Antisense modulation of estrogen receptor alpha expression

Details Antisense modulation of estrogen receptor alpha expression Antisense modulation of estrogen receptor alpha expression

2001-12-18

2003-09-09

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, C435S325000, C536S023100, C536S024500

Reexamination Certificate

active

06617162

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of estrogen receptor alpha. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding estrogen receptor alpha. Such compounds have been shown to modulate the expression of estrogen receptor alpha.
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 preinitiation complex affecting its stability and ultimately, the rate of transcription of the target genes. Members of the nuclear receptor family share several structural features including a central, highly conserved DNA-binding domain which targets the receptor to specific DNA sequences known as hormone response elements (Kliewer et al.,
Science
, 1999, 284, 757-760).
Estrogens are steroid hormones that exert a wide range of effects throughout the body. They are required for normal female sexual maturation and promote growth and differentiation of the breast, uterus, fallopian tubes, vagina and ovaries. Male reproductive tissues such as testis and prostate are also estrogen target tissues. Furthermore, estrogens are important for bone maintenance and have a protective role in the cardiovascular system. In the brain, estrogens appear to modulate the regulation and reproduction of autonomic and reproductive neuroendocrine systems, mood and cognition (Osterlund and Hurd,
Prog. Neurobiol
., 2001, 64, 251-267).
Similar to the other steroid hormone receptors, the estrogen receptors consist of several structural domains that exhibit different functional features. Both the estrogen receptor alpha and estrogen receptor beta consist of five different domains, the N-terminal domain (called A/B), the DNA-binding domain (C), a short hinge region (E) and a short C-terminal domain (F) (Osterlund and Hurd,
Prog. Neurobiol
., 2001, 64, 251-267).
Estrogen receptor alpha (also known as estrogen receptor 1, ESR1, ESR-alpha and NR3A1) was first cloned and sequenced from the MCF-7 breast cancer cell line in 1985 (Green et al.,
J. Steroid Biochem
., 1986, 24, 77-83; Greene et al.,
Science
, 1986, 231, 1150-1154; Walter et al.,
Proc. Natl. Acad. Sci. U.S.A
., 1985, 82, 7889-7893). Greene et al. noted significant homology of estrogen receptor alpha to the v-erbA oncogene product (Greene et al.,
Science
, 1986, 231, 1150-1154).
Estrogen receptors constitute the only known members of the family of steroid receptors that have multiple subtypes. The involvement of mutations and splice variants of estrogen receptor alpha in breast cancer is well.known in the art (Balleine et al.,
J. Clin. Endocrinol. Metab
., 1999, 84, 1370-1377; Fuqua et al.,
J. Cell Biochem
., 1993, 51, 135-139; Garcia et al.,
Mol. Endocrinol
., 1988, 2, 785-791; McGuire et al.,
J. Steroid Biochem. Mol. Biol
., 1992, 43, 243-247; McGuire et al.,
Mol. Endocrinol
., 1991, 5, 1571-1577; Murphy et al.,
J. Clin. Endocrinol. Metab
., 1996, 81, 1420-1427; Scott et al.,
J. Clin. Invest
., 1991, 88, 700-706). Additionally, splice variants of estrogen receptor alpha have been identified in pituitary tumors (Chaidarun and Alexander,
Mol. Endocrinol
., 1998, 12, 1355-1366; Chaidarun et al.,
J. Clin. Endocrinol. Metab
., 1997, 82, 1058-1065; Shupnik et al.,
J. Clin. Endocrinol. Metab
., 1998, 83, 3965-3972) and in normal endometrial tissue (Daffada and Dowsett,
J. Steroid Biochem. Mol. Biol
., 1995, 55, 413-421). Ye et al. have identified a novel estrogen receptor alpha/fatty acid synthase fusion transcript in a variety of human cancers including prostate, breast, cervical and bladder cancer cell lines (Ye et al.,
Biochim. Biophys. Acta
