Antisense modulation of PTP1B expression

Details Antisense modulation of PTP1B expression Antisense modulation of PTP1B expression

2000-01-18

2001-07-17

Schwartzman, Robert A. (Department: 1635)

Chemistry: molecular biology and microbiology

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

Method of regulating cell metabolism or physiology

C435S006120, C435S091100, C435S458000, C435S366000, C536S023100, C536S024500, C536S025300

Reexamination Certificate

active

06261840

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of PTP1B. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding PTP1B. Such oligonucleotides have been shown to modulate the expression of PTP1B.
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 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 of, or functional mutations in, kinases and phosphatases. Consequently, considerable attention has been devoted recently to the characterization of tyrosine kinases and tyrosine phosphatases.
PTP1B (also known as protein phosphatase 1B and PTPN1) is an endoplasmic reticulum (ER)-associated enzyme originally isolated as the major protein tyrosine phosphatase of the human placenta (Tonks et al.,
J. Biol. Chem.,
1988, 263, 6731-6737; Tonks et al.,
J. Biol. Chem.,
1988, 263, 6722-6730).
An essential regulatory role in signaling mediated by the insulin receptor has been established for PTP1B. PTP1B interacts with and dephosphorylates the activated insulin receptor both in vitro and in intact cells resulting in the downregulation of the signaling pathway (Goldstein et al.,
Mol. Cell. Biochem.,
1998, 182, 91-99; Seely et al.,
Diabetes,
1996, 45, 1379-1385). In addition, PTP1B modulates the mitogenic actions of insulin (Goldstein et al.,
Mol. Cell. Biochem.,
1998, 182, 91-99). In rat adipose cells overexpressing PTP1B, the translocation of the GLUT4 glucose transporter was inhibited, implicating PTP1B as a negative regulator of glucose transport as well (Chen et al.,
J. Biol. Chem.,
1997, 272, 8026-8031). Mouse knockout models lacking the PTP1B gene also point toward the negative regulation of insulin signaling by PTP1B.
Mice harboring a disrupted PTP1B gene showed increased insulin sensitivity, increased phosphorylation of the insulin receptor and when placed on a high-fat diet, PTP1B −/− mice were resistant to weight gain and remained insulin sensitive (Elchebly et al.,
Science,
1999, 283, 1544-1548). These studies clearly establish PTP1B as a therapeutic target in the treatment of diabetes and obesity.
PTP1B, which is differentially regulated during the cell cycle (Schievella et al.,
Cell. Growth Differ.,
1993, 4, 239-246), is expressed in insulin sensitive tissues as two different isoforms that arise from alternate splicing of the pre-mRNA (Shifrin and Neel,
J. Biol. Chem.,
1993, 268, 25376-25384). It was recently demonstrated that the ratio of the alternatively spliced products is affected by growth factors such as insulin and differs in various tissues examined (Sell and Reese,
Mol. Genet. Metab.,
1999, 66, 189-192). In these studies it was also found that the levels of the variants correlated with the plasma insulin concentration and percentage body fat and may therefore be used as a biomarker for patients with chronic hyperinsulinemia or type 2 diabetes.
Liu and Chernoff have shown that PTP1B binds to and serves as a substrate for the epidermal growth factor receptor (EGFR) (Liu and Chernoff,
Biochem. J.,
1997, 327, 139-145). Furthermore, in A431 human epidermoid carcinoma cells, PT1B was found to be inactivated by the presence of H
2
O
2
generated by the addition of EGF. These studies indicate that PTP1B can be negatively regulated by the oxidation state of the cell, which is often deregulated during tumorigenesis (Lee et al.,
J. Biol. Chem.,
1998, 273, 15366-15372).
Overexpression of PTP1B has been demonstrated in malignant ovarian cancers and this correlation was accompanied by a concomitant increase in the expression of the associated growth factor receptor (Wiener et al.,
Am. J. Obstet. Gynecol.,
1994, 170, 1177-1183).
PTP1B has been shown to suppress transformation in NIH3T3 cells induced by the neu oncogene (Brown-Shimer et al.,
Cancer Res.,
1992, 52, 478-482), as well as in rat 3Y1 fibroblasts induced by v-srk, v-src, and v-ras (Liu et al.,
Mol. Cell. Biol.,
1998, 18, 250-259) and rat-1 fibroblasts induced by bcr-abl (LaMontagne et al.,
Proc. Natl. Acad. Sci. U. S. A.,
1998, 95, 14094-14099). It has also been shown that PTP1B promotes differentiation of K562 cells, a chronic myelogenous leukemia cell line, in a similar manner as does an inhibitor of the bcr-abl oncoprotein. These studies describe the possible role of PTP1B in controlling the pathogenesis of chronic myeloid leukemia (LaMontagne et al.,
Proc. Natl. Acad. Sci. U. S. A.,
1998, 95, 14094-14099).
PTP1B negatively regulates integrin signaling by interacting with one or more adhesion-dependent signaling components and repressing integrin-mediated MAP kinase activation (Liu et al.,
Curr. Biol.,
1998, 8, 173-176). Other studies designed to study integrin signaling, using a catalytically inactive form of PTP1B, have shown that PTP1B regulates cadherin-mediated cell adhesion (Balsamo et al.,
J. Cell. Biol.,
1998, 143, 523-532) as well as cell spreading, focal adhesion and stress fiber formation and tyrosine phosphorylation (Arregui et al.,
J. Cell. Biol.,
1998, 143, 861-873).
Currently, therapeutic agents designed to inhibit the synthesis or action of PTP1B include small molecules (Ham et al.,
Bioorg. Med. Chem. Lett.,
1999, 9, 185-186; Skorey et al.,
J. Biol. Chem.,
1997, 272, 22472-22480; Taing et al.,
Biochemistry,
1999, 38, 3793-3803; Taylor et al.,
Bioorg. Med. Chem.,
1998, 6, 1457-1468; Wang et al.,
Bioorg. Med. Chem. Lett.,
1998, 8, 345-350; Wang et al.,
Biochem. Pharmacol.,
1997, 54, 703-711; Yao et al.,
Bioorg. Med. Chem.,
1998, 6, 1799-1810) and peptides (Chen et al.,
Biochemistry,
1999, 38, 384-389; Desmarais et al.,
Arch. Biochem. Biophys.,
1998, 354, 225-231; Roller et al.,
Bioorg. Med. Chem. Lett.,
1998, 8, 2149-2150). In addition, disclosed in the PCT publication WO 97/32595 are phosphopeptides and antibodies that inhibit the association of PTP1B with the activated insulin receptor for the treatment of disorders associated with insulin resistance. Antisense nucleotides against PTP1B are also generally disclosed (Olefsky, 1997).
There remains a long felt need for additional agents capable of effectively inhibiting PTP1B function and antisense technology is emerging as an effective means for reducing the expression of specific gene products. This technology may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of PTP1B expression.
The present invention, therefore, provides compositions and methods for modulating PTP1B expression, including modulation of the alternatively spliced form of PTP1B.
SUMMARY OF THE INVENTION
The present invention is directed to antisense compounds, particularly oligonucleotides, which are targeted to a nucleic acid encoding PTP1B, and which modulate the expression of PTP1B. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. Further provided are methods of modulating the expression of PTP1B in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds

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