Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
2000-09-14
2001-08-21
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
C435S325000, C435S006120, C435S091100, C536S023100, C536S024500, C536S024300, C536S024310, C536S024330
Reexamination Certificate
active
06277636
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of MADH6. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding MADH6. Such compounds have been shown to modulate the expression of MADH6.
BACKGROUND OF THE INVENTION
The transforming growth factor-beta (TGF-&bgr;) superfamily of cytokines regulate a diverse array of physiologic functions including cell proliferation and growth, cell migration, differentiation, development and apoptosis. This large family includes the TGF-&bgr;s, activins, and bone-morphogenic proteins (BMPs) and each subgroup initiates a unique signaling cascade activated by ligand-induced serine/threonine kinase receptor complex formation (Wrana,
Miner. Electrolyte Metab.,
1998, 24, 120-130). These complexes, once formed, recruit and phosphorylate members of a family of cytosolic proteins, known as SMADs. SMADs exist as monomers in unstimulated cells but homo- or heterodimerize and translocate to the nucleus activating target gene transcription upon ligand binding. SMADs, therefore, connect the pathway of TGF-&bgr; signaling from the cell membrane to the nucleus.
To date, nine vertebrate SMADs have been identified and these have been divided into subgroups based on their functional role in various pathways. SMAD1, 5, and MADH6, which is 80% homologous to SMAD1, all mediate signal transduction from BMPs while SMAD2 and 3 mediate signal transduction from TGF-&bgr;s and activins. Collectively, these SMADs are known as the pathway-restricted SMADs and can form homo or heterodimers. SMAD4 has been shown to be a shared hetero-oligomerization partner to the pathway-restricted SMADs and is known as the common mediator. The last two members of the family, SMAD6 and 7, act to inhibit the SMAD signaling cascades often by forming unproductive dimers with other SMADs and are therefore classified as antagonistic SMADs (Heldin et al.,
Nature,
1997, 390, 465-471; Kretzschmar and Massague,
Curr. Opin. Genet. Dev.,
1998, 8, 103-111).
MADH6 (also known as MADH9 and SMAD9) is a putative member of a subgroup of SMAD family transcription factors which are regulated by bone morphogenic proteins (BMPs).
The MADH6 gene was first isolated from a human fetal brain cDNA library and shown to encode two alternatively transcribed mRNAs, termed MADH6a and MADH6b. This gene was found to reside on chromosome 13q12-q14 between BRCA2 and RB, a region that displays allelic losses in breast, liver and prostate cancers. It is therefore believed that MADH6 may be involved in growth inhibition and tumor suppression (Watanabe et al.,
Genomics,
1997, 42, 446-451). Northern blot analysis showed that expression of MADH6 isoforms was found in adult and fetal tissue to the same extent, with the strongest expression in the brain, lung and kidney.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of MADH6. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting MADH6 function. Therefore, antisense oligonucleotides may provide a promising new pharmaceutical tool for the effective and specific modulation of MADH6 expression.
SUMMARY OF THE INVENTION
The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding MADH6, and which modulate the expression of MADH6. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of MADH6 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 MADH6 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 MADH6, ultimately modulating the amount of MADH6 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding MADH6. As used herein, the terms “target nucleic acid” and “nucleic acid encoding MADH6” encompass DNA encoding MADH6, 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 MADH6. 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 MADH6. 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 MADH6, regardless of the sequence(s) of such codons.
It is also known in the art that a translation termination
Cowsert Lex M.
Monia Brett P.
ISIS Pharmaceuticals Inc.
Lacourciere Karen A
LeGuyader John L.
Licata & Tyrrell P.C.
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