Antisense modulation of calreticulin expression

Details Antisense modulation of calreticulin expression Antisense modulation of calreticulin expression

2000-10-30

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

06426220

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of calreticulin. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding human calreticulin. Such oligonucleotides have been shown to modulate the expression of calreticulin.
BACKGROUND OF THE INVENTION
Calreticulin, originally isolated from endoplasmic reticulum, is a ubiquitous multifunctional protein shown to participate in calcium storage, signaling, cell adhesion, gene expression and protein processing. The multifunctionality of this protein arises from the presence of distinct structural and functional domains within the protein reviewed in (Coppolino and Dedhar,
Int. J. Biochem. Cell Biol.,
1998, 30, 553-558; Nash et al.,
Mol. Cell. Biochem.,
1994, 135, 71-78; Sontheimer et al.,
J. Investig. Med.,
1995, 43, 362-370). Through these domains, calreticulin has been shown to act as a protein chaperone which orchestrates the correct folding of glycosylated proteins (Svaerke and Houen,
Acta. Chem. Scand.,
1998, 52, 942-949), as a regulator of Ca2+homeostasis (Fliegel et al.,
Biochim. Biophys. Acta.,
1989, 982, 1-8; Milner et al.,
J. Biol. Chem.,
1991, 266, 7155-7165; Waser et al.,
J. Cell Biol.,
1997, 138, 547-557) and in steroid receptor superfamily DNA binding (Perrone et al.,
J. Biol. Chem.,
1999, 274, 4640-4645; Sela-Brown et al.,
Mol. Endocrinol.,
1998, 12, 1193-1200; Shago et al.,
Exp. Cell. Res.,
1997, 230, 50-60). Recently it has been demonstrated that calreticulin plays a significant role in integrin-mediated cell adhesion (Coppolino et al.,
Nature,
1997, 386, 843-847; Dedhar,
Trends Biochem. Sci.,
1994, 19, 269-271) and in thrombosis (Dai et al.,
Arterioscler. Thromb. Vasc. Biol.,
1997, 17, 2359-2368).
Calreticulin has been isolated under several different names which reflect its role as either a calcium binding protein (calregulin, CRP55, CaBP3, HACBP, CAB-63, calsequestrin-like protein) or its cellular localization (ERp60 and reticulin) (Khanna et al.,
Methods Enzymol.,
1987, 139, 36-50; Macer and Koch,
J. Cell. Sci.,
1988, 91, 61-70; Mazzarella et al.,
Gene,
1992, 120, 217-225; Ostwald and MacLennan,
J. Biol. Chem.,
1974, 249, 974-979; Waisman et al.,
J. Biol. Chem.,
1985, 260, 1652-1660). By convention, the official name, calreticulin, was assigned to reflect both properties of the protein (Michalak et al.,
Biochem. J.,
1992, 285, 681-692).
Cellular localization dictates the role of calreticulin as it displays different functions when associated with proteins in the cytoplasm, nucleus or extracellular compartments. Extracellular calreticulin binds to and mediates the mitogenic effects of fibrinogen and intracellularly the protein binds rubella virus RNA, regulates perforin mediated lysis and regulates duodenal iron uptake (Coppolino and Dedhar,
Int. J. Biochem. Cell Biol.,
1998, 30, 553-558).
Overexpression or upregulation of calreticulin has been associated with several disease conditions including malignant melanoma, congenital heart block, viral warts, rubella, schistosomiasis and osteoporosis. This upregulation can be triggered by cell stress, heat shock or exposure to heavy metals (Sontheimer et al.,
J. Investig. Med.,
1995, 43, 362-370).
Calreticulin also plays a role in autoimmunity, being targeted by autoantibodies and binding to the Ro/SS-A antigen complex, a common target for autoimmune responses (Eggleton and Llewellyn,
Scand. J. Immunol.,
1999, 49, 466-473). Calreticulin is an autoantigen prevalent in the sera of patients with rheumatoid arthritis, Sjogren's syndrome, mixed connective tissue disease and systemic lupus erythromatosus (Eggleton et al.,
Lupus,
1997, 6, 564-571). In addition autoantibodies to the protein are also found in the sera of persons infected with the parasitic disease, onchocerciasis (Rokeach et al.,
