Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
1998-03-26
2001-05-29
Schwartzman, Robert A. (Department: 1635)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S006120, C435S091100, C435S371000, C536S023100, C536S024310, C536S024330, C536S024500
Reexamination Certificate
active
06238921
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to compositions and methods for modulating expression of the human mdm2 gene, a naturally present cellular gene implicated in abnormal cell proliferation and tumor formation. This invention is also directed to methods for inhibiting hyperproliferation of cells; these methods can be used diagnostically or therapeutically.
Furthermore, this invention is directed to treatment of conditions associated with expression of the human mdm2 gene.
BACKGROUND OF THE INVENTION
Inactivation of tumor suppressor genes leads to unregulated cell proliferation and is a cause of tumorigenesis.
In many tumors, p53 or the retinoblastoma (Rb) protein are inactivated. This can occur either by mutations within these genes, or by overexpression of the mdm2 gene. The mdm2 protein physically associates with both p53 and Rb, inhibiting their function. The levels of mdm2 are maintained through a feedback loop mechanism with p53. Overexpression of mdm2 effectively inactivates p53 and promotes cell proliferation. Amplification of the mdm2 gene is found in many human cancers, including soft tissue sarcomas, astrocytomas, glioblastomas, breast cancers and non-small cell lung carcinomas. In many blood cancers, overexpression of mdm2 can occur with a normal copy number. This has been attributed to enhanced translation of mdm2 mRNA, which is thought to be related to a distinct 5′-untranslated region (5′-UTR) which causes the transcript to be translated more efficiently than the normal mdm2 transcript. Landers et al.,
Cancer Res.
57, 3562, (1997).
Several approaches have been used to disrupt the interaction between p53 and mdm2. Small peptide inhibitors, screened from a phage display library, have been shown in ELISA assays to disrupt this interaction [Bottger et al.,
J. Mol. Biol.,
269, 744 (1997)]. Microinjection of an anti-mdm2 antibody targeted to the p53-binding domain of mdm2 increased p53-dependent transcription [Blaydes et al.,
Oncogene,
14, 1859 (1997)].
A vector-based antisense approach has been used to study the function of mdm2. Using a rhabdomyosarcoma model, Fiddler et al. [
Mol. Cell Biol.,
16, 5048 (1996)] demonstrated that amplified mdm2 inhibits the ability of MyoD to function as a transcription factor. Furthermore, expression of full-length antisense mdm2 from a cytomegalovirus promoter-containing vector restores muscle-specific gene expression. Antisense oligonucleotides have also been useful in understanding the role of mdm2 in regulation of p53. An antisense oligonucleotide directed to the mdm2 start codon allowed cisplatin-induced p53-mediated apoptosis to occur in a cell line overexpressing mdm2 [Kondo et al.,
Oncogene,
10, 2001 1995). The same oligonucleotide was found to inhibit the expression of P-glycoprotein [Kondo et al.,
Br. J. Cancer,
74, 1263 (1996)]. P-glycoprotein was shown to be induced by mdm2. Teoh et al [
Blood,
90, 1982 (1997)] demonstrated that treatment with an identical mdm2 antisense oligonucleotide or a shorter version within the same region in a tumor cell line decreased DNA synthesis and cell viability and triggered apoptosis.
Chen et al. [
Proc. Natl. Acad. Sci. USA,
95, 195 (1998)] disclose antisense oligonucleotides targeted to the coding region of mdm2. A reduction in mdm2 RNA and protein levels was seen, and transcriptional activity from a p53-responsive promoter was increased after oligonucleotide treatment of JAR (choriocarcinoma) or SJSA (osteosarcoma) cells.
WO 93/20238 and WO 97/09343 disclose, in general, the use of antisense constructs, antisense oligonucleotides, ribozymes and triplex-forming oligonucleotides to detect or to inhibit expression of mdm2.
There remains a long-felt need for improved compositions and methods for inhibiting mdm2 gene expression.
SUMMARY OF THE INVENTION
The present invention provides oligonucleotides which are targeted to nucleic acids encoding human mdm2 and are capable of inhibiting mdm2 expression. The present invention also provides chimeric oligonucleotides targeted to nucleic acids encoding human mdm2. The oligonucleotides of the invention are believed to be useful both diagnostically and therapeutically, and are believed to be particularly useful in the methods of the present invention.
The present invention also comprises methods of inhibiting the expression of human mdm2, particularly the increased expression resulting from amplification of mdm2. These methods are believed to be useful both therapeutically and diagnostically as a consequence of the association between mdm2 expression and hyperproliferation. These methods are also useful as tools, for example, for detecting and determining the role of mdm2 expression in various cell functions and physiological processes and conditions and for diagnosing conditions associated with mdm2 expression.
The present invention also comprises methods of inhibiting hyperproliferation of cells using oligonucleotides of the invention. These methods are believed to be useful, for example, in diagnosing mdm2-associated cell hyperproliferation. Methods of treating abnormal proliferative conditions associated with mdm2 are also provided. These methods employ the oligonucleotides of the invention. These methods are believed to be useful both therapeutically and as clinical research and diagnostic tools.
DETAILED DESCRIPTION OF THE INVENTION
Tumors often result from genetic changes in cellular regulatory genes. Among the most important of these are the tumor suppressor genes, of which p53 is the most widely studied. Approximately half of all human tumors have a mutation in the p53 gene. This mutation disrupts the ability of the p53 protein to bind to DNA and act as a transcription factor. Hyperproliferation of cells occurs as a result.
Another mechanism by which p53 can be inactivated is through overexpression of mdm2, which regulates p53 activity in a feedback loop. The mdm2 protein binds to p53 in its DNA binding region, preventing its activity. Mdm2 is amplified in some human tumors, and this amplification is diagnostic of neoplasia or the potential therefor. Over one third of human sarcomas have elevated mdm2 sequences. Elevated expression may also be involved in other tumors including but not limited to those in which p53 inactivation has been implicated. These include colorectal carcinoma, lung cancer and chronic myelogenous leukemia.
Many abnormal proliferative conditions, particularly hyperproliferative conditions, are believed to be associated with mdm2 expression and are, therefore believed to be responsive to inhibition of mdm2 expression. Examples of hyperproliferative conditions are cancers, psoriasis, blood vessel stenosis (e.g., restenosis or atherosclerosis), and fibrosis, e.g., of the lung or kidney.
The present invention employs antisense compounds, particularly oligonucleotides, for use in inhibiting the function of nucleic acid molecules encoding mdm2, ultimately modulating the amount of mdm2 produced. This is accomplished by providing oligonucleotides which specifically hybridize with nucleic acids, preferably mRNA, encoding mdm2.
This relationship between an antisense compound such as an oligonucleotide and its complementary nucleic acid target, to which it hybridizes, is commonly referred to as “antisense”. “Targeting” an oligonucleotide to a chosen nucleic acid target, in the context of this invention, is a multistep process. The process usually begins with identifying a nucleic acid sequence whose function is to be modulated. This may be, as examples, a cellular gene (or mRNA made from the gene) whose expression is associated with a particular disease state, or a foreign nucleic acid from an infectious agent. In the present invention, the target is a nucleic acid encoding mdm2; in other words, a mdm2 gene or RNA expressed from a mdm2 gene. mdm2 mRNA is presently the preferred target. The targeting process also includes determination of a site or sites within the nucleic acid sequence for the antisense interaction to occur such th
Graham Mark J.
Miraglia Loren J.
Monia Brett P.
Nero Pamela
ISIS Pharmaceuticals Inc.
Law Offices of Jane Massey Licata
Schwartzman Robert A.
Shibuya Mark L.
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