Antisense inhibition of SHP-2 expression

Details Antisense inhibition of SHP-2 expression Antisense inhibition of SHP-2 expression

1999-07-21

2001-03-13

Elliott, George C. (Department: 1635)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

C536S024500, C536S023100, C514S04400A, C435S006120, C435S091100, C435S455000

Reexamination Certificate

active

06200807

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of SHP-2. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding human SHP-2. Such oligonucleotides have been shown to modulate the expression of SHP-2.
BACKGROUND OF THE INVENTION
The process of phosphorylation, defined as the attachment of a phosphate moiety to a biological molecule through the action of enzymes called kinases, represents one course by which intracellular signals are propagated from molecule to molecule resulting finally in a cellular response. Within the cell, proteins can be phosphorylated on serine, threonine or tyrosine residues and the extent of phosphorylation is regulated by the opposing action of phosphatases, which remove the phosphate moieties. While the majority of protein phosphorylation within the cell is on serine and threonine residues, tyrosine phosphorylation is modulated to the greatest extent during oncogenic transformation and growth factor stimulation (Zhang,
Crit. Rev. Biochem. Mol. Biol.,
1998, 33, 1-52).
Because phosphorylation is such a ubiquitous process within cells and because cellular phenotypes are largely influenced by the activity of these pathways, it is currently believed that a number of disease states and/or disorders are a result of either aberrant activation or functional mutations in kinases and phosphatases. Consequently, considerable attention has been devoted recently to the characterization of tyrosine kinases and tyrosine phosphatases.
SHP-2 (also known as Syp, SHPTP2, PTP2C, PTPN11, PTP1D and BPTP3) is a member of the family of non-membrane tyrosine phosphatases and is ubiquitously expressed in all tissues examined with the highest levels in the heart and brain (Ahmad et al.,
Proc. Natl. Acad. Sci. U.S.A.,
1993, 90, 2197-2201; Bastien et al.,
Biochem. Biophys. Res. Commun.,
1993, 196, 124-133; Freeman et al.,
Proc. Natl. Acad. Sci. U.S.A.,
1992, 89, 11239-11243).
Disclosed in U.S. Pat. No. 5,589,375 is a purified nucleic acid molecule that encodes SHP-2 or hybridizes to the complement of the nucleic acid sequence that encodes SHP-2 (Ullrich and Vogel, 1996,). Further disclosed in this U.S. patent are nucleotide sequences that encode variants of SHP-2 that lack domains within the protein and nucleotide sequences that hybridize to these variants (Ullrich and Vogel, 1996,).
The SHP-2 protein contains two src homology 2 (SH2) domains, conserved regions of approximately 100 amino acids originally identified in Src protein tyrosine kinases, that promote protein-protein interactions through phosphotryosyl residue binding (Neel,
Semin. Cell. Biol.,
1993, 4, 419-432). These two domains have been shown to display differential functions in the regulation of the SHP-2 phosphatase and consequently affect different signaling pathways. The N-terminal SH2 domain serves as a regulatory and recruiting domain, producing an autoinhibitory effect through intramolecular interactions with the internal catalytic phosphatase domain. While the C-terminal SH2 domain acts merely to recruit other proteins for intermolecular interactions necessary for signal transduction (Pei et al.,
Proc. Natl. Acad. Sci. U.S.A.,
1996, 93, 1141-1145).
The SHP-2 protein is disclosed in U.S. Pat. No. 5,831,009 as are fusion proteins containing the SHP-2 protein sequence and peptide fragments of SHP-2 comprising the signal peptide domain, the catalytic phosphatase domain or the ezrin-like domain (Ullrich et al., 1998,).
The SHP-2 protein interacts with several different types of signaling molecules within the cell through the SH2 domains. These include the &bgr;c subunit of the human interleukin 3 receptor (Bone et al.,
J. Biol. Chem.,
1997, 272, 14470-14476), the high affinity IgE receptor (Kimura et al.,
J. Immunol.,
1997, 159, 4426-4434), and killer cell inhibitory receptors (Olcese et al.,
J. Immunol.,
1996, 156, 4531-4534). It has also been shown to interact with biliary glycoprotein, an immunoglobulin like cytohesion molecule with tumor suppressor function, in epithelial cells (Huber et al.,
J. Biol. Chem.,
1999, 274, 335-344).
Many signaling cascades have been shown to flow through SHP-2, either via a phosphorylation event or through protein-protein interactions. The p85 subunit of PI3 kinase has been shown to associate with SHP-2 in response to cytokine signaling in hematopoietic cells (Zhang et al.,
Biochem. Biophys. Res. Commun.,
1999, 254, 440-445). SHP-2 also couples platelet derived growth factor signaling to Ras activation by binding to the receptor and becoming phosphorylated thereby recruiting other signaling proteins such as Grb2 (Bennett et al.,
Proc. Natl. Acad. Sci. U.S.A.,
1994, 91, 7335-7339).
In concert with focal adhesion kinase, SHP-2 has been shown to regulate chemotaxis in human breast adenocarcinoma cells. In these cells, it was demonstrated that, upon growth factor stimulation, focal adhesion kinase was dephosphorylated by SHP-2 which in turn increased cell adhesion. Alternatively, expression of a dominant negative mutant of SHP-2, lacking its phosphatase activity, resulted in the increase of focal adhesions and diminished migration capability indicating that SHP-2 regulates motility in these cells (Manes et al.,
Mol. Cell. Biol.,
1999, 19, 3125-3135). further studies of SHP-2 and integrin signaling demonstrated that SHP-2 acts upstream of MAP kinase pathways that determine cell shape. In these studies, cells expressing SHP-2 mutants lacking exon 3, which encodes the N-terminal SH2 domain necessary for protein-protein interactions, were investigated for their ability to affect cell spreading. Cells expressing the mutant SHP-2 protein lacked the ability to spread on fibronectin, while those expressing the wild type protein displayed the spreading phenotype (Oh et al.,
Mol. Cell. Biol.,
1999, 19, 3205-3215).
It has also been shown that SHP-2 plays a critical role in the allergic response system. Interleukin 5 promotes eosinophil survival and an antisense oligonucleotide targeting SHP-2 was shown to inhibit this response indicating that SHP-2 plays a positive role in the activation of eosinophils (Pazdrak et al.,
J. Exp. Med.,
1997, 186, 561-568).
Aberrant expression or activation of SHP-2 has also been implicated in disease states such as diabetes. Kuhne et al. demonstrated that the insulin receptor substrate 1 is dephosphorylated by SHP-2, an event that terminates insulin action (Kuhne et al.,
J. Biol. Chem.,
1994, 269, 15833-15837). Overexpression of SHP-2, therefore, would dampen the cells response to insulin and may lead to the development of diabetes.
To date, strategies aimed at modulating SHP-2 function have involved the use of antibodies, dominant negative mutants and gene knock-outs in mice.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of SHP-2. Consequently, there remains a long felt need for agents capable of effectively inhibiting SHP-2 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of SHP-2 expression.
SUMMARY OF THE INVENTION
The present invention is directed to antisense compounds, particularly oligonucleotides, which are targeted to a nucleic acid encoding SHP-2, and which modulate the expression of SHP-2. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. Further provided are methods of modulating the expression of SHP-2 in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further provided are methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of SHP-2 by administering a therapeutically or prophylactically effectiv

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