, 2000, 1493, 373-377).
The nucleic acid sequence of estrogen receptor alpha is disclosed in U.S. Pat. No. 6,235,872 (Bredesen and Rabizadeh, 2001).
Disclosed and claimed in PCT publications WO 01/00823 and WO 01/62969 are nucleic acid molecules encoding isoforms of human estrogen receptor alpha (Gannon et al., 2001; Kalush et al., 2001). Additionally disclosed and claimed in PCT publication WO 01/00823 are nucleic acid molecules hybridizing to said nucleic acid molecules encoding estrogen receptor alpha isoforms, under stringent conditions (Gannon et al., 2001).
The involvement of estrogen receptor alpha in cell proliferation indicates that its selective inhibition may prove to be a useful target for therapeutic intervention in a variety of cancers.
Because estrogens are thought to support breast cancer, estrogen antagonists have been developed which act through the estrogen receptor. Selective estrogen receptor modulators (SERMs) are small molecules which manifest variable agonist and antagonist properties when examined in the context of estrogen-dependent responses occurring in various tissues. Examples of SERMs include raloxifene and tamoxifen (Dutertre. and Smith,
J. Pharmacol. Exp. Ther
., 2000, 295, 431-437).
Disclosed and claimed in PCT publications WO 00/74485 and WO 99/54459 are catalytic nucleic acids (known as ribozymes), capable of modulating the expression of estrogen receptor alpha (Roy et al., 2000; Thompson et al., 1999).
Double-stranded DNA fragments containing 5′ upstream sequences of the human estrogen receptor alpha gene have been used to modulate the transcriptional activity of estrogen receptor alpha (Penolazzi et al.,
Breast Cancer Res. Treat
., 1998, 49, 227-235).
A series of studies have employed antisense estrogen receptor alpha RNA transfections to investigate effects of downregulation of estrogen receptor alpha expression in MCF-7 human breast cancer cells (Defazio et al.,
Cell Growth Differ
., 1997, 8, 903-911; Jeng et al.,
Cancer Lett
., 1994, 82, 123-128; Jiang and Jordan,
J. Natl. Cancer Inst
., 1992, 84, 580-591; Lazennec et al.,
Mol. Endocrinol
., 1999, 13, 969-980; Madden et al.,
Eur. J. Cancer
, 2000, 36, S34-S35; Williard et al.,
Gene
, 1994, 149, 21-24). In two of the aforementioned investigations, paradoxical upregulation of estrogen receptor alpha was observed (Jeng et al.,
Cancer Lett
., 1994, 82, 123-128; Jiang and Jordan,
J. Natl. Cancer Inst
., 1992, 84, 580-591).
Antisense oligodeoxynucleotides have also been used to decrease levels of estrogen receptor alpha in rat brain (Inamdar et al.,
J. Appl. Physiol
., 2001, 91, 1886-1892; McCarthy et al.,
Endocrinology
, 1993, 133, 433-439) and in a mouse colon cancer cell line (Xu and Thomas,
Mol. Cell Endocrinol
., 1994, 105, 197-201).
Antisense 18-mer oligonucleotides targeting positions −117 to −99, −3 to 15 (encompassing the start codon), 302 to 320, 495 to 512, 539 to 557, 1140 to 1157 and 4134 to 4151, of estrogen receptor alpha have been used to inhibit its expression in MCF-7 human breast cancer cells (Santagati et al.,
Mol. Endocrinol
., 1997, 11, 938-949; Taylor et al.,
Antisense Nucleic Acid Drug Dev
., 2001, 11, 219-231). In a separate investigation, also using MCF-7 breast cancer cells, a 15-mer methylphosphonate-phosphodiester antisense oligonucleotide was used to target the start codon of estrogen receptor alpha (Madden et al.,
Eur. J. Cancer
, 2000, 36, S34-S35).
To date, investigative strategies aimed at modulating estrogen receptor alpha expression have involved the use of small molecule SERMs, ribozymes, double-stranded DNA decoys, antisense RNA transfections and antisense oligonucleotides. There remains a long felt need for additional agents capable of effectively inhibiting estrogen receptor alpha 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 modul

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