Antisense modulation of integrin-linked kinase expression

Details Antisense modulation of integrin-linked kinase expression Antisense modulation of integrin-linked kinase expression

1999-10-26

2001-01-23

Guzo, David (Department: 1635)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

C435S006120, C435S377000, C435S455000, C536S023100, C536S024100, C536S024500, C514S04400A

Reexamination Certificate

active

06177273

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of Integrin-linked kinase. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding human Integrin-linked kinase. Such oligonucleotides have been shown to modulate the expression of Integrin-linked kinase.
BACKGROUND OF THE INVENTION
The primary contacts made between cells and the surrounding environment, including contacts with other cells, are mediated by the transmembrane proteins known as integrins. These contacts are critical for the bidirectional transduction of signals to the interior of the cell and consequently to the modulation of biochemical pathways. Integrins are heterodimeric cation-dependent membrane-spanning glycoproteins that mediate cell adhesion, migration and signal transduction. Integrins are composed of an alpha and beta subunit and to date, 8 beta and 15 alpha subunits have been identified which combine to form over 20 different &agr;&bgr; heterodimers. Integrins have been found in all tissues examined and consist of a large extracellular domain, a transmembrane domain and a smaller cytoplasmic domain. It is the extracellular domain of the integrin that acts as a receptor for various matrix proteins, while the cytoplasmic domain has been shown to interact with actin filaments of the cytoskeleton and with cytoplasmic proteins such as talin, paxillin, filamin and focal adhesion kinase (FAK) (LaFlamme et al.,
Matrix Biol.,
1997, 16, 153-163). Recently, four additional proteins that interact with &bgr;-integrin subunit cytoplasmic domains were reported. Of the four, only the ankyrin repeat containing serine/threonine protein integrin-linked kinase (ILK) has been shown to bind multiple forms of the integrin beta subunit (Dedhar and Hannigan,
Curr. Opin. Cell. Biol.,
1996, 8, 657-669; Hannigan et al.,
Nature,
1996, 379, 91-96).
Integrin-linked kinase (also known as ILK and p59ILK) was originally identified from a two-hybrid screen of a human placental cDNA library by its ability to bind to and phosphorylate the &bgr;1-integrin cytoplasmic domain (Hannigan et al.,
Nature,
1996, 379, 91-96). Characterization of integrin-linked kinase in these studies also revealed that overexpression leads to disrupted epithelial morphology of IEC-18 cells, decreased cell adhesion to extracellular matrix substrates as well as anchorage-independent growth (Hannigan et al.,
Nature,
1996, 379, 91-96). Others have shown that overexpression of integrin-linked kinase leads to stimulation of the cell cycle, fibronectin matrix assembly, reduced expression of E-cadherin and malignant transformation (Radeva et al.,
J. Biol. Chem.,
1997, 272, 13937-13944; Wu et al.,
J. Biol. Chem.,
1998, 273, 528-536). Interestingly, the integrin-linked kinase gene, which maps to chromosome 11p15.5, is located in a region associated with genomic imprinting, whereby the expression level of the alleles of a gene depends upon their parental origin and loss of heterozygosity in certain tumor types (Hannigan et al.,
Genomics,
1997, 42, 177-179).
The expression pattern of integrin-linked kinase is distributed among most human tissues and has been shown to be overexpressed in certain tumors, those being Ewing's sarcoma, primitive neuroectodermal tumor (PNET), medulloblastoma and neuroblastoma (Chung et al.,
Virchows Arch.,
1998, 433, 113-117). Recently it was demonstrated that integrin-linked kinase expression is regulated by erbB-2, a member of the epidermal growth factor receptor family, which plays a pivotal role in epidermal growth and differentiation. The investigators showed that overexpression of erbB-2 led to a specific increase in integrin-linked kinase expression in several regions of epidermal tissue (Xie et al.,
Am. J. Pathol.,
1998, 153, 367-372). These studies implicate integrin-linked kinase in skin development and the pathogenesis of skin diseases.
Integrin-linked kinase also triggers the LEF-1/beta catenin signaling pathway when overexpressed, indicating a role in the activation of transcription within the Wnt signaling cascade (Novak et al.,
Proc. Natl. Acad. Sci. U.S.A.,
1998, 95, 4374-4379). The activity of integrin-linked kinase has been shown to be modulated within other signaling pathways including those involving G-proteins (Tu et al.,
Mol. Cell. Biol.,
1999, 19, 2425-2434) phosphotidylinositol 3-kinase, protein kinase B and glycogen synthase kinase 3 (Delcommenne et al.,
Proc. Natl. Acad. Sci. U.S.A.,
1998, 95, 11211-11216). These results indicate that integrin-linked kinase may play a role in insulin-dependent responses in the cell and possible in the development of diabetes.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of integrin-linked kinase. Consequently, there remains a long felt need for agents capable of effectively inhibiting integrin-linked kinase 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 integrin-linked kinase expression. The present invention provides compositions and methods for modulating integrin-linked kinase expression using antisense technology.
SUMMARY OF THE INVENTION
The present invention is directed to antisense compounds, particularly oligonucleotides, which are targeted to a nucleic acid encoding Integrin-linked kinase, and which modulate the expression of Integrin-linked kinase. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. Further provided are methods of modulating the expression of Integrin-linked 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 Integrin-linked 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 antisense compounds, particularly oligonucleotides, for use in modulating the function of nucleic acid molecules encoding Integrin-linked kinase, ultimately modulating the amount of Integrin-linked kinase produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding Integrin-linked kinase. As used herein, the terms “target nucleic acid” and “nucleic acid encoding Integrin-linked kinase” encompass DNA encoding Integrin-linked 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 Integrin-linked 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 tar

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