 2340 gtgcagtttt ccaggaatag gcatttgcct aattcctggc atgacactct agtgacttcc 2400 tggtgaggcc cagcctgtcc tggtacagca gggtcttgct gtaactcaga cattccaagg 2460 gtatgggaag ccatattcac acctcacgct ctggacatga tttagggaag cagggacacc 2520 ccccgccccc cacctttggg atcagcctcc gccattccaa gtcgacactc ttcttgagca 2580 gaccgtgatt tggaagagag gcacctgctg gaaaccacac ttcttgaaac agcctgggtg 2640 acggtccttt aggcagcctg ccgccgtctc tgtcccggtt caccttgccg agagaggcgc 2700 gtctgcccca ccctcaaacc ctgtggggcc tgatggtgct cacgactctt cctgcaaagg 2760 gaactgaaga cctccacatt aagtggcttt ttaacatgaa aaacacggca gctgtagctc 2820 ccgagctact ctcttgccag cattttcaca ttttgccttt ctcgtggtag aagccagtac 2880 agagaaattc tgtggtggga acattcgagg tgtcaccctg cagagctatg gtgaggtgtg 2940 gataaggctt aggtgccagg ctgtaagcat tctgagctgg cttgttgttt ttaagtcctg 3000 tatatgtatg tagtagtttg ggtgtgtata tatagtagca tttcaaaatg gacgtactgg 3060 tttaacctcc tatccttgga gagcagctgg ctctccacct tgttacacat tatgttagag 3120 aggtagcgag ctgctctgct atgtccttaa gccaatattt actcatcagg tcattatttt 3180 ttacaatggc catggaataa accattttta caaaa 3215 10 435 PRT Homo sapiens 10 Met Glu Met Glu Lys Glu Phe Glu Gln Ile Asp Lys Ser Gly Ser Trp 1 5 10 15 Ala Ala Ile Tyr Gln Asp Ile Arg His Glu Ala Ser Asp Phe Pro Cys 20 25 30 Arg Val Ala Lys Leu Pro Lys Asn Lys Asn Arg Asn Arg Tyr Arg Asp 35 40 45 Val Ser Pro Phe Asp His Ser Arg Ile Lys Leu His Gln Glu Asp Asn 50 55 60 Asp Tyr Ile Asn Ala Ser Leu Ile Lys Met Glu Glu Ala Gln Arg Ser 65 70 75 80 Tyr Ile Leu Thr Gln Gly Pro Leu Pro Asn Thr Cys Gly His Phe Trp 85 90 95 Glu Met Val Trp Glu Gln Lys Ser Arg Gly Val Val Met Leu Asn Arg 100 105 110 Val Met Glu Lys Gly Ser Leu Lys Cys Ala Gln Tyr Trp Pro Gln Lys 115 120 125 Glu Glu Lys Glu Met Ile Phe Glu Asp Thr Asn Leu Lys Leu Thr Leu 130 135 140 Ile Ser Glu Asp Ile Lys Ser Tyr Tyr Thr Val Arg Gln Leu Glu Leu 145 150 155 160 Glu Asn Leu Thr Thr Gln Glu Thr Arg Glu Ile Leu His Phe His Tyr 165 170 175 Thr Thr Trp Pro Asp Phe Gly Val Pro Glu Ser Pro Ala Ser Phe Leu 180 185 190 Asn Phe Leu Phe Lys Val Arg Glu Ser Gly Ser Leu Ser Pro Glu His 195 200 205 Gly Pro Val Val Val His Cys Ser Ala Gly Ile Gly Arg Ser Gly Thr 210 215 220 Phe Cys Leu Ala Asp Thr Cys Leu Leu Leu Met Asp Lys Arg Lys Asp 225 230 235 240 Pro Ser Ser Val Asp Ile Lys Lys Val Leu Leu Glu Met Arg Lys Phe 245 250 255 Arg Met Gly Leu Ile Gln Thr Ala Asp Gln Leu Arg Phe Ser Tyr Leu 260 265 270 Ala Val Ile Glu Gly Ala Lys Phe Ile Met Gly Asp Ser Ser Val Gln 275 280 285 Asp Gln Trp Lys Glu Leu Ser His Glu Asp Leu Glu Pro Pro Pro Glu 290 295 300 His Ile Pro Pro Pro Pro Arg Pro Pro Lys Arg Ile Leu Glu Pro His 305 310 315 320 Asn Gly Lys Cys Arg Glu Phe Phe Pro Asn His Gln Trp Val Lys Glu 325 330 335 Glu Thr Gln Glu Asp Lys Asp Cys Pro Ile Lys Glu Glu Lys Gly Ser 340 345 350 Pro Leu Asn Ala Ala Pro Tyr Gly Ile Glu Ser Met Ser Gln Asp Thr 355 360 365 Glu Val Arg Ser Arg Val Val Gly Gly Ser Leu Arg Gly Ala Gln Ala 370 375 380 Ala Ser Pro Ala Lys Gly Glu Pro Ser Leu Pro Glu Lys Asp Glu Asp 385 390 395 400 His Ala Leu Ser Tyr Trp Lys Pro Phe Leu Val Asn Met Cys Val Ala 405 410 415 Thr Val Leu Thr Ala Gly Ala Tyr Leu Cys Tyr Arg Phe Leu Phe Asn 420 425 430 Ser Asn Thr 435 11 4127 DNA Rattus norvegicus 11 agccgctgct ggggaggttg gggctgaggt ggtggcgggc gacgggcctc gagacgcgga 60 gcgacgcggc ctagcgcggc ggacggccga gggaactcgg gcagtcgtcc cgtcccgcca 120 tggaaatgga gaaggaattc gagcagatcg ataaggctgg gaactgggcg gctatttacc 180 aggatattcg acatgaagcc agtgacttcc catgcagaat agcgaaactt cctaagaaca 240 aaaaccggaa caggtaccga gatgtcagcc cttttgacca cagtcggatt aaattgcatc 300 aggaagataa tgactatatc aatgccagct tgataaaaat ggaggaagcc cagaggagct 360 atatcctcac ccagggccct ttaccaaaca cgtgcgggca cttctgggag atggtgtggg 420 agcagaagag caggggcgtg gtcatgctca accgcatcat ggagaaaggc tcgttaaaat 480 gtgcccagta ttggccacag aaagaagaaa aagagatggt cttcgatgac accaatttga 540 agctgacact gatctctgaa gatgtcaagt catattacac agtacggcag ttggagttgg 600 agaacctggc tacccaggag gctcgagaga tcctgcattt ccactacacc acctggcctg 660 actttggagt ccctgagtca cctgcctctt tcctcaattt cctattcaaa gtccgagagt 720 caggctcact cagcccagag cacggcccca ttgtggtcca ctgcagtgct ggcattggca 780 ggtcagggac cttctgcctg gctgacacct gcctcttact gatggacaag aggaaagacc 840 cgtcctctgt ggacatcaag aaagtgctgt tggagatgcg caggttccgc atggggctca 900 tccagacggc cgaccaactg cgcttctcct acctggctgt gatcgagggt gcaaagttca 960 tcatgggcga ctcgtcagtg caggatcagt ggaaggagct ttcccatgaa gacctggagc 1020 ctccccctga gcacgtgccc ccacctcccc ggccacccaa acgcacattg gagcctcaca 1080 atggcaagtg caaggagctc ttctccaacc accagtgggt gagcgaggag agctgtgagg 1140 atgaggacat cctggccaga gaggaaagca gagccccctc aattgctgtg cacagcatga 1200 gcagtatgag tcaagacact gaagttagga aacggatggt gggtggaggt cttcaaagtg 1260 ctcaggcatc tgtccccact gaggaagagc tgtccccaac cgaggaggaa caaaaggcac 1320 acaggccagt tcactggaag cccttcctgg tcaacgtgtg catggccacg gccctggcga 1380 ctggcgcgta cctctgttac cgggtatgtt ttcactgaca gactgctgtg aggcatgagc 1440 gtggtgggcg ctgccactgc ccaggttagg atttggtctg cggcgtctaa cctggtgtag 1500 aagaaacaac agcttacaag cctgtggtgg aactggaagg gccagcccca ggaggggcat 1560 ctgtgcactg ggctttgaag gagcccctgg tcccaagaac agagtctaat ctcagggcct 1620 taacctgttc aggagaagta gaggaaatgc caaatactct tcttgctctc acctcactcc 1680 tcccctttct ctggttcgtt tgtttttgga aaaaaaaaaa aaagaattac aacacattgt 1740 tgtttttaac atttataaag gcaggttttt gttattttta gagaaaacaa aagatgctag 1800 gcactggtga gattctcttg tgccctttgg catgtgatca gattcacgat ttacgtttat 1860 ttccggggga gggtcccacc tgtcaggact gtaaagttcc tgctggcttg gtcagccccc 1920 ccaccccccc accccgagct tgcaggtgcc ctgctgtgag gagagcagca gcagaggctg 1980 cccctggaca gaagcccagc tctgcttccc tcaggtgtcc ctgcgtttcc atcctccttc 2040 tttgtgaccg ccatcttgca gatgacccag tcctcagcac cccacccctg cagatgggtt 2100 tctccgaggg cctgcctcag ggtcatcaga ggttggctgc cagcttagag ctggggcttc 2160 catttgattg gaaagtcatt actattctat gtagaagcca ctccactgag gtgtaaagca 2220 agactcataa aggaggagcc ttggtgtcat ggaagtcact ccgcgcgcag gacctgtaac 2280 aacctctgaa acactcagtc ctgctgcagt gacgtccttg aaggcatcag acagatgatt 2340 tgcagactgc caagacttgt cctgagccgt gatttttaga gtctggactc atgaaacacc 2400 gccgagcgct tactgtgcag cctctgatgc tggttggctg aggctgcggg gaggtggaca 2460 ctgtgggtgc atccagtgca gttgcttttg tgcagttggg tccagcagca cagcccgcac 2520 tccagcctca gctgcaggcc acagtggcca tggaggccgc cagagcgagc tggggtggat 2580 gcttgttcac ttggagcagc cttcccagga cgtgcagctc ccttcctgct ttgtccttct 2640 gcttccttcc ctggagtagc aagcccacga gcaatcgtga ggggtgtgag ggagctgcag 2700 aggcatcaga gtggcctgca gcggcgtgag gccccttccc ctccgacacc cccctccaga 2760 ggagccgctc cactgttatt tattcacttt gcccacagac acccctgagt gagcacaccc 2820 tgaaactgac cgtgtaaggt gtcagcctgc acccaggacc gtcaggtgca gcaccgggtc 2880 agtcctaggg ttgaggtagg actgacacag ccactgtgtg gctggtgctg gggcaggggc 2940 aggagctgag ggtcttagaa gcaatcttca ggaacagaca acagtggtga catgtaaagt 3000 ccctgtggct actgatgaca tgtgtaggat gaaggctggc ctttctccca tgactttcta 3060 gatcccgttc cccgtctgct ttccctgtga gttagaaaac acacaggctc ctgtcctggt 3120 ggtgccgtgt gcttgacatg ggaaacttag atgcctgctc actggcgggc acctcggcat 3180 cgccaccact cagagtgaga gcagtgctgt ccagtgccga ggccgcctga ctcccggcag 3240 gactcttcag gctctggcct gccccagcac accccgctgg atctcagaca ttccacaccc 3300 acacctcatt ccctggacac ttgggcaagc aggcccgccc ttccacctct ggggtcagcc 3360 cctccattcc gagttcacac tgctctggag caggccagga ccggaagcaa ggcagctggt 3420 gaggagcacc ctcctgggaa cagtgtaggt gacagtcctg agagtcagct tgctagcgct 3480 gctggcacca gtcaccttgc tcagaagtgt gtggctcttg aggctgaaga gactgatgat 3540 ggtgctcatg actcttctgt gaggggaact tgaccttcac attgggtggc tttttttaaa 3600 ataagcgaag gcagctggaa ctccagtctg cctcttgcca gcacttcaca ttttgccttt 3660 cacccagaga agccagcaca gagccactgg ggaaggcgat ggccttgcct gcacaggctg 3720 aggagatggc tcagccggcg tccaggctgt gtctggagca gggggtgcac agcagcctca 3780 caggtggggg cctcagagca ggcgctgccc tgtcccctgc cccgctggag gcagcaaagc 3840 tgctgcatgc cttaagtcaa tacttactca gcagggcgct ctcgttctct ctctctctct 3900 ctctctctct ctctctctct ctctctctct ctctaaatgg ccatagaata aaccatttta 3960 caaaaataaa agccaacaac aaagtgctct ggaatagcac ctttgcagga gcggggggtg 4020 tctcagggtc ttctgtgacc tcaccgaact gtccgactgc accgtttcca acttgtgtct 4080 cactaatggg tctgcattag ttgcaacaat aaatgttttt aaagaac 4127 12 432 PRT Rattus norvegicus 12 Met Glu Met Glu Lys Glu Phe Glu Gln Ile Asp Lys Ala Gly Asn Trp 1 5 10 15 Ala Ala Ile Tyr Gln Asp Ile Arg His Glu Ala Ser Asp Phe Pro Cys 20 25 30 Arg Ile Ala Lys Leu Pro Lys Asn Lys Asn Arg Asn Arg Tyr Arg Asp 35 40 45 Val Ser Pro Phe Asp His Ser Arg Ile Lys Leu His Gln Glu Asp Asn 50 55 