Antisense inhibition of clusterin expression

Details Antisense inhibition of clusterin expression Antisense inhibition of clusterin expression

2000-09-11

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

C435S325000, C435S006120, C435S091100, C536S024500, C536S024300, C536S024310, C536S024330

Reexamination Certificate

active

06383808

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of clusterin. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding clusterin. Such compounds have been shown to modulate the expression of clusterin.
BACKGROUND OF THE INVENTION
Clusterin is an amphipathic glycoprotein that was first isolated from the male reproductive system (Bettuzzi et al.,
Biochem. J.,
1989, 257, 293-296; O'Bryan et al.,
J. Clin. Invest.,
1990, 85, 1477-1486). Subsequently, it has been shown that clusterin is ubiquitously distributed among tissues, having a wide range of biologic properties. Investigators from several disciplines, therefore, have isolated clusterin homologs under more than ten different names reviewed in (Bailey and Griswold,
Mol. Cell. Endocrinol.,
1999, 151, 17-23; Koch-Brandt and Morgans,
Prog. Mol. Subcell. Biol.,
1996, 16, 130-149; Meri and Jarva,
Vox. Sang.,
1998, 74, 291-302; Silkensen et al.,
Biochem. Cell. Biol.,
1994, 72, 483-488).
The clusterin protein consists of two non-identical subunits of 34 kDa and 47 kDa, designated alpha and beta, respectively. Clusterin expression is induced almost exclusively as a result of cellular injury, death, or pathology.
Among its many roles, clusterin is a component of the soluble SCb-5 complement complex which is assembled in the plasma upon activation of the complement cascade (Choi et al.,
Mol. Immunol.,
1989, 26, 835-840; Kirszbaum et al.,
Embo J.,
1989, 8, 711-718; Murphy et al.,
Int. Immunol.,
1989, 1, 551-554; Tschopp and French,
Clin. Exp. Immunol.,
1994, 97
Suppl
2, 11-14). Binding of clusterin has been shown to abolish the membranolytic potential of complement complexes and it has therefore been termed complement lysis inhibitor (CLI) (Jenne and Tschopp,
Proc. Natl. Acad. Sci.
U. S. A., 1989, 86, 7123-7127).
Further investigations of clusterin demonstrated that it circulates in plasma as a high density lipoprotein (HDL) complex which serves not only as an inhibitor of the lytic complement cascade, but as a regulator of lipid transport and local lipid redistribution (Jenne et al.,
J. Biol. Chem.,
1991, 266, 11030-11036). In this capacity, clusterin isolated and characterized by de Silva et al. and was given the name Apolipoprotein J (ApoJ) (de Silva et al.,
Biochemistry,
1990, 29, 5380-5389; de Silva et al.,
J. Biol. Chem.,
1990, 265, 13240-13247; de Silva et al.,
J. Biol. Chem.,
1990, 265, 14292-14297). In these studies, clusterin (ApoJ) was shown to play a role in cholesterol transport in the liver and in the regulation of vascular smooth muscle cell differentiation (de Silva et al.,
J. Biol. Chem.,
1990, 265, 13240-13247; Moulson and Millis,
J. Cell. Physiol.,
1999, 180, 355-364). A link between the modulation of HDL and complement activity is provided by studies by James et al. that characterize the association of a high density lipoprotein, NA1/NA2, with apolipoprotein A-I (ApoA-I). This novel protein NA1/NA2, was subsequently shown to be clusterin (James et al.,
Arterioscler. Thromb.,
1991, 11, 645-652).
Clusterin has also been shown to participate in the cellular process of programmed cell death or apoptosis. Clusterin expression demarcates cells undergoing apoptosis (Buttyan et al.,
Mol. Cell. Biol.,
1989, 9, 3473-3481) and in studies of the kidney, the onset of hydronephrosis following unilateral obstruction is associated with the increased expression of proteins encoded by the clusterin gene (Connor et al.,
Kidney Int.,
1991, 39, 1098-1103). In both of these studies, clusterin is referred to by two other synonyms, sulfated glycoprotein-2 gene (SGP-2) and testosterone-repressed prostate message-2 (TRPM-2) (Buttyan et al.,
Mol. Cell. Biol.,
1989, 9, 3473-3481; Connor et al.,
Kidney Int.,
10 1991, 39, 1098-1103).
