Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-03-08
2001-08-28
Wortman, Donna C (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S023000, C435S320100
Reexamination Certificate
active
06280940
ABSTRACT:
TECHNICAL AND INDUSTRIAL APPLICABILITY OF INVENTION
A cell-based assay system in which the detection of reporter gene activity (secreted alkaline phosphatase or SEAP) is dependent upon active Hepatitis C virus (HCV) NS3 protease. The assay system is useful in the in vitro screening, in a mammalian cell-based assay, of potential protease inhibiting molecules useful in the treatment of HCV. The advantages of using SEAP over more routinely used reporter genes such as beta-galactosidase or luciferase, is that a cell lysis step is not required since the SEAP protein is secreted out of the cell. The absence of a cell lysis step decreases intra- and inter-assay variability as well as makes the assay easier to perform then earlier assays.
BACKGROUND OF THE INVENTION
HCV is one of the major causes of parenterally transmitted non-A, non-B hepatitis worldwide. HCV is now known as the etiologic agent for Non-A Non-B hepatitis throughout the world. Mishiro et al., U.S. Pat. No. 5,077,193; Mishiro et al., U.S. Pat. No. 5,176,994; Takahashi et al, U.S. Pat. No. 5,032,511; Houghton et al., U.S. Pat. Nos. 5,714,596 and 5,712,088; as well as (M. Houghton,
Hepatitis C Viruses
, p.1035-1058 in B. N. Fields et al.(eds.),
Field's Virology
(3d. ed. 1996). HCV infection is characterized by the high rate (>70%) with which acute infection progresses to chronic infection (Alter, M. J. 1995. Epidemiology of hepatitis C in the west. Sem. Liver Dis. 15:5-14.). Chronic HCV infection may lead to progressive liver injury, cirrhosis, and in some cases, hepatocellular carcinoma. Currently, there are no specific antiviral agents available for the treatment of HCV infection. Although alpha interferon therapy is often used in the treatment of HCV-induced moderate or severe liver disease, only a minority of patients exhibit a sustained response Saracco, G. et al.,
J. Gastroenterol. Hepatol
. 10:668-673 1995. Additionally, a vaccine to prevent HCV infection is not yet available and it remains uncertain whether vaccine development will be complicated by the existence of multiple HCV genotypes as well as viral variation within infected individuals Martell, M. et al.,
J. Virol
. 66:3225-3229 1992; Weiner, et al.,
Proc. Natl. Acad. Sci
. 89:3468-3472 1992. The presence of viral heterogeneity may increase the likelihood that drug resistant virus will emerge in infected individuals unless antiviral therapy effectively suppresses virus replication. Most recently, several of the HCV encoded enzymes, specifically the NS3 protease and NS5B RNA polymerase, have been the focus of intensive research, in vitro screening, and/or rational drug design efforts.
HCV has been classified in the flavivirus family in a genus separate from that of the flaviviruses and the pestiviruses. Rice, C. M., in B. N. Fields and P. M. Knipe (eds.), Virology, 3rd edn., p. 931-959;1996 Lippincott-Raven, Philadelphia, Pa. Although the study of HCV replication is limited by the lack of an efficient cell-based replication system, an understanding of replicative events has been inferred from analogies made to the flaviviruses, pestiviruses, and other positive strand RNA viruses. The HCV virus has a 9.4 kb single positive-strand RNA genome encoding over 3,000 amino acids. The genome expresses over 10 structural and non-structural proteins. Post-translational processing of the viral genome requires cleavage by two proteases. As in the pestiviruses, translation of the large open reading frame occurs by a cap-independent mechanism and results in the production of a polyprotein of 3010-3030 amino acids. Proteolytic processing of the structural proteins (the nucleocapsid protein or core (C)) and two envelope glycoproteins, E1 and E2 is accomplished by the action of host cell signal peptidases. Santolini, E., et al.,
J. Virol
. 68:3631-3641, 1994; Ralston, R., et al.,
J. Virol
. 67:6753-6761 1993. Cleavage of the nonstructural proteins (NS4A, NS4B, NS5A, and NS5B) is mediated by the action of the NS2/3 protease or the NS3 protease. Grakoui, A. et al.,
J. Virol
. 67:2832-2843 1993; Hirowatari, Y., et al.,
Anal. Biochem
. 225:113-120 1995; Bartenschlager, R. et al.,
J. Virol
. 68:5045-5055 1994; Eckart, M. R., et al.,
Biochem. Biophys. Res. Comm
. 192:399-406 1993; Grakoui, A., et al.,
J. Virol
. 67:2832-2843 1993; Tomei, L., et al.,
J. Virol
. 67:4017-40261993; NS4A is a cofactor for NS3 and NS5B is an RNA dependent RNA polymerase. Bartenschlager, R. et al., (1994); Failla, C., et al.,
J. Virol
. 68:3753-3760 1994; Lin, C. et al.,
Proc. Natl. Acad. Sci
. 92:7622-7626 1995; Behrens, S.-E., et al.,
EMBO J
. 15:12-22 1996. Functions for the NS4B and NS5A proteins have yet to be defined.