60 Asp Tyr Ile Asn Ala Ser Leu Ile Lys Met Glu Glu Ala Gln Arg Ser 65 70 75 80 Tyr Ile Leu Thr Gln Gly Pro Leu Pro Asn Thr Cys Gly His Phe Trp 85 90 95 Glu Met Val Trp Glu Gln Lys Ser Arg Gly Val Val Met Leu Asn Arg 100 105 110 Ile Met Glu Lys Gly Ser Leu Lys Cys Ala Gln Tyr Trp Pro Gln Lys 115 120 125 Glu Glu Lys Glu Met Val Phe Asp Asp Thr Asn Leu Lys Leu Thr Leu 130 135 140 Ile Ser Glu Asp Val Lys Ser Tyr Tyr Thr Val Arg Gln Leu Glu Leu 145 150 155 160 Glu Asn Leu Ala Thr Gln Glu Ala Arg Glu Ile Leu His Phe His Tyr 165 170 175 Thr Thr Trp Pro Asp Phe Gly Val Pro Glu Ser Pro Ala Ser Phe Leu 180 185 190 Asn Phe Leu Phe Lys Val Arg Glu Ser Gly Ser Leu Ser Pro Glu His 195 200 205 Gly Pro Ile Val Val His Cys Ser Ala Gly Ile Gly Arg Ser Gly Thr 210 215 220 Phe Cys Leu Ala Asp Thr Cys Leu Leu Leu Met Asp Lys Arg Lys Asp 225 230 235 240 Pro Ser Ser Val Asp Ile Lys Lys Val Leu Leu Glu Met Arg Arg Phe 245 250 255 Arg Met Gly Leu Ile Gln Thr Ala Asp Gln Leu Arg Phe Ser Tyr Leu 260 265 270 Ala Val Ile Glu Gly Ala Lys Phe Ile Met Gly Asp Ser Ser Val Gln 275 280 285 Asp Gln Trp Lys Glu Leu Ser His Glu Asp Leu Glu Pro Pro Pro Glu 290 295 300 His Val Pro Pro Pro Pro Arg Pro Pro Lys Arg Thr Leu Glu Pro His 305 310 315 320 Asn Gly Lys Cys Lys Glu Leu Phe Ser Asn His Gln Trp Val Ser Glu 325 330 335 Glu Ser Cys Glu Asp Glu Asp Ile Leu Ala Arg Glu Glu Ser Arg Ala 340 345 350 Pro Ser Ile Ala Val His Ser Met Ser Ser Met Ser Gln Asp Thr Glu 355 360 365 Val Arg Lys Arg Met Val Gly Gly Gly Leu Gln Ser Ala Gln Ala Ser 370 375 380 Val Pro Thr Glu Glu Glu Leu Ser Pro Thr Glu Glu Glu Gln Lys Ala 385 390 395 400 His Arg Pro Val His Trp Lys Pro Phe Leu Val Asn Val Cys Met Ala 405 410 415 Thr Ala Leu Ala Thr Gly Ala Tyr Leu Cys Tyr Arg Val Cys Phe His 420 425 430 13 4127 DNA Rattus norvegicus 13 agccgctgct ggggaggttg gggctgaggt ggtggcgggc gacgggcctc gagacgcgga 60 gcgacgcggc ctagcgcggc ggacggccga gggaactcgg gcagtcgtcc cgtcccgcca 120 tggaaatgga gaaggaattc gagcagatcg ataaggctgg gaactgggcg gctatttacc 180 aggatattcg acatgaagcc agtgacttcc catgcagaat agcgaaactt cctaagaaca 240 aaaaccggaa caggtaccga gatgtcagcc cttttgacca cagtcggatt aaattgcatc 300 aggaagataa tgactatatc aatgccagct tgataaaaat ggaggaagcc cagaggagct 360 atatcctcac ccagggccct ttaccaaaca cgtgcgggca cttctgggag atggtgtggg 420 agcagaagag caggggcgtg gtcatgctca accgcatcat ggagaaaggc tcgttaaaat 480 gtgcccagta ttggccacag aaagaagaaa aagagatggt cttcgatgac accaatttga 540 agctgacact gatctctgaa gatgtcaagt catattacac agtacggcag ttggagttgg 600 agaacctggc tacccaggag gctcgagaga tcctgcattt ccactacacc acctggcctg 660 actttggagt ccctgagtca cctgcctctt tcctcaattt cctattcaaa gtccgagagt 720 caggctcact cagcccagag cacggcccca ttgtggtcca ctgcagtgct ggcattggca 780 ggtcagggac cttctgcctg gctgacacct gcctcttact gatggacaag aggaaagacc 840 cgtcctctgt ggacatcaag aaagtgctgt tggagatgcg caggttccgc atggggctca 900 tccagacggc cgaccaactg cgcttctcct acctggctgt gatcgagggt gcaaagttca 960 tcatgggcga ctcgtcagtg caggatcagt ggaaggagct ttcccatgaa gacctggagc 1020 ctccccctga gcacgtgccc ccacctcccc ggccacccaa acgcacattg gagcctcaca 1080 atggcaagtg caaggagctc ttctccaacc accagtgggt gagcgaggag agctgtgagg 1140 atgaggacat cctggccaga gaggaaagca gagccccctc aattgctgtg cacagcatga 1200 gcagtatgag tcaagacact gaagttagga aacggatggt gggtggaggt cttcaaagtg 1260 ctcaggcatc tgtccccact gaggaagagc tgtccccaac cgaggaggaa caaaaggcac 1320 acaggccagt tcactggaag cccttcctgg tcaacgtgtg catggccacg gccctggcga 1380 ctggcgcgta cctctgttac cgggtatgtt ttcactgaca gactgctgtg aggcatgagc 1440 gtggtgggcg ctgccactgc ccaggttagg atttggtctg cggcgtctaa cctggtgtag 1500 aagaaacaac agcttacaag cctgtggtgg aactggaagg gccagcccca ggaggggcat 1560 ctgtgcactg ggctttgaag gagcccctgg tcccaagaac agagtctaat ctcagggcct 1620 taacctgttc aggagaagta gaggaaatgc caaatactct tcttgctctc acctcactcc 1680 tcccctttct ctggttcgtt tgtttttgga aaaaaaaaaa aaagaattac aacacattgt 1740 tgtttttaac atttataaag gcaggttttt gttattttta gagaaaacaa aagatgctag 1800 gcactggtga gattctcttg tgccctttgg catgtgatca gattcacgat ttacgtttat 1860 ttccggggga gggtcccacc tgtcaggact gtaaagttcc tgctggcttg gtcagccccc 1920 ccaccccccc accccgagct tgcaggtgcc ctgctgtgag gagagcagca gcagaggctg 1980 cccctggaca gaagcccagc tctgcttccc tcaggtgtcc ctgcgtttcc atcctccttc 2040 tttgtgaccg ccatcttgca gatgacccag tcctcagcac cccacccctg cagatgggtt 2100 tctccgaggg cctgcctcag ggtcatcaga ggttggctgc cagcttagag ctggggcttc 2160 catttgattg gaaagtcatt actattctat gtagaagcca ctccactgag gtgtaaagca 2220 agactcataa aggaggagcc ttggtgtcat ggaagtcact ccgcgcgcag gacctgtaac 2280 aacctctgaa acactcagtc ctgctgcagt gacgtccttg aaggcatcag acagatgatt 2340 tgcagactgc caagacttgt cctgagccgt gatttttaga gtctggactc atgaaacacc 2400 gccgagcgct tactgtgcag cctctgatgc tggttggctg aggctgcggg gaggtggaca 2460 ctgtgggtgc atccagtgca gttgcttttg tgcagttggg tccagcagca cagcccgcac 2520 tccagcctca gctgcaggcc acagtggcca tggaggccgc cagagcgagc tggggtggat 2580 gcttgttcac ttggagcagc cttcccagga cgtgcagctc ccttcctgct ttgtccttct 2640 gcttccttcc ctggagtagc aagcccacga gcaatcgtga ggggtgtgag ggagctgcag 2700 aggcatcaga gtggcctgca gcggcgtgag gccccttccc ctccgacacc cccctccaga 2760 ggagccgctc cactgttatt tattcacttt gcccacagac acccctgagt gagcacaccc 2820 tgaaactgac cgtgtaaggt gtcagcctgc acccaggacc gtcaggtgca gcaccgggtc 2880 agtcctaggg ttgaggtagg actgacacag ccactgtgtg gctggtgctg gggcaggggc 2940 aggagctgag ggtcttagaa gcaatcttca ggaacagaca acagtggtga catgtaaagt 3000 ccctgtggct actgatgaca tgtgtaggat gaaggctggc ctttctccca tgactttcta 3060 gatcccgttc cccgtctgct ttccctgtga gttagaaaac acacaggctc ctgtcctggt 3120 ggtgccgtgt gcttgacatg ggaaacttag atgcctgctc actggcgggc acctcggcat 3180 cgccaccact cagagtgaga gcagtgctgt ccagtgccga ggccgcctga ctcccggcag 3240 gactcttcag gctctggcct gccccagcac accccgctgg atctcagaca ttccacaccc 3300 acacctcatt ccctggacac ttgggcaagc aggcccgccc ttccacctct ggggtcagcc 3360 cctccattcc gagttcacac tgctctggag caggccagga ccggaagcaa ggcagctggt 3420 gaggagcacc ctcctgggaa cagtgtaggt gacagtcctg agagtcagct tgctagcgct 3480 gctggcacca gtcaccttgc tcagaagtgt gtggctcttg aggctgaaga gactgatgat 3540 ggtgctcatg actcttctgt gaggggaact tgaccttcac attgggtggc tttttttaaa 3600 ataagcgaag gcagctggaa ctccagtctg cctcttgcca gcacttcaca ttttgccttt 3660 cacccagaga agccagcaca gagccactgg ggaaggcgat ggccttgcct gcacaggctg 3720 aggagatggc tcagccggcg tccaggctgt gtctggagca gggggtgcac agcagcctca 3780 caggtggggg cctcagagca ggcgctgccc tgtcccctgc cccgctggag gcagcaaagc 3840 tgctgcatgc cttaagtcaa tacttactca gcagggcgct ctcgttctct ctctctctct 3900 ctctctctct ctctctctct ctctctctct ctctaaatgg ccatagaata aaccatttta 3960 caaaaataaa agccaacaac aaagtgctct ggaatagcac ctttgcagga gcggggggtg 4020 tctcagggtc ttctgtgacc tcaccgaact gtccgactgc accgtttcca acttgtgtct 4080 cactaatggg tctgcattag ttgcaacaat aaatgttttt aaagaac 4127 14 432 PRT Rattus norvegicus 14 Met Glu Met Glu Lys Glu Phe Glu Gln Ile Asp Lys Ala Gly Asn Trp 1 5 10 15 Ala Ala Ile Tyr Gln Asp Ile Arg His Glu Ala Ser Asp Phe Pro Cys 20 25 30 Arg Ile Ala Lys Leu Pro Lys Asn Lys Asn Arg Asn Arg Tyr Arg Asp 35 40 45 Val Ser Pro Phe Asp His Ser Arg Ile Lys Leu His Gln Glu Asp Asn 50 55 60 Asp Tyr Ile Asn Ala Ser Leu Ile Lys Met Glu Glu Ala Gln Arg Ser 65 70 75 80 Tyr Ile Leu Thr Gln Gly Pro Leu Pro Asn Thr Cys Gly His Phe Trp 85 90 95 Glu Met Val Trp Glu Gln Lys Ser Arg Gly Val Val Met Leu Asn Arg 100 105 110 Ile Met Glu Lys Gly Ser Leu Lys Cys Ala Gln Tyr Trp Pro Gln Lys 115 120 125 Glu Glu Lys Glu Met Val Phe Asp Asp Thr Asn Leu Lys Leu Thr Leu 130 135 140 Ile Ser Glu Asp Val Lys Ser Tyr Tyr Thr Val Arg Gln Leu Glu Leu 145 150 155 160 Glu Asn Leu Ala Thr Gln Glu Ala Arg Glu Ile Leu His Phe His Tyr 165 170 175 Thr Thr Trp Pro Asp Phe Gly Val Pro Glu Ser Pro Ala Ser Phe Leu 180 185 190 Asn Phe Leu Phe Lys Val Arg Glu Ser Gly Ser Leu Ser Pro Glu His 195 200 205 Gly Pro Ile Val Val His Cys Ser Ala Gly Ile Gly Arg Ser Gly Thr 210 215 220 Phe Cys Leu Ala Asp Thr Cys Leu Leu Leu Met Asp Lys Arg Lys Asp 225 230 235 240 Pro Ser Ser Val Asp Ile Lys Lys Val Leu Leu Glu Met Arg Arg Phe 245 250 255 Arg Met Gly Leu Ile Gln Thr Ala Asp Gln Leu Arg Phe Ser Tyr Leu 260 265 270 Ala Val Ile Glu Gly Ala Lys Phe Ile Met Gly Asp Ser Ser Val Gln 275 280 285 Asp Gln Trp Lys Glu Leu Ser His Glu Asp Leu Glu Pro Pro Pro Glu 290 295 300 His Val Pro Pro Pro Pro Arg Pro Pro Lys Arg Thr Leu Glu Pro His 305 310 315 320 Asn Gly Lys Cys Lys Glu Leu Phe Ser Asn His Gln Trp Val Ser Glu 325 330 335 Glu Ser Cys Glu Asp Glu Asp Ile Leu Ala Arg Glu Glu Ser Arg Ala 340 345 350 