Antisense inhibition of cytohesin-1 expression

Details Antisense inhibition of cytohesin-1 expression Antisense inhibition of cytohesin-1 expression

2000-10-30

2002-05-07

Wang, Andrew (Department: 1635)

Chemistry: molecular biology and microbiology

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

Method of regulating cell metabolism or physiology

C435S006120, C435S325000, C435S366000, C536S023100, C536S024500, C514S04400A

Reexamination Certificate

active

06383809

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of cytohesin-1. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding human cytohesin-1. Such oligonucleotides have been shown to modulate the expression of cytohesin-1.
BACKGROUND OF THE INVENTION
G-proteins are heterotrimeric GTP-binding proteins that, via their association with cell-surface receptors, relay external signals from biologically active molecules including growth factors, cytokines, neurotransmitters and hormones to a variety of cellular effectors.
G-proteins have been shown to mediate the processes of cell growth, proliferation, signal transduction, membrane trafficking and cytoskeletal interactions. They do so by cycling between an active GTP-bound form and an inactive GDP-bound form. Regulation of the on/off rates of nucleotides for G-proteins is controlled by two classes of molecules; GTPase activating proteins (GAPs) which hydrolyze GTP and guanine nucleotide exchange factors (GEFs) that activate G-proteins by catalyzing the replacement of bound GDP with GTP.
ADP ribosylation factors (ARFs) are a subfamily of small monomeric G-proteins, belonging to the Ras superfamily, which regulate critical vesicular traffic pathways. ARFs initiate the secretion of proteins through the endoplasmic reticulum and Golgi by binding to the donor membrane, which in turn, recruits coatomers, leads to bud formation and eventual vesicle release. ARFs also regulate endocytosis, synaptic vesicle formation, peroxisome biogenesis, and phospholipase D activation (Exton,
Eur. J. Biochem.,
1997, 243, 10-20; Exton,
Physiol. Rev.,
1997, 77, 303-320; Roth and Sternweis,
Curr. Opin. Cell Biol.,
1997, 9, 519-526; Schurmann et al.,
J. Biol. Chem.,
1999, 274, 9744-9751; Vaughan and Moss,
Adv. Exp. Med. Biol.,
1997, 419, 315-320).
A small family of cytosolic adapter proteins have been isolated and shown to function as guanine nucleotide exchange factors for the ARF proteins. These include cytohesin-1 (Liu and Pohajdak,
Biochim. Biophys. Acta.,
1992, 1132, 75-78), cytohesin-2 (Chardin et al.,
Nature,
1996, 384, 481-484), ARNO3 (Franco et al.,
Proc. Natl. Acad. Sci. U.S.A.,
1998, 95, 9926-9931; Klarlund et al.,
Science,
1997, 275, 1927-1930) and EFA6 (Franco et al.,
Embo J.,
1999, 18, 1480-1491). All of the members of this family contain conserved domain structures that include a pleckstrin homology (PH) domain responsible for signal-dependent phospholipid binding (Hemmings,
Science,
1997, 275, 1899) and a Sec7 domain that mediates guanine nucleotide exchange. Disclosed in patent application EP 0763597 are peptides encoding the pleckstrin homology domain of cytohesin-1 and cytohesin-2 as well as DNA encoding the peptides and the full-length proteins, cells that express the peptides, and the use of the peptides to treat various disorders (Kolanus and Ostner, 1997). Also disclosed are DNA molecules that hybridize to the cytohesin domains under stringent conditions (Kolanus and Ostner, 1997).
Cytohesin-1 (also known as PSCD1, B2-1, Sec7p-like protein and mSec7-1) was the first ARF GEF member to be isolated and was initially identified in natural killer and cytotoxic T-cells (Liu and Pohajdak,
Biochim. Biophys. Acta.,
1992, 1132, 75-78). Overexpression of the full-length cytosolic protein or the Sec7 domain of cytohesin-1 in Jurkat cells, a T-cell leukemia cell line, has been shown to give rise to constitutive activation of lymphocyte function-associated antigen 1 (LFA-1). However, expression of the PH domain of cytohesin-1 served as a dominant-negative regulator of integrin function in lymphocytes. These finding suggest that the overall function of cytohesin-1 in this system is to regulate cell adhesion through LFA-1 and to function as an inside-out signaling molecule through interactions with integrins (Kolanus et al.,
Cell,
1996, 86, 233-242). It was recently confirmed that the inside-out signaling by &bgr;2 integrins, mediated by the membrane recruitment of cytohesin-1 via the PH and polybasic c domains, is activated by phosphotidylinositide 3-OH kinase (Nagel et al.,
Mol. Biol. Cell,
1998, 9, 1981-1994; Nagel et al.,
J. Biol. Chem.,
1998, 273, 14853-14861). In addition, adherence of monocytes to endothelial cells, mediated in part by LFA-1, is necessary for localization to the site of inflammation and, recently, Hmama et al. demonstrated that monocyte adherence induced by lipopolysacharides (LPS) involves cytohesion-1 (Hmama et al.,
J. Biol. Chem.,
1999, 274, 1050-1057). Treatment of THP-1 cells with an antisense phosphorothioate oligonucleotide targeting the initiation region of cytohesin-1, resulted in a loss of LPS-induced adherence of monocytes (Hmama et al.,
J. Biol. Chem.,
1999, 274, 1050-1057). These studies indicate a role for cytohesin-1 in the process of inflammation.
Cytohesin-1 has been shown to act as a GEF for several other ARF members, including ARF1 and ARF3 (Meacci et al.,
Proc. Natl. Acad. Sci. U.S.A.,
1997, 94, 1745-1748; Pacheco-Rodriguez et al.,
J. Biol. Chem.,
1998, 273, 26543-26548). In rat, microinjection and overexpression of the rat cytohesin-1 homologue, msec7-1, into Xenopus neuromuscular junctions led to the translocation of ARF6 and an increase in basal synaptic transmission. These studies implicate cytohesin-1 in the presynaptic cycling of synaptic vesicles (Ashery et al.,
Proc. Natl. Acad. Sci. U.S.A.,
1999, 96, 1094-1099).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of cytohesin-1.
To date, strategies aimed at modulating cytohesin-1 function have involved the use of molecules that block upstream entities such as the PI3 kinase enzyme, microinjection strategies and antisense oligonucleotides. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting cytohesin-1 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 cytohesin-1 expression. Therefore, the present invention provides compositions and methods for modulating cytohesin-1 expression.
SUMMARY OF THE INVENTION
The present invention is directed to antisense compounds, particularly oligonucleotides, which are targeted to a nucleic acid encoding cytohesin-1, and which modulate the expression of cytohesin-1. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. Further provided are methods of modulating the expression of cytohesin-1 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 cytohesin-1 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 cytohesin-1, ultimately modulating the amount of cytohesin-1 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding cytohesin-1. As used herein, the terms “target nucleic acid” and “nucleic acid encoding cytohesin-1” encompass DNA encoding cytohesin-1, 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

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