Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-12-22
2002-09-17
Wang, Andrew (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S325000, C435S375000, C536S024500
Reexamination Certificate
active
06451538
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of CHK2. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding human CHK2. Such oligonucleotides have been shown to modulate the expression of CHK2.
BACKGROUND OF THE INVENTION
In eukaryotes, the regulated progression through the cell cycle is controlled by a system of checkpoint pathways that monitor the integrity and status of the DNA. These checkpoint pathways provide a mechanism to arrest the cell cycle while the cell undergoes DNA replication or repair. Failure to arrest the cell cycle during these processes results in genetic instability and can contribute to the development of pathological conditions such as cancer (Russell,
Trends Biochem. Sci.,
1998, 23, 399-402).
The principal regulatory proteins of the cell cycle are the cyclin dependent kinases (Cdks) and their associated cyclins. Upon DNA damage, a signaling cascade is triggered that results in the inactivation of the heterodimeric Cdk-cyclin complexes and the ultimate arrest of the cell cycle.
CHK2 (also known as checkpoint kinase 2 and Rad53/cds1 in yeast) is a recently identified checkpoint protein kinase that functions in the regulation of the cell cycle by participating in the transduction of DNA damage and replication stress signals to the cyclin dependent kinases (Blasina et al.,
Curr. Biol.,
1999, 9, 1-10; Matsuoka et al.,
Science,
1998, 282, 1893-1897). It has been demonstrated that CHK2 phosphorylates several cyclin dependent kinases including Cdc25A, Cdc25B and Cdc25C as well as being autophosphorylated. In kinetic analysis studies it was shown that CHK2 mobility on SDS-PAGE gels is not modified during the progression of the cell cycle in the absence of DNA damage but is modified within 15 minutes of gamma irradiation, hydroxyurea treatment or ultraviolet light exposure (Matsuoka et al.,
Science,
1998, 282, 1893-1897).
The activation of the CHK2 kinase is dependent on the presence of a functional ATM (ataxia-telangiectasia mutated) gene. The ATM gene encodes a protein that controls cell cycle arrest in G1 and G2 by preventing DNA synthesis (Westphal,
Curr. Biol.,
1997, 7, R789-792).
Northern blot studies revealed a broad range of CHK2 mRNA expression with the highest being in the testis, spleen, colon and peripheral blood leukocytes (Matsuoka et al.,
Science,
1998, 282, 1893-1897).
WO 99/20747 (Luyten et al.) generally discloses and claims antisense molecules capable of hybridizing to the coding region of CHK2 under high stringency conditions.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis or function of CHK2. It is likely that cancer cells treated with agents that inhibit CHK2 could render those cells more susceptible to DNA damaging agents currently being used to treat several types of cancer. Consequently, there remains a long felt need for agents capable of effectively inhibiting CHK2 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 CHK2 expression.
SUMMARY OF THE INVENTION
The present invention is directed to antisense compounds, particularly oligonucleotides, which are targeted to a nucleic acid encoding CHK2, and which modulate the expression of CHK2. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. Further provided are methods of modulating the expression of CHK2 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 CHK2 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 antisense compounds, particularly oligonucleotides, for use in modulating the function of nucleic acid molecules encoding CHK2, ultimately modulating the amount of CHK2 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding CHK2. As used herein, the terms “target nucleic acid” and “nucleic acid encoding CHK2” encompass DNA encoding CHK2, 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 CHK2. 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 infectious agent. In the present invention, the target is a nucleic acid molecule encoding CHK2. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed MRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding CHK2, regardless of the sequence(s) of such codons.
It
ISIS Pharmaceuticals Inc.
Licata & Tyrrell P.C.
Wang Andrew
LandOfFree
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