Pro Ser Ile Ala Val His Ser Met Ser Ser Met Ser Gln Asp Thr Glu 355 360 365 Val Arg Lys Arg Met Val Gly Gly Gly Leu Gln Ser Ala Gln Ala Ser 370 375 380 Val Pro Thr Glu Glu Glu Leu Ser Pro Thr Glu Glu Glu Gln Lys Ala 385 390 395 400 His Arg Pro Val His Trp Lys Pro Phe Leu Val Asn Val Cys Met Ala 405 410 415 Thr Ala Leu Ala Thr Gly Ala Tyr Leu Cys Tyr Arg Val Cys Phe His 420 425 430 15 36 DNA Artificial Sequence Oligonucleotide primer mPTP1B-sense 15 gggggggatc catggagatg gagaaggagt tcgagg 36 16 35 DNA Artificial Sequence Oligonucleotide primer mPTP1B-anti sense 16 gggggaattc tcagtgaaaa cacacccggt agcac 35 17 21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.1 17 gaagcccaga ggagcuauan n 21 18 19 RNA Artificial Sequence Small interfering RNA - mPTP1B1.1 18 gaagcccaga ggagcuaua 19 19 19 RNA Artificial Sequence Small interfering RNA - mPTP1B1.1 19 uauagcuccu cugggcuuc 19 20 21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.1 20 gaagcccaga ggagcuauan n 21 21 21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.1 21 nnuauagcuc cucugggcuu c 21 22 21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.2 22 cuacaccaca uggccugacn n 21 23 19 RNA Artificial Sequence Small interfering RNA - mPTP1B1.2 23 cuacaccaca uggccugac 19 24 19 RNA Artificial Sequence Small interfering RNA - mPTP1B1.2 24 gucaggccau gugguguag 19 25 21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.2 25 cuacaccaca uggccugacn n 21 26 21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.2 26 nngucaggcc auguggugua g 21 27 21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.3 27 gacugccgac cagcugcgcn n 21 28 19 RNA Artificial Sequence Small interfering RNA - mPTP1B1.3 28 gacugccgac cagcugcgc 19 29 19 RNA Artificial Sequence Small interfering RNA - mPTP1B1.3 29 gcgcagcugg ucggcaguc 19 30 21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.3 30 gacugccgac cagcugcgcn n 21 31 21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.3 31 nngcgcagcu ggucggcagu c 21 32 21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.4 32 gguaccgaga ugucagcccn n 21 33 19 RNA Artificial Sequence Small interfering RNA - mPTP1B1.4 33 gguaccgaga ugucagccc 19 34 19 RNA Artificial Sequence Small interfering RNA - mPTP1B1.4 34 gggcugacau cucgguacc 19 35 21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.4 35 gguaccgaga ugucagcccn n 21 36 21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.4 36 nngggcugac aucucgguac c 21 37 21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.5 37 ugacuauauc aaugccagcn n 21 38 19 RNA Artificial Sequence Small interfering RNA - mPTP1B1.5 38 ugacuauauc aaugccagc 19 39 19 RNA Artificial Sequence Small interfering RNA - mPTP1B1.5 39 gcuggcauug auauaguca 19 40 21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.5 40 ugacuauauc aaugccagcn n 21 41 21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.5 41 nngcuggcau ugauauaguc a 21 42 21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.6 42 agaagaaaag gagauggucn n 21 43 19 RNA Artificial Sequence Small interfering RNA - mPTP1B1.6 43 agaagaaaag gagaugguc 19 44 19 RNA Artificial Sequence Small interfering RNA - mPTP1B1.6 44 gaccaucucc uuuucuucu 19 45 21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.6 45 agaagaaaag gagauggucn n 21 46 21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.6 46 nngaccaucu ccuuuucuuc u 21 47 21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.7 47 cgggaagugc aaggagcucn n 21 48 19 RNA Artificial Sequence Small interfering RNA - mPTP1B1.7 48 cgggaagugc aaggagcuc 19 49 19 RNA Artificial Sequence Small interfering RNA - mPTP1B1.7 49 gagcuccuug cacuucccg 19 50 21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.7 50 cgggaagugc aaggagcucn n 21 51 21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.7 51 nngagcuccu ugcacuuccc g 21 52 21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.8 52 ggaucagugg aaggagcucn c 21 53 19 RNA Artificial Sequence Small interfering RNA - mPTP1B1.8 53 ggaucagugg aaggagcuc 19 54 19 RNA Artificial Sequence Small interfering RNA - mPTP1B1.8 54 gagcuccuuc cacugaucc 19 55 21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.8 55 ggaucagugg aaggagcucn n 21 56 21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.8 56 nngagcuccu uccacugauc c 21 57 21 RNA Artificial Sequence Small interfering RNA - rPTP1B1.1 57 agaagaaaaa gagauggucn n 21 58 19 RNA Artificial Sequence Small interfering RNA - rPTP1B1.1 58 agaagaaaaa gagaugguc 19 59 19 RNA Artificial Sequence Small interfering RNA - rPTP1B1.1 59 gaccaucucu uuuucuucu 19 60 21 RNA Artificial Sequence Small interfering RNA - rPTP1B1.1 60 agaagaaaaa gagauggucn n 21 61 21 RNA Artificial Sequence Small interfering RNA - rPTP1B1.1 61 nngaccaucu cuuuuucuuc u 21 62 21 RNA Artificial Sequence Small interfering RNA - rPTP1B1.2 62 cggauggugg guggaggucn n 21 63 19 RNA Artificial Sequence Small interfering RNA - rPTP1B1.2 63 cggauggugg guggagguc 19 64 19 RNA Artificial Sequence Small interfering RNA - rPTP1B1.2 64 gaccuccacc caccauccg 19 65 21 RNA Artificial Sequence Small interfering RNA - rPTP1B1.2 65 cggauggugg guggaggucn n 21 66 21 RNA Artificial Sequence Small interfering RNA - rPTP1B1.2 66 nngaccucca cccaccaucc g 21 67 21 RNA Artificial Sequence Small interfering RNA - rPTP1B1.3 67 uggcaagugc aaggagcucn n 21 68 19 RNA Artificial Sequence Small interfering RNA - rPTP1B1.3 68 uggcaagugc aaggagcuc 19 69 19 RNA Artificial Sequence Small interfering RNA - rPTP1B1.3 69 gagcuccuug cacuugcca 19 70 21 RNA Artificial Sequence Small interfering RNA - rPTP1B1.3 70 uggcaagugc aaggagcucn n 21 71 21 RNA Artificial Sequence Small interfering RNA - rPTP1B1.3 71 nngagcuccu ugcacuugcc a 21 72 21 RNA Artificial Sequence Small interfering RNA - rPTP1B1.4 72 cuacaccacc uggccugacn n 21 73 19 RNA Artificial Sequence Small interfering RNA - rPTP1B1.4 73 cuacaccacc uggccugac 19 74 19 RNA Artificial Sequence Small interfering RNA - rPTP1B1.4 74 gucaggccag gugguguag 19 75 21 RNA Artificial Sequence Small interfering RNA - rPTP1B1.4 75 cuacaccacc uggccugacn n 21 76 21 RNA Artificial Sequence Small interfering RNA - rPTP1B1.4 76 nngucaggcc agguggugua g 21 77 21 RNA Artificial Sequence Small interfering RNA - hPTP1B1.1 77 cuauaccaca uggccugacn n 21 78 19 RNA Artificial Sequence Small interfering RNA - hPTP1B1.1 78 cuauaccaca uggccugac 19 79 19 RNA Artificial Sequence Small interfering RNA - hPTP1B1.1 79 gucaggccau gugguauag 19 80 21 RNA Artificial Sequence Small interfering RNA - hPTP1B1.1 80 cuauaccaca uggccugacn n 21 81 21 RNA Artificial Sequence Small interfering RNA - hPTP1B1.1 81 nngucaggcc augugguaua g 21 82 21 RNA Artificial Sequence Small interfering RNA - hPTP1B1.2 82 gcccaaagga guuacauucn n 21 83 19 RNA Artificial Sequence Small interfering RNA - hPTP1B1.2 83 gcccaaagga guuacauuc 19 84 19 RNA Artificial Sequence Small interfering RNA - hPTP1B1.2 84 gaauguaacu ccuuugggc 19 85 21 RNA Artificial Sequence Small interfering RNA - hPTP1B1.2 85 gcccaaagga guuacauucn n 21 86 21 RNA Artificial Sequence Small interfering RNA - hPTP1B1.2 86 nngaauguaa cuccuuuggg c 21 87 21 RNA Artificial Sequence Small interfering RNA - hPTP1B1.3 87 ggaagaaaaa ggaagccccn n 21 88 19 RNA Artificial Sequence Small interfering RNA - hPTP1B1.3 88 ggaagaaaaa ggaagcccc 19 89 19 RNA Artificial Sequence Small interfering RNA - hPTP1B1.3 89 ggggcuuccu uuuucuucc 19 90 21 RNA Artificial Sequence Small interfering RNA - hPTP1B1.3 90 ggaagaaaaa ggaagccccn n 21 91 21 RNA Artificial Sequence Small interfering RNA - hPTP1B1.3 91 nnggggcuuc cuuuuucuuc c 21 92 21 RNA Artificial Sequence Small interfering RNA - hPTP1B1.4 92 caaugggaaa ugcagggagn n 21 93 19 RNA Artificial Sequence Small interfering RNA - hPTP1B1.4 93 caaugggaaa ugcagggag 19 94 19 RNA Artificial Sequence Small interfering RNA - hPTP1B1.4 94 cucccugcau uucccauug 19 95 21 RNA Artificial Sequence Small interfering RNA - hPTP1B1.4 95 caaugggaaa ugcagggagn n 21 96 21 RNA Artificial Sequence misc_feature 1, 2 n = A,T,C,U or G 96 nncucccugc auuucccauu g 21 97 21 RNA Artificial Sequence Small interfering RNA - hPTP1B1.5 97 ggaucagugg aaggagcuun c 21 98 19 RNA Artificial Sequence Small interfering RNA - hPTP1B1.5 98 ggaucagugg aaggagcuu 19 99 19 RNA Artificial Sequence Small interfering RNA - hPTP1B1.5 99 aagcuccuuc cacugaucc 19 100 21 RNA Artificial Sequence Small interfering RNA - hPTP1B1.5 100 ggaucagugg aaggagcuun n 21 101 21 RNA Artificial Sequence