Antisense modulation of MEKK3 expression

Details Antisense modulation of MEKK3 expression Antisense modulation of MEKK3 expression

2000-09-08

2002-12-24

McGarry, Sean (Department: 1635)

Chemistry: molecular biology and microbiology

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

Method of regulating cell metabolism or physiology

C435S377000, C435S006120, C536S023100, C536S024100, C536S024500, C514S04400A

Reexamination Certificate

active

06498035

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of MEKK3. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding MEKK3. Such compounds have been shown to modulate the expression of MEKK3.
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. Four distinct subgroups of the MAP kinase pathways have been identified and each of these consists of a specific three-module set of downstream kinases. These four are known as the extracellular signal regulated kinase (ERK) family, the Jun N-terminal kinase/Stress activated protein kinase (JNK/SAPK) family, the p38 kinase family and the Big Mitogen-activated protein kinase (BMK1/ERK5) family. Each of these families is activated by distinct signaling events resulting in unique biological functions (Widmann et al.,
Physiol. Rev.,
1999, 79, 143-180). Briefly, the JNK/SAPK and p38 pathways are activated primarily by proinflammatory cytokines such as interleukins and tumor necrosis factor (TNF) and various stress signals such as UV irradiation, osmotic or heat shock and wound stress. In contrast, the ERK pathway is activated by mitogenic stimuli such as growth and/or differentiation factors. Finally, the BMK/ERK5 family, the newest pathway to be identified, is activated by growth factors, oxidative stress and hyperosmolar conditions (Widmann et al.,
Physiol. Rev.,
1999, 79, 143-180).
It is evident from the similarity in activating stimuli that cross-talk between families does occur and while there are unique kinases found in every family, one kinase, MEKK3, has been identified which is involved in the regulation of all four signaling cascades.
MEKK3 (also known as MAP/ERK kinase kinase 3 and MAPKKK3) is a ubiquitously expressed serine-threonine kinase first isolated in the mouse and originally shown to activate only the ERK and JNK/SAPK pathways (Blank et al.,
J. Biol. Chem.,
1996, 271, 5361-5368; Ellinger-Ziegelbauer et al.,
J. Biol. Chem.,
1997, 272, 2668-2674). Recently, it has been demonstrated that MEKK3 also mediates the p38 (Deacon and Blank,
J. Biol. Chem.,
1999, 274, 16604-16610) and the BMK/ERK5 pathways (Chao et al.,
J. Biol. Chem.,
1999, 274, 36035-36038). Furthermore, it has been shown that MEKK3 can activate the downstream transcription factor, NFkB (Zhao and Lee,
J. Biol. Chem.,
1999, 274, 8355-8358).
Disclosed in the PCT application WO 99/47686 are the nucleic acid sequence of MEKK3 as well as vectors comprising the MEKK3 nucleic acid molecule and host cells containing said vector. Further disclosed are monoclonal and polyclonal antibodies to human MEKK proteins as well as transgenic animals carrying a transgene that encodes a human MEKK protein. In addition, methods are disclosed for modulating human MEKK activity in the cell (Johnson, 1999). The MEKK3 protein, like other MEKK proteins, contains distinct catalytic and regulatory domains and these are disclosed in U.S. Pat. No. 5,854,043 (Johnson, 1998).
Considering the diverse role of MEKK3 in the regulation of all MAP kinase pathways identified to date, and the variety of pathologic conditions resulting in the deregulation of these pathways, the pharmacological modulation of MEKK3 activity and/or expression may be an appropriate point of therapeutic intervention.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of MEKK3 and to date, investigative strategies aimed at modulating MEKK3 function have involved the use of antibodies and compounds that interfere in signal transduction pathways.
Disclosed in U.S. Pat. No. 5,910,417 are methods to treat allergic inflammation in humans, comprising administering an effective amount of a regulatory compound that interacts with a MEKK/JNKK signal transduction molecule from the group consisting of MEKK1, MEKK2, MEKK3, MEKK4, JNKK1, JNKK2 and JNK1. Further disclosed are methods to screen for the modulation of cytokine production after the treatment of hematopoietic cells with said regulatory compounds (Gelfand and Johnson, 1999).
Disclosed in PCT application WO 98/54203 are methods to increase cancer cell sensitivity to cancer therapy by contacting said cells with a SAPK pathway inhibitor, specifically an inhibitor of MEKK1. These inhibitors include ribozymes, antisense nucleic acid molecules targeting a SAPK kinase kinase, or dominant negative mutants of an SAPK kinase kinase (Mercola, 1998). However, within this PCT publication, the composition of these inhibitors is not disclosed.
Disclosed in U.S. Pat. No. 5,981,265 are methods for regulating MEKK protein activity by transfecting or transforming a cell with a nucleic acid molecule capable of hybridizing with a nucleic acid molecule consisting of any of the known MEKK proteins, MEKK1, MEKK2, MEKK3, MEKK4, MEKK5 or MEKK6 (Johnson, 1999).
However, these strategies are untested as therapeutic protocols and consequently there remains a long felt need for additional agents capable of effectively inhibiting MEKK3 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 MEKK3 expression.
The present invention provides compositions and methods for modulating MEKK3 expression.
SUMMARY OF THE INVENTION
The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding MEKK3, and which modulate the expression of MEKK3. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of MEKK3 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 MEKK3 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 compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding MEKK3, ultimately modulating the amount of MEKK3 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding MEKK3. As used herein, the terms “target nucleic acid” and “nucleic acid encoding MEKK3” encompass DNA encoding MEKK3, 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 interfer

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