Antisense inhibition of src-c expression

Details Antisense inhibition of src-c expression Antisense inhibition of src-c expression

2001-05-18

2003-12-02

Lacourciere, Karen (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, C435S091200, C536S023100, C536S023200, C536S024300, C536S024310, C536S024330, C536S024500

Reexamination Certificate

active

06656732

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of src-c. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding src-c. Such compounds have been shown to modulate the expression of src-c.
BACKGROUND OF THE INVENTION
Cells in higher animals normally divide only when they are stimulated by growth factors produced by other cells and act by binding to receptor tyrosine kinases on dividing cells. Cancer cells proliferate excessively because, as a result of accumulated mutations, they are able to divide without stimulation from other cells and therefore are no longer subject to normal controls on cell proliferation. The mutated genes which led to excessive proliferation were originally called oncogenes before their origin as normal genes was understood. The normal genes, from which they arise, are thus often referred to as proto-oncogenes.
The v-src gene encoded by the Rous sarcoma virus was the first discovered as a transmissible agent found to induce tumors in chickens (Irby and Yeatman,
Oncogene
, 2000, 19, 5636-5642). The protein product of this gene, v-src, is a tyrosine kinase with a cellular homolog known as src-c (also known as c-src, SRC and pp60c-src) (Irby and Yeatman,
Oncogene
, 2000, 19, 5636-5642). The structure of the two proteins is similar but the regulatory carboxyl-terminus of v-src is truncated (Irby and Yeatman,
Oncogene
, 2000, 19, 5636-5642). Found in normal cells and presumed to be a proto-oncogene, src-c is a tyrosine kinase which regulates cell growth via phosphorylation of transcription factors, members of signal transduction cascades and growth factor receptors (Irby and Yeatman,
Oncogene
, 2000, 19, 5636-5642).
While elevation of src-c protein levels is common to a large number of cancers, this elevation is often modest when compared to the increases in src-c kinase activity that have been observed (Irby and Yeatman,
Oncogene
, 2000, 19, 5636-5642). These data indicate the importance of src-c activation in human tumor development and progression.
Several mechanisms of activation of src-c in cancer have been proposed including: (i) activation of src-c by receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) and human growth factor (HGF) (Mao et al.,
Oncogene
, 1997, 15, 3083-3090), (ii) post translational activation (for example, via insufficient phosphorylation of src-c by protein tyrosine kinase Csk) (Lutz et al.,
Biochem. Biophys. Res. Commun
., 1998, 243, 503-508), (iii) activation via mutations (Irby et al.,
Nat. Genet
., 1999, 21, 187-190) and (iv) loss of function of src-c regulatory proteins (Irby and Yeatman,
Oncogene
, 2000, 19, 5636-5642).
Activated src-c has been implicated in the progression and metastasis of many human cancers. Increased src-c activity has been shown to increase the growth rate of cells (Mao et al.,
Oncogene
, 1997, 15, 3083-3090), reduce the adhesive contacts between cells (Hamaguchi et al.,
Embo J
., 1993, 12, 307-314) and increase the metastatic potential of cells (Irby et al.,
Nat. Genet
., 1999, 21, 187-190).
In human mammary carcinomas, src-c kinase activity has been detected at levels 4-20 times higher than that found in normal tissue (Irby and Yeatman,
Oncogene
, 2000, 19, 5636-5642). Likewise, elevation of src-c kinase activity is increased 5-8 fold in the majority of colon tumors (Irby and Yeatman,
Oncogene
, 2000, 19, 5636-5642). Activated src-c was found to be instrumental in promoting the growth of pancreatic tumor cells by increasing the number of insulin-like growth factor receptor molecules and thus enhancing insulin growth factor-dependent growth (Flossmann-Kast et al.,
Cancer Res