Small interfering RNA - hPTP1B1.5 101 nnaagcuccu uccacugauc c 21 102 50 DNA Artificial Sequence Hairpin vector - hPTP1B H1.2-HP4 102 tttgcccaaa ggagttacat tcgtaagaat gtaactcctt tgggcttttt 50 103 50 DNA Artificial Sequence Hairpin vector - hPTP1B H1.2-HP4 103 ctagaaaaag cccaaaggag ttacattctt acgaatgtaa ctcctttggg 50 104 50 RNA Artificial Sequence Hairpin vector - hPTP1B H1.2-HP4 104 uuugcccaaa ggaguuacau ucguaagaau guaacuccuu ugggcuuuuu 50 105 50 RNA Artificial Sequence Hairpin vector - hPTP1B H1.2-HP4 105 cuagaaaaag cccaaaggag uuacauucuu acgaauguaa cuccuuuggg 50 106 55 DNA Artificial Sequence Hairpin vector - hPTP1B H1.2-HP9 106 tttgcccaaa ggagttacat tccctgggta agaatgtaac tcctttgggc ttttt 55 107 55 DNA Artificial Sequence Hairpin vector - hPTP1B H1.2-HP9 107 ctagaaaaag cccaaaggag ttacattctt acccagggaa tgtaactcct ttggg 55 108 55 RNA Artificial Sequence Hairpin vector - hPTP1B H1.2-HP9 108 uuugcccaaa ggaguuacau ucccugggua agaauguaac uccuuugggc uuuuu 55 109 55 RNA Artificial Sequence Hairpin vector - hPTP1B H1.2-HP9 109 cuagaaaaag cccaaaggag uuacauucuu acccagggaa uguaacuccu uuggg 55 110 50 DNA Artificial Sequence Hairpin vector - mPTP1B M1.1-HP4 110 tttgaagccc agaggagcta taagaatata gctcctctgg gcttcttttt 50 111 50 DNA Artificial Sequence Hairpin vector - mPTP1B M1.1-HP4 111 ctagaaaaag aagcccagag gagctatatt cttatagctc ctctgggctt 50 112 50 RNA Artificial Sequence Hairpin vector - mPTP1B M1.1-HP4 112 uuugaagccc agaggagcua uaagaauaua gcuccucugg gcuucuuuuu 50 113 50 RNA Artificial Sequence Hairpin vector - mPTP1B M1.1-HP4 113 cuagaaaaag aagcccagag gagcuauauu cuuauagcuc cucugggcuu 50 114 55 DNA Artificial Sequence Hairpin vector - mPTP1B M1.1-HP9 114 tttgaagccc agaggagcta tagggtgaga atatagctcc tctgggcttc ttttt 55 115 55 DNA Artificial Sequence Hairpin vector - mPTP1B M1.1-HP9 115 ctagaaaaag aagcccagag gagctatatt ctcaccctat agctcctctg ggctt 55 116 55 RNA Artificial Sequence Hairpin vector - mPTP1B M1.1-HP9 116 uuugaagccc agaggagcua uagggugaga auauagcucc ucugggcuuc uuuuu 55 117 55 RNA Artificial Sequence Hairpin vector - mPTP1B M1.1-HP9 117 cuagaaaaag aagcccagag gagcuauauu cucacccuau agcuccucug ggcuu 55 118 19 RNA Artificial Sequence Small interfering RNA 118 aaggaguucg agcagaucg 19 119 19 RNA Artificial Sequence Small interfering RNA 119 aggaguucga gcagaucga 19 120 19 RNA Artificial Sequence Small interfering RNA 120 ggaguucgag cagaucgac 19 121 19 RNA Artificial Sequence Small interfering RNA 121 gccagugacu ucccaugua 19 122 19 RNA Artificial Sequence Small interfering RNA 122 ccgaaauagg uacagagac 19 123 19 RNA Artificial Sequence Small interfering RNA 123 auagguacag agacgucag 19 124 19 RNA Artificial Sequence Small interfering RNA 124 uagguacaga gacgucagu 19 125 19 RNA Artificial Sequence Small interfering RNA 125 aauggaagaa gcccaaagg 19 126 19 RNA Artificial Sequence Small interfering RNA 126 auggaagaag cccaaagga 19 127 19 RNA Artificial Sequence Small interfering RNA 127 uggaagaagc ccaaaggag 19 128 19 RNA Artificial Sequence Small interfering RNA 128 gaagcccaaa ggaguuaca 19 129 19 RNA Artificial Sequence Small interfering RNA 129 gcccaaagga guuacauuc 19 130 19 RNA Artificial Sequence Small interfering RNA 130 cacaugcggu cacuuuugg 19 131 19 RNA Artificial Sequence Small interfering RNA 131 cagagugaug gagaaaggu 19 132 19 RNA Artificial Sequence Small interfering RNA 132 gguucguuaa aaugcgcac 19 133 19 RNA Artificial Sequence Small interfering RNA 133 aaugcgcaca auacuggcc 19 134 19 RNA Artificial Sequence Small interfering RNA 134 augcgcacaa uacuggcca 19 135 19 RNA Artificial Sequence Small interfering RNA 135 ugcgcacaau acuggccac 19 136 19 RNA Artificial Sequence Small interfering RNA 136 cccaagaaac ucgagagau 19 137 19 RNA Artificial Sequence Small interfering RNA 137 ucaccagccu cauucuuga 19 138 19 RNA Artificial Sequence Small interfering RNA 138 ccuucugucu ggcugauac 19 139 19 RNA Artificial Sequence Small interfering RNA 139 gaggaaagac ccuucuucc 19 140 19 RNA Artificial Sequence Small interfering RNA 140 agacccuucu uccguugau 19 141 19 RNA Artificial Sequence Small interfering RNA 141 gacccuucuu ccguugaua 19 142 19 RNA Artificial Sequence Small interfering RNA 142 augaggaagu uucggaugg 19 143 19 RNA Artificial Sequence Small interfering RNA 143 ggugccaaau ucaucaugg 19 144 19 RNA Artificial Sequence Small interfering RNA 144 ggagcuuucc cacgaggac 19 145 19 RNA Artificial Sequence Small interfering RNA 145 acgaauccug gagccacac 19 146 19 RNA Artificial Sequence Small interfering RNA 146 cgaauccugg agccacaca 19 147 19 RNA Artificial Sequence Small interfering RNA 147 uccuggagcc acacaaugg 19 148 19 RNA Artificial Sequence Small interfering RNA 148 ugggaaaugc agggaguuc 19 149 19 RNA Artificial Sequence Small interfering RNA 149 ggaagagacc caggaggau 19 150 19 RNA Artificial Sequence Small interfering RNA 150 gagacccagg aggauaaag 19 151 19 RNA Artificial Sequence Small interfering RNA 151 agacugcccc aucaaggaa 19 152 19 RNA Artificial Sequence Small interfering RNA 152 gacugcccca ucaaggaag 19 153 19 RNA Artificial Sequence Small interfering RNA 153 ggaagaaaaa ggaagcccc 19 154 19 RNA Artificial Sequence Small interfering RNA 154 aaggaagccc cuuaaaugc 19 155 19 RNA Artificial Sequence Small interfering RNA 155 aggaagcccc uuaaaugcc 19 156 19 RNA Artificial Sequence Small interfering RNA 156 ggaagccccu uaaaugccg 19 157 19 RNA Artificial Sequence Small interfering RNA 157 gccccuuaaa ugccgcacc 19 158 19 RNA Artificial Sequence Small interfering RNA 158 augccgcacc cuacggcau 19 159 19 RNA Artificial Sequence Small interfering RNA 159 agcaugaguc aagacacug 19 160 19 RNA Artificial Sequence Small interfering RNA 160 gcaugaguca agacacuga 19 161 19 RNA Artificial Sequence Small interfering RNA 161 ggacgaggac caugcacug 19 162 19 RNA Artificial Sequence Small interfering RNA 162 gcccuuccug gucaacaug 19 163 19 RNA Artificial Sequence Small interfering RNA 163 caugugcgug gcuacgguc 19 164 19 RNA Artificial Sequence Small interfering RNA 164 cagcaacaca uagccugac 19 165 19 RNA Artificial Sequence Small interfering RNA 165 cacauagccu gacccuccu 19 166 19 RNA Artificial Sequence Small interfering RNA 166 aacccaucuu ccccggaug 19 167 19 RNA Artificial Sequence Small interfering RNA 167 acccaucuuc cccggaugu 19 168 19 RNA Artificial Sequence Small interfering RNA 168 cccaucuucc ccggaugug 19 169 19 RNA Artificial Sequence Small interfering RNA 169 agagaguacc augcuggcg 19 170 19 RNA Artificial Sequence Small interfering RNA 170 gagaguacca ugcuggcgg 19 171 19 RNA Artificial Sequence Small interfering RNA 171 cagccccccc cuugaaucu 19 172 19 RNA Artificial Sequence Small interfering RNA 172 aggcauccau agugcacua 19 173 19 RNA Artificial Sequence Small interfering RNA 173 ggcauccaua gugcacuag 19 174 19 RNA Artificial Sequence Small interfering RNA 174 ggaggacggu uguaagcag 19 175 19 RNA Artificial Sequence Small interfering RNA 175 ucacugcucc cccgugugu 19 176 19 RNA Artificial Sequence Small interfering RNA 176 ggucuucuug uguccugau 19 177 19 RNA Artificial Sequence Small interfering RNA 177 ugugccccau guccaaguc 19 178 19 RNA Artificial Sequence Small interfering RNA 178 guccaaccug ccugugcau 19 179 19 RNA Artificial Sequence Small interfering RNA 179 ccugccugug caugaccug 19 180 19 RNA Artificial Sequence Small interfering RNA 180 gccuguugcu gaagucauu 19 181 19 RNA Artificial Sequence Small interfering RNA 181 gucauugucg cucagcaau 19 182 19 RNA Artificial Sequence Small interfering RNA 182 uuccuggcau gacacucua 19 183 19 RNA Artificial Sequence Small interfering RNA 183 gccauauuca caccucacg 19 184 19 RNA Artificial Sequence Small interfering RNA 184 gucaacacuc uucuugagc 19 185 19 RNA Artificial Sequence Small interfering RNA 185 cacucuucuu gagcagacc 19 186 19 RNA Artificial Sequence Small interfering RNA 186 gagaggcacc ugcuggaaa 19 187 19 RNA Artificial Sequence Small interfering RNA 187 ccacacuucu ugaaacagc 19 188 19 RNA Artificial Sequence Small interfering RNA 188 gaccuccaca uuaaguggc 19 189 19 RNA Artificial Sequence Small interfering RNA 189 caugaaaaac acggcagcu 19 190 19 RNA Artificial Sequence Small interfering RNA 190 aaacacggca gcuguagcu 19 191 19 RNA Artificial Sequence Small interfering RNA 191 aacacggcag cuguagcuc 19 192 19 RNA Artificial Sequence Small interfering RNA 192 acacggcagc uguagcucc 19 193 19 RNA Artificial Sequence Small interfering RNA 193 cauucgaggu gucacccug 19 194 19 RNA Artificial Sequence Small interfering RNA 194 ggcuuaggug ccaggcugu 