Link:  Pharm/Biotech Resources

Title:  Self-replicating RNA molecule from hepatitis C virus

United States Patent:  6,956,117

Issued:  October 18, 2005

Inventors:  Kukolj; George (Laval, CA); Pause; Arnim (Laval, CA)

Assignee:  Boehringer Ingelheim (Canada) Ltd. (Laval, CA)

Appl. No.:  309561

Filed:  December 4, 2002

A unique HCV RNA molecule is provided having an enhanced efficiency of establishing cell culture replication. Novel adaptive mutations have been identified within the HCV non-structural region that improves the efficiency of establishing persistently replicating HCV RNA in cell culture. This self-replicating polynucleotide molecule contains, contrary to all previous reports, a 5′-NTR that can be either an A as an alternative to the G already disclosed and therefore provides an alternative to existing systems comprising a self-replicating HCV RNA molecule. The G->A mutation gives rise to HCV RNA molecules that, in conjunction with mutations in the HCV non-structural region, such as the G(2042)C/R mutations, possess greater efficiency of transduction and/or replication. These RNA molecules when transfected in a cell line are useful for evaluating potential inhibitors of HCV replication.

FIELD OF THE INVENTION

The present invention relates generally to a HCV RNA molecule that self-replicates in appropriate cell lines, particularly to a self-replicating HCV RNA construct having an enhanced efficiency of establishing cell culture replication.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV) is the major etiological agent of post-transfusion and community-acquired non-A non-B hepatitis worldwide. It is estimated that over 200 million people worldwide are infected by the virus. A high percentage of carriers become chronically infected and many progress to chronic liver disease, so called chronic hepatitis C. This group is in turn at high risk for serious liver disease such as liver cirrhosis, hepatocellular carcinoma and terminal liver disease leading to death. The mechanism by which HCV establishes viral persistence and causes a high rate of chronic liver disease has not been thoroughly elucidated. It is not known how HCV interacts with and evades the host immune system. In addition, the roles of cellular and humoral immune responses in protection against HCV infection and disease have yet to be established.

Various clinical studies have been conducted with the goal of identifying pharmaceutical compounds capable of effectively treating HCV infection in patients afflicted with chronic hepatitis C. These studies have involved the use of interferon-alpha, alone and in combination with other antiviral agents such as ribavirin. Such studies have shown that a substantial number of the participants do not respond to these therapies, and of those that do respond favorably, a large proportion were found to relapse after termination of treatment. To date there are no broadly effective antiviral compounds for treatment of HCV infection.

HCV is an enveloped positive strand RNA virus in the Flaviviridae family. The single strand HCV RNA genome is of positive polarity and comprises one open reading frame (ORF) of approximately 9600 nucleotides in length, which encodes a linear polyprotein of approx. 3010 amino acids. In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce structural and non-structural (NS) proteins. The structural proteins (C, E1, E2 and E2-p7) comprise polypeptides that constitute the virus particle (Hijikata, M. et al., 1991, Proc. Natl. Acad. Sci. USA. 88, 5547-5551; Grakoui et al., 1993(a), J. Virol. 67, 1385-1395). The non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B) encode for enzymes or accessory factors that catalyze and regulate the replication of the HCV RNA genome. Processing of the structural proteins is catalyzed by host cell proteases (Hijikata, M. et al., 1991, Proc. Natl. Acad. Sci. USA. 88, 5547-5551). The generation of the mature non-structural proteins is catalyzed by two virally encoded proteases. The first is the NS2/3 zinc-dependent metalloprotease which auto-catalyses the release of the NS3 protein from the polyprotein. The released NS3 contains a N-terminal serine protease domain (Grakoui et al., 1993(b), Proc Natl Acad Sci USA, 90, 10583-7; Hijikata, M. et al., 1993, J. Virol. 67, 4665-4675) and catalyzes the remaining cleavages from the polyprotein. The released NS4A protein has at least two roles. First, forming a stable complex with NS3 protein and assisting in the membrane localization of the NS3/NS4A complex (Kim et al., Arch Virol. 1999, 144, 329-343) and second, acting as a cofactor for NS3 protease activity. This membrane-associated complex, in turn catalyzes the cleavage of the remaining sites on the polyprotein, thus effecting the release of NS4B, NS5A and NS5B (Bartenschlager, R. et al., 1993, J. Virol., 67, 3835-3844; Grakoui et al., 1993(a), J. Virol. 67, 1385-1395; Hijikata, M. et al., 1993, J. Virol. 67, 4665-4675; Love, R. A. et al., 1996, Cell, 87, 331-342; reviewed in Kwong et al., 1998 Antiviral Res., 40, 1-18). The C-terminal segment of the NS3 protein also harbors nucleoside triphosphatase and RNA helicase activity (Kim et al., 1995, Biochem. Biophys. Res. Comm., 215, 160-166.). The function of the protein NS4B is unknown. NS5A, a highly phosphorylated protein, seems to be responsible for the Interferon resistance of various HCV genotypes (Gale Jr. et al. 1997 Virology 230, 217; Reed et al., 1997 J. Virol. 71, 7187. NS5B is an RNA-dependent RNA polymerase (RdRp) that is involved in the replication of HCV.

