Antisense inhibition of cyclin D2 expression

Details Antisense inhibition of cyclin D2 expression Antisense inhibition of cyclin D2 expression

2001-08-01

2002-12-10

Wang, Andrew (Department: 1635)

Chemistry: molecular biology and microbiology

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

Method of regulating cell metabolism or physiology

C435S006120, C536S024500, C536S024300

Reexamination Certificate

active

06492173

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of Cyclin D2. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding Cyclin D2. Such compounds have been shown to modulate the expression of Cyclin D2.
BACKGROUND OF THE INVENTION
In eukaryotes, the regulated progression through the cell cycle is controlled by a group of genes whose expression fluctuates throughout the cycle. Cyclin dependent kinases (CDKs) and their associated regulatory subunits, the cyclins, are the primary regulators of the cell cycle. These heterodimeric complexes act by phosphorylating downstream targets that, in turn, trigger signaling events that liberate nuclear proteins necessary for entry into subsequent phases of the cell cycle. It has been shown that mitogens enhance the assembly of these complexes and that overexpression of cyclins eliminates the need for mitogen-dependent stimulation resulting in uncontrolled cellular development and often tumorigenesis. As a result of this finding, much effort has been focused on the study of cyclin expression and regulation as they relate to the development of the cancerous phenotype.
Cyclin D2 is a member of the cyclin family of cell cycle regulators. There are eight major classes of cyclins, each been focused on the study of cyclin expression and regulation as they relate to the development of the cancerous phenotype.
Cyclin D2 is a member of the cyclin family of cell cycle regulators. There are eight major classes of cyclins, each with a tissue specific pattern of expression within the phases of the cell cycle. Cyclins D1-D3 reach a maximal activity in the G
1
phase and regulate the transition to S phase thus determining whether a new cell cycle will occur. These cyclins have also been shown to play a role as developmental regulators during gastrulation and neuralation (Wianny et al.,
Dev. Dyn.,
1998, 212, 49-62). Therefore, since cell proliferation is primarily regulated in the G
1
phase and since deregulation of this transition can result in neoplasia, the controlled expression of the D-cyclins is critical to normal cellular development.
Cyclin D2 was originally isolated from B- and T-lymphocytes in a search for genes related to Cyclin D1, another cyclin which has been shown to be involved in the development of several human cancers (Palmero et al.,
Oncogene,
1993, 8, 1049-1054). Since then, the regulation and expression of Cyclin D2 has been characterized in many human cell lines with both normal and carcinomal phenotypes. In these studies, it was shown that the expression of Cyclin D2 is more restricted than Cyclin D1 and that the levels of Cyclin D2 undergo cell cycle-dependent oscillations with a peak in late G
1
, indicating that it is necessary for cell cycle progression (Lukas et al.,
Oncogene,
1995, 10, 2125-2134). Cyclin D2 has been shown to be a key player in several cell signaling events including those involving the MAP kinases (Vadiveloo et al.,
The Journal of Biological Chemistry,
1998, 273, 23104-23109) and those triggered by transforming growth factor beta (TGF-&bgr;) (Arvanitakis et al.,
J. Immunol.,
1995, 155, 1047-1056; Ohtsuki et al.,
Br. J. Haematol.,
1997, 98, 520-527; Yamato et al.,
Mol. Endocrinol.,
1997, 11, 1044-1052).
Aberrant expression of Cyclin D2 has been implicated in several disease processes. The expression of Cyclin D2 has been shown to be deregulated in male germ cell tumorigenesis (Houldsworth et al.,
Cell Growth Differ.,
1997, 8, 293-299) and chronic B-cell malignancies including leukemia and lymphomas (Delmer et al.,
Blood,
1995, 85, 2870-2876). In addition, Cyclin D2 was shown to be overexpressed in human T-cell leukemia virus (HTLV-I) infected cells (Akagi et al.,
Oncogene,
1996, 12, 1645-1652).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of Cyclin D2 and strategies aimed at investigating Cyclin D2 function have involved the use of antibodies, antisense expression vectors, antisense therapy, and gene knock-outs in mice.
In investigations of cyclin expression during lung development, injury and repair, Wu et al. used antisense oligonucleotides directed against the mouse Cyclin D2 cDNA sequence (−6 to +13), in which there is only one nucleotide mismatch between the human and the mouse cDNAs. These studies revealed a stage-specific expression of Cyclin D2 during normal development (Wu et al.,
Am. J. Respir. Cell. Mol. Biol.,
1995, 12, 95-103).
Mouse knockouts of Cyclin D2 have also been investigated. Cyclin D2 deficient females are sterile due to the inability of the granulosa cells to proliferate in response to follicle stimulating hormone (FSH) whereas males display hypoplastic testes (Sicinski et al.,
Nature,
1996, 384, 470-474). Further investigations of human cells derived from corresponding tissues showed that 10 of 12 ovarian granulosa cell tumors as well as human testicular germ-cell tumors (TCG) expressed high levels of Cyclin D2 (Sicinski et al.,
Nature,
1996, 384, 470-474).
Current strategies for the selective inhibition of Cyclin D2 expression are inappropriate as therapeutic protocols. Consequently there remains a long felt need for additional agents capable of effectively inhibiting Cyclin D2 function.
Antisense technology, however, 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 Cyclin D2 expression.
SUMMARY OF THE INVENTION
The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding Cyclin D2, and which modulate the expression of Cyclin D2. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of Cyclin D2 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 Cyclin D2 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 Cyclin D2, ultimately modulating the amount of Cyclin D2 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding Cyclin D2. As used herein, the terms “target nucleic acid” and “nucleic acid encoding Cyclin D2” encompass DNA encoding Cyclin D2, 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 Cyclin D2. 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 inv

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