Modulation of TCPTP signal transduction by RNA interference

BACKGROUND OF THE INVENTION

[0003] 1. Technical Field

[0004] 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, TCPTP, 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.

[0005] 2. Description of the Related Art

[0006] 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.

[0007] 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.

[0008] 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.

[0009] 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.

[0010] Human T cell protein tyrosine phosphatase (TCPTP) is a nontransmembrane PTP. Alternative mRNA splicing results in variation in the sequence at the extreme C terminus of TCPTP and generates a 45-kDa form (TC45) that is targeted to the nucleus and a 48-kDa variant (TC48) associated with membranes of the endoplasmic reticulum (Hao, et al., J. Biol. Chem. 272:29322-29 (1997). The alternatively spliced forms, TC45 and TC48, share the same catalytic domain but differ at their extreme carboxy-termini (Mosinger et al., Proc. Natl. Acad. Sci. USA 89:499-503 (1992)). TC45 is characterized by the presence of bipartite nuclear localization signal and it is rapidly translocated from the nucleus to the cytoplasm following EGF stimulation. In the cytoplasm, TC45 dephosphorylates the EGF receptor, as well as downstream adapters including p52Shc, at the plasma membrane, thereby regulating growth factor signaling (Tiganis et al., Mol. Cell Biol. 18:1622-34 (1998); Tiganis et al, J. Biol. Chem. 274:27768:75 (1999)). Overexpression of TC45 has been implicated in inducing p53-dependent, serum starvation independent, and caspase-mediated apoptosis (Radha et al., FEBS Lett. 453:308-12 (1999).

[0011] 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).

[0012] 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); 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).

[0013] 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).

[0014] 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. Ser. No. 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.

[0015] 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.

[0016] Currently, therefore, desirable goals for therapeutic regulation of biological signal transduction include modulation of PTP (e.g., TCPTP, PTP1B, DSP-3, or other PTP)-mediated cellular events include, inter alia, inhibition or potentiation of interactions among PTP-binding molecules, substrates and binding partners, or of other agents that regulate PTP activities. Accordingly, a need exists in the art for an improved ability to intervene in the regulation of phosphotyrosine signaling, including regulating by altering PTP catalytic activity, PTP binding to substrate molecules, and/or -encoding gene expression. An increased ability to so regulate 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

[0017] The present invention relates to compositions and methods, including specific siRNA polynucleotides comprising nucleotide sequences disclosed herein, for modulating TCPTP. It is therefore an aspect of the invention to provide an isolated small interfering RNA (siRNA) polynucleotide, comprising at least one nucleotide sequence selected from SEQ ID NOS:28-31, 33-36, 38-41, and 68-71, which in certain further embodiments comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71 and the complementary polynucleotide thereto, and which siRNA polynucleotide in certain further embodiments is capable of interfering with expression of a TCPTP polypeptide, wherein the TCPTP polypeptide comprises an amino acid sequence as set forth in a sequence selected from GenBank Acc. Nos. M25393, NM—002828, NM—080422, and SEQ ID NOS:4, 12, and 14. In another embodiment the siRNA polynucleotide differs by one, two, three or four nucleotides at any of positions 1-19 of a sequence selected from the sequences set forth in SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71. In another embodiment the siRNA polynucleotide differs by at least two, three or four nucleotides at any of positions 1-19 of a sequence selected from the sequences set forth in SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71. The invention provides in other embodiments an isolated siRNA polynucleotide comprising a nucleotide sequence according to SEQ ID NO: 28, or the complement thereof; SEQ ID NO: 33, or the complement thereof; SEQ ID NO: 38, or the complement thereof; and SEQ ID NO: 68, or the complement thereof. In certain embodiments the siRNA polynucleotide comprises at least one synthetic nucleotide analogue of a naturally occurring nucleotide. In another embodiment the siRNA polynucleotide is linked to a detectable label, which in certain further embodiments is a reporter molecule that in certain further embodiments is a dye, a radionuclide, a luminescent group, a fluorescent group, or biotin. In a further embodiment the fluorescent group is fluorescein isothiocyanate, and in a distinct further embodiment the detectable label is a magnetic particle. The invention also provides a pharmaceutical composition comprising any of the above described siRNA polynucleotides and a physiologically acceptable carrier, which carrier in a further embodiment comprises a liposome.

[0018] It is another aspect of the invention to provide 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: 28-31, 33-36, 38-41, and 68-71, 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 the 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 another embodiment the 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 another embodiment the siRNA is capable of interfering with expression of a TCPTP polypeptide, wherein the TCPTP polypeptide comprises an amino acid sequence as set forth in a sequence selected from GenBank Acc. Nos. M25393, NM—002828, and NM—080422, SEQ ID NOS:4, 12, and 14.

[0019] According to another embodiment of the invention 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 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: 28-31, 33-36, 38-41, and 68-71, 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 a further embodiment the siRNA comprises an overhang of at least one and no more than four nucleotides, the overhang being located immediately 3′ to (iii). In another embodiment the spacer sequence comprises at least 9 nucleotides, and in another embodiment 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. In another related embodiment the invention provides a host cell transformed or transfected with the recombinant nucleic acid constructs just described.

[0020] Turning to another embodiment, there is provided by the present invention 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 the group consisting of SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71, (ii) an RNA polynucleotide that comprises at least one nucleotide sequence selected from SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71and 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: 28-31, 33-36, 38-41, and 68-71, 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: 28-31, 33-36, 38-41, and 68-71. In another related embodiment the carrier comprises a liposome.

