Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
2001-02-09
2002-10-15
LeGuyader, John L. (Department: 1635)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S006120, C435S091100, C435S325000, C435S366000, C536S023100, C536S024310, C536S024330, C536S024500
Reexamination Certificate
active
06465250
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of Protein Phosphatase 2 catalytic subunit alpha. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding Protein Phosphatase 2 catalytic subunit alpha. Such compounds have been shown to modulate the expression of Protein Phosphatase 2 catalytic subunit alpha.
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 (Wera and Hemings,
Biochemistry Journal
, 1995, 311, 17-29), 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 these enzymes.
The enzyme protein phosphatase 2A (also known as PPP2A and PP2A) is one of four major protein phosphatases identified in the cytosol of eukaryotic cells which are responsible for the dephosphorylation of serine and threonine residues in proteins. These four enzymes have overlapping substrate specificities and are distinguished by their regulation and dependence on metal ions. Protein phosphatase 2A activity is independent of metal ions and appears to play a role in the regulation of major metabolic pathways, as well as the processes of translation, transcription, platelet activation and control of the cell cycle (Goldberg,
Biochem. Pharmacol
., 1999, 57, 321-328; Millward et al.,
Trends Biochem. Sci
., 1999, 24, 186-191; Toyoda et al.,
Thromb. Haemost
., 1996, 76, 1053-1062). More specifically, Protein Phosphatase 2A participates as a negative regulator in many kinase signal transduction pathways, including those involving MAP kinase, JNK kinase, ERK kinase, CaM kinase, and casein kinase. In addition, Protein Phosphatase 2A also interacts with many cellular and viral proteins (Millward et al.,
Trends Biochem. Sci
., 1999, 24, 186-191). The enzyme has been shown to be activated by ceramide, a metabolic product of sphingomyelin hydrolysis and mediator of the biological effects of hormones, cytokines and growth factors (Dobrowsky et al.,
J. Biol. Chem
., 1993, 268, 15523-15530).
The mammalian protein phosphatase 2A enzyme is a heterotrimer composed of a catalytic subunit of 36 kD complexed to two regulatory subunits, one of mass 65 kD and one of variable mass. In addition, two isoforms of the catalytic subunit of protein phosphatase 2A, alpha and beta, are demonstrable in many species. The structures of these catalytic subunits show high evolutionary conservation supporting the idea that they may serve crucial functions (Goldberg,
Biochem. Pharmacol
., 1999, 57, 321-328; Millward et al.,
Trends Biochem. Sci
., 1999, 24, 186-191).
Protein Phosphatase 2 catalytic subunit alpha (also known as PPP2CA) was originally isolated from lung and lung fibroblast libraries (Stone et al.,
Nucleic Acids Res
., 1988, 16, 11365), while the gene was isolated from a human leukocyte library (Khew-Goodall et al.,
Biochemistry
, 1991, 30, 89-97). Northern analysis has revealed that the alpha subunit is expressed at relatively high levels compared to the beta subunit in all tissues examined. The structural characterization of the two genes implies that this is due in part to the different strengths of the promoters (Khew-Goodall et al.,
Biochemistry
, 1991, 30, 89-97).
The catalytic subunit of Protein Phosphatase 2A has been linked to both insulin signaling (Klarlund et al.,
J. Biol. Chem
., 1991, 266, 4052-4055; Kowluru et al.,
Endocrinology
, 1996, 137, 2315-2323) and to retinoic acid-induced cellular differentiation of HL-60 cells, an acute promyelocytic leukemia cell line (Nishikawa et al.,
Cancer Res
., 1994, 54, 4879-4884; Tawara et al.,
FEBS Lett
., 1993, 321, 224-228). The pharmacological modulation of the catalytic subunit of Protein Phosphatase 2A activity and/or expression may therefore be an appropriate point of therapeutic intervention in pathological conditions such as diabetes and cancer.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of the alpha isoform of Protein Phosphatase 2A catalytic subunit, and to date, investigative strategies aimed at modulating activity of Protein Phosphatase 2A function have involved the use of antibodies, molecules that block upstream entities, chemical inhibitors and gene knock-outs in mice.
Disclosed in U.S. Pat. Nos. 5,925,660 and 5,700,821 are compounds useful as phosphatase inhibitors and methods of making such inhibitors (Lazo et al., 1999; Lazo et al., 1997). It has also been reported that the compound, Fostriecin and compounds structurally related to it are effective serine/threonine phosphatase inhibitors. These are disclosed in the PCT publication WO 98/14606 (Honkanen and Downey, 1998). Disclosed in the PCT publication WO 99/27134 are antisense oligonucleotides targeting serine/threonine phosphatases, PP5, PP4 and PP1&ggr;1 none of which target or hybridize to the Protein Phosphatase 2A isoforms (Honkanen and Dean, 1999).
In addition, at the protein level, there are compounds that interact with and consequently modulate the activity of the Protein Phosphatase 2A enzyme. These compounds and methods to identify these compounds are disclosed in the PCT publication WO 97/37037 (Hemmings, 1997).
Finally, homozygous null mutant mice are embryonically lethal, demonstrating that the alpha subunit gene is an essential gene (Gotz et al.,
Proc. Natl. Acad. Sci. U. S. A
., 1998, 95, 12370-12375).
These strategies are untested as therapeutic protocols and consequently there remains a long felt need for additional agents capable of effectively inhibiting Protein Phosphatase 2A catalytic subunit alpha 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 Protein Phosphatase 2A catalytic subunit alpha expression.
The present invention provides compositions and methods for modulating Protein Phosphatase 2A catalytic subunit alpha expression.
SUMMARY OF THE INVENTION
The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding Protein Phosphatase 2 catalytic subunit alpha, and which modulate the expression of Protein Phosphatase 2 catalytic subunit alpha. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of Protein Phosphatase 2 catalytic subunit alpha 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 Protein Phosphatase 2 catalytic subunit alpha by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the inv
ISIS Pharmaceuticals Inc.
LeGuyader John L.
Licata & Tyrrell P.C.
Schmidt M
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