19 195 19 RNA Artificial Sequence Small interfering RNA 195 uggacguacu gguuuaacc 19 196 19 RNA Artificial Sequence Small interfering RNA 196 ccuccuaucc uuggagagc 19 197 19 RNA Artificial Sequence Small interfering RNA 197 ugaagaagca gcagcggcu 19 198 19 RNA Artificial Sequence Small interfering RNA 198 ggauauccga caugaagcc 19 199 19 RNA Artificial Sequence Small interfering RNA 199 ugaagccagu gacuuccca 19 200 19 RNA Artificial Sequence Small interfering RNA 200 gugacuuccc auguagagu 19 201 19 RNA Artificial Sequence Small interfering RNA 201 uguagugugg ccaagcuuc 19 202 19 RNA Artificial Sequence Small interfering RNA 202 gagacgucag ucccuuuga 19 203 19 RNA Artificial Sequence Small interfering RNA 203 gucccuuuga ccauagucg 19 204 19 RNA Artificial Sequence Small interfering RNA 204 ugcucaacag agugaugga 19 205 19 RNA Artificial Sequence Small interfering RNA 205 acagagugau ggagaaagg 19 206 19 RNA Artificial Sequence Small interfering RNA 206 gagugaugga gaaagguuc 19 207 19 RNA Artificial Sequence Small interfering RNA 207 gugcgacagc uagaauugg 19 208 19 RNA Artificial Sequence Small interfering RNA 208 acccaagaaa cucgagaga 19 209 19 RNA Artificial Sequence Small interfering RNA 209 cuauaccaca uggccugac 19 210 19 RNA Artificial Sequence Small interfering RNA 210 cauggccuga cuuuggagu 19 211 19 RNA Artificial Sequence Small interfering RNA 211 uggccugacu uuggagucc 19 212 19 RNA Artificial Sequence Small interfering RNA 212 ccagccucau ucuugaacu 19 213 19 RNA Artificial Sequence Small interfering RNA 213 ggcaucggca ggucuggaa 19 214 19 RNA Artificial Sequence Small interfering RNA 214 ucggcagguc uggaaccuu 19 215 19 RNA Artificial Sequence Small interfering RNA 215 ggucuggaac cuucugucu 19 216 19 RNA Artificial Sequence Small interfering RNA 216 agaggaaaga cccuucuuc 19 217 19 RNA Artificial Sequence Small interfering RNA 217 gcugcgcuuc uccuaccug 19 218 19 RNA Artificial Sequence Small interfering RNA 218 ggaucagugg aaggagcuu 19 219 19 RNA Artificial Sequence Small interfering RNA 219 guggaaggag cuuucccac 19 220 19 RNA Artificial Sequence Small interfering RNA 220 cccaaacgaa uccuggagc 19 221 19 RNA Artificial Sequence Small interfering RNA 221 aacgaauccu ggagccaca 19 222 19 RNA Artificial Sequence Small interfering RNA 222 cacaauggga aaugcaggg 19 223 19 RNA Artificial Sequence Small interfering RNA 223 caaugggaaa ugcagggag 19 224 19 RNA Artificial Sequence Small interfering RNA 224 augggaaaug cagggaguu 19 225 19 RNA Artificial Sequence Small interfering RNA 225 ggaggauaaa gacugcccc 19 226 19 RNA Artificial Sequence Small interfering RNA 226 aggaagaaaa aggaagccc 19 227 19 RNA Artificial Sequence Small interfering RNA 227 cccuacggca ucgaaagca 19 228 19 RNA Artificial Sequence Small interfering RNA 228 ugcacugagu uacuggaag 19 229 19 RNA Artificial Sequence Small interfering RNA 229 cugaguuacu ggaagcccu 19 230 19 RNA Artificial Sequence Small interfering RNA 230 acaugugcgu ggcuacggu 19 231 19 RNA Artificial Sequence Small interfering RNA 231 ugugcguggc uacgguccu 19 232 19 RNA Artificial Sequence Small interfering RNA 232 gguuccuguu caacagcaa 19 233 19 RNA Artificial Sequence Small interfering RNA 233 acagcaacac auagccuga 19 234 19 RNA Artificial Sequence Small interfering RNA 234 gcaacacaua gccugaccc 19 235 19 RNA Artificial Sequence Small interfering RNA 235 acacauagcc ugacccucc 19 236 19 RNA Artificial Sequence Small interfering RNA 236 cauagccuga cccuccucc 19 237 19 RNA Artificial Sequence Small interfering RNA 237 uagccugacc cuccuccac 19 238 19 RNA Artificial Sequence Small interfering RNA 238 cuccaccucc acccacugu 19 239 19 RNA Artificial Sequence Small interfering RNA 239 ggcaugccgc gguagguaa 19 240 19 RNA Artificial Sequence Small interfering RNA 240 cuaaaaccca ucuuccccg 19 241 19 RNA Artificial Sequence Small interfering RNA 241 ucuuccccgg auguguguc 19 242 19 RNA Artificial Sequence Small interfering RNA 242 acagcccccc ccuugaauc 19 243 19 RNA Artificial Sequence Small interfering RNA 243 aaggcaucca uagugcacu 19 244 19 RNA Artificial Sequence Small interfering RNA 244 aucacugcuc ccccgugug 19 245 19 RNA Artificial Sequence Small interfering RNA 245 cugcuccccc guguguauu 19 246 19 RNA Artificial Sequence Small interfering RNA 246 uguccaaguc caaccugcc 19 247 19 RNA Artificial Sequence Small interfering RNA 247 aguccaaccu gccugugca 19 248 19 RNA Artificial Sequence Small interfering RNA 248 accugccugu gcaugaccu 19 249 19 RNA Artificial Sequence Small interfering RNA 249 uuacauggcu gugguuccu 19 250 19 RNA Artificial Sequence Small interfering RNA 250 uggcuguggu uccuaagcc 19 251 19 RNA Artificial Sequence Small interfering RNA 251 ugacacucua gugacuucc 19 252 19 RNA Artificial Sequence Small interfering RNA 252 cucuagugac uuccuggug 19 253 19 RNA Artificial Sequence Small interfering RNA 253 gccuguccug guacagcag 19 254 19 RNA Artificial Sequence Small interfering RNA 254 uauucacacc ucacgcucu 19 255 19 RNA Artificial Sequence Small interfering RNA 255 caccucacgc ucuggacau 19 256 19 RNA Artificial Sequence Small interfering RNA 256 ccucacgcuc uggacauga 19 257 19 RNA Artificial Sequence Small interfering RNA 257 cgcucuggag augauuuag 19 258 19 RNA Artificial Sequence Small interfering RNA 258 gccuccgcca uuccaaguc 19 259 19 RNA Artificial Sequence Small interfering RNA 259 acacucuucu ugagcagac 19 260 19 RNA Artificial Sequence Small interfering RNA 260 cucuucuuga gcagaccgu 19 261 19 RNA Artificial Sequence Small interfering RNA 261 gaccgugauu uggaagaga 19 262 19 RNA Artificial Sequence Small interfering RNA 262 ccugcuggaa accacacuu 19 263 19 RNA Artificial Sequence Small interfering RNA 263 cacuucuuga aacagccug 19 264 19 RNA Artificial Sequence Small interfering RNA 264 ugaaaaacac ggcagcugu 19 265 19 RNA Artificial Sequence Small interfering RNA 265 gcuguagcuc ccgagcuac 19 266 19 RNA Artificial Sequence Small interfering RNA 266 cauuuugccu uucucgugg 19 267 19 RNA Artificial Sequence Small interfering RNA 267 uucgaggugu cacccugca 19 268 19 RNA Artificial Sequence Small interfering RNA 268 cccugcagag cuaugguga 19 269 19 RNA Artificial Sequence Small interfering RNA 269 gagcuauggu gaggugugg 19 270 19 RNA Artificial Sequence Small interfering RNA 270 ggcuguaagc auucugagc 19 271 19 RNA Artificial Sequence Small interfering RNA 271 gcuggcucuc caccuuguu 19 272 19 RNA Artificial Sequence Small interfering RNA 272 ccaggauauc cgacaugaa 19 273 19 RNA Artificial Sequence Small interfering RNA 273 uccgacauga agccaguga 19 274 19 RNA Artificial Sequence Small interfering RNA 274 aaaccgaaau agguacaga 19 275 19 RNA Artificial Sequence Small interfering RNA 275 gagacgucag ucccuuuga 19 276 19 RNA Artificial Sequence Small interfering RNA 276 uuaacauuga ucucugaag 19 277 19 RNA Artificial Sequence Small interfering RNA 277 uuauacagug cgacagcua 19 278 19 RNA Artificial Sequence Small interfering RNA 278 gugcgacagc uagaauugg 19 279 19 RNA Artificial Sequence Small interfering RNA 279 agaaacucga gagaucuua 19 280 19 RNA Artificial Sequence Small interfering RNA 280 gaaacucgag agaucuuac 19 281 19 RNA Artificial Sequence Small interfering RNA 281 aacucgagag aucuuacau 19 282 19 RNA Artificial Sequence Small interfering RNA 282 acucgagaga ucuuacauu 19 283 19 RNA Artificial Sequence Small interfering RNA 283 cucgagagau cuuacauuu 19 284 19 RNA Artificial Sequence Small interfering RNA 284 ucuuacauuu ccacuauac 19 285 19 RNA Artificial Sequence Small interfering RNA 285 ggcaucggca ggucuggaa 19 286 19 RNA Artificial Sequence Small interfering RNA 286 agacccuucu uccguugau 19 287 19 RNA Artificial Sequence Small interfering RNA 287 gacccuucuu ccguugaua 19 288 19 RNA Artificial Sequence Small interfering RNA 288 cccuucuucc guugauauc 19 289 19 RNA Artificial Sequence Small interfering RNA 289 agaaagugcu guuagaaau 19 290 19 RNA Artificial Sequence Small interfering RNA 290 gaaagugcug uuagaaaug 19 291 19 RNA Artificial Sequence Small interfering RNA 291 cgaauccugg agccacaca 19 292 19 RNA Artificial Sequence Small interfering RNA 292 ugagucaaga cacugaagu 19 293 19 RNA Artificial Sequence Small interfering RNA 293 gaaggacgag gaccaugca 19 294 19 RNA Artificial Sequence Small interfering RNA 294 auaaauccuc agguaguac 19 295 19 RNA Artificial Sequence Small interfering RNA 295 uaaauccuca gguaguacu 19 296 19 RNA Artificial Sequence Small interfering RNA 296 gguaguacug ggaauggaa 19 297 19 RNA Artificial Sequence Small interfering RNA 297 aggaggacgg uuguaagca 19 298 19 RNA Artificial