The open reading frame of the HCV RNA genome is flanked on its 5′ end by a non-translated region (NTR) of approx. 340 nucleotides that functions as the internal ribosome entry site (IRES), and on its 3′ end by a NTR of approximately 230 nucleotides. Both the 5′ and 3′ NTRs are important for RNA genome replication. The genomic sequence variance is not evenly distributed over the genome and the 5′NTR and parts of the 3′NTR are the most highly conserved portions. The authentic, highly conserved 3′NTR is the object of U.S. Pat. No. 5,874,565 granted to Rice et al.

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. Four viral enzyme activities provide possible targets such as (1) the NS2/3 protease; (2) the NS3/4A protease complex, (3) the NS3 Helicase and (4) 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. Protein Science, 7, 837, 1998; Love, R. A. et al., 1996, Cell, 87, 331-342; 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). The NS5B RNA dependent RNA polymerase has also been crystallized to reveal a structure reminiscent of other nucleic acid polymerases (Bressanelli et al. 1999, Proc. Natl. Acad. Sci, USA 96, 13034-13039; Ago et al. 1999, Structure 7, 1417-1426; Lesburg et al. 1999, Nat. Struct. Biol. 6, 937-943).

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 and HTS, the development of therapy has one major deficiency, namely the lack of cell culture systems or simple animal models, which allow direct and reliable propagation of HCV viruses. The lack of an efficient cell culture system is still the main reason to date that an understanding of HCV replication remains elusive.

Although flavi- and pestivirus self-replicating RNAs have been described and used for the replication in different cell lines with a relatively high yield, similar experiments with HCV have not been successful to date (Khromykh et al., 1997, J. Virol. 71, 1497; Behrens et al., 1998, J. Virol. 72, 2364; Moser et al., 1998 J. Virol. 72, 5318). It is known from different publications that cell lines or primary cell cultures can be infected with high-titer patient serum containing HCV (Lanford et al. 1994 Virology 202, 606; Shimizu et al. 1993 PNAS, USA 90, 6037-6041; Mizutani et al. 1996 J. Virol. 70, 7219-7223; Ikda, et al. 1998, Virus Res. 56, 157; Fourner et al. 1998, J. Gen. Virol. 79, 2376; Ito et al. 1996, J. Gen. Virol. 77, 1043-1054). However, these virus-infected cell lines or cell cultures do not allow the direct detection of HCV-RNA or HCV antigens.

It is also known from the publications of Yoo et al. 1995 J. Virol., 69, 32-38; and of Dash et al., 1997, Am. J. Pathol., 151, 363-373; that hepatoma cell lines can be transfected with synthetic HCV-RNA obtained through in vitro transcription of the cloned HCV genome. In both publications the authors started from the basic idea that the viral HCV genome is a plus-strand RNA functioning directly as mRNA after being transfected into the cell, permitting the synthesis of viral proteins in the course of the translation process, and so new HCV particles could form HCV viruses and their RNA detected through RT-PCR. However the published results of the RT-PCR experiments indicate that the HCV replication in the described HCV transfected hepatoma cells is not particularly efficient and not sufficient to measure the quality of replication, let alone measure the modulations in replication after exposure to potential antiviral drugs. Furthermore it is now known that the highly conserved 3′ NTR is essential for the virus replication (Yanagi et al., 1999 Proc. Natl. Acad. Sci. USA, 96, 2291-95). This knowledge strictly contradicts the statements of Yoo et al. J. Virol., 69, 32-38(supra) and Dash et al., 1997, Am. J. Pathol., 151, 363-373. (supra), who used for their experiments only HCV genomes with shorter 3′ NTRs and not the authentic 3′ end of the HCV genome.