[0021] According to another aspect of the invention, there is provided a method for interfering with expression of a TCPTP polypeptide, or variant thereof, comprising contacting a subject that comprises at least one cell which is capable of expressing a TCPTP polypeptide with a siRNA polynucleotide for a time and under conditions sufficient to interfere with TCPTP polypeptide expression, wherein (a) the PTP1B polypeptide comprises an amino acid sequence as set forth in a sequence selected from GenBank Acc. Nos. M25393, NM—002828, and NM—080422, SEQ ID NOS:4, 12, and 14, (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: 28-31, 33-36, 38-41, and 68-71, (ii) an RNA polynucleotide that comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71and 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: 28-31, 33-36, 38-41, and 68-71, 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: 28-31, 33-36, 38-41, and 68-71.

[0022] In another embodiment there is provided a method for interfering with expression of a TCPTP polypeptide that comprises an amino acid sequence as set forth in a sequence selected from the group consisting of GenBank Acc. Nos. M25393, NM—002828, and NM—080422, SEQ ID NOS:4, 12, and 14, or a variant of said TCPTP polypeptide, said method comprising contacting, under conditions and for a time sufficient to interfere with TCPTP polypeptide expression, (i) a subject that comprises at least one cell that is capable of expressing the TCPTP polypeptide, and (ii) a recombinant nucleic acid construct according to the above described aspects and embodiments.

[0023] The invention also provides in certain embodiments a method for identifying a component of a TCPTP 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 TCPTP polypeptide, or a variant of said TCPTP polypeptide, under conditions and for a time sufficient for TCPTP expression when the siRNA polynucleotide is not present, wherein (1) the TCPTP polypeptide comprises an amino acid sequence as set forth in a sequence selected from GenBank Acc. Nos. M25393, NM—002828, and NM—080422, SEQ ID NOS:4, 12, and 14, (2) the siRNA polynucleotide is selected from (i) an RNA polynucleotide which comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71, (ii) an RNA polynucleotide that comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71and 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: 28-31, 33-36, 38-41, and 68-71, 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: 28-31, 33-36, 38-41, and 68-71; 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 TCPTP signal transduction pathway. In certain embodiments the signal transduction pathway comprises a Jak2 kinase.

[0024] Another aspect of the 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 TCPTP polypeptide, wherein (a) the TCPTP polypeptide comprises an amino acid sequence as set forth in a sequence selected from the group consisting of GenBank Acc. Nos. M25393, NM—002828, and NM—080422, SEQ ID NOS:4, 12, and 14, (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: 28-31, 33-36, 38-41, and 68-71, or the complements thereof, (ii) an RNA polynucleotide that comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71and 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: 28-31, 33-36, 38-41, and 68-71, 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: 28-31, 33-36, 38-41, and 68-71; 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.

[0025] Another aspect of the invention pertains to 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 TCPTP polypeptide, wherein (a) the TCPTP polypeptide comprises an amino acid sequence as set forth in a sequence selected from GenBank Acc. Nos. M25393, NM—002828, and NM—080422, SEQ ID NOS:4, 12, and 14, (b) the siRNA polynucleotide is selected from (i) an RNA polynucleotide which comprises at least one nucleotide sequence selected from SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71, or the complements thereof, (ii) an RNA polynucleotide that comprises at least one nucleotide sequence selected from SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71 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: 28-31, 33-36, 38-41, and 68-71, 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: 28-31, 33-36, 38-41, and 68-71; 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. In another embodiment there is provided 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 TCPTP polypeptide, or a variant thereof. In a further embodiment the Jak2-associated disorder is selected from the group consisting of diabetes, obesity, hyperglycemia-induced apoptosis, inflammation, and 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:28-31, 33-36, 38-41, and 68-71. 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.

[0026] 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 200125.441 and 200125.449, respectively), which have been filed concurrently.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]
FIG. 1 illustrates insulin-induced activation of PKB/Akt in HepG2 cells following ablation of TC45 by RNA interference. FIG. 1A represents an immunoblot of serum-deprived Rat-1 and HEPG2 cells that were exposed to varying concentrations of insulin (INS) as shown. The insulin receptor (IR) was immunoprecipitated from cell lysates with an anti-IR-β antibody followed by immunoblotting with an anti-phosphotyrosine antibody (pY) (top panel); an anti-pYpY1162/1163-IR-β antibody (middle panel); and an anti-IR β antibody. FIG. 1B represents an immunoblot of HepG2 cell lysates prepared from cells that were untransfected (control) or transfected with TCPTP1 siRNA (SEQ ID NO:______) (+siRNA). The lysates were immunoblotted with an anti-phospho-PKB/Akt antibody (p-AKT) (first immunoblot); anti-PKB/Akt antibody (AKT) (second immunoblot); anti-TC45 (TC45) antibody (third immunoblot); and an anti-PTP1B antibody (PTP1B). FIG. 1C represents a densitometric analysis of the gel image to illustrate the ratio of phosphorylated PKB/Akt to total PKB/Akt.

