Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
2001-04-27
2002-12-10
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, C536S063000
Reexamination Certificate
active
06492172
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of GU Protein. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding GU Protein. Such compounds have been shown to modulate the expression of GU Protein.
BACKGROUND OF THE INVENTION
RNA helicases represent a large family of proteins that have been detected in almost all biological systems in which RNA plays a central role. They are ubiquitously distributed over a wide range of organisms and are involved in diverse cellular functions such as nuclear and mitochondrial RNA splicing, RNA editing, ribosome assembly and processing, initiation of translation, spermatogenesis, embryogenesis, and cell growth and division, nuclear mRNA export, and mRNA degradation. RNA helicases are essential factors in cell development and differentiation, and some of them play a role in transcription and replication of viral single-stranded RNA genomes.
Sequence homologies among these proteins place them in two families, the D-E-A-D box protein family (D-E-A-D=Asp-Glu-Ala-Asp) and the D-E-A-D/H box protein family (D-E-A-D/H =Asp-Glu-Ala-Asp/His). Some of the better characterized members have been shown to possess ATP-binding and hydrolyzing activities as well as ATP-dependent RNA helicase activities (Luking et al.,
Crit. Rev. Biochem. Mol. Biol
., 1998, 33, 259-296).
GU protein (also known as GURDB, DDX21 and RH-II/GU) is a nucleolar RNA helicase of the DEAD/H family of helicases. This protein was originally identified as an autoantigen in the serum of a patient with water-melon stomach disease (Valdez et al.,
Nucleic Acids Res
., 1996, 24, 1220-1224). It was subsequently characterized as a nucleolar RNA helicase having distinct functional domains for RNA unwinding, RNA folding and nuclear localization and targeting (Ou et al.,
Exp. Cell. Res
., 1999, 247, 389-398; Valdez et al.,
Biochem. Biophys. Res. Commun
., 1997, 234, 335-340; Valdez et al.,
Eur. J. Biochem
., 1997, 250, 800-807). These domains have been shown to be necessary for cell growth and cell cycle progression (Ou et al.,
Exp. Cell. Res
., 1999, 247, 389-398).
Autoantibodies to GU protein have also been identified in patients with connective tissue diseases such as systemic sclerosis, systemic lupus erythematosus and undifferentiated connective tissue disease (Arnett et al.,
Arthritis Rheum
., 1997, 40, 1487-1492).
The pharmacological modulation of GU protein activity and/or expression is therefore believed to be an appropriate point of therapeutic intervention in pathological conditions involving an autoimmune response, especially those involving connective tissues.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of GU protein and investigative strategies aimed at modulating GU protein function have involved the use of antibodies (Valdez et al.,
Eur. J. Biochem
., 1997, 250, 800-807) and Adriamycin, an anticancer drug (Zhu et al.,
Biochem. Biophys. Res. Commun
., 1999, 266, 361-365). Adriamycin acts to inhibit GU protein activity by binding to its RNA substrate in a dose-dependent manner.
This effect is not specific, however, as Adriamycin has been shown to interact with RNAs in several experimental protocols. Therefore, this strategy is untested as a therapeutic protocol as well as being non-specific to GU protein. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting GU protein function.
Antisense technology is emerging as an effective means an 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 GU protein expression.
The present invention provides compositions and methods for modulating GU protein expression.
SUMMARY OF THE INVENTION
The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding GU Protein, and which modulate the expression of GU Protein. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of GU Protein 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 GU Protein 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 GU Protein, ultimately modulating the amount of GU Protein produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding GU Protein. As used herein, the terms “target nucleic acid” and “nucleic acid encoding GU Protein” encompass DNA encoding GU Protein, 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 GU Protein. 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 GU Protein. 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 s
Bennett C. Frank
Busch Harris
Wyatt Jacqueline
ISIS Pharmaceuticals Inc.
LeGuyader John L.
Licata & Tyrrell P.C.
Schmidt M
LandOfFree
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