Antisense modulation of ARA70 expression

Details Antisense modulation of ARA70 expression Antisense modulation of ARA70 expression

2000-01-21

2001-07-03

McGarry, Sean (Department: 1635)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

C435S006120, C435S455000, C536S023100, C536S024100, C536S024500

Reexamination Certificate

active

06255110

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of ARA70. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding ARA70. Such oligonucleotides have been shown to modulate the expression of ARA70.
BACKGROUND OF THE INVENTION
Steroid, thyroid and retinoid hormones produce a diverse array of physiologic effects through the regulation of gene expression. Upon entering the cell, these hormones bind to a unique group of intracellular nuclear receptors which have been characterized as ligand-dependent transcription factors. This complex then moves into the nucleus where the receptor and its cognate ligand interact with the transcription pre-initiation complex affecting its stability and ultimately the rate of transcription of the target genes. The interactions of the liganded receptor with the specific elements in the promoter region are mediated by two classes of molecules; corepressors, which inhibit transactivation and coactivators, which enhance transactivation (Shibata et al.,
Recent Prog. Horm. Res
., 1997, 52, 141-164).
ARA70 (also known as androgen receptor (AR) activator, ELE1, RET/PCT3, RFG for RET-fused gene and NCOA4 for nuclear receptor coactivator 4) is a member of the growing family of transcriptional coactivators. ARA70 was first isolated as a coactivator for the androgen receptor (AR) in human prostate cancer cells and has since been shown to act as a ligand-enhanced coactivator for the peroxisome proliferator-activated receptor gamma, another nuclear hormone receptor, in these same cells (Heinlein et al.,
J. Biol. Chem
., 1999, 274, 16147-16152; Yeh and Chang,
Proc. Natl. Acad. Sci. U.S.A
., 1996, 93, 5517-5521). ARA70 shows a broad tissue distribution of expression, including adipose tissue (Yeh and Chang,
Proc. Natl. Acad. Sci. U.S.A
., 1996, 93, 5517-5521) and the DNA and protein sequence are disclosed in the PCT publication WO 97/44490 (Chang and Yeh, 1997).
Studies of androgen receptor regulation in prostate cancer cell lines have revealed a pivotal role for ARA70 in sex hormone signaling. By properly interacting with the androgen receptor, it has been suggested that ARA70 confers specificity of action between androgen and estrogen in the prostate (Miyamoto et al.,
Proc. Natl. Acad. Sci. U.S.A
., 1998, 95, 11083-11088; Miyamoto et al.,
Proc. Natl. Acad. Sci. U.S.A
., 1998, 95, 7379-7384; Yeh et al.,
Proc. Natl. Acad. Sci. U.S.A
., 1998, 95, 5527-5532).
Furthermore, cotransfection of ARA70 with the retinoblastoma protein (Rb), a tumor suppressor, in the prostate cancer cell line DU 145 has been shown to additively induce androgen receptor transcriptional activity by 13-fold with a concomitant reduction of cell growth (Yeh et al.,
Biochem. Biophys. Res. Commun
., 1998, 248, 361-367).
ARA70 has also been identified in thyroid cancer cells, where chromosomal rearrangement resulted in the fusion of the genomic region of the RET tyrosine kinase to the 5′ terminal region of the ELE1 gene. The resulting protein was designated in this context as RET/PTC3 or RET-fused gene (Bongarzone et al.,
Genomics
, 1997, 42, 252-259; Minoletti et al.,
Genes Chromosomes Cancer
, 1994, 11, 51-57; Santoro et al., Oncogene, 1994, 9, 509-516). More recently, it has been discovered that there are two isoforms of the ELE1 gene to which the RET tyrosine kinase can be fused to produce ARA70. The expression of these isoforms, designated alpha and beta (beta being a deletion variant most likely caused by the removal of one or more exons), thereby produce two isoforms of the ARA70 coactivator (Alen et al.,
Mol. Endocrinol
., 1999, 13, 117-128).
Given the suggested role and expression pattern of ARA70 in prostate and thyroid carcinomas, the pharmacological modulation of ARA70 activity and/or expression may be an appropriate point of therapeutic intervention in pathological conditions.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of ARA70. Consequently, there remains a long felt need for agents capable of effectively inhibiting ARA70 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 ARA70 expression.
The present invention provides compositions and methods for modulating ARA70 expression, including modulation of the expression of the extended mRNA form of ARA70, known as RET-fused gene (RFG).
SUMMARY OF THE INVENTION
The present invention is directed to antisense compounds, particularly oligonucleotides, which are targeted to a nucleic acid encoding ARA70, and which modulate the expression of ARA70. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. Further provided are methods of modulating the expression of ARA70 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 ARA70 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 antisense compounds, particularly oligonucleotides, for use in modulating the function of nucleic acid molecules encoding ARA70, ultimately modulating the amount of ARA70 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding ARA70. As used herein, the terms “target nucleic acid” and “nucleic acid encoding ARA70” encompass DNA encoding ARA70, 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 functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of ARA70. In the context of the present invention, “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target.
It is preferred to target specific nucleic acids for antisense. “Targeting” an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding ARA70. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred intragenic site is the region encompassing the translatio

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