Antisense modulation of E2F transcription factor 2 expression

Details Antisense modulation of E2F transcription factor 2 expression Antisense modulation of E2F transcription factor 2 expression

2000-09-08

2002-09-03

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, C435S377000, C536S023100, C536S024100, C536S024500, C514S04400A

Reexamination Certificate

active

06444464

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of E2F transcription factor 2. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding E2F transcription factor 2. Such compounds have been shown to modulate the expression of E2F transcription factor 2.
BACKGROUND OF THE INVENTION
Transcription factors represent a group of molecules within the cell that function to connect the pathways from extracellular signals to intracellular responses. Immediately after an environmental stimulus, these proteins which reside predominantly in the cytosol are translocated to the nucleus where they bind to specific DNA sequences in the promoter elements of target genes and activate the transcription of these target genes. One family of transcription factors, E2F transcription factors, regulates the expression of an extensive panel of genes that control DNA synthesis and cellular proliferation in a cell cycle-dependent manner reviewed in (Dyson,
Genes Dev
., 1998, 12, 2245-2262; Lavia and Jansen-Durr,
BioEssays
, 1999, 21, 221-230; Yamasaki,
Results Probl. Cell. Differ
., 1998, 22, 199-227).
The “E2F” designation given to these proteins refers to a multigene family of transcription factors that heterodimerize with members of the DP gene family (Helin and Harlow,
J. Virol
., 1994, 68, 5027-5035; Magae et al.,
J. Cell. Sci
., 1996, 109, 1717-1726). E2F/DP heterodimers can mediate both transcriptional activation and repression and, to date, six members of the E2F family and two members of the DP family have been isolated.
The E2F family has been further subdivided into three subfamilies based on their interaction with the product of the retinoblastoma tumor suppressor gene (RB), a transcriptional repressor whose deregulation has been shown to result in oncogenesis. Consequently, the deregulation of the expression or function of E2F proteins alone or in conjunction with DP proteins has been implicated in the development of certain pathologic conditions, including cancer (Sladek,
Cell. Prolif
., 1997, 30, 97-105; Yamasaki,
Biochim. Biophys. Acta
, 1999, 1423, M9-15).
The first subfamily of E2Fs consists of E2F transcription factor 1, 2 and 3 (E2F-1, E2F-2 and E2F-3) which bind with high affinity to the retinoblastoma protein, pRB (Arroyo and Raychaudhuri,
Nucleic Acids Res
., 1992, 20, 5947-5954; Nevins,
Science
, 1992, 258, 424-429).
The second subfamily comprises E2F transcription factors 4 and 5 (E2F-4 and E2F-5) and these bind to the pRB homologues, p107 and p130 while the third subfamily contains one member, E2F transcription factor 6 (E2F-6) that does not bind to any of the pRB family members.
While relatively little is known about the specific properties of the individual members of the E2F family, the best characterized are those of the first subfamily (E2F-1, E2F-2 and E2F-3). Because E2F binding sites are found in the promoters of many genes, these transcription factors mediate a multitude of cellular processes including apoptosis, cell cycle control, DNA replication, and proliferation (Bernards,
Biochim. Biophys. Acta
, 1997, 1333, M33-40; Helin,
Curr. Opin. Genet. Dev
., 1998, 8, 28-35; Nevins,
Cell. Growth Differ
., 1998, 9, 585-593).
It has been shown that when expressed alone in rat fibroblasts any of the three members of this subfamily can induce S-phase entry in serum starved cells (Lukas et al.,
Mol. Cell. Biol
., 1996, 16, 1047-1057). In U343 astrocytoma cells, Dirks et al. have demonstrated that deregulated expression of any of the E2F transcription factors overcame cell cycle arrest and affects the expression of a distinct array of cell cycle regulatory proteins. However, it was further demonstrated that only the expression of E2F-1 or E2F-2 resulted in potent cell death (Dirks et al.,
Oncogene
, 1998, 17, 867-876).
E2F transcription factors have been shown to regulate keratinocyte proliferation, implying a role in skin tumor development (Jones et al.,
J. Invest. Dermatol
., 1997, 109, 187-193). In fact, it has been demonstrated that expression of five of the six members of the E2F family of transcription factors (excluding E2F-6) is upregulated in mouse skin tumors (Balasubramanian et al., Int.
J. Oncol
., 1999, 15, 387-390).
E2F transcription factor 2 (also known as E2F-2) is the second member of the E2F family identified from a low-stringency hybridization screen designed to isolate additional E2F-1-related genes (Ivey-Hoyle et al.,
Mol. Cell. Biol
., 1993, 13, 7802-7812; Lees et al.,
Mol. Cell. Biol
., 1993, 13, 7813-7825). In these studies, Lees et al. have shown that the three E2F members all share four regions of protein sequence homology, each having a specific function. They also demonstrate that, while all three transcription factors are expressed in all cell lines examined, E2F-2 and E2F-3 are expressed at consistently lower levels than E2F-1. Upon examining human tissues, it was found that E2F-2 was expressed at highest levels in placenta, lung and kidney (Lees et al.,
Mol. Cell. Biol
., 1993, 13, 7813-7825). E2F-2 expression has been shown to be up-regulated during epithelial development in mice and various regions in the E2F-2 promoter have been identified which control this expression in response to cell growth signals (Dagnino et al.,
Cell. Growth Differ
., 1997, 8, 553-563; Sears et al.,
Mol. Cell. Biol
., 1997, 17, 5227-5235). The pharmacological modulation of E2F transcription factor 2 activity and/or expression may therefore be an appropriate point of therapeutic intervention in pathological conditions.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of E2F transcription factor 2 and to date, investigative strategies aimed at modulating E2F transcription factor 2 function have involved the use of antibodies. Consequently, there remains a long felt need for agents capable of effectively inhibiting E2F transcription factor 2 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 E2F transcription factor 2 expression.
The present invention provides compositions and methods for modulating E2F transcription factor 2 expression.
SUMMARY OF THE INVENTION
The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding E2F transcription factor 2, and which modulate the expression of E2F transcription factor 2. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of E2F transcription factor 2 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 E2F transcription factor 2 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 compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding E2F transcription factor 2, ultimately modulating the amount of E2F transcription factor 2 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding E2F transcription factor 2. As used herein, the terms “target nucleic acid” and “nucleic acid encoding E2F transcription factor 2” encompass DNA encoding E2F transcription factor 2, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric comp

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