Antisense modulation of cot oncogene expression

Details Antisense modulation of cot oncogene expression Antisense modulation of cot oncogene expression

2000-01-20

2001-07-24

McGarry, Sean (Department: 1635)

Chemistry: molecular biology and microbiology

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

Method of regulating cell metabolism or physiology

C536S024500, C514S04400A

Reexamination Certificate

active

06265216

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of cot oncogene. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding cot oncogene. Such oligonucleotides have been shown to modulate the expression of cot oncogene.
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 highly regulated and often overlapping as evidenced by the existence of many protein kinases as well as protein phosphatases. It is currently believed that a number of disease states and/or disorders are a result of either aberrant expression or functional mutations in the molecular components of kinase cascades. Consequently, considerable attention has been devoted to the characterization of these proteins.
The cot oncogene (also known as Tpl-2 and est) was first identified in a rearranged form in embryonic SHOK (Syrian hamster osaka kanazawa) cells transformed by transfection with genomic DNA from human thyroid carcinoma cells (Miyoshi et al.,
Mol. Cell. Biol
., 1991, 11, 4088-4096). The gene encoding the normal cellular product of the cot oncogene was later isolated from a Ewing sarcoma cell line and found to reside on the long arm of human chromosome 10 (Chan et al.,
Oncogene
, 1993, 8, 1329-1333).
The cot oncogene protein has been characterized as a serine/threonine kinase of the MAP kinase kinase kinase family of enzymes and, as such, has been shown to participate in several kinase pathways including the MAPK (Kim et al.,
J. Cell. Biochem
., 1998, 71, 286-301), JNK/SAPK (Hagemann et al.,
Oncogene
, 1999, 18, 1391-1400; Troppmair et al.,
J. Biol. Chem
., 1994, 269, 7030-7035) and NF-kappa-B kinases (Belich et al., Nature, 1999, 397, 363-368; Lin et al.,
Immunity
, 1999, 10, 271-280). Ectopic expression of the cot oncogene has been shown to induce the transcription of interleukin-2 in T-cells, suggesting that cot oncogene may also be involved in the CD3/CD28 costimulatory pathway (Ballester et al.,
J. Immunol
., 1997, 159, 1613-1618). More recently, it has been demonstrated that the cot oncogene activates tumor necrosis factor (TNF)-alpha gene expression in a cyclosporin A-resistant manner. In these studies, overexpression of cot oncogene in Jurkat cells (a human T-cell leukemia cell line) promoted TNF-alpha production while a kinase-inactive form of the enzyme did not (Ballester et al.,
J. Biol. Chem
., 1998, 273, 14099-14106).
The cot enzyme is localized to the cytoplasm and exists in two different forms, a long form and a truncated or short form, which are products of alternative initiation mechanisms. These two forms have differing transforming activities and it is rearrangement of the C-terminus that increases cellular transformation (Aoki et al.,
Oncogene
, 1991, 6, 1515-1519; Aoki et al.,
J. Biol. Chem
., 1993, 268, 22723-22732). Expression of cot oncogene is increased upon treatment of cells with interleukin-1, a cytokine involved in inflammation or okadaic acid, a well-known tumor promoter (Chan et al.,
Oncogene
, 1993, 8, 1329-1333). In the rat, it has been demonstrated that the cot oncogene is involved in mouse mammary tumor virus (MMTV) associated transformation of mammary gland cells (Erny et al.,
Oncogene
, 1996, 13, 2015-2020). The pharmacological modulation of cot oncogene activity and/or expression may therefore be an appropriate point of therapeutic intervention in pathological conditions such as inflammation, cancer and disorders of the immune system.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of cot oncogene and to date, investigative strategies aimed at modulating cot oncogene function have involved the use of antibodies and kinase-inactive mutants. Consequently, there remains a long felt need for agents capable of effectively inhibiting cot oncogene 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 cot oncogene expression.
The present invention provides compositions and methods for modulating cot oncogene expression, including modulation of the alternative short form of the cot oncogene.
SUMMARY OF THE INVENTION
The present invention is directed to antisense compounds, particularly oligonucleotides, which are targeted to a nucleic acid encoding cot oncogene, and which modulate the expression of cot oncogene. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. Further provided are methods of modulating the expression of cot oncogene 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 cot oncogene by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention employs oligomeric antisense compounds, particularly oligonucleotides, for use in modulating the function of nucleic acid molecules encoding cot oncogene, ultimately modulating the amount of cot oncogene produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding cot oncogene. As used herein, the terms “target nucleic acid” and “nucleic acid encoding cot oncogene” encompass DNA encoding cot oncogene, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as “antisense”. The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of cot oncogene. In the context of the resent invention, “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target.
It is preferred to target specific nucleic acids for antisense. “Targeting” an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding cot oncogene. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of e

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