Antisense inhibition of replication protein a p70 subunit

Details Antisense inhibition of replication protein a p70 subunit Antisense inhibition of replication protein a p70 subunit

1999-09-10

2001-10-30

McGarry, Sean (Department: 1635)

Chemistry: molecular biology and microbiology

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

Method of regulating cell metabolism or physiology

C435S325000, C435S091100, C435S006120, C536S023100, C536S024500, C536S024300, C536S024310, C536S024330

Reexamination Certificate

active

06309882

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to compositions and methods for modulating expression of the replication protein A 70 kDa subunit gene, a naturally present cellular gene encoding a subunit of a complex implicated in multiple processes of DNA metabolism. These compositions and methods can be used diagnostically or therapeutically. Furthermore, this invention is directed to treatment of cancer via specific inhibition of the replication protein A 70 kDa subunit gene.
BACKGROUND OF THE INVENTION
Human replication protein A (RPA), also human single-stranded DNA-binding protein (HSSB), is a single-stranded DNA-binding protein that also interacts and regulates the function of a number of other cellular proteins. This heterotrimeric protein is composed of three subunits, the 70 kDa subunit (RPA70 or p70), p34 (RPA34) and p11 (RPA11) encoded by genes located on different chromosomes (sometimes referred to as RPA1, RPA2 and RPA3 genes, respectively). Each of the three subunits of RPA has been shown to be essential for DNA replication, homologous recombination and nucleotide excision repair in vitro (He, Z., et al.,
Nature,
1995, 374, 566-569), and disruption of any of the three subunits in yeast is lethal (Brill, S. J. and Stillman, B.,
Genes and Development,
1991, 5, 1589-1600).
RPA70 binds to single-stranded DNA and certain double-stranded sequences with high affinity (Lao, Y., et al.,
Biochemistry,
1999, 38, 3974-3984) and possesses unwindase activity. RPA also binds cisplatin (Patrick, S. M., and Turchi, J. J.,
Biochemistry,
1998, 37, 8808-8815) and UV-damaged DNA. In addition to its DNA binding, RPA interacts with various proteins involved in DNA replication, repair, recombination, transcription and cell regulation. RPA70 interacts with DNA polymerase &agr;, during initiation of replication and elongation, and DNA polymerase &dgr; (Longhese, M. P., et al.,
Mol. Cell. Biol.,
1994, 14, 7884-7890). Both RPA70 and RPA34 interact with the Xeroderma Pigmentosum group A complementing protein (XPA) on damaged DNA recruiting endonucleases involved in DNA repair (Stigger, E., et al., J. Biol. Chem., 1998, 273, 9337-9343). RPA also interacts with transcriptional activators including the tumor suppressor gene, p53 (Miller, S. D., et al.,
Mol. Cell. Biol.,
1997, 17, 2194-2201).
The function of the RPA70 subunit has been studied primarily with mutant RPA1 genes (Longhese, M. P., et al.,
Mol. Cell. Biol.,
1994, 14, 7884-7890) and deletion analysis.
RPA70 is believed to be an attractive target for cancer therapeutics. In a preferred embodiment, such a therapy can take advantage of natural genetic variation within the genome in combination with loss of heterozygosity (LOH) in cancer cells. This approach is based on allele-specific targeting and is described in WO 98/41648, herein incorporated by reference in its entirety.
It is estimated that natural genetic variation occurs in approximately one nucleotide in 300 throughout the genome (Cooper, D. N., et al.,
Human Genetics,
1985, 69, 201-205). Because of the large number of polymorphisms or sequence variances found in the human genome, most individuals are heterozygous for one or more sequence variances in genes of normal tissues, including many genes that are essential for cell survival. LOH reduces many of these genes to hemizygosity in cancer cells, eliminating heterozygosity, and creating a large number of absolute genetic differences between tumor and normal cells (Cavenee, W. K., et al.,
Mutat. Res.,
1991, 247, 199-202; Schwechheimer, K. and Cavenee, W. K.,
Clin. Investig.,
1993, 71, 488-502).
An early event in the clonal evolution of cancers is the loss of large chromosomal regions or even whole chromosomes (Lengauer, C., et al.,
Nature,
1998, 396, 643-649). Presumably, these losses are driven, in part, by positive selection for cells in which LOH leads to the loss of tumor suppressor functions. LOH in certain cancers can involve more than 20% of the total genome (Lengauer, C., et al.,
Nature,
1998, 396, 643-649) and it is evident that thousands of genes are also lost from cancer cells due to LOH. Based on current estimates of human gene number this suggests that 15,000 to 20,000 genes, that are not tumor suppressor genes, are also reduced to hemizygosity in cancer cells by LOH. Among these genes are many that are essential for cell survival. The RPA70 gene has been mapped to chromosome 17p13.3 in close proximity to the tumor suppressor gene p53 at position 17p13.1 (Umbricht, C. B., et al.,
Genomics,
1993, 20, 249-257). This segment of the genome is affected by LOH in many common epithelial cancers (Rodriguez, E., et al.,
Cancer Res.,
1994, 54, 3398-3406). Thus, RPA70 represents an attractive target for allele-specific therapy.
By exploiting the absolute genetic differences in RPA70 genes between cancer cells and normal cells that arise as a consequence of normal genetic variation and LOH, RPA70 can be an effective target for cancer therapy. Inhibitors, especially antisense compounds, are identified that inactivate one or more variant forms of the target gene, but not the normal form that is present in the general population. Inhibitors specific for the remaining allele expressed in the cancer cells, when administered to patients, would be selectively toxic to the cancer cells. Normal cells and tissues, which express both the sensitive and insensitive alleles, would escape significant toxicity.
There remains a long-felt need for improved compositions and methods for inhibiting RPA 70 kDa subunit gene expression.
SUMMARY OF THE INVENTION
The present invention provides antisense compounds which are targeted to nucleic acids encoding human replication protein A 70 kDa subunit and are capable of inhibiting replication protein A 70 kDa subunit expression. In preferred embodiments, the antisense compounds are targeted to a variant form of RPA70. 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 replication protein A 70 kDa subunit. These methods are believed to be useful both therapeutically and diagnostically as a consequence of the association between replication protein A 70 kDa subunit inhibition and its essential roles in DNA metabolism and, consequently, cell viability. These methods are also useful as tools, for example, for determining the role of replication protein A 70 kDa subunit expression in various cell functions and physiological processes and conditions and for detecting and diagnosing conditions associated with replication protein A 70 kDa subunit and its variant forms.
Methods of treating cancer 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
Replication protein A is an essential protein that plays a major role in multiple processes in DNA metabolism. It has both DNA binding activity and protein binding activity. The RPA70 subunit is required in many processes involving DNA replication, repair and recombination.
Since natural sequence variations occur within this gene as well as throughout the genome, and LOH at this gene locus is associated with many cancers, the RPA70 gene is an attractive target for cancer therapeutics based on allele-specific targeting. Nucleic acid based therapeutics, such as exemplified by antisense approach, are ideal for this targeting.
The 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

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