[0028]
FIG. 2 provides an immunoblot indicating that tyrosine phosphorylated IR-β is a substrate of TC45. HepG2 cells overexpressing wild-type (WT) or substrate trapping mutant (DA) forms of PTP1B (1B) and TC45 were either not treated with insulin (−INS) or stimulated with insulin for 5 minutes (+INS), lysed, separated by SDS-PAGE, and immunoprecipitated with anti-PTP1B antibody (FG6) or anti-TC45 antibody (CF4). The immunoprecipitates were immunoblotted with an anti-IR-β antibody (top panel, FIG. 2A); anti-PTP1B antibody FG6 (middle panel, FIG. 2A); and anti-TCPTP antibody CF4 (bottom panel, FIG. 2A). FIG. 2B depicts immunoblots of HepG2 cells that were serum-starved and untransfected (control) or transfected with TC45 siRNA (100 nM) and then stimulated with 10 nM insulin (INS) for the indicated times. The insulin receptor was immunoprecipitated from cell lysates with an anti-IR-β antibody, which was then immunoblotted with the following antibodies: anti-phosphotyrosine (p-Tyr) (first immunoblot); anti-pY972-IR-β (second immunoblot); anti-pYpY1162/1163-IR-β (third immunoblot); and anti-IR-β (fourth immunoblot). FIG. 2C presents densitometric analyses of the gel image to show the ratio of phosphorylated IR-β, to total IR-β for total phosphotyrosine (top panel); phosphorylation of Tyr 972 (middle panel); and phosphorylation of the activation loop tyrosines 1162 and 1163 (lower panel).

[0029]
FIG. 3 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 hTCPTP1.4 siRNA (TC1.4, SEQ ID NO:______) (FIG. 3A) and mPTP1B1.1b siRNA (M1.1, SEQ ID NO:______) (FIG. 3B). 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.

DETAILED DESCRIPTION OF THE INVENTION

[0030] 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 TC-PTP polypeptide, such as a human or murine TC-PTP polypeptide (e.g., GenBank Accession Nos. M25393 (SEQ ID NOS: ______-______); M81478 (SEQ ID NO: ______); M80737 (SEQ ID NO: ______); M81477 (SEQ ID NOS: ______-______); X58828 (SEQ ID NOS: ______-______); NM—002828; and TC45 (e.g. NM—080422 (SEQ ID NOS: ______ and ______); 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 TCPTP 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.

[0031] In preferred embodiments of the invention, the siRNA polynucleotide interferes with expression of a TCPTP 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 TCPTP.

[0032] PTP associated disorders include, for example, diabetes mellitus, obesity, impaired glucose tolerance and other metabolic disorders wherein alteration of a TCPTP or of a TCPTP signaling pathway component is associated with such a disorder. The protein tyrosine phosphatase TCPTP exists in two alternatively spliced forms, TC45 and TC48, that share the same catalytic domain but differ at their extreme carboxy-termini (Mosinger et al., Proc. Natl. Acad. Sci. USA 89:499-503 (1992)). Insulin-induced oxidation and inactivation of TC45 suggests that it may function as a negative regulator of insulin signaling (see U.S. Ser. No. 10/366,547; see also, Galic et al., Mol. Cell Biol. 23:2096-108 (2003)). 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. As used herein, TCPTP is understood to include TCPTP and its alternatively spliced forms.

SiRNA Polynucleotides

[0033] 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 TCPTP polypeptide, or a variant of the TCPTP polypeptide, wherein a single strand of the siRNA comprises a portion of a RNA polynucleotide sequence that encodes the TCPTP 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 TCPTP polypeptide, or a variant of the TCPTP 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 TCPTP 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.

[0034] 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.

[0035] 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 TCPTP polypeptide. These polynucleotides may also find uses as probes or primers.

[0036] 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)).

[0037] 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 TCPTP. The percent identity may be readily determined by comparing sequences of the polynucleotides to the corresponding portion of the TCPTP polynucleotide, 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.

[0038] Certain siRNA polynucleotide variants are substantially homologous to a portion of a native TCPTP 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 TCPTP 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 TCPTP polypeptide for which interference with expression is desired, and in certain other embodiments the sequence (or its complement) may be shared by TCPTP and one or more PTPs for which interference with polypeptide expression is desired.

[0039] 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.

[0040] 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., TCPTP 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 polynucleotide 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.pdf.) 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.

[0041] It should be appreciated that not all siRNAs designed using the above methods will be effective at silencing or interfering with expression of a TCPTP 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 the target. Accordingly, identification of specific siRNA polynucleotide sequences that are capable of interfering with expression of a TCPTP polypeptide requires production and testing of each siRNA, as demonstrated in greater detail below (see Examples).

[0042] 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 TCPTP polypeptide expression and/or cellular compensatory mechanisms that are induced in response to RNAi-mediated inhibition of a specific target polypeptide expression.

[0043] 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.

[0044] Polynucleotides, including target polynucleotides, 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.

[0045] 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.

[0046] 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.

[0047] A number of specific siRNA polynucleotide sequences useful for interfering with TCPTP 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. Further, siRNAs may be chemically modified or conjugated to improve theur 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. Modifications in a polynucleotide sequence may also be introduced using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis. 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.

[0048] Accordingly, a siRNA polynucleotide that is complementary to at least a portion of a TCPTP 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 TCPTP. 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)).

[0049] 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.

[0050] 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. Ser. No. 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 TCPTP 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.

[0051] 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. Ser. No. 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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).

