Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
1999-12-29
2001-02-13
Elliott, George C. (Department: 1635)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S006120, C536S023100, C536S024100, C536S024500, C514S04400A
Reexamination Certificate
active
06187586
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of Akt-3. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding human Akt-3. Such oligonucleotides have been shown to modulate the expression of Akt-3.
BACKGROUND OF THE INVENTION
One of the principal mechanisms by which cellular regulation is effected is through the transduction of extracellular signals across the membrane that in turn modulate biochemical pathways within the cell. Protein phosphorylation represents one course by which intracellular signals are propagated from molecule to molecule resulting finally in a cellular response. These signal transduction cascades are highly regulated and often overlapping as evidenced by the existence of many protein kinases as well as phosphatases. Phosphorylation of proteins occurs predominantly at serine, threonine, or tyrosine residues and protein kinases have therefore been classified by their specificity of phosphorylation site i.e. serine/threonine kinases and tyrosine kinases. Because phosphorylation is such a ubiquitous process within cells and because cellular phenotypes are largely influenced by the activity of these pathways, it is currently believed that a number of disease states and/or disorders are a result of either aberrant activation or functional mutations in the molecular components of kinase cascades. Consequently, considerable attention has been devoted to the characterization of these proteins.
Akt-3 (also known as PKB gamma and RAC-PK gamma) is a member of the AKT/PKB family of serine/threonine kinases first isolated from a rat brain cDNA and shown to be expressed predominantly in the central nervous system and the testis (Konishi et al.,
Biochem. Biophys. Res. Commun
., 1995, 216, 526-534).
Akt-3, like other members of the AKT/PKB family, is located in the cytosol of unstimulated cells and translocates to the plasma membrane following stimulation by several ligands including mitogens and survival factors (Meier et al.,
J. Biol. Chem
., 1997, 272, 30491-30497). Other studies have shown that this activation is through PI3 kinase which is wortmannin sensitive (Franke et al.,
Science
, 1997, 275, 665-668). It is through the pleckstrin homology domain (PH) within the protein that Akt-3 binds to the lipid products of PI3 kinase allowing presentation of Akt-3 to its upstream activators by directing its translocation to the membrane. Phosphorylation of Akt-3 is necessary for its activation and the kinase responsible for this activation has been identified as PDK1 (Cohen et al.,
FEBS Lett
., 1997, 410, 3-10).
Once localized to the membrane, Akt-3 mediates several functions within the cell including the metabolic effects of insulin (Walker et al.,
Biochem. J
., 1998, 331, 299-308).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of Akt-3. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting Akt-3 function.
To date, strategies aimed at inhibiting Akt-3 function have involved the use of antibodies to Akt-3 and inhibitors of the upstream PI3 kinase, including a dominant-negative form of the PI3 kinase. However, these upstream inhibitors are not specific to Akt-3 and disrupt several divergent cellular pathways. Antisense oligonucleotides, therefore, provide a promising new pharmaceutical tool for the effective modification of the expression of specific genes including Akt-3.
SUMMARY OF THE INVENTION
The present invention is directed to antisense compounds, particularly oligonucleotides, which are targeted to a nucleic acid encoding Akt-3, and which modulate the expression of Akt-3. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. Further provided are methods of modulating the expression of Akt-3 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 Akt-3 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 Akt-3, ultimately modulating the amount of Akt-3 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding Akt-3. As used herein, the terms “target nucleic acid” and “nucleic acid encoding Akt-3” encompass DNA encoding Akt-3, 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 Akt-3. 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 Akt-3. 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 th
Cowsert Lex M.
Monia Brett P.
Roth Richard A.
Elliott George C.
ISIS Pharmaceuticals Inc.
Law Offices of Jane Massey Licata
McGarry Sean
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