Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Utility Patent
1999-07-23
2001-01-02
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
C435S006120, C435S091100, C435S366000, C536S023100, C536S024310, C536S024330, C536S024500
Utility Patent
active
06168950
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of MEKK1. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding human MEKK1. Such oligonucleotides have been shown to modulate the expression of MEKK1.
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.
Nearly all cell surface receptors use one or more of the mitogen-activated protein kinase (MAP kinase) cascades during signal transduction. Three distinct subgroups of the MAP kinases have been identified and each of these consists of a specific module of downstream kinases. One subgroup of the MAP kinases is the Jun N-terminal kinase/Stress activated protein kinase (JNK/SAPK) cascade. This pathway was originally identified as an oncogene- and ultraviolet light-stimulated kinase pathway but is now known to be activated by growth factors, cytokines and T-cell costimulation (Moriguchi et al.,
Adv. Pharmacol
., 1996, 36, 121-137; Su and Karin,
Curr. Opin. Immunol
., 1996, 8, 402-411).
MEKK1 (also known as mitogen-activated protein kinase kinase kinase 1, MEK kinase 1 and MAP/ERK kinase kinase 1) is a dual specific serine/threonine kinase that functions to mediate cellular responses to mitogenic stimuli. The MEKK1 protein has been shown to regulate signaling events associated with programmed cell death or apoptosis and NF-kappa-B, both of which have been associated with the development of hyperproliferative conditions such as cancer.
Apoptosis is not only a normal biological process but contributes to the pathogenesis of many diseases when deregulated. It has recently been shown that expression of the kinase domain of MEKK1 can potentiate the apoptotic response to external mitogenic stimuli in fibrosarcoma cells (Widmann et al.,
Oncogene
, 1997, 15, 2439-2447). Further support of MEKK1 involvement in apoptosis comes from studies of Bcl-2, an intracellular protein that prevents apoptosis. These studies showed that Bcl-2 prevented the mitogen-induced stimulation of MEKK1 in neuroglioma cells, indicating that MEKK1 lies directly downstream of Bcl-2 in an apoptosis signaling cascade (Park et al.,
J. Biol. Chem
., 1997, 272, 16725-16728).
Finally, studies involving the expression of mutant forms of MEKK1 protein corroborate the role for MEKK1 in apoptosis. Overexpression of a dominant negative form of MEKK1 was shown to prevent apoptosis induced by the topoisomerase inhibitor, &bgr;-lapachone (Shiah et al.,
Cancer Res
., 1999, 59, 391-398) and expression of a constitutively active mutant of MEKK1 has been shown to potentiate p53-dependent apoptosis (Fuchs et al.,
Proc. Natl. Acad. Sci. U. S. A
., 1998, 95, 10541-10546).
MEKK1 has also been shown to play a critical role in the control of NF-kappa-B-mediated transcription at multiple points in the cascade (Meyer et al.,
J. Biol. Chem
., 1996, 271, 8971-8976). MEKK1 was shown to induce transcription from the human immunodeficiency virus-1 long terminal repeat and the interleukin-2R&agr; promoters in a kappa-B dependent fashion (Meyer et al.,
J. Biol. Chem
., 1996, 271, 8971-8976). It was also shown to activate the I-kappa-B&agr; kinase complex, an inhibitor of NF-kappa-B, through phosphorylation (Lee et al., Cell, 1997, 88, 213-222).
MEKK1 also participates in the signaling pathway of keratinocyte differentiation (Efimova et al.,
J. Biol. Chem
., 1998, 273, 24387-24395), tumor necrosis factor-alpha production in mast cells (Ishizuka et al.,
Proc. Natl. Acad. Sci. U. S. A
., 1997, 94, 6358-6363) and neurite outgrowth in PC12 cells (Leppa et al.,
Embo. J
., 1998, 17, 4404-4413) and has been shown to form complexes with JNK/SAPK and Grb2, through the MEKK1 noncatalytic N-terminus (Pomerance et al.,
J. Biol. Chem
., 1998, 273, 24301-24304; Xu and Cobb,
J. Biol. Chem
., 1997, 272, 32056-32060).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of MEKK1 and to date, strategies aimed at modulating MEKK1 function have involved the use of antibodies, dominant negative and dominant active mutants of the protein and antisense oligonucleotides. Disclosed in U.S. Pat. No. 5,405,941 are the protein encoded by the MEKK1 gene, antibodies to the protein (Johnson, 1995). Disclosed in PCT application WO 98/54203 are methods to increase cancer cell sensitivity to cancer therapy by contacting said cells with a MEKK1 inhibitor. These inhibitors being ribozymes, antisense nucleic acid molecules targeting MEKK1, or dominant negative mutants of MEKK1 (Mercola, 1998, ). However, within this PCT publication, the composition of these inhibitors is not disclosed.
Therefore, there remains a long felt need for additional agents capable of effectively inhibiting MEKK1 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 MEKK1 expression.
SUMMARY OF THE INVENTION
The present invention is directed to antisense compounds, particularly oligonucleotides, which are targeted to a nucleic acid encoding MEKK1, and which modulate the expression of MEKK1. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. Further provided are methods of modulating the expression of MEKK1 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 MEKK1 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 MEKK1, ultimately modulating the amount of MEKK1 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding MEKK1. As used herein, the terms “target nucleic acid” and “nucleic acid encoding MEKK1” encompass DNA encoding MEKK1, 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 MEKK1. In th
Cowsert Lex M.
Gaarde William
Monia Brett P.
Ward Donna T.
Elliott George C.
ISIS Pharmaceuticals Inc.
Law Offices of Jane Massey Licata
Schmidt Melissa
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