Antisense modulation of WRN expression

Details Antisense modulation of WRN expression Antisense modulation of WRN expression

2001-02-23

2002-09-10

LeGuyader, John L. (Department: 1635)

Chemistry: molecular biology and microbiology

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

Method of regulating cell metabolism or physiology

C435S006120, C435S091100, C435S325000, C435S366000, C536S023100, C536S024310, C536S024330, C536S024500

Reexamination Certificate

active

06448080

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of WRN. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding WRN. Such compounds have been shown to modulate the expression of WRN.
BACKGROUND OF THE INVENTION
Genomic integrity is critical to the health and survival of any organisms and cells have evolved multiple pathways for the repair of DNA damage.
One class of enzymes involved in the maintenance of genomic integrity and stability are DNA helicases. These proteins play important roles in DNA replication, repair, recombination and transcription by unwinding duplex genomic strands allowing the repair machinery access to damaged or mispaired DNA. For example, the RecQ family of helicases has been shown to be important players in linking cell cycle checkpoint responses to recombination repair (Chakraverty and Hickson,
BioEssays
, 1999, 21, 286-294; Frei and Gasser,
J. Cell Sci
., 2000, 113, 2641-2646; Wu et al.,
Curr. Biol
., 1999, 9, R518-520). More recently, these helicases have been implicated in the process of posttranscriptional gene silencing (PTGS) (Cogoni and Macino,
Science
, 1999, 286, 2342-2344). In this process, the helicase is required to separate the double-stranded DNA (dsDNA) before any hybridization and silencing mechanism could be initiated.
The RecQ family consists of five members and can be divided into two distinct groups according to whether they contain an additional carboxy- or amino-terminus group. One class containing the longest members of the family include genes known to be defective in several syndromes including the BLM gene in Bloom's syndrome, the WRN gene in Werner's syndrome and the RECQ4 gene in Rothmund-Thompson syndrome. Mutations in these genes lead to an increase in the incidence of cancer as well as other physiologic abnormalities (Karow et al.,
Curr. Opin. Genet. Dev
., 2000, 10, 32-38; Kawabe et al.,
Oncogene
, 2000, 19, 4764-4772).
The second class contains the RECQL gene and the RECQ5 gene which encode little more than the central helicase domain and have not been associated with any human disease.
WRN (also known as RECQL3) was originally identifed by positional cloning as the gene responsible for Werner's syndrome and localized to chromosome 8p12 (Yu et al.,
Science
, 1996, 272, 258-262.). Werner's syndrome is a rare autosomal recessive disorder characterized by symptoms similar to premature aging including atherosclerosis, osteoporosis, type II diabetes, cataracts and cancers (Goto,
Clin Exp. Rheumatol
., 2000, 18, 760-766; Oshima, BioEssays, 2000, 22, 894-901; Shen and Loeb,
Trends Genet
., 2000, 16, 213-220).
In an effort to better define the role of the WRN gene, Marciniak et al. determined the subcellular localization of the protein using indirect immunofluorescence and a 30 polyclonal antibody. These studies revealed a predominant nuclear localization and no difference in localization was detected in normal compared to transformed human cell lines (Marciniak et al.,
Proc. Natl. Acad. Sci. U. S. A
., 1998, 95, 6887-6892). In addition to having helicase activity, WRN also contains intrinsic exonuclease activity and has been shown to bind single-stranded DNA with higher affinity than double-stranded DNA (Orren et al., Nucleic Acids Res., 1999, 27, 3557-3566; Shen et al.,
J. Biol. Chem
., 1998, 273, 34139-34144).
Disclosed in U.S. Pat. No. 6,090,620 are the nucleic acid molecules encoding the WRN gene as well as WRN gene products, expression vectors, viral vectors and host cells suitable for expressing the WRN gene products (Fu et al., 2000).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of WRN. There are reports of DNA minor groove-binding drugs which inhibit the helicase activity of WRN (Brosh et al.,
Nucleic Acids Res
., 2000, 28, 2420-2430). WRN-deficient mice display reduced embryonic survival while live-born mice otherwise appear normal during the first year of life (Lebel and Leder,
Proc. Natl. Acad. Sci. U. S. A
., 1998, 95, 13097-13102).
While mutations and targeted disruptions resulting in altered protein expression in the WRN gene are responsible for Werner's syndrome, the normal function of the WRN gene product and its regulation are still unclear. It is, however, believed to be involved in DNA metabolism and is therefore a potential therapeutic target in conditions involving the production of aberrant DNA products, including the recognition of foreign DNA products as is the case upon viral infection. Consequently, there remains a long felt need for agents capable of effectively inhibiting and/or modulating WRN 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 WRN expression.
The present invention provides compositions and methods for modulating WRN expression.
SUMMARY OF THE INVENTION
The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding WRN, and which modulate the expression of WRN. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of WRN 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 WRN 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 WRN, ultimately modulating the amount of WRN produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding WRN. As used herein, the terms “target nucleic acid” and “nucleic acid encoding WRN” encompass DNA encoding WRN, 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 WRN. 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 invention, inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target.
It is preferred to target specific nucleic acids for antisense. “Targeting” an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an i

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