Antisense modulation of RIP2 expression

Details Antisense modulation of RIP2 expression Antisense modulation of RIP2 expression

2001-08-01

2002-07-30

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

06426221

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of RIP2. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding RIP2. Such compounds have been shown to modulate the expression of RIP2.
BACKGROUND OF THE INVENTION
Apoptosis, or programmed cell death, is a naturally occurring process that has been strongly conserved during evolution to prevent uncontrolled cell proliferation. This form of cell suicide plays a crucial role in ensuring the development and maintenance of multicellular organisms by eliminating superfluous or unwanted cells. However, if this process becomes overstimulated, cell loss and degenerative disorders including neurological disorders such as Alzheimer's, Parkinsons, ALS, retinitis pigmentosa and blood cell disorders can result. Stimuli which can trigger apoptosis include growth factors such as tumor necrosis factor (TNF), Fas and transforming growth factor beta (TGF&bgr;), neurotransmitters, growth factor withdrawal, loss of extracellular matrix attachment and extreme fluctuations in intracellular calcium levels (Afford and Randhawa,
Mol. Pathol.,
2000, 53, 55-63).
Alternatively, insufficient apoptosis, triggered by growth factors, extracellular matrix changes, CD40 ligand, viral gene products neutral amino acids, zinc, estrogen and androgens, can contribute to the development of cancer, autoimmune disorders and viral infections (Afford and Randhawa,
Mol. Pathol.,
2000, 53, 55-63). Consequently, apoptosis is regulated under normal circumstances by the interaction of gene products that either induce or inhibit cell death and several gene products which modulate the apoptotic process have now been identified.
The most well-characterized apoptotic signaling cascade to date is that orchestrated by a family of cysteine proteases known as caspases. These enzymes activate apoptosis through proteolytic events triggered by one of several described mechanisms; including ligand binding to the cell surface death receptors of either the TNF or NGF receptor families, changes in mitochondrial integrity or chemical induction (Thornberry,
British Medical Bulletin,
1997, 53, 478-490).
The proteins that control the other less understood caspase activation pathways often exist as families that can be recognized based on their amino acid sequence and/or structural similarity. Moreover, interactions among these proteins are commonly mediated by domains that are intimately associated with apoptosis regulation including caspase-associated recruitment domains (CARDs), death domains (DDs) and death effector domains (DEDs) (Reed,
Am. J. Pathol.,
2000, 157, 1415-1430).
The overall structure of the CARD is comprised of six alpha helices (Reed,
Am. J. Pathol.,
2000, 157, 1415-1430). Homotypic interactions among CARD-carrying proteins play important roles in caspase activation throughout evolution (Reed,
Am. J. Pathol.,
2000, 157, 1415-1430). Several pro-caspases contain N-terminal CARDs in their pro-domains including caspases 1, 2, 4, 5 and 9 in humans and caspases 1, 2, 9, 11 and 12 in mice (Reed,
Am. J. Pathol.,
2000, 157, 1415-1430). Additional CARD-carrying proteins involved in caspase activation include: CED-4, ApafI, Nod-1 (CARD4) and Bcl-10 (huE10, CIPER) (Reed,
Am. J. Pathol.,
2000, 157, 1415-1430).
RIP2 (also known as receptor interacting protein 2, RICK, CARDIAK, CARD-containing ICE-associated kinase and RIP-like interacting CLARP kinase) contains a CARD as well as a serine/threonine kinase domain. (Inohara et al.,
J. Biol. Chem.,
1998, 273, 12296-12300). It was cloned in 1998 and identified in multiple human tissues including heart, brain, placenta, lung, pancreas, spleen, lymph node and peripheral lymphocytes. Its mRNA transcripts of 2.5 and 1.8 kb differ due to alternative polyadenylation (Inohara et al.,
J. Biol. Chem.,
1998, 273, 12296-12300; McCarthy et al.,
J. Biol. Chem.,
1998, 273, 16968-16975; Thome et al.,
Curr. Biol.,
1998, 8, 885-888).
Expression of RIP2 has been demonstrated to potentiate Fas apoptosis signaling via interactions with CLARP, a caspase-like protein with homology to caspase-8 (Inohara et al.,
J. Biol. Chem.,
1998, 273, 12296-12300). McCarthy et al have shown that RIP2 is a component of both the tumor necrosis factor receptor-1 (TNFR-1) and CD40 signaling complexes (McCarthy et al.,
J. Biol. Chem.,
1998, 273, 16968-16975). Thome et al. proposed a potential role for RIP2 in NF-&kgr;B/JNK signaling and in the generation of the proinflammatory cytokine IL-1&bgr; through activation of caspase-1 (Thome et al.,
Curr. Biol.,
1998, 8, 885-888).
Increased levels of RIP2 have been found in the frontal cortex in brains of patients with Alzheimer's disease thus strengthening the proposed role of caspase-8 in mediating apoptotic death in this disorder (Engidawork et al.,
Biochem. Biophys. Res. Commun.,
2001, 281, 84-93).
The role of RIP2 in apoptotic signaling pathways makes its selective inhibition a potential strategy with which to derive treatments for disorders arising from aberrant apoptosis. To date, specific inhibitors for RIP2 have yet to be developed or identified. Consequently, there remains a long felt need for agents capable of effectively and selectively inhibiting the function of RIP2.
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 expression of RIP2.
The present invention provides compositions and methods for modulating expression of RIP2, including alternative mRNA transcripts of RIP2.
SUMMARY OF THE INVENTION
The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding RIP2, and which modulate the expression of RIP2. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of RIP2 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 RIP2 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 RIP2, ultimately modulating the amount of RIP2 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding RIP2. As used herein, the terms “target nucleic acid” and “nucleic acid encoding RIP2” encompass DNA encoding RIP2, 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 RIP2. In the context of the present invention, “modulation” means either an increase (stimulation) or a decrease

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