Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
2000-05-24
2001-09-04
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, C435S366000, C536S023100, C536S024310, C536S024330, C536S024500
Reexamination Certificate
active
06284538
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of PTEN. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding human PTEN. Such oligonucleotides have been shown to modulate the expression of PTEN.
BACKGROUND OF THE INVENTION
One of the principal mechanisms by which cellular regulation is effected is through the transduction of extracellular signals across the membrane that in turn modulate biochemical pathways within the cell. Protein phosphorylation represents one course by which intracellular signals are propagated from molecule to molecule resulting finally in a cellular response. These signal transduction cascades are tightly regulated and often overlap as evidenced by the existence of multiple protein kinase and phosphatase families and isoforms.
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 the molecular components of these cascades. Consequently, considerable attention has been devoted to the characterization of proteins exhibiting either kinase or phosphatase enzymatic activity.
PTEN (also known as MMAC1 and TEP1) is a dual-specificity protein phosphatase recently implicated as a phosphoinositide phosphatase in the insulin-signaling pathway. In studies of human 293 cells, PTEN was shown to dephosphorylate phosphatidylinositol 3,4,5-triphosphate (PIP3), an acidic lipid that is involved in cellular growth signaling (Maehama and Dixon,
J. Biol. Chem
., 1998, 273, 13375-13378). In Drosophila, studies of PTEN activation and overexpression demonstrated that PTEN affects both cell size and cell cycle progression during eye development. In addition, the authors demonstrated that PTEN acts in the insulin signaling pathway and that all signals from the insulin receptor can be antagonized by PTEN. These data suggest that modulation of PTEN may represent a means for modulating altered insulin signaling (Huang et al.,
Development
, 1999, 126, 5365-5372).
PIP3 is an important second messenger generated specifically by the actions of phosphatidylinositol 3-kinase (PI3-kinase) following insulin binding (Stephens et al.,
Science
, 1998, 279, 710-714). overexpression of PTEN was shown to reduce the levels of PIP3 in insulin treated cells without affecting the activity of PI3-kinase (Maehama and Dixon,
J. Biol. Chem
., 1998, 273, 13375-13378). These results establish a role for PTEN as a regulator of the downstream pathways initiated by insulin binding. In the nematode,
Caenorhabditis elegans
, the PTEN homolog, daf-18, has been cloned and shown to antagonize signaling cascades associated with PI3-kinase (Gil et al.,
Proc. Natl. Acad. Sci. USA
, 1999, 96, 2925-2930). The authors suggest that this may indicate that PTEN may play a role in mammalian glucose homeostasis, and that PTEN may be a rational pharmacological target for Type II diabetes.
The PTEN protein also contains an amino terminal domain homologous to tensin and auxilin, proteins that interact with actin filaments and are involved in synaptic vesicle transport, respectively (Li and Sun,
Cancer Res
., 1997, 57, 2124-2129; Li et al.,
Science
, 1997, 275, 1943-1947; Steck et al.,
Nat. Genet
., 1997, 15, 356-362). In addition, PTEN is also downregulated by transforming growth factor beta (TGF-&bgr;), a cytokine involved in the regulation of cell adhesion and motility (Li and Sun,
Cancer Res
., 1997, 57, 2124-2129). Taken together these data suggest that PTEN plays a dual role within the cell by mediating the activity of protein kinases while regulating cell motility (Tamura et al.,
Science
, 1998, 280, 1614-1617).
Finally, a large number of naturally occurring point and germ-line mutations have been identified in PTEN. These mutations have been isolated from several cancerous solid tumors and cell lines including brain, breast, prostate, ovary, skin, thyroid, lung, bladder and colon (Teng et al.,
Cancer Res
., 1997, 57, 5221-5225) and have led to the classification of PTEN as a tumor suppressor gene. Disclosed in the PCT publication WO 99/02704 are PTEN proteins and altered PTEN proteins and the nucleic acids encoding them. Also disclosed are methods of diagnosis and treatment utilizing compositions comprising PTEN or altered PTEN proteins or nucleic acid molecules.
The most common mutations found in tumor specimens were frameshift mutations (1 in 17 breast carcinomas), missense variants (1 in 10 melanomas), nonsense mutations and splice variants (2 in 5 pediatric glioblastomas). In tumor cell lines exhibiting loss of heterozygosity (LOH), 11 homozygous deletions affecting the coding region were detected. Two cell lines had lost all 9 exons and nine cell lines had homozygous deletions of portions of the coding regions. The remaining 65 cell lines contained 3 frameshift, one nonsense and 8 nonconservative missense mutations (Teng et al.,
Cancer Res
., 1997, 57, 5221-5225).
The known germ-line mutations in PTEN give rise to three distinct autosomal dominant disorders known as Cowden disease (CD) (Liaw et al.,
Nat. Genet
., 1997, 16, 64-67; Nelen et al.,
Hum. Mol. Genet
., 1997, 6, 1383-1387; Tsou et al.,
Hum. Genet
., 1998, 102, 467-473), Lhermitte-Duclos disease (LDD) (Liaw et al.,
Nat. Genet
., 1997, 16, 64-67) and Bannayan-Zonana syndrome (BZS, also known as Bannayan-Riley-Ruvalcaba syndrome, Ruvalcaba-Myhre-Smith syndrome and Riley-Smith syndrome) (Arch et al.,
Am. J. Med. Genet
., 1997, 71, 489-493; Marsh et al.,
Nat. Genet
., 1997, 16, 333-334). All of these conditions are characterized by the presence of gastrointestinal polyps, increased tumor susceptibility and developmental defects.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of PTEN, and strategies aimed at inhibiting and/or investigating PTEN function have involved the use of gene knock-outs in mice and ribozyme- and vector-based antisense-mediated regulation of PTEN expression.
Di Cristofano et al. demonstrated that the complete disruption of the mouse PTEN gene by homologous recombination resulted in embryonic lethality (Di Cristofano et al.,
Nat. Genet
., 1998, 19, 348-355). By contrast, PTEN +/− chimeric mice were phenotypically identical to their wild-type littermates. However, post-mortem analysis revealed abnormal pathological conditions similar to those observed in human diseases.
Other studies involving the targeted disruption of exons 3 and 5 in mice demonstrated that homozygous mice died by day 9.5 of development and that immortalized cells from these embryos showed decreased sensitivity to various apoptotic stimuli (Stambolic et al.,
Cell
, 1998, 95, 29-39). These cells also displayed constitutively elevated activity of the PKB/Akt kinases. Taken together these results suggest that PTEN acts by negatively regulating the PI3-kinase/PKB/Akt pathway.
Devlin and Clawson identified ribozyme-accessible sites on full length PTEN cDNA and, using these results, designed a ribozyme construct for the purpose of regulating PTEN transcripts.
Proc. Am. Assoc. Cancer Res
., 1999, 40, 438.
Tamura et al. established stable transfectant lines of mouse 3T3 cells in which the expression of PTEN was up- or down-regulated using expression plasmids containing full length sense PTEN or full-length antisense PTEN. The antisense construct enhanced cell migration.
Science
, 1998, 280, 1614-1617.
There remains a long felt need for agents capable of effectively inhibiting PTEN 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 PTEN expression.
SUMMARY OF THE INVENTION
The present invention is di
Cowsert Lex M.
McKay Robert
Monia Brett P.
ISIS Pharmaceuticals Inc.
Licata & Tyrrell P.C.
Schmidt M
Schwartzman Robert A.
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