Antisense inhibition of HPK/GCK-like kinase expression

Details Antisense inhibition of HPK/GCK-like kinase expression Antisense inhibition of HPK/GCK-like kinase expression

2000-08-29

2002-02-12

McGarry, Sean (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, C435S091300, C536S023100, C536S023200, C536S024500, C536S024300, C536S024330, C536S024310

Reexamination Certificate

active

06346416

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of HPK/GCK-like kinase. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding HPK/GCK-like kinase. Such compounds have been shown to modulate the expression of HPK/GCK-like kinase.
BACKGROUND OF THE INVENTION
Nearly all cell surface receptors use one or more of the mitogen-activated protein kinase (MAP kinase) cascades during signal transduction. Three distinct subgroups of the MAP kinases have been identified, and each of these consists of a specific module of downstream kinases that mediate a diverse array of effects including cell proliferation, transformation, differentiation and apoptosis. One subgroup of the MAP kinases is the Jun N-terminal kinase/Stress activated protein kinase (JNK/SAPK) cascade. This pathway was originally identified as an oncogene- and ultraviolet light-stimulated kinase pathway but is now known to be activated by several stressors such as heat and osmotic shock, DNA-damaging agents and protein synthesis inhibitors as well as growth factors, cytokines and T-cell costimulation (Su and Karin,
Curr. Opin. Immunol.,
1996, 8, 402-411). It is currently believed that a number of disease states and/or disorders are a result of either aberrant expression or functional mutations in the molecular components of kinase cascades. Consequently, considerable attention has been devoted to the characterization of these proteins and their associated activators and suppressors.
HPK/GCK-like kinase (HGK) (also known as NIK for Nck-interacting kinase) is a recently described serine/threonine kinase that mediates tumor necrosis factor alpha (TNF-&agr;) signaling by activating the JNK pathway (Yao et al.,
J. Biol. Chem.,
1999, 274, 2118-2125). HGK exerts its role in the cell by activating the kinases upstream of JNK, specifically TGF-beta activated kinase 1 (TAK1) (Yao et al.,
J. Biol. Chem.,
1999, 274, 2118-2125). HGK is most homologous to the sterile 20 (STE20) family of protein kinases in Drosophila and the mouse homologue has been shown to interact with the oncoprotein effector molecule, Nck (Su et al.,
Embo. J.,
1997, 16, 1279-1290; Su et al.,
Genes Dev.,
1998, 12, 2371-2380). Nck has been shown to regulate multiple intracellular signal transduction events that, when deregulated, can lead to cellular transformation (McCarty,
BioEssays,
1998, 20, 913-921).
HGK is expressed as two isoforms in human tissues, a long and short form. The long form is predominantly localized to the brain while a shorter form lacking a proline-rich region of 77 amino acids is found in the liver, skeletal muscle and placenta (Yao et al.,
J. Biol. Chem.,
1999, 274, 2118-2125). It is currently believed that the presence of two forms of the protein, both of which can activate the JNK pathway, suggests that HGK has cell-specific activities.
The expression of HGK in several transformed cell lines has also been investigated and it was shown that HGK is expressed in MCF-7, HeLa, 293T, PC3 and LNCaP which are breast, cervical, kidney and prostate cancer cell lines, respectively (Yao et al.,
J. Biol. Chem.,
1999, 274, 2118-2125).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of HGK. Consequently, there remains a long felt need for agents capable of effectively inhibiting HGK 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 HGK expression.
SUMMARY OF THE INVENTION
The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding HPK/GCK-like kinase, and which modulate the expression of HPK/GCK-like kinase. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of HPK/GCK-like kinase 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 HPK/GCK-like kinase 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 HPK/GCK-like kinase, ultimately modulating the amount of HPK/GCK-like kinase produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding HPK/GCK-like kinase. As used herein, the terms “target nucleic acid” and “nucleic acid encoding HPK/GCK-like kinase” encompass DNA encoding HPK/GCK-like kinase, 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 HPK/GCK-like kinase. 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 HPK/GCK-like kinase. 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

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