[0057] As noted above, according to certain embodiments of the invention compositions and methods are provided that relate to altering or altered TCPTP expression, and/or to a TCPTP associated disorder. A TCPTP associated disorder includes any disease, disorder, condition, syndrome, pathologic or physiologic state, or the like, wherein at least one undesirable deviation or departure from a physiological norm causes, correlates with, is accompanied by or results from an inappropriate alteration (i.e., a statistically significant change) to the structure, activity, function, expression level, physicochemical or hydrodynamic property, or stability of TCPTP or of a molecular component of a biological signal transduction pathway that comprises a TCPTP. In preferred embodiments the molecular component may be a protein, peptide or polypeptide, and in certain other preferred embodiments the alteration may be an altered level of TCPTP expression. In certain other preferred embodiments the alteration may be manifest as an atypical or unusual phosphorylation state of a protein under particular conditions, for example, hypophosphorylation or hyperphosphorylation of a phosphoprotein, wherein those familiar with the art will appreciate that phosphorylated proteins typically comprise one or more phosphotyrosine, phosphoserine, or phosphothreonine residues.

[0058] TCPTP associated disorders therefore include, for example, diabetes mellitus, obesity, impaired glucose tolerance and other metabolic disorders wherein alteration of TCPTP or of a TCPTP signaling pathway component is associated with the disorder. 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. The invention is not intended, however, to be so limited and contemplates other disorders, such as JNK-associated disorders (e.g., cancer, cardiac hypertrophy, ischemia, diabetes, hyperglycemia-induced apoptosis, inflammation, neurodegenerative disorders), and other disorders associated with different signal transduction pathways, for instance, cancer, autoimmunity, cellular proliferative disorders, neurodegenerative disorders, and infectious diseases (see, e.g., Fukada et al., 2001 J. Biol. Chem. 276:25512; Tonks et al., 2001 Curr. Opin. Cell Biol. 13:182; Salmeen et al., 2000 Mol. Cell 6:1401; Hu et al., J. Neurochem. 85:432-42 (2003); and references cited therein). Persons skilled in the art will be familiar with an array of criteria according to which it may be recognized what are, for instance, biological, physiological, pathological and/or clinical signs and/or symptoms of TCPTP associated and other disorders as provided herein.

[0059] 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.

[0060] 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).

[0061] 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 colorimetric) 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.

TCPTP

[0062] TCPTPs for use in the present invention include the following: (see, e.g., Andersen et al., 2001 Mol. Cell. Biol.; GenBank Accession Nos. M25393 (SEQ ID NOS: ______-______); M81478 (SEQ ID NO: ______); M80737 (SEQ ID NO: ______); M81477 (SEQ ID NOS: ______-______); X58828 (SEQ ID NOS: ______-______); NM—002828; and TC45 (e.g., NM—080422 (SEQ ID NOS: ______ and ______)). The invention also contemplates using variants or mutated forms of TCPTP, which may include a TCPTP polypeptide that contains single nucleotide polymorphisms (SNPs), or may include allelic forms.

[0063] 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.

[0064] In particular, portions of two TCPTP polypeptide sequences are regarded as “corresponding” amino acid sequences, regions, fragments or the like, based on a convention of numbering one TCPTP 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 TCPTP-encoding DNA sequence according to a convention for numbering nucleic acid sequence positions in the known wildtype TCPTP DNA sequence, whereby the candidate PTP DNA sequence is aligned with the known TCPTP 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 TCPTP DNA sequence. In certain particularly preferred embodiments, a portion, region or fragment of a candidate PTP DNA sequence is identical to a corresponding known TCPTP 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.

[0065] 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., TCPTP). 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.

[0066] 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 TCPTP 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).

[0067] 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 polynucleotide segment that may be transcribed to yield TCPTP 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.

[0068] 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 TCPTP 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 1 9-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 H1 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.

[0069] TCPTP polypeptides can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters, providing ready systems for evaluation 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).

[0070] 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.

[0071] 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, Sireptomyces, 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.

[0072] 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.

[0073] 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.

[0074] 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 TCPTP 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.

[0075] 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.

[0076] 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 lac, 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 TCPTP polypeptide is described herein.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] The producer cell line generates infectious retroviral vector particles that include the nucleic acid sequence(s) encoding the TCPTP 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.

[0081] In another aspect, the present invention relates to host cells containing the above described recombinant TCPTP 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.

[0082] 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 Sf9; 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.

[0083] 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 TCPTP 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.

[0084] 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.

Samples

[0085] According to the present invention, a method is provided for interfering with expression of a PTP polypeptide such as TCPTP, or a variant thereof, as provided herein. A method is also provided for interfering with expression of a TCPTP polypeptide, comprising contacting a siRNA polynucleotide with a cell that is capable of expressing TCPTP, typically in a biological sample or in a subject or biological source. A “sample” as used herein refers to a biological sample containing at least one desired target protein, 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 TCPTP polypeptide, and in certain particularly preferred embodiments the cell comprises an inducible biological signaling pathway, at least one component of which is TCPTP. 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 TCPTP knockout animals and which may be transfected with human insulin receptor (HIR), for example, 1 BKO mouse embryo fibroblasts.

[0086] In certain other preferred embodiments the sample is a cell that includes, for example, a cell line that is derived from a tumor cell. The cell line may be a primary tumor cell line, that is, a cell line prepared directly from a tumor sample removed from a human or a non-human animal. Alternatively, the cell line may be one of several established tumor cell lines known in the art, including but not limited to MCF7, T47D, SW620, HS578T, MDA-MB-435, MDA MB 231, HCT-116, HT-29, HeLa, Raji, Ramos, and the like (see ATCC collection).

[0087] 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 (NAC) 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.

[0088] 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] 10 Oct. 2000 Science's stke, Internet:URL<www.stke.org/cgl/content/full/ OC_sigtrans;2000/53/pe1>), and references cited therein).

[0089] 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 colorimetric) 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.

[0090] In preferred embodiments, a TCPTP substrate may be any naturally or non-naturally occurring phosphorylated peptide, polypeptide or protein that can specifically bind to and/or be dephosphorylated by a TCPTP polypeptide.

[0091] Identification and selection of TCPTP 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. TCPTP 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)).

[0092] The TCPTP substrate peptides of the present invention may therefore be derived from TCPTP substrate proteins, polypeptides and peptides as provided herein having amino acid sequences that are identical or similar to tyrosine phosphorylated TCPTP substrate sequences known in the art. For example by way of illustration and not limitation, peptide sequences derived from the known TCPTP 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).

[0093] In certain preferred embodiments of the present invention, the siRNA polynucleotide and/or the TCPTP 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 TCPTP-specific siRNA polynucleotide-magnetic particle conjugates may offer certain advantages in the further characterization or regulation of PTP signaling pathways.

[0094] In certain embodiments of the present invention, particular TCPTP-specific siRNA polynucleotides of interest may be identified by contacting a candidate siRNA polynucleotide with a sample comprising a cell that comprises a TCPTP gene and that is capable of TCPTP gene transcription or expression (e.g., translation), under conditions and for a time sufficient to detect TCPTP gene transcription or expression, and comparing TCPTP transcription levels, TCPTP polypeptide expression and/or TCPTP functional expression (e.g., TCPTP catalytic activity) in the absence and presence of the candidate siRNA polynucleotide. Preferably TCPTP transcription or expression is decreased in the presence of the siRNA polynucleotide, thereby providing an alternative to TCPTP active site directed approaches to modulating TCPTP activity. (The invention need not be so limited, however, and contemplates other embodiments wherein one or more 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 TCPTP-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.)

[0095] For a cell that expresses TCPTP 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 TCPTP levels through interference with TCPTP 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 PI3 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. NM—004972, AF058925, AF005216, NM—031514, and NM—008261) 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.

[0096] TCPTP 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 TCPTP and that have been transfected with a TCPTP-specific siRNA polynucleotide that is either known or suspected of being capable of interfering with TCPTP 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 TCPTP may be added to such a system, as described above, to evaluate their effect on TCPTP activity.

[0097] Within other aspects, the present invention provides animal models in which an animal, by virtue of introduction of an appropriate TCPTP-specific siRNA polynucleotide, for example, as a transgene, does not express (or expresses a significantly reduced amount of) a functional TCPTP. 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.

[0098] Therapeutic Methods

[0099] One or more siRNA polynucleotides capable of interfering with TCPTP polypeptide expression and identified according to the above-described methods may also be used to modulate (e.g., inhibit or potentiate) TCPTP 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 TCPTP 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 TCPTP activity include any disorder associated with cell proliferation, including cancer, graft-versus-host disease (GVHD), autoimmune diseases, allergy or other conditions in which unregulated TCPTP activity may be involved, metabolic diseases (including e.g., diabetes mellitus, obesity, impaired glucose tolerance), abnormal cell growth or proliferation and cell cycle abnormalities.

[0100] 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.

[0101] 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, intracavemous, 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] The following Examples are offered by way of illustration and not by way of limitation.

EXAMPLE 1

Interference of TCPTP Expression by Specific siRNA

[0108] This Example describes the effect on expression of TCPTP expression in cells transfected with sequence-specific siRNA polynucleotides.

[0109] The siRNA nucleotide sequences specific for each TCPTP 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 TCPTP 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.

[0110] 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.

[0111] In each of the examples, each siRNA sequence represents the sense strand of the siRNA polynucleotide and its corresponding sequence identifier. “Related sequence identifiers” referred to in the Examples identify sequences in the sequence listing that contain the same nucleotides at positions 1-19 of the siRNA sequence with and without two additional nucleotides (NN) at the 3′ end (which would correspond to a two-nucleotide overhang in a double stranded polynucleotide), and the reverse complement of each. (It is noted that each 21-mer sequence thus 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.) 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.

[0112] Interference with Expression of Murine TCPTP by siRNA in Co-Transfection Assays

[0113] A co-transfection assay was performed in which 1BKO+HIR murine fibroblasts were co-transfected with an expression vector comprising a polynucleotide sequence (SEQ ID NO:______) encoding murine TCPTP (SEQ ID NO:______) and siRNA mTCPTP1.1 (5′-guugucaugcuaaaccgaact-3′ (SEQ ID NO:______)) (1 nM) or mTCPTP1.2 (5′-cagaacagagugaugguugag-3′ (SEQ ID NO:______)) (20 nM). The level of TCPTP expression was determined by immunoblotting with an anti-human TCPTP antibody (Curt Diltz, CEPTYR, Inc.). A co-transfection assay was performed in which 1BKO+HIR murine 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. The transfected cells were incubated 22-24 hours at 37°.

[0114] 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-human TCPTP antibody (Curt Diltz, CEPTYR, Inc.) 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.).

[0115] The siRNA mTCPTP1.2 did not interfere with expression of murine TCPTP. Expression of murine TCPTP decreased more than 95% in cells transfected with siRNA, mTCPTP1.1.

