Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2000-03-31
2003-10-07
Wortman, Donna C (Department: 1648)
Chemistry: molecular biology and microbiology
Vector, per se
C435S005000, C435S069100, C435S067000, C435S239000, C435S370000, C536S023400, C536S023720
Reexamination Certificate
active
06630343
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a hepatitis C virus (HCV) cell culture system, which comprises mainly eukaryotic cells containing transfected HCV specific genetic material, which means they are transfected with HCV specific genetic material.
2. Background Information
The hepatitis C virus (HCV) is one of the main causes worldwide of chronic and sporadic liver diseases. The history of most HCV infections does not involve any obvious clinical signs, but 80-90% of the infected people become chronic carriers of the virus and 50% of these chronic carriers of the virus develop chronic hepatitis with different degrees of severity. Approx. 20% of the chronically infected develop a cirrhosis of the liver over 10 to 20 years, based on what a primary hepatocellular carcinoma can develop. Nowadays chronic hepatitis C is the main indication for liver transplantation. One currently available therapy involves high-dose administration of Interferon alpha or a combination of Interferon alpha and the purine nucleoside analogue Ribavirin. However, only approx. 60% of all treated persons respond to this therapy and with these, a new viraemia occurs in more than half of all cases after the discontinuation of the treatment.
Due to the high prevalence, especially in industrialized countries, the serious effects of chronic infections and the lack of effective therapy, the development of a HCV specific chemotherapy is an important goal of pharmaceutical research and development. Such a goal, however, has been hampered by the lack of a suitable cell culture system, which enables the study of virus replication and pathogenesis in eukaryotic cells.
Due to the small amount of virus in blood or tissue, the lack of suitable cell culture systems or animal models (the chimpanzee is still the only possible experimental animal) as well as the lack of efficient systems for producing virus-like particles, it has been difficult to analyze the molecular composition of the HCV particle in-depth. The information currently available can be summarized as follows: HCV is an enveloped plus-strand RNA virus with a particle diameter of 50-60 nm and a medium density of 1.03-1.1 g/ml. It was molecularly cloned and characterized for the first time in 1989 (Choo et al., 1989, Science, 244, 359-362). The HCV-RNA has a length of approx. 9.6 kb (=9600 nucleotides), a positive polarity and comprises one open reading frame (ORF), which encodes a linear polyprotein of approx. 3010 amino acids (see Rice 1996, in Virology, B. N. Fields, D. M. Knipe, P. M. Howley, Eds. (Lippincott-Raven, Philadelphia, Pa., 1996), vol. 1, pp. 931-960; Clarke 1997,
J. Gen. Virol.
78, 2397; and Bartenschlager 1997,
Intervirology
40, 378 and see FIG.
1
A). During the replication of the virus the polyprotein is cleaved into the mature and functionally active proteins by cellular and viral proteases.
Within the polyprotein the proteins are arranged as follows (from the amino- to the carboxy terminus) : Core-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. The core protein is the main component of the nucleocapsid. The glycoproteins E1 and E2 are transmembrane proteins and the main components of the viral envelope. They probably play an important role during the attachment of the virus to the host cell. These three proteins core, E1, and E2 constitute the viral particle and are therefore called structural proteins. The function of the protein p7 is still not clear. The protein NS2 is probably the catalytic domain of the NS2-3 protease, which is responsible for the processing between the proteins NS2 and NS3. The protein NS3 has two functions, one is a protease activity in the amino terminal domain, which is essential for the polyprotein processing, and the other a NTPase/helicase function in the carboxy terminal domain, which is probably important during the replication of the viral RNA. The protein NS4A is a co-factor of the NS3 protease. The function of the protein NS4B is unknown.
The open reading frame is flanked on its 5′ end by a non-translated region (NTR) approx. 340 nucleotides in length, which functions as the internal ribosome entry site (IRES), and on its 3′ end by a NTR approx. 230 nucleotides in length, which is most likely important for the genome replication. A 3′to NTR such as this is the object of patent application PCT/US 96/14033. The structural proteins in the amino terminal quarter of the polyprotein are cleaved by host cell signal peptidase. The non-structural proteins (NS) 2 to (NS) 5B are processed by two viral enzymes, namely the NS2-3 and the NS3/4A protease. The NS3/4A protease is required for all cleavages beyond the carboxy terminus of NS3. The function of NS4B is unknown. NS5A, a highly phosphorylated protein, seems to be responsible for the Interferon resistance of various HCV genotypes (see Enomoto et al. 1995,
J. Clin. Invest.
96, 224; Enomoto et al. 1996,
N. Engl. J. Med.
334, 77; Gale Jr. et al. 1997,
Virology
230, 217; Kaneko et al. 1994,
Biochem. Biophys. Res. Commun.
205, 320; Reed et al., 1997,
J. Virol.
71, 7187), and NS5B has been identified as the RNA-dependent RNA polymerase.
First diagnostic systems have been developed from these findings, which are either based on the detection of HCV specific antibodies in patient serum or the detection of HCV specific RNA using the reverse transcription polymerase chain reaction (RT-PCR), and which are routinely used with all blood and blood products and/or according to the regulations.
Since the first description of the genome in 1989 several partial and complete sequences of the HCV have been cloned and characterized using the PCR method. A comparison of these sequences shows a high variability of the viral genome in particular in the area of the NS5B gene, which eventually resulted in the classification of 6 genotypes, which are again subdivided into the subtypes a, b, and c.
The genomic variance is not evenly distributed over the genome. The 5′to NTR and parts of the 3′to NTR are highly conserved, while certain encoded sequences vary a lot, in particular the envelope proteins E1 and E2.
The cloned and characterized partial and complete sequences of the HCV genome have also been analyzed with regard to appropriate targets for a prospective antiviral therapy. In the course of this, three viral enzymes have been discovered, which may provide a possible target. These include (1) the NS3/4A protease complex, (2) the NS3 Helicase and (3) the NS5B RNA-dependent RNA polymerase. The NS3/4A protease complex and the NS3 Helicase have already been crystallized and their three-dimensional structure determined (Kim et al., 1996, Cell, 87,343; Yem et al., 1998, Protein Science, 7, 837; Love et al., 1996, Cell, 87, 311; Kim et al., 1998, Structure, 6, 89; Yao et al., 1997, Nature Structural Biology, 4, 463, Cho et al., 1998, J. Biol. Chem., 273, 15045). it has not been successful until now with the NS5B RNA-dependent RNA polymerase.
Even though important targets for the development of a therapy for chronic HCV infection have been defined with these enzymes and even though a worldwide intensive search for suitable inhibitors is ongoing with the aid of rational drug design as well as high throughput screening, the development of a therapy has one major deficiency, namely the lack of cell culture systems or simple animal models, which allow direct, reliable identification of HCV-RNA or HCV antigens with simple methods which are common in the laboratory. The lack of these cell culture systems is also the main reason that to date the comprehension of HCV replication is still incomplete and mainly hypothetical.
Although it has been reported that a close evolutionary relationship exists between HCV and the flavi- and pestiviruses, and self-replicating RNAs have been described for these, which can be used for the replication in different cell lines with a relatively high yield, (see Khromykh et al., 1997,
J. Virol.
71, 1497; Behrens et al., 1998,
J. Virol.
72, 2364; Moser et al., 1998,
J. Virol.
72, 5318)
Pendorf & Cutliff
Wortman Donna C
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