Sequence Small interfering RNA 298 ggacgguugu aagcaguug 19 299 19 RNA Artificial Sequence Small interfering RNA 299 uauugugggu aacgugaga 19 300 19 RNA Artificial Sequence Small interfering RNA 300 augaacacgu ggguauuua 19 301 19 RNA Artificial Sequence Small interfering RNA 301 caugauguga gauuacuuu 19 302 19 RNA Artificial Sequence Small interfering RNA 302 gauuacuuug ucccgcuua 19 303 19 RNA Artificial Sequence Small interfering RNA 303 uuacuuuguc ccgcuuauu 19 304 19 RNA Artificial Sequence Small interfering RNA 304 gaucuaguuc ucaaucacu 19 305 19 RNA Artificial Sequence Small interfering RNA 305 gaaugcaugu aaggucuuc 19 306 19 RNA Artificial Sequence Small interfering RNA 306 ugcauguaag gucuucuug 19 307 18 RNA Artificial Sequence Small interfering RNA 307 ucauuacaug gcuguggu 18 308 19 RNA Artificial Sequence Small interfering RNA 308 uuccuggcau gacacucua 19 309 19 RNA Artificial Sequence Small interfering RNA 309 cgcucuggac augauuuag 19 310 19 RNA Artificial Sequence Small interfering RNA 310 ucagccuccg ccauuccaa 19 311 19 RNA Artificial Sequence Small interfering RNA 311 gccuccgcca uuccaaguc 19 312 19 RNA Artificial Sequence Small interfering RNA 312 gaccgugauu uggaagaga 19 313 19 RNA Artificial Sequence Small interfering RNA 313 acugaagacc uccacauua 19 314 19 RNA Artificial Sequence Small interfering RNA 314 gcuacucucu ugccagcau 19 315 19 RNA Artificial Sequence Small interfering RNA 315 uggugaggug uggauaagg 19 316 19 RNA Artificial Sequence Small interfering RNA 316 ggugccaggc uguaagcau 19 317 19 RNA Artificial Sequence Small interfering RNA 317 uaugccuuaa gccaauauu 19 318 19 RNA Artificial Sequence Small interfering RNA 318 uuuacucauc aggucauua 19 319 19 RNA Artificial Sequence Small interfering RNA 319 ccaaacggac aacccauag 19 320 19 RNA Artificial Sequence Small interfering RNA 320 acggacaacc cauaguacc 19 321 19 RNA Artificial Sequence Small interfering RNA 321 cggacaaccc auaguaccc 19 322 19 RNA Artificial Sequence Small interfering RNA 322 cccauaguac ccgaagaca 19 323 19 RNA Artificial Sequence Small interfering RNA 323 ccagacaauc guaagcuug 19 324 19 RNA Artificial Sequence Small interfering RNA 324 gcuugauggu guuuucccu 19 325 19 RNA Artificial Sequence Small interfering RNA 325 gcaucucaug aaugucagc 19 326 19 RNA Artificial Sequence Small interfering RNA 326 ugucagccaa auuccguac 19 327 19 RNA Artificial Sequence Small interfering RNA 327 auuccguaca guucggugc 19 328 19 RNA Artificial Sequence Small interfering RNA 328 uuccguacag uucggugcg 19 329 19 RNA Artificial Sequence Small interfering RNA 329 cgaaacaccu ccuguacca 19 330 19 RNA Artificial Sequence Small interfering RNA 330 acaccuccug uaccagguu 19 331 19 RNA Artificial Sequence Small interfering RNA 331 caccuccugu accagguuc 19 332 19 RNA Artificial Sequence Small interfering RNA 332 cuucagaauc auccaggcu 19 333 19 RNA Artificial Sequence Small interfering RNA 333 ucauccaggc uucaucaug 19 334 19 RNA Artificial Sequence Small interfering RNA 334 ggugagagcc accacagag 19 335 19 RNA Artificial Sequence Small interfering RNA 335 cugguaggcu gaacccaug 19 336 19 RNA Artificial Sequence Small interfering RNA 336 cccaugcuga agcuccacc 19 337 19 RNA Artificial Sequence Small interfering RNA 337 caugcagaag ccgcugcug 19 338 19 RNA Artificial Sequence Small interfering RNA 338 ggaguucgag gagaucgac 19 339 19 RNA Artificial Sequence Small interfering RNA 339 gccagcgacu ucccaugca 19 340 19 RNA Artificial Sequence Small interfering RNA 340 agucgcgaag cuuccuaag 19 341 19 RNA Artificial Sequence Small interfering RNA 341 gucgcgaagc uuccuaaga 19 342 19 RNA Artificial Sequence Small interfering RNA 342 gaacaaaaac cggaacagg 19 343 19 RNA Artificial Sequence Small interfering RNA 343 caaaaaccgg aacagguac 19 344 19 RNA Artificial Sequence Small interfering RNA 344 aaaccggaac agguaccga 19 345 19 RNA Artificial Sequence Small interfering RNA 345 aaccggaaca gguaccgag 19 346 19 RNA Artificial Sequence Small interfering RNA 346 accggaacag guaccgaga 19 347 19 RNA Artificial Sequence Small interfering RNA 347 ccggaacagg uaccgagau 19 348 19 RNA Artificial Sequence Small interfering RNA 348 cagguaccga gaugucagc 19 349 19 RNA Artificial Sequence Small interfering RNA 349 aaauggaaga agcccagag 19 350 19 RNA Artificial Sequence Small interfering RNA 350 aauggaagaa gcccagagg 19 351 19 RNA Artificial Sequence Small interfering RNA 351 auggaagaag cccagagga 19 352 19 RNA Artificial Sequence Small interfering RNA 352 uggaagaagc ccagaggag 19 353 19 RNA Artificial Sequence Small interfering RNA 353 gaagcccaga ggagcuaua 19 354 19 RNA Artificial Sequence Small interfering RNA 354 gcccagagga gcuauauuc 19 355 19 RNA Artificial Sequence Small interfering RNA 355 ccgcaucaug gagaaaggc 19 356 19 RNA Artificial Sequence Small interfering RNA 356 aggcucguua aaaugugcc 19 357 19 RNA Artificial Sequence Small interfering RNA 357 ggcucguuaa aaugugccc 19 358 19 RNA Artificial Sequence Small interfering RNA 358 aaugugccca guauuggcc 19 359 19 RNA Artificial Sequence Small interfering RNA 359 augugcccag uauuggcca 19 360 19 RNA Artificial Sequence Small interfering RNA 360 ugugcccagu auuggccac 19 361 19 RNA Artificial Sequence Small interfering RNA 361 ggagaugguc uuugaugac 19 362 19 RNA Artificial Sequence Small interfering RNA 362 aaccugacua ccaaggaga 19 363 19 RNA Artificial Sequence Small interfering RNA 363 accugacuac caaggagac 19 364 19 RNA Artificial Sequence Small interfering RNA 364 ccugacuacc aaggagacu 19 365 19 RNA Artificial Sequence Small interfering RNA 365 ggagacucga gagauccug 19 366 19 RNA Artificial Sequence Small interfering RNA 366 aguccgagag ucaggcuca 19 367 19 RNA Artificial Sequence Small interfering RNA 367 guccgagagu caggcucac 19 368 19 RNA Artificial Sequence Small interfering RNA 368 gaggaaagac ccaucuucc 19 369 19 RNA Artificial Sequence Small interfering RNA 369 agacccaucu uccguggac 19 370 19 RNA Artificial Sequence Small interfering RNA 370 gacccaucuu ccguggaca 19 371 19 RNA Artificial Sequence Small interfering RNA 371 gaaaguacug cuggagaug 19 372 19 RNA Artificial Sequence Small interfering RNA 372 aguacugcug gagaugcgc 19 373 19 RNA Artificial Sequence Small interfering RNA 373 guacugcugg agaugcgca 19 374 19 RNA Artificial Sequence Small interfering RNA 374 acgcacacug gagccucac 19 375 19 RNA Artificial Sequence Small interfering RNA 375 cgcacacugg agccucaca 19 376 19 RNA Artificial Sequence Small interfering RNA 376 gugcaaggag cucuucucc 19 377 19 RNA Artificial Sequence Small interfering RNA 377 ggagcucuuc uccagccac 19 378 19 RNA Artificial Sequence Small interfering RNA 378 ggcagagccc agucaagug 19 379 19 RNA Artificial Sequence Small interfering RNA 379 gugccaugca cagcgugag 19 380 19 RNA Artificial Sequence Small interfering RNA 380 guuaggagac ggauggugg 19 381 19 RNA Artificial Sequence Small interfering RNA 381 agugcucagg cgucugucc 19 382 19 RNA Artificial Sequence Small interfering RNA 382 gagcuguccu ccacugagg 19 383 19 RNA Artificial Sequence Small interfering RNA 383 cacaaggcac auuggccaa 19 384 19 RNA Artificial Sequence Small interfering RNA 384 ggcacauugg ccaagucac 19 385 19 RNA Artificial Sequence Small interfering RNA 385 gucacuggaa gcccuuccu 19 386 19 RNA Artificial Sequence Small interfering RNA 386 gcccuuccug gucaaugug 19 387 19 RNA Artificial Sequence Small interfering RNA 387 ugugugcaug gccacgcuc 19 388 19 RNA Artificial Sequence Small interfering RNA 388 caacaacucg caagccugc 19 389 19 RNA Artificial Sequence Small interfering RNA 389 caacucgcaa gccugcucu 19 390 19 RNA Artificial Sequence Small interfering RNA 390 cucgcaagcc ugcucugga 19 391 19 RNA Artificial Sequence Small interfering RNA 391 gccugcucug gaacuggaa 19 392 19 RNA Artificial Sequence Small interfering RNA 392 ccuguucagg agaaguaga 19 393 19 RNA Artificial Sequence Small interfering RNA 393 uacucuucuu gcucucacc 19 394 19 RNA Artificial Sequence Small interfering RNA 394 cauuuauaaa ggcaggccc 19 395 19 RNA Artificial Sequence Small interfering RNA 395 cgggaagugc aaggagcuc 19 396 19 RNA Artificial Sequence Small interfering RNA 396 ugacuauauc aaugccagc 19 397 19 RNA Artificial Sequence Small interfering RNA 397 cauuccuagu uagcagugc 19 398 19 RNA Artificial Sequence Small interfering RNA 398 gugcauacuc aucagacug 19 399 19 RNA Artificial Sequence Small interfering RNA 399 aacggacaac ccauaguac 19 400 19 RNA Artificial Sequence Small interfering RNA 400 acccauagua cccgaagac 19 401 19 RNA Artificial Sequence Small interfering RNA 401 gccaaauucc guacaguuc 19 402 19 RNA Artificial Sequence Small interfering RNA 402 aauuccguac aguucggug 19 403 19 RNA Artificial Sequence Small interfering RNA 403 guucggugcg gauccgaac 19 404 19 RNA Artificial Sequence Small interfering RNA 404 ccuccuguac cagguuccc 19 405 19 RNA Artificial Sequence Small interfering RNA 405 gguucccgug ucgcucuca 19 406 19 RNA Artificial Sequence Small interfering RNA 406 gaaucaucca ggcuucauc 19 407 19 RNA Artificial Sequence Small interfering RNA 407 ggcuucauca uguuuuccc 19 408 19 RNA Artificial Sequence Small interfering RNA 408 ucauguuuuc ccaccucca 19 409 19 RNA Artificial Sequence Small interfering RNA 409 uguuuuccca ccuccagca 19 410 19 RNA Artificial Sequence Small interfering RNA 410 ccuccagcaa gaaccgagg 19 411 19 RNA Artificial Sequence Small interfering RNA 411 ugaaggugag agccaccac 19 412 19 RNA Artificial Sequence Small interfering RNA 412 ccacagagga gacgcaugg 19 413 19 RNA Artificial Sequence Small interfering RNA 413 cagacgauga cgaagacgc 19 414 19 RNA Artificial Sequence Small interfering RNA 414 gacgaugacg aagacgcgc 19 415 19 RNA Artificial Sequence Small interfering RNA 415 cguguggaac ugguaggcu 19 416 19 RNA Artificial Sequence Small interfering RNA 416 ugcugaagcu ccacccgua 19 417 19 RNA Artificial Sequence Small interfering RNA 417 ggcauggcgg aggcuagau 19 418 19 RNA Artificial Sequence Small interfering RNA 418 uccagaacau gcagaagcc 19 419 19 RNA Artificial Sequence Small interfering RNA 419 gaacaugcag aagccgcug 19 420 19 RNA Artificial Sequence Small interfering RNA 420 uggagaugga gaaggaguu 19 421 19 RNA Artificial Sequence Small interfering RNA 421 ggacauucga caugaagcc 19 422 19 RNA Artificial Sequence Small interfering RNA 422 uucgacauga agccagcga 19 423 19 RNA Artificial Sequence Small interfering RNA 423 ugaagccagc gacuuccca 19 424 19 RNA Artificial Sequence Small interfering RNA 424 gcgacuuccc augcaaagu 19 425 19 RNA Artificial Sequence Small interfering RNA 425 ugcaaagucg cgaagcuuc 19 426 19 RNA Artificial Sequence Small interfering RNA 426 aagucgcgaa gcuuccuaa 19 427 19 RNA Artificial Sequence Small interfering RNA 427 aaaaccggaa cagguaccg 19 428 19 RNA Artificial Sequence Small interfering RNA 428 gguaccgaga ugucagccc 19 429 19 RNA Artificial Sequence Small interfering RNA 429 gcccuuuuga ccacagucg 19 430 19 RNA Artificial Sequence Small interfering RNA 430 gaggagcuau auucucacc 19 431 19 RNA Artificial Sequence Small interfering RNA 431 ugcucaaccg caucaugga 19 432 19 RNA Artificial Sequence Small interfering RNA 432 accgcaucau ggagaaagg 19 433 19 RNA Artificial Sequence Small interfering RNA 433 ucauggagaa aggcucguu 19 434 19 RNA Artificial Sequence Small interfering RNA 434 guauuggcca cagcaagaa 19 435 19 RNA Artificial Sequence Small interfering RNA 435 caguacgaca guuggaguu 19 436 19 RNA Artificial Sequence Small interfering RNA 436 guacgacagu uggaguugg 19 437 19 RNA Artificial Sequence Small interfering RNA 437 guuggaguug gaaaaccug 19 438 19 RNA Artificial Sequence Small interfering RNA 438 aggagacucg agagauccu 19 439 19 RNA Artificial Sequence Small interfering RNA 439 uuuccacuac accacaugg 19 440 19 RNA Artificial Sequence Small interfering RNA 440 cuacaccaca uggccugac 19 441 19 RNA Artificial Sequence Small interfering RNA 441 ccacauggcc ugacuuugg 19 442 19 RNA Artificial Sequence Small interfering RNA 442 cauggccuga cuuuggagu 19 443 19 RNA Artificial Sequence Small interfering RNA 443 uggccugacu uuggagucc 19 444 19 RNA Artificial Sequence Small interfering RNA 444 ccggcuucuu uccucaauu 19 445 19 RNA Artificial Sequence Small interfering RNA 445 aaguccgaga gucaggcuc 19 446 19 RNA Artificial Sequence Small interfering RNA 446 uggccccauu gugguccac 19 447 19 RNA Artificial Sequence Small interfering RNA 447 uuguggucca cugcagcgc 19 448 19 RNA Artificial Sequence Small interfering RNA 448 ccugccucuu acugaugga 19 449 19 RNA Artificial Sequence Small interfering RNA 449 agaggaaaga cccaucuuc 19 450 19 RNA Artificial Sequence Small interfering RNA 450 ucuuccgugg acaucaaga 19 451 19 RNA Artificial Sequence Small interfering RNA 451 uccagacugc cgaccagcu 19 452 19 RNA Artificial Sequence Small interfering RNA 452 gcugcgcuuc uccuaccug 19 453 19 RNA Artificial Sequence Small interfering RNA 453 gugcaggauc aguggaagg 19 454 19 RNA Artificial Sequence Small interfering RNA 454 ggaucagugg aaggagcuc 19 455 19 RNA Artificial Sequence Small interfering RNA 455 cccaaacgca cacuggagc 19 456 19 RNA Artificial Sequence Small interfering RNA 456 aacgcacacu ggagccuca 19 457 19 RNA Artificial Sequence Small interfering RNA 457 cacuggagcc ucacaacgg 19 458 19 RNA Artificial Sequence Small interfering RNA 458 aggagcucuu cuccagcca 19 459 19 RNA Artificial Sequence Small interfering RNA 459 gagaggaagg cagagccca 19 460 19 RNA Artificial Sequence Small interfering RNA 460 gagcccaguc aagugccau 19 461 19 RNA Artificial Sequence Small interfering RNA 461 gucaagugcc augcacagc 19 462 19 RNA Artificial Sequence Small interfering RNA 462 agugccaugc acagcguga 19 463 19 RNA Artificial Sequence Small interfering RNA 463 ugcacagcgu gagcagcau 19 464 19 RNA Artificial Sequence Small interfering RNA 464 cagcgugagc agcaugagu 19 465 19 RNA Artificial Sequence Small interfering RNA 465 gcgugagcag caugagucc 19 466 19 RNA Artificial Sequence Small interfering RNA 466 gcaugagucc agacacuga 19 467 19 RNA Artificial Sequence Small interfering RNA 467 ugaguccaga cacugaagu 19 468 19 RNA Artificial Sequence Small interfering RNA 468 gacacugaag uuaggagac 19 469 19 RNA Artificial Sequence Small interfering RNA 469 cugaaguuag gagacggau 19 470 19 RNA Artificial Sequence Small interfering RNA 470 aagugcucag gcgucuguc 19 471 19 RNA Artificial Sequence Small interfering RNA 471 ccgaggaaga gcuguccuc 19 472 19 RNA Artificial Sequence Small interfering RNA 472 cugaggagga acacaaggc 19 473 19 RNA Artificial Sequence Small interfering RNA 473 caaggcacau uggccaagu 19 474 19 RNA Artificial Sequence Small interfering RNA 474 aggcacauug gccaaguca 19 475 19 RNA Artificial Sequence Small interfering RNA 475 cauuggccaa gucacugga 19 476 19 RNA Artificial Sequence Small interfering RNA 476 uuggccaagu cacuggaag 19 477 19 RNA Artificial Sequence Small interfering RNA 477 agucacugga agcccuucc 19 478 19 RNA Artificial Sequence Small interfering RNA 478 cuggaagccc uuccugguc 19 479 19 RNA Artificial Sequence Small interfering RNA 479 augugugcau ggccacgcu 19 480 19 RNA Artificial Sequence Small interfering RNA 480 ccggcgcgua cuugugcua 19 481 19 RNA Artificial Sequence Small interfering RNA 481 cugccacugc ccagcuuag 19 482 19 RNA Artificial Sequence Small interfering RNA 482 cugcccagcu uaggaugcg 19 483 19 RNA Artificial Sequence Small interfering RNA 483 gcuuaggaug cggucugcg 19 484 19 RNA Artificial Sequence Small interfering RNA 484 acaacucgca agccugcuc 19 485 19 RNA Artificial Sequence Small interfering RNA 485 acucgcaagc cugcucugg 19 486 19 RNA Artificial Sequence Small interfering RNA 486 agccugcucu ggaacugga 19 487 19 RNA Artificial Sequence Small interfering RNA 487 ggagaaguag aggaaaugc 19 488 19 RNA Artificial Sequence Small interfering RNA 488 ccucacuccu ccccuuucu 19 489 19 RNA Artificial Sequence Small interfering RNA 489 cuccuccccu uucucugau 19 490 19 RNA Artificial Sequence Small interfering RNA 490 uuuauaaagg caggcccga 19 491 19 RNA Artificial Sequence Small interfering RNA 491 gguaccgaga ugucagccc 19 492 19 RNA Artificial Sequence Small interfering RNA 492 agaagaaaag gagaugguc 19 493 19 RNA Artificial Sequence Small interfering RNA 493 gacugccgac cagcugcgc 19 494 19 RNA Artificial Sequence Small interfering RNA 494 accaaacgga caacccaua 19 495 19 RNA Artificial Sequence Small interfering RNA 495 ccaaacggac aacccauag 19 496 19 RNA Artificial Sequence Small interfering RNA 496 accagacaau cguaagcuu 19 497 19 RNA Artificial Sequence Small interfering RNA 497 gacaaucgua agcuugaug 19 498 19 RNA Artificial Sequence Small interfering RNA 498 caaucguaag cuugauggu 19 499 19 RNA Artificial Sequence Small interfering RNA 499 ucguaagcuu gaugguguu 19 500 19 RNA Artificial Sequence Small interfering RNA 500 guugaagcau cucaugaau 19 501 19 RNA Artificial Sequence Small interfering RNA 501 gccaaauucc guacaguuc 19 502 19 RNA Artificial Sequence Small interfering RNA 502 gguucccgug ucgcucuca 19 503 19 RNA Artificial Sequence Small interfering RNA 503 ggcugaaccc augcugaag 19 504 19 RNA Artificial Sequence Small interfering RNA 504 ggcauggcgg