[0116] Interference with Expression of Human TCPTP by siRNA in Co-Transfection Assays

[0117] Co-transfection assays were performed essentially as described above to determine siRNA inhibition of human TCPTP expression. A recombinant expression construct was prepared that encodes wild-type human TC45. The following oligonucleotide primers were used for the wild-type construct. The sequences of the BamHI and EcoRi restriction sites are underlined.

1Human TC45 sense (TC45 5′BamHI)5′-GGGGGGATCCATGCCCACCACCATCGAGCGGGAGTT-3′(SEQ ID NO_)Human TC45 antisense (TC45 3′EcoRI)5′-GGGGAATTCTTAGGTGTCTGTCAATCTTGGCCTTTTTCTTTTTCGTTCA-3′(SEQ ID NO:_)

[0118] 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) 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.

[0119] Vectors for expression of TC45 wild type were prepared as follows: The TC45 construct was subcloned into a GATEWAY™ entry vector pENTR3C™ (Invitrogen Life Technologies) by digesting 10 μl of the TC45 cDNA with 1 μl of BamHI (New England Biolabs), 1 μl of EcoRI (New England Biolabs), 3 μl 10×EcoRI buffer (New England Biolabs), 3 μl 10×BSA (New England Biolabs), and 12 μl distilled water for 3 hours at 37° C. Two microliters of the pENTR3C™ vector was digested with 0.5 μl of BamHI (New England Biolabs), 0.5 μl of EcoRI (New England Biolabs), 2 μl 10×EcoRI buffer (New England Biolabs), 2 μl 10×BSA (New England Biolabs), and 13 μl distilled water for 3 hours at 37° C., followed by an incubation of 30 minutes at 37° C. with calf intestinal phosphatase (New England Biolabs). Digested DNA was run on a 1% agarose gel, digested bands were excised and gel purified using a QIAGEN Gel Extraction kit (QIAGEN, Inc.). Four microliters of the TC45 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 Life Technologies), and 2 μl distilled water. The construct was transformed into LIBRARY EFFICIENCY® DH5α™ cells. The FLAG® epitope-tagged TC45 construct was prepared by cloning the pENTR3C™ TC45 WT construct into the GWpCMVTag2 vector. The pENTR3C™ construct containing the TC45 polynucleotide was linearized by digesting the construct with Pvu I (New England Biolabs)) at 37° C. for 2 hours. The DNA was purified using a QIAGEN PCR Purification kit (QIAGEN, Inc.). Two microliters (150 ng/μl) of the GWpCMVTag2 vector were combined in a GATEWAY™ LR reaction with 3 μl linearized pENTR3C™ TC45 WT, 5 μl TE buffer, 4 μl Clonase™ Enzyme, and 4 μl LR reaction buffer (Invitrogen Life Technologies) overnight 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.

[0120] Cells (1BKO+HIR murine embryo fibroblasts) were co-transfected (see method above) with an expression vector containing a nucleotide sequence encoding human TCPTP (SEQ ID NO:______) and siRNAs, hTCPTP1.4 (5′-guugucaugcugaaccgcatt-3′ (SEQ ID NO:______)) (20 nM); hTCPTP1.5 (5′-gcccauaugaucacagucgtg-3′ (SEQ ID NO:______)) (10 nM); hTCPTP1.6 (5′-ucgguuaaaugugcacaguac-3′ (SEQ ID NO:______)) (10 nM); or hTCPTP1.7 (5′-ugacuauccucauagaguggg-3′ (SEQ ID NO:______)) (20 nM). Additional human TCPTP specific siRNA polynucleotides were prepared; the sequences of each are as follows: hTCPTP1.1 (5′-agugagagaaucuggcucctt-3′ (SEQ ID NO:______)); hTCPTP1.2 (5′-ggaagacuuaucuccugcctt-3′ (SEQ ID NO:______)); and hTCPTP1.3 (5′-ggugaccgauguacaggactt-3′ (SEQ ID NO:______)). The level of TCPTP expression was determined by immunoblotting with an anti-human TCPTP antibody. The level of expression of human TCPTP was not affected by siRNA hTCPTP1.7. Expression levels decreased more than 95% in the cells co-transfected with hTCPTP1.4; 80% in cells co-transfected with hTCPTP1.5; and greater than 90% in cells transfected with hTCPTP1.6.

[0121] Interference of Endogenous Expression of Human TCPTP by siRNA

[0122] 293-HEK HIR cells were transfected with either hTCPTP1.4 (SEQ ID NO:______) or rPTP1B1.2, a rat PTP1B sequence specific siRNA (5′-cggaugguggguggagguctt-3′ (SEQ ID NO:______), which was included as a nonspecific siRNA control, at concentrations of 2, 5, 10, 20, and 50 nM. Cells were plated one day prior to transfection in either a 24-well or 6-well format. The siRNA polynucleotide to be added to each well was diluted into 50 μl OPTIMEM® to the final concentrations listed above. In a separate vessel, 3 μl Oligofectamine™ (Invitrogen Life Technologies) was diluted into 12 μl OPTIMEM®. Each solution was incubated for 7 minutes at room temperature, after which the solutions were mixed and incubated for 22 minutes. The final volume of the mixed solution was brought to 100 μl with OPTIMEM®. Cells were transfected with the specific siRNA polynucleotides, non-specific siRNA polynucleotides, and annealing buffer alone, and incubated at 37° for 48-72 hours. Endogenous expression of human TCPTP in the cells transfected with sequence specific hTCPTP1.4 decreased 90%.

EXAMPLE 2

Regulatory Role of TCPTP in Insulin Signaling

[0123] The protein tyrosine phosphatase TC-PTP exists in two alternatively spliced forms, TC45 and TC48, that share the same catalytic domain but differ at their extreme carboxy-termini (Mosinger et al., Proc. Natl. Acad. Sci. USA 89:499-503 (1992)). Insulin-induced oxidation and inactivation of TC45 suggested that it functions as a negative regulator of insulin signaling (see U.S. Ser. No. 10/366,547). This Example examines the regulatory role of TC45 in insulin signaling by inhibiting expression of the PTP by RNAi.

[0124] The specific siRNA duplexes were designed by first scanning through the open reading frame of TC45 mRNA and selecting sequences of 5′AA(N19)3′ (N=any nucleotide) for further characterization. The following 2 oligonucleotides were chosen: 5′-AACAGAUACAGAGAUGUAAGC-3′ (TCPTP1) (SEQ ID NO:______) and 5′-AAGCCCA UAUGAUC ACAGUCG -3′ (TCPTP2) (SEQ ID NO:______). These sequences were submitted to a BLAST search against human, rat, and mouse genome databases to ensure specificity for TC-PTP. The 21-nt siRNA duplexes were obtained in a deprotected and desalted form (Dharmacon Research). Rat-1 fibroblasts (Fischer rat fibroblast 3T3 like cell line) and HepG2 (human hepatocellular carcinoma) cells (American Type Culture Collection (ATCC), Manasass, Va.) were transfected with each siRNA at 100 nM. Both siRNA oligonucleotides suppressed expression of endogenous TC45 in the transfected HepG2 cells and Rat-1 fibroblasts, with TCPTP1 being more efficient.

[0125] Rat-1 (fibroblasts) and HepG2 (human hepatocellular carcinoma) cells were routinely maintained in DMEM supplemented with 10% FBS, 1% glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. For stimulation with insulin, cells were plated in media containing 10% FBS for 48 hours, then serum-starved for 16 hours before treatment. For transient transfection, cells were plated in DMEM supplemented with 10% FBS for 16 hours, then in OptiMEM (Invitrogen) without serum, after which the plasmid (5 μg/dish for Rat-1, 30 μg/dish for HepG2) was introduced by LipofectAMINE and PLUS reagents (Invitrogen), according to the manufacture's recommendations. The transfection efficiency was routinely 40%. For RNAi experiments, cells were plated as above and the TCPTP siRNA duplexes were introduced by Oligofectamine (Invitrogen) according to the guidelines provided by Dharmacon Research Inc.

[0126] The potential regulatory role of TC45 in insulin signaling was investigated by examining the phosphorylation status of PKB/Akt, which is a critical effector in the PI3 kinase pathway that mediates various intracellular responses to insulin, following ablation of the PTP by RNAi. The human hepatoma cell line HepG2 has been used extensively as a model to study insulin signaling (see Huang et al., J. Biol. Chem. 277:18151-60 (2002); Haj et al., Science 295 1708-11 (2002)). Serum-deprived Rat-1 and HepG2 cells were exposed to 10 or 50 nM insulin for 5 min and lysed. The insulin receptor (IR) was immunoprecipitated from 500 μg of cell lysate with anti-IR-β antibody 29B4 (Santa Cruz Biotechnology), then immunoblotted with anti-phosphotyrosine, anti-pYpY1162/1162-IR-β (Biosource International, Camarillo, Calif.) and anti-IR-β (C-19) (Santa Cruz Biotechnology) antibodies. HepG2 cells expressed higher levels of IR-β than Rat-1 cells as shown in FIG. 1A and displayed a robust response to insulin stimulation, as shown by the overall tyrosine phosphorylation level of IR-β and autophosphorylation of the activation loop tyrosines 1162 and 1163 (see FIG. 1A).

[0127] For the RNAi experiment, HepG2 cells were untransfected (control) or transfected (+siRNA) with 100 nM siRNA TCPTP1 oligonucleotide. Two days after transfection, cells were serum-starved for 16 hours and then stimulated with 10 nM insulin for 0, 1, 2, 5, 10, and 20 minutes. Total lysates (30 μg) were immunoblotted with anti-phospho-PKB/Akt (Cell Signaling Technology, Beverly, Mass.); anti-PKB/Akt (Cell Signaling Technology); anti-TC45 (1910H (Lorenzen et al., J. Cell. Biol. 131:631-43 (1995))); and anti-PTP1B (FG6 (LaMontagne et al., Mol. Cell. Biol. 18:2965-75 (1998))) antibodies. The results presented in FIG. 1B indicate that depletion of TC45 enhanced both the intensity and duration of the signaling response. FIG. 1C illustrates a densitometric analysis of the gel image to show the ratio of phosphorylated PKB/Akt relative to total PKB/Akt. Similar results were observed in three independent experiments.

