Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
2001-05-09
2002-06-25
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, C435S366000, C536S023100, C536S024310, C536S024330, C536S024500
Reexamination Certificate
active
06410325
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of Phospholipase A2, group VI (Ca2+-independent). In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding Phospholipase A2, group VI (Ca
2+
-independent). Such compounds have been shown to modulate the expression of Phospholipase A2, group VI (Ca2+-independent).
BACKGROUND OF THE INVENTION
The enzymes of the phospholipase A2 (PLA2) family catalyze hydrolysis of the sn-2 fatty acid bond of phospholipids to liberate free fatty acids and lysophospholipids. These metabolites are involved in diverse cellular processes including signal transduction, host defense (including antibacterial effects), formation of platelet activating cofactor, membrane remodeling and general lipid metabolism (Dennis,
J. Biol. Chem.,
1994, 269, 13057-13060; Dennis,
Trends Biochem. Sci,
1997, 22, 1-2). While the human PLA2 enzymes are of greatest interest, most of the current understanding of PLA2s has been obtained by studying enzymes from non-human sources. Since most of the human PLA2s have essentially identical non-human counterparts, the knowledge obtained from the non-human enzymes is equally applicable to the human enzymes (Dennis,
J. Biol. Chem.,
1994, 269, 13057-13060).
PLA2s are a diverse class of enzymes with regard to function, localization, regulation, mechanism, structure and dependence on divalent metal ions for activity (Dennis,
J. Biol. Chem.,
1994, 269, 13057-13060; Dennis,
Trends Biochem. Sci,
1997, 22, 1-2). The PLA2s have been divided into ten groups (denoted using Roman numerals I through X) (Cupillard et al.,
J. Biol. Chem.,
1997, 272, 15745-15752; Dennis,
Trends Biochem. Sci,
1997, 22, 1-2). The PLA2s of groups IV and VI are intracellular, high molecular weight enzymes which have not been as extensively studied as the secreted PLA2s (groups I-III, V and X) (Balsinde and Dennis,
J. Biol. Chem.,
1997, 272, 16069-16072.). Group IV requires calcium for activity whereas the activity of group VI is calcium-independent.
First cloned from Chinese hamster ovary cells in 1997 (Tang et al.,
J. Biol. Chem.,
1997, 272, 8567-8575.), the calcium-independent phospholipase A2 (PLA2 group VI, also known as iPLA2 and PLA2G6) plays a housekeeping role in phospholipid remodeling (Balsinde and Dennis,
J. Biol. Chem.,
1997, 272, 16069-16072.). Signaling roles in generation of substrate for leukotriene biosynthesis (Larsson Forsell et al.,
FEBS Lett.,
1998, 434, 295-299.), generation of lipid messengers involved in regulation of ion channel activity (Ma et al.,
J. Biol. Chem.,
1997, 272, 11118-11127.) and apoptosis (Atsumi et al.,
J. Biol. Chem.,
1998, 273, 13870-13877.) have also been proposed for this enzyme.
Human calcium-independent phospholipase A2 (PLA2 group VI) expressed in B-cells was found to undergo extensive alternative splicing, generating multiple isoforms that contribute to a novel catalytic control mechanism (Larsson et al.,
J. Biol. Chem.,
1998, 273, 207-214.). In addition, Ma et al. have mapped human calcium-independent phospholipase A2 (PLA2, group VI) to chromosome 22q13.1 and identified mRNA encoding two catalytically active isoforms in pancreatic islet cells. The long 88 KDa isoform is known as LH-iPLA2 while the shorter, 85 KDa protein (SH-iPLA2) is a splice variant produced by an exon-skipping mechanism (Ma et al.,
J. Biol. Chem.,
1999, 274, 9607-9616.).
Functional impairment of pancreatic islet beta cells is implicated in both type I and type II diabetes mellitus (Ma et al.,
J. Biol. Chem.,
1999, 274, 9607-9616.). In rodent islets, calcium-independent phospholipase A2 (PLA2 group VI) has been proposed to play a signaling role in glucose-induced insulin secretion (Tang et al.,
J. Biol. Chem.,
1997, 272, 8567-8575.) and in experimentally induced beta cell apoptosis (Zhou et al.,
J.Clin. Invest.,
1998, 101, 1623-1632.).
A number of small molecules have been employed as inhibitors of calcium-independent phospholipase A2 (PLA2 group VI), including: arachidonyl trifluoromethyl ketone, arachidonyl tricarbonyl and methyl arachidonyl fluorophosphonate which function as transition state analogues and bromoenol lactone which acts an irreversible mechanism-based inhibitor (Balsinde and Dennis,
J. Biol. Chem.,
1997, 272, 16069-16072.).
An antisense phosphorothioate oligonucleotide targeting nucleotides 59-78 of murine calcium-independent phospholipase A2 (PLA2 group VI) mRNA was used to inhibit the process of phospholipid fatty acid remodeling in murine P388D1 macrophages (Balsinde et al.,
J. Biol. Chem.,
1997, 272, 29317-29321.).
Pharmacological modulation of calcium-independent phospholipase A2 (PLA2 group VI) activity and/or expression may be an appropriate point for therapeutic intervention in pathologic conditions such as diabetes mellitus types I and II and conditions related to abnormal apoptosis. To date, investigative strategies aimed at inhibiting the action of calcium-independent phospholipase A2 (PLA2 group VI) have been limited to the previously cited studies involving small molecule inhibitors and the single antisense oligonucleotide. Consequently, there remains a need for additional agents which modulate the function of this enzyme.
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 calcium-independent phospholipase A2 (PLA2 group VI) expression.
The present invention provides compositions and methods for modulating calcium-independent phospholipase A2 (PLA2 group VI) expression, including modulation of the splice variant forms of calcium-independent phospholipase A2 (PLA2 group VI).
SUMMARY OF THE INVENTION
The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding Phospholipase A2, group VI (Ca2+-independent), and which modulate the expression of Phospholipase A2, group VI (Ca2+-independent). Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of Phospholipase A2, group VI (Ca2+-independent) 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 Phospholipase A2, group VI (Ca2+-independent) 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 Phospholipase A2, group VI (Ca2+-independent), ultimately modulating the amount of Phospholipase A2, group VI (Ca2+-independent) produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding Phospholipase A2, group VI (Ca2+-independent). As used herein, the terms “target nucleic acid” and “nucleic acid encoding Phospholipase A2, group VI (Ca2+-independent)” encompass DNA encoding Phospholipase A2, group VI (Ca2+-independent), 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
Bennett C. Frank
Freier Susan M.
Watt Andrew T.
ISIS Pharmaceuticals Inc.
LeGuyader John L.
Licata & Tyrrell P.C.
Schmidt M
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