aggcuagau 19 505 19 RNA Artificial Sequence Small interfering RNA 505 ggcuagaugc cgccaauca 19 506 19 RNA Artificial Sequence Small interfering RNA 506 ucgacaaggc ugggaacug 19 507 19 RNA Artificial Sequence Small interfering RNA 507 aagucgcgaa gcuuccuaa 19 508 19 RNA Artificial Sequence Small interfering RNA 508 agucgcgaag cuuccuaag 19 509 19 RNA Artificial Sequence Small interfering RNA 509 gucgcgaagc uuccuaaga 19 510 19 RNA Artificial Sequence Small interfering RNA 510 ccggaacagg uaccgagau 19 511 19 RNA Artificial Sequence Small interfering RNA 511 ccacagucgg auuaaauug 19 512 19 RNA Artificial Sequence Small interfering RNA 512 aauugcacca ggaagauaa 19 513 19 RNA Artificial Sequence Small interfering RNA 513 auugcaccag gaagauaau 19 514 19 RNA Artificial Sequence Small interfering RNA 514 gaagcccaga ggagcuaua 19 515 19 RNA Artificial Sequence Small interfering RNA 515 agcccagagg agcuauauu 19 516 19 RNA Artificial Sequence Small interfering RNA 516 ugcucaaccg caucaugga 19 517 19 RNA Artificial Sequence Small interfering RNA 517 accgcaucau ggagaaagg 19 518 19 RNA Artificial Sequence Small interfering RNA 518 ucauggagaa aggcucguu 19 519 19 RNA Artificial Sequence Small interfering RNA 519 uggagaaagg cucguuaaa 19 520 19 RNA Artificial Sequence Small interfering RNA 520 gauggucuuu gaugacaca 19 521 19 RNA Artificial Sequence Small interfering RNA 521 gguuugaagu ugacacuaa 19 522 19 RNA Artificial Sequence Small interfering RNA 522 ucucugaaga ugucaaguc 19 523 19 RNA Artificial Sequence Small interfering RNA 523 gucauauuac acaguacga 19 524 19 RNA Artificial Sequence Small interfering RNA 524 caguacgaca guuggaguu 19 525 19 RNA Artificial Sequence Small interfering RNA 525 ccugacuacc aaggagacu 19 526 19 RNA Artificial Sequence Small interfering RNA 526 gacucgagag auccugcau 19 527 19 RNA Artificial Sequence Small interfering RNA 527 uccugcauuu ccacuacac 19 528 19 RNA Artificial Sequence Small interfering RNA 528 cugauggaca agaggaaag 19 529 19 RNA Artificial Sequence Small interfering RNA 529 cccaucuucc guggacauc 19 530 19 RNA Artificial Sequence Small interfering RNA 530 ucaugggcga cucgucagu 19 531 19 RNA Artificial Sequence Small interfering RNA 531 cucgucagug caggaucag 19 532 19 RNA Artificial Sequence Small interfering RNA 532 cgcacacugg agccucaca 19 533 19 RNA Artificial Sequence Small interfering RNA 533 cagcgugagc agcaugagu 19 534 19 RNA Artificial Sequence Small interfering RNA 534 gcaugagucc agacacuga 19 535 19 RNA Artificial Sequence Small interfering RNA 535 uaaaggcagg cccgaauuc 19 536 19 RNA Artificial Sequence Small interefering RNA 536 ucgauaaggc ugggaacug 19 537 19 RNA Artificial Sequence Small interefering RNA 537 gaauagcgaa acuuccuaa 19 538 19 RNA Artificial Sequence Small interefering RNA 538 auagcgaaac uuccuaaga 19 539 19 RNA Artificial Sequence Small interefering RNA 539 ccacagucgg auuaaauug 19 540 19 RNA Artificial Sequence Small interefering RNA 540 cagucggauu aaauugcau 19 541 19 RNA Artificial Sequence Small interefering RNA 541 gucggauuaa auugcauca 19 542 19 RNA Artificial Sequence Small interefering RNA 542 uugcaucagg aagauaaug 19 543 19 RNA Artificial Sequence Small interefering RNA 543 gcccagagga gcuauaucc 19 544 19 RNA Artificial Sequence Small interefering RNA 544 uccucaccca gggcccuuu 19 545 19 RNA Artificial Sequence Small interefering RNA 545 accgcaucau ggagaaagg 19 546 19 RNA Artificial Sequence Small interefering RNA 546 ucauggagaa aggcucguu 19 547 19 RNA Artificial Sequence Small interefering RNA 547 uggagaaagg cucguuaaa 19 548 19 RNA Artificial Sequence Small interefering RNA 548 guauuggcca cagaaagaa 19 549 19 RNA Artificial Sequence Small interefering RNA 549 agagaugguc uucgaugac 19 550 19 RNA Artificial Sequence Small interefering RNA 550 caccaauuug aagcugaca 19 551 19 RNA Artificial Sequence Small interefering RNA 551 ccaauuugaa gcugacacu 19 552 19 RNA Artificial Sequence Small interefering RNA 552 uuacacagua cggcaguug 19 553 19 RNA Artificial Sequence Small interefering RNA 553 caguacggca guuggaguu 19 554 19 RNA Artificial Sequence Small interefering RNA 554 ggcucgagag auccugcau 19 555 19 RNA Artificial Sequence Small interefering RNA 555 cugauggaca agaggaaag 19 556 19 RNA Artificial Sequence Small interefering RNA 556 caucaagaaa gugcuguug 19 557 19 RNA Artificial Sequence Small interefering RNA 557 gggugcaaag uucaucaug 19 558 19 RNA Artificial Sequence Small interefering RNA 558 ucaugggcga cucgucagu 19 559 19 RNA Artificial Sequence Small interefering RNA 559 cucgucagug caggaucag 19 560 19 RNA Artificial Sequence Small interefering RNA 560 cgcacauugg agccucaca 19 561 19 RNA Artificial Sequence Small interefering RNA 561 agugcaagga gcucuucuc 19 562 19 RNA Artificial Sequence Small interefering RNA 562 gcaugagcag uaugaguca 19 563 19 RNA Artificial Sequence Small interefering RNA 563 guaugaguca agacacuga 19 564 19 RNA Artificial Sequence Small interefering RNA 564 gucaagacac ugaaguuag 19 565 19 RNA Artificial Sequence Small interefering RNA 565 uggugggugg aggucuuca 19 566 19 RNA Artificial Sequence Small interefering RNA 566 aaggcacaca ggccaguuc 19 567 19 RNA Artificial Sequence Small interefering RNA 567 aggcacacag gccaguuca 19 568 19 RNA Artificial Sequence Small interefering RNA 568 ggcacacagg ccaguucac 19 569 19 RNA Artificial Sequence Small interefering RNA 569 agcccuuccu ggucaacgu 19 570 19 RNA Artificial Sequence Small interefering RNA 570 uuuggucugc ggcgucuaa 19 571 19 RNA Artificial Sequence Small interefering RNA 571 gaagaaacaa cagcuuaca 19 572 19 RNA Artificial Sequence Small interefering RNA 572 agaaacaaca gcuuacaag 19 573 19 RNA Artificial Sequence Small interefering RNA 573 gucuaaucuc agggccuua 19 574 19 RNA Artificial Sequence Small interefering RNA 574 augccaaaua cucuucuug 19 575 19 RNA Artificial Sequence Small interefering RNA 575 ucagauucac gauuuacgu 19 576 19 RNA Artificial Sequence Small interefering RNA 576 gccacuccac ugaggugua 19 577 19 RNA Artificial Sequence Small interefering RNA 577 cuccacugag guguaaagc 19 578 19 RNA Artificial Sequence Small interefering RNA 578 gccuuggugu cauggaagu 19 579 19 RNA Artificial Sequence Small interefering RNA 579 acaaccucug aaacacuca 19 580 19 RNA Artificial Sequence Small interefering RNA 580 gucuggacuc augaaacac 19 581 19 RNA Artificial Sequence Small interefering RNA 581 aacaccgccg agcgcuuac 19 582 19 RNA Artificial Sequence Small interefering RNA 582 acaccgccga gcgcuuacu 19 583 19 RNA Artificial Sequence Small interefering RNA 583 gccgcuccac uguuauuua 19 584 19 RNA Artificial Sequence Small interefering RNA 584 uucacuuugc ccacagaca 19 585 19 RNA Artificial Sequence Small interefering RNA 585 cagacaacag uggugacau 19 586 19 RNA Artificial Sequence Small interefering RNA 586 gacaacagug gugacaugu 19 587 19 RNA Artificial Sequence Small interefering RNA 587 acagugguga cauguaaag 19 588 19 RNA Artificial Sequence Small interefering RNA 588 cugaugacau guguaggau 19 589 19 RNA Artificial Sequence Small interefering RNA 589 cucccggcag gacucuuca 19 590 19 RNA Artificial Sequence Small interefering RNA 590 ccucauuccc uggacacuu 19 591 19 RNA Artificial Sequence Small interefering RNA 591 ccagucaccu ugcucagaa 19 592 19 RNA Artificial Sequence Small interefering RNA 592 gucaccuugc ucagaagug 19 593 19 RNA Artificial Sequence Small interefering RNA 593 uaagcgaagg cagcuggaa 19 594 19 RNA Artificial Sequence Small interefering RNA 594 aagcugcugc augccuuaa 19 595 19 RNA Artificial Sequence Small interefering RNA 595 agcugcugca ugccuuaag 19 596 19 RNA Artificial Sequence Small interefering RNA 596 caacaaagug cucuggaau 19 597 19 RNA Artificial Sequence Small interefering RNA 597 cuguccgacu gcaccguuu 19 598 19 RNA Artificial Sequence Small interefering RNA 598 cugcaccguu uccaacuug 19 599 19 RNA Artificial Sequence Small interefering RNA 599 acuugugucu cacuaaugg 19

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Classifications
U.S. Classification514/44.00A, 536/23.1
International ClassificationA61K38/00, C12N15/113
Cooperative ClassificationC12N2310/111, C12N2310/53, A61K38/00, C12Y301/03048, C12N2310/3517, C12N2310/14, C12N15/1137
European ClassificationC12Y301/03048, C12N15/113D
Legal Events
DateCodeEventDescription
Sep 15, 2003ASAssignment
Owner name: CEPTYR, INC., WASHINGTON
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:LEWIS, STEPHEN PATRICK;KLINGHOFFER, RICHARD;WILSON, LINDA K.;REEL/FRAME:014486/0880
Effective date: 20030708