[0128] The role of TC45 in insulin signaling was further investigated by preparing a TC45 substrate trapping mutant. Substitution of an alanine residue for the invariant aspartate, which functions as a general acid in catalysis, into the vector expressing TC45 and into a vector expressing PTP1B was performed by standard site-directed mutagenesis protocols. HepG2 cells overexpressing wild type (WT) or trapping mutant (DA) forms of PTP1B and TC45 were either left untreated (−INS) or stimulated with 10 nM insulin for 5 min (+INS), then lysed in trapping buffer (20 mM Tris (pH 7.4), 1% NP-40, 150 mM NaCl, 10% glycerol, 10 mM IAA and 25 μg/ml each of aprotinin and leupeptin). Aliquots (1 mg) of cell lysate were incubated with anti-PTP1B antibody (FG6) or anti-TC45 antibody (CF4). The immunocomplexes were washed with lysis buffer, subjected to SDS-PAGE then immunoblotted with anti-IR-β (C-19) antibody. An aliquot of lysate (30 μg) was immunoblotted with anti-PTP1B antibody (FG6) or anti-TC-PTP antibody (CF4) to verify PTP expression. The data are shown in FIG. 2A and are representative of three independent experiments. These data suggest that TC45 recognizes IR-β as a substrate.

[0129] Serum starved, untransfected (control) or TC45 siRNA (100 nM) transfected (+siRNA) HepG2 cells were stimulated with 10 nM insulin for 0, 1, 2, 5, 10, and 20 minutes. The insulin receptor was immunoprecipitated from 750 μg of cell lysate with anti-IR-β antibody 29B4 and immunoblotted with anti-phosphotyrosine (G104), anti-pY972-IR-β (Biosource), anti-pYpY1162/1163-IR-β, and anti-IR-β (C-19) antibodies as shown in FIG. 2B. FIG. 2C illustrates densitometric analyses of the gel image to show the ratio of phosphorylated IR-β relative to total IR-β for total phosphotyrosine (upper panel), phosphorylation of Tyr 972 (middle panel), and phosphorylation of the activation loop tyrosines 1162 and 1163 (lower panel). Similar results were observed in two independent experiments.

EXAMPLE 3

Effect of siRNAs Specific for TCPTP on Insulin Receptor Tyrosine Phosphorylation

[0130] This example illustrates the effect of RNAi on the function of components in a cell signaling pathway.

[0131] The effect of human TCPTP siRNA on the level of phosphorylation of IR-β was evaluated by ELISA. 292-HEK HIR cells were transfected with 0, 0.5, 3, or 10 nM siRNAs. The siRNA polynucleotides transfected into the cells included mPTP1B1.1 (SEQ ID NO:______) and hTCPTP1.4 (SEQ ID NO:______). Seventy-two hours after transfection, cells were exposed to insulin for 7 minutes at concentrations of 0, 5, 10, 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.).

[0132] An ELISA was performed as follows. Dynex Immulon HB4X 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 H2SO4 and then mixed. The optical density of each well was measured at 450 nM in a Spectramax plate reader (Molecular Devices Corp., Sunnyvale, Calif.). As shown in FIG. 3 increased phosphorylation of the insulin receptor was observed in cells transfected with hTCPTP1.4.

Additional References

[0133] Agami et al., Cell 102:55-66 (2000)

[0134] Bass, Brenda L., Cell 101:235:238 (2000)

[0135] Brummelkamp et al., Science 296:550-53 (2002)

[0136] Carthew, Richard W., Current Opinion in Cell Biology 13:244-248 (2001)

[0137] Clemens et al., Proc. Natl. Acad. Sci. USA 97:6499-6503 (2000)

[0138] Elbashir et al., Genes & Development 15:188-200 (2001)

[0139] Elbashir, et al., Nature 411:494-498 (2001)

[0140] Fire et al., Nature 391:806-11 (1993)

[0141] Flint et al., Proc. Natl. Acad. Sci. USA 94:1680-1685 (1997)

[0142] Fukada et al., J. Biol. Chem. 276:25512-25519 (2001)

[0143] Harborth et al., J. Cell Sci. 114:4557-4565 (2001)

[0144] Hutvagner et al., Curr. Opin. Gen. & Dev. 12:225-232 (2002)

[0145] Kisielow et al., Biochem. J. 363:1-5 (2002)

[0146] Paddison et al., Genes & Development 16:948-958 (2002)

[0147] Salmeen et al., Moleular Cell 6:1401-1412 (2000)

[0148] Scadden et al., EMBO Reports 2:1107-1111 (2001)

[0149] Sharp, Phillip A., Genes & Development 13:139-141 (1999)

[0150] Sharp, Phillip A., Genes & Development 15:485-490 (2001)

[0151] Shen et al., Proc. Natl. Acad. Sci. USA 24:13613-13618 (2001)

[0152] Sui et al., Proc. Natl. Acad. Sci. USA 99:5515-5520 (2002)

[0153] Tonks et al, Curr. Opin. Cell Biol. 13:182-195 (2001)

[0154] Tuschl, Thomas, Chembiochem. 2:239-245 (2001)

[0155] Ui-Tei et al., FEBS Letters 479:79-82 (2000)

[0156] Wen et al., Proc. Natl. Acad. Sci. 98:4622-4627 (2001)

[0157] Zamore et al., Cell 101:25-33 (2000)

[0158] EP1 152 056

[0159] U.S. Pat. No. 2001/0029617

[0160] U.S. Pat. No. 2002/0007051

[0161] U.S. Pat. No. 6,326,193

[0162] U.S. Pat. No. 6,342,595

[0163] U.S. Pat. No. 6,506,559

[0164] WO 01/29058

[0165] WO 01/34815

[0166] WO 01/42443

[0167] WO 01/68836

[0168] WO 01/75164

[0169] WO 01/92513

[0170] WO 01/96584

[0171] WO 99/32619

[0172] 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.

	Number	Date	Country
	60462942	Apr 2003	US
	60383249	May 2002	US

Modulation of TCPTP signal transduction by RNA interference

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