Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-12-20
2002-07-23
McGarry, Sean (Department: 1635)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S006120, C435S091100, C536S023100, C536S024500, C536S025300
Reexamination Certificate
active
06423543
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of hepsin. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding human hepsin. Such oligonucleotides have been shown to modulate the expression of hepsin.
BACKGROUND OF THE INVENTION
Proteases are involved in many biological and physiological processes including tissue rearrangement, cell growth and migration, complement activation, blood coagulation, fibrinolysis, processing of enzyme, hormone, and growth factor precursors, and the activation of other proteases. Aberrant expression or activation of these same proteases has been shown to contribute to the development of neoplastic processes such as inflammation, tumor growth and metastasis.
Hepsin (also known as TMPRSS1 and HPN) is a transmembrane serine protease that has been shown to be highly expressed on the surface of human hepatocytes, with the regulation of expression and localization being cell-cycle dependent (Leytus et al.,
Biochemistry,
1988, 27, 1067-1074; Tsuji et al.,
J. Biol. Chem.,
1991, 266, 16948-16953). Human hepsin is localized to chromosome 19q11 and has the molecular orientation of a type II membrane-associated protein, with its catalytic carboxyl terminus localized to the external membrane surface and the amino terminus facing the cytosol (Tsuji et al.,
J. Biol. Chem.,
1991, 266, 16948-16953).
It is believed that, as a protease, hepsin may be involved in the proteolytic digestion of neighboring extracellular matrix components to create spaces for subsequent cell migration and metastasis. Support for this hypothesis is provided by studies of selected cancer cells, including HepG2, Alexander cells, breast cancer, ovarian cancer and nerve cells, which have been shown to express hepsin at very high levels (Kurachi et al.,
Methods Enzymol.,
1994, 244, 100-114; Tanimoto et al.,
Cancer Res.,
1997, 57, 2884-2887). Investigations of 44 ovarian tumors found that levels of hepsin were significantly increased in 7 of 12 low malignant potential tumors and in 27 of 32 carcinomas. By comparisons on Northern blots, the hepsin transcript was almost never expressed in normal tissues (Tanimoto et al.,
Cancer Res.,
1997, 57, 2884-2887).
In addition it has been demonstrated that hepsin is required for mammalian cell growth and morphology (Torres-Rosado et al.,
Proc. Natl. Acad. Sci. U.S.A.,
1993, 90, 7181-7185). In these studies, cells were treated with antisense oligonucleotides designed to the first ATG site, nucleotides 952-971 of the coding region and the 3′ untranslated region (UTR) of human hepsin. Both phosphodiester and their phosphorothioate counterpart oligonucleotides were investigated and those designed to the ATG start site showed large inhibitory effects, resulting in the arrest of cell growth and changes in cell morphology. Those oligonucleotides designed to the 3′ UTR showed no significant inhibitory effects with the oligonucleotides targeted to the downstream coding regions having intermediate effects on cell growth (Torres-Rosado et al.,
Proc. Natl. Acad. Sci. U.S.A.,
1993, 90, 7181-7185).
Hepsin has also been shown to activate human factor VII, a blood coagulation factor, thereby initiating a coagulation pathway on the cell surface that leads to thrombin formation (Kazama et al.,
J. Biol. Chem.,
1995, 270, 66-72).
More recently, a connection between the role of hepsin in coagulation and the neoplastic phenotype was suggested. It was demonstrated that hepsin is highly expressed in renal cell carcinoma, and from these results it was proposed that hepsin might be the initiator of tumor cell-induced thrombin generation in certain tumors that lack tissue factor expression (Zacharski et al.,
Thromb. Haemost.,
1998, 79, 876-877).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of hepsin and, to date, strategies aimed at modulating hepsin function have involved the use of antibodies, antisense oligonucleotides and gene knock-outs in mice.
While antisense inhibition of hepsin resulted in cellular growth arrest, it has been demonstrated that the homozygous deletion of the hepsin gene in mice was not lethal (Wu et al.,
J. Clin. Invest.,
1998, 101, 321-326). Mice lacking the hepsin gene were found to be viable and fertile and grew normally. Liver weight and serum concentrations of liver-derived proteins were also similar between the transgenic animals and their wild-type littermates. The only observable difference was the concentration of bone-derived alkaline phosphatase, with a two-fold higher concentration in the mice lacking the hepsin gene (Wu et al.,
J. Clin. Invest.,
1998, 101, 321-326).
In light of the elevated levels of hepsin expression associated with various cancers, there remains a long felt need for agents capable of effectively inhibiting hepsin 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 hepsin expression.
SUMMARY OF THE INVENTION
The present invention is directed to antisense compounds, particularly oligonucleotides, which are targeted to a nucleic acid encoding hepsin, and which modulate the expression of hepsin. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. Further provided are methods of modulating the expression of hepsin 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 hepsin 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 hepsin, ultimately modulating the amount of hepsin produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding hepsin. As used herein, the terms “target nucleic acid” and “nucleic acid encoding hepsin” encompass DNA encoding hepsin, 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 nRNA 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 hepsin. 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
Cowsert Lex M.
Marcotte Patrick Allen
ISIS Pharmaceuticals Inc.
Licata & Tyrrell P.C.
McGarry Sean
Zara Jane
LandOfFree
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