Antisense inhibition of A20 expression

Details Antisense inhibition of A20 expression Antisense inhibition of A20 expression

2000-09-08

2002-05-14

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, C435S091100, C435S091300, C536S024500, C536S024300, C536S024310, C536S024330, C536S023100, C536S023200

Reexamination Certificate

active

06387699

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of A20. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding A20. Such compounds have been shown to modulate the expression of A20.
BACKGROUND OF THE INVENTION
Cytokines are soluble factors produced by lymphocytes that regulate the survival, proliferation, differentiation, and homeostasis of cells involved in mediating the immune response. These factors not only activate other lymphocytes, they also relay signals to non-lymphoid cells including macrophages, epithelial and stromal cells creating a broad spectrum of cytokine activity that is critical to the maintenance of health.
Tumor necrosis factor (TNF), a cytokine produced by activated macrophages, initiates a variety of cellular responses. These responses include proliferation, differentiation and activation as well as apoptosis, inflammatory and immunoregulatory responses. The most studied effect of TNF is the cellular process of apoptosis.
Apoptosis, or programmed cell death, is a naturally occurring process that has been strongly conserved during evolution to prevent uncontrolled cell proliferation. This form of cell suicide plays a crucial role in the development and maintenance of multicellular organisms by eliminating superfluous or unwanted cells. However, if this process goes awry, excessive apoptosis results in cell loss that can trigger conditions such as blood cell disorders or degenerative disorders such as Alzheimers, Parkinsons, ALS or retinitis pigmentosa, while insufficient apoptosis contributes to the development of cancer, autoimmune disorders and viral infections (Thompson,
Science,
1995, 267, 1456-1462).
Although several stimuli can induce apoptosis, little is known about the intermediate signaling events, including inhibition, that connect the apoptotic signal to a common cell death pathway conserved across many species. Recently, major advances have been made in understanding the signaling pathways mediated by the tumor necrosis factor receptor (TNFR) family and its cytokine ligand, TNF.
A20 (also known as tumor necrosis factor alpha inducible protein and TNFAIP3) is a primary response gene originally identified as a cytokine-inducible gene in human umbilical vein endothelial (HUVEC) cells (Dixit et al.,
J. Biol. Chem.,
1990, 265, 2973-2978; Opipari et al.,
J. Biol. Chem.,
1990, 265, 14705-14708). Expression of A20 has been shown to be induced by several stimuli including treatment with lipopolysacharides (LPS) (Hu et al.,
Blood,
1998, 92, 2759-2765), tumor necrosis factor (TNF) (Krikos et al.,
J. Biol. Chem.,
1992, 267, 17971-17976; Opipari et al.,
J. Biol. Chem.,
1992, 267, 12424-12427), interleukin-1 (Dixit et al.,
J. Biol. Chem.,
1990, 265, 2973-2978), the Epstein-Barr virus LMP1 (Fries et al.,
J.Virol.,
1996, 70, 8653-8659), CD40 (Dixit et al.,
J. Biol. Chem.,
1990, 265, 2973-2978), the human T-cell leukemia virus type I Tax protein and phorbol 12-myristate 13-acetate (PMA) (Laherty et al.,
J. Biol. Chem.,
1993, 268, 5032-5039).
The original function ascribed to A20 was its ability to protect cells from TNF-induced apoptosis, as its expression has been shown to confer resistence in several cell types (Heyninck et al.,
Anticancer Res.,
1999, 19, 2863-2868; Opipari et al.,
J. Biol. Chem.,
1992, 267, 12424-12427). It has since been shown to inhibit TNF-induced endothelial cell activation (Cooper et al.,
J. Biol. Chem.,
1996, 271, 18068-18073; Cooper et al.,
Transplant Proc.,
1997, 29, 881; Ferran et al.,
Transplant. Proc.,
1997, 29, 879-880), to protect HUVEC cells from apoptosis by age-dependent injury (Varani et al.,
