Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
2000-03-02
2002-09-17
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
06451602
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of Poly (ADP-ribose) polymerase (PARP). In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding human PARP. Such oligonucleotides have been shown to modulate the expression of PARP.
BACKGROUND OF THE INVENTION
Posttranslational modifications of proteins are required for many cellular functions including the mediation of protein—protein interactions, enzymatic activity, protein degradation, localization of proteins to cellular compartments and maintenance of protein stability.
Poly (ADP-ribosylation) is a posttranslational modification of nuclear proteins whereby molecules of ADP-ribose are added to acceptor proteins to form branched polymers. The attachment of these polymers to nuclear proteins, which is dependent on the presence of DNA strand breaks, converts the DNA ends into intracellular signals that modulate DNA repair and cell survival programs. ADP-ribosylation, therefore, plays an important role in many cellular processes including chromatin decondensation, DNA replication, DNA repair, gene expression, malignant transformation, cellular differentiation and apoptosis (D'Amours et al.,
Biochem. J
., 1999, 342, 249-268; Pieper et al.,
Trends Pharmacol. Sci
., 1999, 20, 171-181).
The enzymes that catalyze the formation of poly (ADP-ribose) polymers, poly (ADP-ribose) polymerases, also known as PARPS, constitute a gene family with three known members identified to date; PARP, PARP-2 and PARP-3 (Ame et al.,
J. Biol. Chem
., 1999, 274, 17860-17868; Cherney et al.,
Proc. Natl. Acad. Sci. U.S.A
., 1987, 84, 8370-8374; Johansson,
Genomics
, 1999, 57, 442-445).
All three members of the PARP family are ubiquitously expressed in all tissues and cell lines examined (Alkhatib et al.,
Proc. Natl. Acad. Sci. U.S.A
., 1987, 84, 1224-1228; Ame et al.,
J. Biol. Chem
., 1999, 274, 17860-17868; Johansson,
Genomics
, 1999, 57, 442-445). Characterization of the PARP proteins revealed a three-domain structure for PARP-1, having a DNA binding domain, an automodification domain and a catalytic domain (Cherney et al.,
Proc. Natl. Acad. Sci. U.S.A
., 1987, 84, 8370-8374). PARP-2 and PARP-3 lack the first two domains and consist primarily of the catalytic domain which contains the NAD binding site in all three proteins (Ame et al.,
J. Biol. Chem
., 1999, 274, 17860-17868; Johansson,
Genomics
, 1999, 57, 442-445). Functionally, however, PARP-1 has been the best characterized of the three PARPS.
Mice lacking the PARP-1 gene show normal fetal and postnatal development but have inherent genomic instability and are highly sensitive to DNA damage induced by radiation and alkylating agents (Trucco et al.,
Mol. Cell. Biochem
., 1999, 193, 53-60). These mice also demonstrated downregulation and lack of responsiveness of p53 protein to genotoxins (Althaus et al.,
Mol. Cell. Biochem
., 1999, 193, 5-11). p53 is induced by a variety of apoptotic stimuli and is required for apoptosis in many cell systems and recently it was shown that PARP-1 binds to p53 and alters its DNA binding properties (Malanga et al.,
J. Biol. Chem
., 1998, 273, 11839-11843). These results support a role for the PARP enzymes in the regulation of programmed cell death, or apoptosis.
Other studies of knockout mice implicate PARP in the development of the inflammatory response. Oliver et al. showed that PARP-1 deficient mice were extremely resistant to LPS-induced endotoxic shock due to a defect in NFkB transcriptional activation with the consequence of attenuating systemic inflammatory processes (Oliver et al.,
Embo J
., 1999, 18, 4446-4454). In addition, the combination therapy of nicotinic acid, a PARP inhibitor, and thalidomide, a TNF-alpha inhibitor, caused a powerful synergistic inhibition of arthritis in male DBA/I hybrid mice suffering from type II collagen-induced arthritis (Kroger et al.,
Inflammation
, 1996, 20, 203-215; Miesel et al.,
Inflammation
, 1995, 19, 379-387).