., 1998, 58, 3551-3554). Additional cancers with elevated src-c protein levels have been reported in neuroblastomas, retinoblastomas, Kaposi's sarcoma, as well as lung, ovarian, and esophageal cancers (Irby and Yeatman,
Oncogene
, 2000, 19, 5636-5642).
Due to the prevalence of activated src-c in cancer, src-c has been the subject of investigations aimed at inhibition of src-c expression and/or activity.
Investigations of src-c null mice indicate that src-c is not required for general cell viability but does have an essential role in osteoclast function and bone remodeling. It is thus possible that other tyrosine kinases related to src-c may provide overlap in function and reduce the severity of the phenotype (Soriano et al.,
Cell
, 1991, 64, 693-702).
Examples of inhibition of human src-c expression by vectors containing antisense src-c fragments of src-c have been described in a mouse models (Karni et al.,
Oncogene
, 1999, 18, 4654-4662; Wiener et al.,
Clin. Cancer Res
., 1999, 5, 2164-2170), colon cancer cell lines (Ellis et al.,
J. Biol. Chem
., 1998, 273, 1052-1057; Fleming et al.,
Surgery
, 1997, 122, 501-507; Rajala et al.,
Biochem. Biophys. Res. Commun
., 2000, 273, 1116-1120; Staley et al.,
Cell Growth Differ
., 1997, 8, 269-274) and leukemia cell lines (Kitanaka et al.,
Biochem. Biophys. Res. Commun
., 1994, 201, 1534-1540; Waki et al.,
Biochem. Biophys. Res. Commun
., 1994, 201, 1001-1007; Yamaguchi et al.,
Leukemia
, 1997, 11, 497-503).
Inhibition of expression of src-c by antisense phosphorothioate oligonucleotides targeting the start codon of human and mouse src-c has been observed in osteoclasts, osteoblasts and vascular endothelial cells (Chellaiah et al.,
J. Biol. Chem
., 1998, 273, 11908-11916.; Marzia et al.,
J. Cell Biol
., 2000, 151, 311-320; Naruse et al.,
FEBS Lett
., 1998, 441, 111-115; Tanaka et al.,
Nature
, 1996, 383, 528-531).
A 60-mer oligonucleotide targeting the 18-nucleotide brain-specific insert of rat src-c was used to map the expression levels of brain-specific src-c in various brain structures (Ross et al.,
Proc. Natl. Acad. Sci. U. S. A
., 1988, 85, 9831-9835).
An expression construct comprising a tumor suppressor gene and an antisense src-c gene directed to the use of genetic therapy is claimed in PCT publication WO 99/47690 (Almond et al., 1999):
An antisense molecule inhibiting the expression of src-c in combination with a lipid formulation containing other compounds used for treatment of hyperproliferative disease in humans is claimed in PCT WO/71096 (Ramesh et al., 2000).
A therapeutic composition including an antisense oligonucleotide specific for src-c and at least one second antisense oligonucleotide specific for a nuclear oncogene is claimed in U.S. Pat. No. 5,734,039 (Calabretta and Skorski, 1998).
Antisense oligonucleotides corresponding to src-c and in combination with at least one other antisense oligonucleotide corresponding to a different gene are claimed in PCT publication WO 99/13886 (Nyce, 1999).
A therapeutic agent composed of a nucleic acid construct containing antisense RNA for disrupting expression of src-c is claimed in PCT publication WO 01/00791 (Lee, 2001).
Methods for producing recombinant viral vectors containing antisense constructs of src-c are claimed in PCT publication WO 99/27123 and WO 00/32754 (Fang et al., 1999; Zhang et al., 2000).
Disclosed and claimed in U.S. Pat. Nos. 5,264,423 and 5,276,019 are oligodeoxynucleotide compounds capable of inhibiting the replication or apoptotic effect of a foreign nucleic acid in a host. In U.S. Pat. No. 5,264,423, a foreign nucleic acid includes an oncogene nucleic acid (Cohen et al., 1993; Cohen et al., 1994).
Antisense technology is emerging as an effective means of reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic and research applications involving modulation of src-c expression.
The present invention provides compositions and methods for modulating src-c expression including modulation of the currently disclosed 5′ UTR variant of src-c.
SUMMARY OF THE INVENTION
The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding src-c, and which modulate the expression

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