Sensibar et al. showed that cell death in the prostate, induced by tumor necrosis factor alpha, could be prevented by overexpressing clusterin. In these studies, transfection of LNCaP cells with any of four 21-mer antisense phosphorothioate oligonucleotides targeting the clusterin coding region resulted in an increase of cell death (Sensibar et al.,
Cancer Res.,
1995, 55, 2431-2437).
Miyake et al. further demonstrated the role of clusterin as an anti-apoptotic gene in the Shionogi tumor model, a model used for the study of castration-induced apoptosis (Miyake et al.,
Cancer Res.,
2000, 60, 170-176). In this model, androgen-dependent mammary carcinoma xenograft tumors in male mice undergo regression after castration but recur as apoptosis-induced tumors after one month. Using a phosphorothioate 21-mer antisense oligonucleotide to the mouse clusterin gene targeting the translation initiation site, Miyake et al. were able to show that treatment with the clusterin antisense oligonucleotide of mice with Shionogi tumors resulted in a more rapid onset of apoptosis and time to complete regresssion. There was also a significant delay of emergence of androgen-independent recurrent tumors compared to control oligonucleotide treated controls.
Using the same oligonucleotide in an experiment designed to test the efficacy of the oligonucleotide in combination with paclitaxel, Miyake et al. showed that the combination of antisense oligonucleotide and paclitaxel induced apoptosis in Shionogi tumors better than either agent alone. These studies suggest that the antisense oligonucleotide may be useful in enhancing the effects of cytotoxic chemotherapy in hormone-refractory prostate cancer (Miyake et al.,
Cancer Res.,
2000, 60, 2547-2554).
Another antisense oligonucleotide, targeting the AUG initiation codon of clusterin was used to investigate the role of clusterin in endothelial cell activation. In these studies, it was shown that clusterin expression is upregulated upon laminar shear stress and that reduction of clusterin levels via antisense treatment increased endothelial cell activation (Urbich et al.,
Circulation,
2000, 101, 352-355).
The level of clusterin is increased in the hippocampus and frontal cortex of the brains of Alzheimer's disease patients. It is currently believed that clusterin, by binding to beta-amyloid, a protein known to aggregate in the brains of these patients, acts to link the progression of this disease to the complement system (Choi-Miura and Oda,
Neurobiol. Aging,
1996, 17, 717-722).
Most recently, clusterin has been isolated as a KU70 binding protein. KU binding proteins (KUBs) are involved in DNA repair pathways. Clusterin (KUB1) was identified as an autoantigen in serum of patients with scleroderma-polymyositis syndrome and shown to dimerize with KUP80 to form an ATP dependent helicase and a regulatory component of a DNA dependent protein kinase (PRKDC) involved in double-strand break repair and V(D)J recombination (Yang et al.,
Nucleic Acids Res.,
1999, 27, 2165-2174).
Clusterin is overexpressed in many disease states including neurodegenerative disorders, gliomas, retinitis pigmentosa and expression is induced in acute and chronic models of renal injury and disease, following ureter obstruction, ischemia/reperfusion, and atherosclerosis reviewed in (Silkensen et al.,
Biochem. Cell. Biol.,
1994, 72, 483-488). The pharmacological modulation of clusterin activity and/or expression may therefore be an appropriate point of therapeutic intervention in pathological conditions.
The expression of clusterin, or variants thereof, has been used as a means of differentiating normal versus abnormal cells in the study of male infertility. A method of assessing acrosomal status of sperm morphology comprising contacting a sperm sample with an immunologically reactive molecule which binds to one form of clusterin and not another is disclosed in WO 95/16916 (Baker and Murphy, 1995, ).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of clusterin and to date, investigative strategies aimed at modulating clusterin function have involved the use of antibodies, antisense oligon

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