MPTP is a neurotoxin that causes parkinsonism in humans and animals, and mice lacking the PARP-1 gene are dramatically spared from MPTP-induced neurotoxicity. This study indicates that limiting PARP activity represents a potential therapeutic target for the reduction of dopaminergic neuronal loss (Mandir et al.,
Proc. Natl. Acad. Sci. U.S.A
., 1999, 96, 5774-5779). Other neurologic conditions associated with the activation of PARP include meningitis-associated intracranial complications and ischemia (Koedel and Pfister,
Brain Pathol
., 1999, 9, 57-67; Love,
Brain Pathol
., 1999, 9, 119-131).
PARP knockout mice have also been studied in the context of the development of diabetes. Burkhart et al. have shown that mice lacking the PARP-1 gene are completely resistant to the development of diabetes induced by the beta-cell toxin, streptozocin (Burkart et al.,
Nat. Med
., 1999, 5, 314-319).
The pharmacological modulation of PARP activity and/or expression may therefore be an appropriate point of therapeutic intervention in pathological conditions such as diabetes, cancer, cellular injury resulting from oxidative stress and inflammatory conditions.
To date, investigative strategies aimed at modulating PARP function have involved the use of antibodies (Duriez et al.,
Biochim. Biophys. Acta
., 1997, 1334, 65-72; Prasad et al.,
Electrophoresis
, 1999, 20, 618-625), inhibitory peptides and peptidomimetics including trans-dominant inhibition by the DNA binding domain of the protein (Kupper et al.,
Cancer Res
., 1996, 56, 2715-2717; Molinete et al.,
Embo J
., 1993, 12, 2109-2117), small molecule inhibitors (Gale, J.
Pediatr. Endocrinol. Metab
., 1996, 9, 375-379; Richardson et al.,
Adv. Exp. Med. Biol
., 1999, 457, 267-279; Sheng et al.,
Cancer Detect. Prev
., 1998, 22, 284-292; Shiokawa et al.,
FEBS Lett
., 1997, 413, 99-103; Weltin et al.,
Int. J. Radiat. Biol
., 1997, 72, 685-692), antisense expression vectors (Simbulan-Rosenthal et al.,
Biochemistry
, 1998, 37, 9363-9370; Simbulan-Rosenthal et al.,
Biochem. Biophys. Res. Comm
., 1998, 253, 864-868; Simbulan-Rosenthal et al.,
Biochemistry
, 1996, 35, 11622-11633; Simbulan-Rosenthal et al.,
J. Biol. Chem
., 1998, 273, 13703-13712; Simbulan-Rosenthal et al.,
Mol. Cell. Biochem
., 1999, 193, 137-148) and gene knock-outs in mice (Althaus et al.,
Mol. Cell. Biochem
., 1999, 193, 5-11; Burkart et al.,
Nat. Med
., 1999, 5, 314-319; Mandir et al.,
Proc. Natl. Acad. Sci. U.S.A
., 1999, 96, 5774-5779; Oliver et al.,
Embo J
., 1999, 18, 4446-4454; Trucco et al.,
Mol. Cell. Biochem
., 1999, 193, 53-60).
Currently, there are no known therapeutic agents that effectively inhibit the synthesis of PARP and consequently, there remains a long felt need for these.
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 PARP expression.
The present invention provides compositions and methods for modulating human PARP expression, including that of PARP-1, PARP-2 and/or PARP-3.
SUMMARY OF THE INVENTION
The present invention is directed to antisense compounds, particularly oligonucleotides, which are targeted to a nucleic acid encoding human PARP, and which modulate the expression of human PARP. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. Further provided are methods of modulating the expression of PARP in human 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 a human suspected of having or being prone to a disease or condition associated with expression of PARP by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.
DETAILED DESCRIPTION OF
Cowsert Lex M.
Popoff Ian
ISIS Pharmaceuticals Inc.
LeGuyader John L.
Licata & TYrrell P.C.
Schmidt M
LandOfFree
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