J. Immunol.,
1991, 147, 3031-3039) and in the inflammatory condition of the gut, celiac disease (Krupickov et al.,
Gut,
1999, 44, 168-173).
An antisense phosphorothioate oligonucleotide targeted to the coding region for amino acids 83-88 of mouse calreticulin has been shown to increase the sensitivity of mouse neuroblastoma cells to ionomycin and reduce apoptotic resistance (Johnson et al.,
Brain Res. Mol. Brain Res.,
1998, 53, 104-111; Liu et al.,
J. Biol. Chem.,
1994, 269, 28635-28639). A phosphorothioate antisense oligonucleotide targeted to the translation initiation codon of human calreticulin was shown to inhibit cell attachment in several human cancer cell lines (Leung-Hagesteijn et al.,
J. Cell. Sci.,
1994, 107, 589-600). Both these oligonucleotides were shown to decrease calreticulin expression and render both LNCap and PC-3 prostate cancer cells sensitive to ionophore induced apoptosis (Zhu and Wang,
Cancer Res.,
1999, 59, 1896-1902).
Mice lacking the calreticulin gene die in utero at day 14.5 due to lack of cardiac morphogenesis (Mesaeli et al.,
J. Cell Biol.,
1999, 144, 857-868). Disclosed in the PCT publication WO 98/48003 are calreticulin-deficient cells either heterozygous or homozygous in nature and methods to identify substances that affect cell integrin-mediated cell adhesion (Coppolino et al.,
Nature,
1997, 386, 843-847; Dedhar and St-Arnaud, 1998).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of calreticulin but it has been reported that administration of exogenous calreticulin can have therapeutic benefit for certain ailments.
For example, it has been reported that administration of an amount calreticulin or fragments thereof alone or in conjunction with other therapeutic agents can promote wound healing (disclosed in U.S. Pat. No. 5,591,716 and PCT publication WO 95/13828) (Siebert et al., 1997; Siebert et al., 1995), prevent or treat thrombosis (disclosed in U.S. Pat. No. 5,426,097) (Stern et al., 1995), and inhibit restenosis (disclosed in PCT publication WO 96/36643) (Michalak and Lucas, 1996). One calreticulin fragment, vasostatin, has been shown to inhibit angiogenesis and suppress tumor growth of human Burkitt lymphoma and colon cancer in athymic mice (Pike et al.,
J. Exp. Med.,
1998, 188, 2349-2356).
Disclosed in Canadian Patent Application number 2,140,814 are the nucleic acid and protein sequences for human calreticulin as well as methods of treating disease by modulating hormone responsiveness whereby a therapeutic amount of the calreticulin protein or fragments thereof are administered to a patient (Dedhar, 1996). Furthermore, pharmaceutical compositions for the modulation of hormone responsiveness, consisting of peptides that bind calreticulin, have also been disclosed in U.S. Pat. No. 5,854,202 (Dedhar, 1998) and in the PCT publication, WO 96/23001 (Dedhar and St-Arnaud, 1996).
To date, investigative strategies aimed at modulating calreticulin function have involved the use of antibodies, the two antisense oligonucleotides previously cited and compounds that affect the calcium status of the cell. However, these strategies are untested as therapeutic protocols. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting calreticulin 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 calreticulin expression.
The present invention provides compositions and methods for modulating calreticulin expression.
SUMMARY OF THE INVENTION
The present invention is directed to antisense compounds, particularly oligonucleotides, which are targeted to a nucleic acid encoding calreticulin, and which modulate the expression of calreticulin. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. Further provided are methods of modulating the expression of calreticulin in cells or tissues comprising contacting said c

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