Lab. Invest.,
1995, 73, 851-858), and H1299 epithelial cells from p53-mediated apoptosis (Fries et al.,
J.Virol.,
1996, 70, 8653-8659), and to protect TNF-sensitive fibroblast L929 cells from TNF-induced necrotic death (Heyninck et al.,
Anticancer Res.,
1999, 19, 2863-2868). In smooth muscle cells, A20 has been shown to inhibit activation and proliferation (Arvelo et al.,
Transplant Proc.,
1999, 31, 858-859).
Overexpression of A20, via adenoviral mediated gene transfer, has also been shown to inhibit IL-1 beta-induced production of nitric oxide by rat islets. This is believed to occur by the blockade of the NFkB pathway and consequently induction of the synthetase, iNOS, which generates the nitric oxide radical (Grey et al.,
Transplant Proc.,
1999, 31, 789).
Characterization of the protein demonstrated that A20 is a zinc finger protein containing cysteine-rich repeat elements in the C-terminus (Opipari et al.,
J. Biol. Chem.,
1990, 265, 14705-14708). It is through this region that A20 forms protein-protein contacts.
A20 has been shown to form both homo- and heterodimeric complexes by self-association (De Valck et al.,
FEBS Lett.,
1996, 384, 61-64) or by binding to any of several proteins involved in the apoptotic pathways including TRAF1 and TRAF2 (Song et al.,
Proc. Natl. Acad. Sci. U. S. A.,
1996, 93, 6721-6725), TRAF6 (Heyninck and Beyaert,
FEBS Lett.,
1999, 442, 147-150), ABIN, a novel NFkB-inhibiting protein (Heyninck et al.,
J. Cell. Biol.,
1999, 145, 1471-1482), the Epstein-Barr virus LMP1 (Fries et al.,
Virology,
2000, 264, 159-166) and the human T-cell leukemia virus type-I (HTLV-I) Tax binding protein, TXBP151 (De Valck et al.,
Oncogene,
1999, 18, 4182-4190). These interactions suggest a role for A20 not only in apoptosis, but in viral infection and inflammation.
Cellular transformation and acquisition of the metastatic phenotype are the two main changes normal cells undergo during the progression to cancer. Failure to undergo apoptosis is a feature of carcinoma cells and it has been demonstrated that breast carcinoma cell lines that are resistant to TNF-induced cytotoxicity and consequently do not undergo apoptosis, express A20 constitutively (Opipari et al.,
J. Biol. Chem.,
1992, 267, 12424-12427). Furthermore, overexpression of A20 in the MCF-7 breast cancer cell line and the WEHI-S fibrosarcoma cell line was shown to inhibit TNF and IL-1 signaling at a very early point in the transduction pathway indicating that overexpression of A20 can have a broad range of effects on the normal function of the downstream regulation of these pathways (Jaattela et al.,
J. Immunol.,
1996, 156, 1166-1173).
The pharmacological modulation of A20 activity and/or expression is therefore believed to be an appropriate point of therapeutic intervention in pathological conditions of insufficient apoptosis, such as cancer.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of A20 and to date, strategies aimed at modulating A20 function have involved the use of antibodies, molecules that block upstream entities such as the p38 MAP kinase inhibitor, SB202190 (Manthey et al.,
J. Leukoc. Biol.,
1998, 64, 409-417), the proteosome inhibitor N-acetyl-leucinyl-leucinyl-norleucinal-H (ALLN) and the tyrosine kinase inhibitor, herbimycin A (HMA) (Zen et al.,
Exp. Cell Res.,
1998, 243, 425-433).
However, these strategies are untested as therapeutic protocols and consequently, there remains a long felt need for additional agents capable of effectively inhibiting A20 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 A20 expression.
The present invention provides compositions and methods for modulating A20 expression.
SUMMARY OF THE INVENTION
The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding A20, and which modulate the expression of A20. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of A20 in cells or tissues comprising contacting s

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