Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-11-03
2002-11-05
McGarry, Sean (Department: 1635)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S006120, C435S091100, C536S023100, C536S024500, C536S025300
Reexamination Certificate
active
06475797
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of SR-cyp. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding SR-cyp. Such compounds have been shown to modulate the expression of SR-cyp.
BACKGROUND OF THE INVENTION
The production of mature messenger RNA (mRNA) molecules which are capable of being translated into functional proteins requires the highly regulated process of posttranscriptional pre-mRNA processing. This includes removal of introns through splicing followed by polyadenylation of the 3′ end of the transcript. It is currently believed that, while splicing and 3′ end modifications can occur independently of the transcription process, these processes are coupled in vivo.
Recently studies have indicated that splicing factors and the polyadenylation apparatus are associated with the transcriptional machinery through the C-terminal domain (CTD) of the largest subunit of RNA Polymerase II reviewed in (Steinmetz,
Cell,
1997, 89, 491-494). This domain is comprised of tandem repeats of a consensus heptapeptide present in 17-52 copies (depending on the organism) and is rich in phosphorylation sites which play a critical role in the ability of the polymerase to synthesize long mRNA transcripts (Corden and Patturajan,
Trends Biochem. Sci.,
1997, 22, 413-416). Several protein families have been shown to be involved in the coupling of transcription to the posttranscriptional processing of mRNA species through their interaction with the CTD of RNA polymerase II. One such family, characterized by the presence of serine-arginine repeats which have been shown to be important in the selection of splice sites, is the SR-cyclophilins. This family has characteristics of both the SR domain-containing splicing factors and cyclophilins which have been implicated in protein folding, assembly and transport.
SR-cyp (also known as CARS-cyp for Clk-associated RS cyclophilin, CASP10 for CTD associated SR-like protein and Matrin CYP) was recently identified in several species, through a yeast two-hybrid screen, as a protein that interacts with the CTD of RNA polymerase II (Bourquin et al.,
Nucleic Acids Res.,
1997, 25, 2055-2061; Mortillaro and Berezney,
J. Biol. Chem.,
1998, 273, 8183-8192; Nestel et al.,
Gene,
1996, 180, 151-155). SR-cyp is ubiquitously expressed in all human tissues examined but appears to be absent from Natural Killer (NK) cells. These cells express a different SR-cyclophilin family protein, NK-TR1 (Nestel et al.,
Gene,
1996, 180, 151-155).
Characterization of the SR-cyp protein revealed a domain structure which includes, at the N-terminus, a peptidyl-prolyl cis-trans isomerase domain similar to immunophilins/cyclophilins. The protein is localized to the nucleus being distributed to large irregularly shaped nuclear speckles and co-localizes with other known splicing factors (Bourquin et al.,
Nucleic Acids Res.,
1997, 25, 2055-2061).
Taken together, these data indicate that SR-cyp plays a role in pre-mRNA splicing and the pharmacological modulation of SR-cyp activity and/or expression may be an appropriate point of therapeutic intervention in pathological conditions that involve the production of aberrantly spliced gene products.
Currently, there are no known therapeutic compounds that effectively inhibit the synthesis of SR-cyp and consequently there remains a long felt need for these agents. 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 SR-cyp expression.
The present invention provides compositions and methods for modulating SR-cyp expression.
SUMMARY OF THE INVENTION
The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding SR-cyp, and which modulate the expression of SR-cyp. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of SR-cyp 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 SR-cyp 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 SR-cyp, ultimately modulating the amount of SR-cyp produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding SR-cyp. As used herein, the terms “target nucleic acid” and “nucleic acid encoding SR-cyp” encompass DNA encoding SR-cyp, 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 SR-cyp. 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 SR-cyp. 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 formylmethion
ISIS Pharmaceuticals Inc.
Licata & Tyrrell P.C.
McGarry Sean
Zara Jane
LandOfFree
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