The NS2/3 is a metalloprotease and has been shown to mediate cleavage at the 2/3 junction site Grakoui, et al. (1993); Hijikata, M., et al.,
J. Virol
. 67:4665-4675 1993. In contrast, the NS3 protease is required for multiple cleavages within the nonstructural segment of the polyprotein, specifically the 3/4A, 4A/4B, 4B/5A, and 5A/5B junction sites Bartenschlager et al. (1993); Eckart, M. R., et al.,
Biochem. Biophys. Res. Comm
. 192:399-406 1993; Grakoui et al. (1993); Tomei et al. (1994). More recently, it is thought that the NS2/3 protease might actually be part of the HCV NS3 protease complex even though they have two functionally distinct activities. Although NS3 protease is presumed to be essential for HCV viability, definitive proof of its necessity has been hampered by the lack of an infectious molecular clone that can be used in cell-based experiments. However, recently two independent HCV infectious molecular clones have been developed and have been shown to replicate in chimpanzees. Kolykhalov, A. A., et al.,
Science
277:570-574 1997; Yanagi, M., et al.,
Proc. Natl. Acad. Sci
. 94:8738-8743 1997. The requirement for NS3 in the HCV life cycle may be validated in these clones by using oligo nucleotide-mediated site directed mutagenesis to inactivate the NS3 catalytic serine residue and then determining whether infectious virus is produced in chimpanzees. Until these experiments are performed, the necessity of NS3 is inferred from cell-based experiments using the related yellow fever (YFV) and bovine viral diarrhea (BVDV) viruses. Mutagenesis of the YFV and BVDV NS3 protease homologs has shown that NS3 serine protease activity is essential for YFV and BVDV replication. Chambers, T. J., et al.,
Proc. Natl. Acad. Sci
. 87:8898-8902 1990; Xu, J., et al.,
J. Virol
. 71:5312-5322 1997.
In general, when investigators screen potential anti-viral compounds for inhibitory activity, it usually involves initial in vitro testing of putative enzyme inhibitors followed by testing the compounds on actual infected cell lines and animals. It is obvious that working with live virus in large scale screening activities can be inherently dangerous and problematic. While final testing of putative inhibitors in infected cells and animals is still necessary for preclinical drug development, for initial screening of candidate molecules, such work is cost-prohibitive and unnecessary. Furthermore, the inability to grow HCV in tissue culture in a reproducible quantitative manner prevents the evaluation of potential antiviral agents for HCV in a standard antiviral cytopathic effect assay. In response to this real need in the industry, development of non-infectious, cell-based, screening systems is essential.
For example, Hirowatari, et al. developed a reporter assay system, inter alia, that involves the transfection of mammalian cells with two eukaryotic expression plasmids. Hirowatari, et al.,
Anal. Biochem
. 225:113-120 1995. One plasmid has been constructed to express a polyprotein that encompasses the HCV NS2-NS3 domains fused in frame to an NS3 cleavage site followed by the HTLV-1 TAX1 protein. A second plasmid has been constructed to have the expression of the chloramphenicol acetyltransferase (CAT) reporter gene under the control of the HTLV-1 LTR. Thus when COS cells are transfected with both plasmids, NS3-mediated cleavage of the TAX1 protein from the NS2-NS3-TAX
Jackson Roberta Lynn
Patick Amy Karen
Potts Karen Elizabeth
Agouron Pharmaceuticals , Inc.
Wortman Donna C
LandOfFree
Reporter gene system for use in cell-based assessment of... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Reporter gene system for use in cell-based assessment of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Reporter gene system for use in cell-based assessment of... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2493891