In WO 98/39031, Rice et al. disclosed authentic HCV genome RNA sequences, in particular containing: a) the highly conserved 5′-terminal sequence "GCCAGCC"; b) the HCV polyprotein coding region; and c) 3′-NTR authentic sequences.

In WO 99/04008, Purcell et al. disclosed an HCV infectious clone that also contained only the highly conserved 5′-terminal sequence "GCCAGC".

Recently Lohman et al. 1999 (Science 285, 110-113) and Bartenschlager, R. et al., 1993, J. Virol., 67, 3835-3844(in CA 2,303,526, laid-open on Oct. 3, 2000) disclosed a HCV cell culture system where the viral RNA (1377/NS2-3′) self-replicates in the transfected cells with such efficiency that the quality of replication can be measured with accuracy and reproducibility. The Lohman and Bartenschlager, R. et al., 1993, J. Virol., 67, 3835-3844 disclosures were the first demonstration of HCV RNA replication in cell culture that was substantiated through direct measurement by Northern blots. This replicon system and sequences disclosed therein highlight once again the conserved 5′ sequence "GCCAGC". A similar observation highlighting the conservation of the 5′NTR was made by Blight et al. 2000 (Science 290, 1972-1974) and WO 01/89364 published on Nov. 29, 2001.

In addition to the conservation of the 5′ and 3′ untranslated regions in cell culture replicating RNAs, three other publications by Lohman et al. 2001, J. Virol. 1437-1449 Krieger et al. 2001 J. Virol. 4614-4624 and Guo et al., (2001) J. Virol. 8516-8523 have recently disclosed distinct adaptive mutants within the HCV non-structural protein coding region. Specific nucleotide changes that alter the amino acids of the HCV non-structural proteins are shown to enhance the efficiency of establishing stable replicating HCV subgenomic replicons in culture cells.

Applicant has now found that, contrary to all previous reports, the highly conserved 5′-NTR can be mutated by adaptation to give rise to a HCV RNA sequence that, in conjunction with mutations in the HCV non-structural region, provides for a greater efficiency of transduction and/or replication.

Applicant has also identified novel adaptive mutations within the HCV non-structural region that improves the efficiency of establishing persistently replicating HCV RNA in cell culture.

One advantage of the present invention is to provide an alternative to these existing systems comprising a HCV RNA molecule that self-replicates. Moreover, the present invention demonstrates that the initiating nucleotide of the plus-strand genome can be either an A as an alternative to the G already disclosed.

A further advantage of the present invention is to provide a unique HCV RNA molecule that transduces and/or replicates with higher efficiency. The Applicant demonstrates the utility of this specific RNA molecule in a cell line and its use in evaluating a specific inhibitor of HCV replication.

SUMMARY OF THE INVENTION

In a first embodiment, the present invention provides a 5′-non translated region of the hepatitis C virus wherein its highly conserved guanine at position 1 is substituted for adenine.

Particularly, the present invention provides a hepatitis C virus polynucleotide comprising adenine at position 1 as numbered according to the 1377/NS2-3′ construct (Lohmann et al. 1999, Science 285, 110-113, Accession #AJ242651). Particularly, the invention provides a HCV self-replicating polynucleotide comprising a 5′-terminus consisting of ACCAGC (SEQ ID NO. 8).

In a second embodiment, the present invention is directed to a HCV self-replicating polynucleotide encoding a polyprotein comprising one or more amino acid substitution selected from the group consisting of: R(1135)K; S(1148)G; S(1560)G; K(1691)R; L(1701)F; 1(1984)V; T(1993)A; G(2042)C; G(2042)R; S(2404)P; L(2155)P; P(2166)L and M(2992)T.

Particularly, the invention is directed to a HCV self-replicating polynucleotide encoding a polyprotein comprising the any one of the amino acid substitutions as described above, further comprising the amino acid substitution E(1202)G.

More particularly, the invention provides a HCV self-replicating polynucleotide encoding a polyprotein comprising a G2042C or a G2042R mutation.

Most particularly, the invention provides for HCV self-replicating polynucleotide comprising a nucleotide substitution G->A at position 1, and said polynucleotide encodes a polyprotein further comprising a G2042C or a G2042R mutation.

Particularly, the polynucleotide of the present invention can be in the form of RNA or DNA that can be transcribed to RNA.

In a third embodiment, the invention also provides for an expression vector comprising a DNA form of the above polynucleotide, operably linked with a promoter.

According to a fourth embodiment, there is provided a host cell transfected with the self-replicating polynucleotide or the vector as described above.

In a fifth embodiment, the present invention provides a RNA replication assay comprising the steps of: