JP2011505159A - HCVNS3 protease replicon shuttle vector - Google Patents

Abstract

The present invention provides a novel HCV NS3 protease replicon shuttle vector useful for cloning HCV polynucleotide sequences from samples of HCV-infected patients and testing the resulting replicons for drug sensitivity.

Description

  The present invention relates to a novel HCV NS3 protease replicon shuttle vector useful for screening, testing and evaluating HCV and other flavivirus protease inhibitors.

  Hepatitis C is a major health problem throughout the world and is also the primary cause of chronic liver disease. (Boyer, N. et al. J. Hepatol. 2000 32: 98-112). Patients infected with HCV are at risk of developing cirrhosis followed by hepatocellular carcinoma, and therefore HCV is a major indication for liver transplantation.

  According to the World Health Organization, there are over 200 million infected individuals worldwide and at least 3-4 million people are infected each year. Once infected, about 20% of people eliminate the virus, but the rest can carry HCV for life. 10-20% of chronically infected individuals eventually develop liver destructive cirrhosis or liver cancer. The viral disease is transmitted sexually and vertically to contaminated blood and blood products, contaminated needles parenterally, or from an infected or carrier mother to the child. Current treatment of HCV infection is limited to immunotherapy using recombinant interferon alpha alone or in combination with the nucleoside analog ribavirin, and its clinical benefit is particularly limited to genotype 1. There is an urgent need for improved therapeutic agents that effectively eradicate chronic HCV infection.

  HCV has been classified as a member of the Flaviviridae family, including the genera Flavivirus, Pestivirus, and Hepacivirus (including hepatitis C virus) (Rice, CM, Flaviviridae: The viruses and their) replication, in: Fields Virology, Editors: Fields, BN, Knipe, DM, and Howley, PM, Lippincott-Raven Publishers, Philadelphia, Pa., Chapter 30, 931-959, 1996). HCV is an enveloped virus that contains a single-stranded RNA genome of about 9.4 kb positive sense strand. The viral genome consists of a 5 'untranslated region (UTR), a long open reading frame encoding a polyprotein precursor of about 3011 amino acids, and a short 3' UTR. The 5'UTR is the most highly conserved part of the HCV genome and is important for the initiation and control of polyprotein translation.

  Genetic analysis of HCV identified six major genotypes whose DNA sequences differed by more than 30%. Each genotype contains a series of more closely related subtypes that show diversity in 20-25% of the nucleotide sequence (Simmonds, P. 2004 J. Gen. Virol. 85: 3173-88). Over 30 subtypes have been identified. In the United States, about 70% of infected individuals have type 1a and 1b infections. Type 1b is the most prevalent subtype in Asia (X. Forns and J. Bukh, Clinics in Liver Disease 1999 3: 693-716; J. Bukh et al., Semin. Liv. Dis. 1995 15: 41-63). Unfortunately, type 1 infection appears to be less responsive to current therapies than either type 2 or type 3 genotype (NN Zein, Clin. Microbiol. Rev., 2000 13: 223-235). ).

  The genetic organization and polyprotein processing of the nonstructural protein portion of the pestivirus and hepacivirus ORF are very similar. These positive-strand RNA viruses have a single large open reading frame (ORF) that encodes all viral proteins required for viral replication. These proteins are expressed as polyproteins, which are processed simultaneously and post-translationally by proteinases encoded by both cells and viruses to produce mature viral proteins. Viral proteins involved in viral genomic RNA replication are located on the carboxy-terminal side. Two-thirds of the ORF are called nonstructural (NS) proteins. The mature nonstructural (NS) protein in both pestiviruses and hepaciviruses is from p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B in order from the amino terminus of the nonstructural protein coding region to the carboxy terminus of the ORF. It is configured.

  The pestivirus and hepacivirus NS proteins share sequence domains that are characteristic of specific protein functions. For example, the NS3 proteins of both groups of viruses have amino acid motifs characteristic of serine proteases and helicases (Gorbalenya et al. Nature 1988 333: 22; Bazan and Fletterick Virology 1989 171: 637-639; Gorbalenya et al. Nucleic Acid Res. 1989 17.3889-3897). Similarly, NS5B proteins of pestiviruses and hepaciviruses have a motif characteristic of RNA-directed RNA polymerase (Koonin, EV and Dolja, VV Crit. Rev. Biochem. Molec. Biol. 1993 28: 375-430 ).

  The actual roles and functions that the pestivirus and hepacivirus NS proteins play in the viral life cycle are very similar. In either case, the NS3 serine protease is involved in all proteolytic processing of the polyprotein precursor downstream of its position in the ORF (Wiskerchen and Collett Virology 1991 184: 341-350; Bartenschlager et al. J Virol. 1993 67: 3835-3844; Eckart et al. Biochem. Biophys. Res. Comm. 1993 192: 399-406; Grakoui et al. J. Virol. 1993 67: 2832-2843; Grakoui et al. Proc. Natl. Acad. Sci. USA 1993 90: 10583-10587; Ilijikata et al. J. Virol. 1993 67: 4665-4675; Tome et al. J. Virol. 1993 67: 4017-4026). In either case, the NS4A protein acts as a cofactor with NS3 serine protease (Bartenschlager et al. J. Virol. 1994 68: 5045-5055; Failla et al. J. Virol. 1994 68: 3753- 3760; Xu et al. J Virol. 1997 71:53 12-5322). NS3 protein of both viruses also functions as a helicase (Kim et al. Biochem. Biophys. Res. Comm. 1995 215: 160-166; Jin and Peterson Arch. Biochem. Biophys. 1995, 323: 47- 53; Warrener and Collett J. Virol. 1995 69: 1720-1726). Finally, pestivirus and hepacivirus NS5B proteins have the predicted RNA-dependent RNA polymerase activity (Behrens et al. EMBO 1996 15: 12-22; Lechmann et al. J. Virol. 1997 71: 8416-8428; Yuan et al. Biochem. Biophys. Res. Comm. 1997 232: 231-235; Hagedorn, PCT WO 97/12033; Zhong et al. J. Virol. 1998 72: 9365-9369).

  HCV NS protein 3 (NS3) contains serine protease activity that helps process most viral enzymes and is therefore considered essential for viral replication and infectivity. It is known that the virus infectivity is reduced by mutation of N3 protease of yellow fever virus (Chambers et. Al., Proc. Natl. Acad. Sci. USA, 1990, 87: 8898-8902). The first 181 amino acids of NS3 (residues 1027 to 1207 of the viral polyprotein) have been shown to contain the serine protease domain of NS3 that processes all four downstream sites of the HCV polyprotein (Lin et al. , J. Virol. 1994 68: 8147-8157). The HCV NS3 serine protease and its associated cofactor, NSA4A, are thought to assist in the processing of all viral enzymes and are therefore essential for viral replication. This processing appears to be similar to that performed by human immunodeficiency virus aspartyl protease, which is also involved in the processing of viral enzymes. HIV protease inhibitors that inhibit the processing of viral proteins are potent antiviral agents in humans, suggesting that inhibiting this stage of the viral life cycle can provide therapeutically effective drugs. Is done. As a result, it is an attractive target for drug discovery.

  Currently, the number of approved therapies currently available for the treatment of HCV infection is small. New and existing approaches for the treatment of HCV and inhibition of HCV NS5B polymerase have been reviewed: RG Gish, Sem. Liver. Dis., 1999 19: 5; Di Besceglie, AM and Bacon, BR, Scientific American, October: 1999 80-85; G. Lake-Bakaar, Current and Future Therapy for Chronic Hepatitis C Virus Liver Disease, Curr. Drug Targ. Infect Dis. 2003 3 (3): 247-253; P. Hoffmann et al., Recent patents on experimental therapy for hepatitis C virus infection (1999-2002), Exp. Opin. Ther. Patents 2003 13 (11): 1707-1723; FF Poordad et al. Developments in Hepatitis C therapy during 2000-2002, Exp. Opin.Emerging Drugs 2003 8 (1): 9-25; MP Walker et al., Promising Candidates for the treatment of chronic hepatitis C, Exp.Opin. Investig. Drugs 2003 12 (8): 1269-1280; S.- L. Tan et al., Hepatitis C Therapeutics: Current Status and Emerging Strategies, Nature Rev. Drug Discov. 2002 1: 867-881; R. De Francesco et al. Approaching a new era for hepatitis C virus t herapy: inhibitors of the NS3-4A serine protease and the NS5B RNA-dependent RNA polymerase, Antiviral Res. 2003 58: 1-16; QM Wang et al. Hepatitis C virus encoded proteins: targets for antiviral therapy, Drugs of the Future 2000 25 (9): 933-8-944; JA Wu and Z. Hong, Targeting NS5B-Dependent RNA Polymerase for Anti-HCV Chemotherapy Cur. Drug Targ.-Inf. Dis. 2003 3: 207-219.

  Despite advances in the elucidation of viral genomic organization and viral protein function, the basic aspects of HCV replication and disease development remain unclear. A major challenge in carrying out HCV replication experimentally is the lack of an effective cell culture system capable of producing infectious viral particles. Although primary cell cultures and infections of certain human cell lines have been reported, the amount of virus produced in such a system and the level of HCV replication are too low for detailed analysis.

Production of selectable subgenomic HCV RNA that replicates with minimal efficiency in the human hepatocytoma cell line Huh-7 has been reported. Lohman et al replicon (I ₃₇₇ / NS3-3 ′) derived from the cloned full length HCV consensus genome (genotype 1b) by deleting the Cp7 or C-NS2 region of the protein coding region. Has been reported (Lohman et al., Science 1999 285: 110-113). This replicon contained the following elements: (i) HCV 5′-UTR fused to 12 amino acids of the capsid coding region; (ii) neomycin phosphotransferase gene (NPTII); (iii) inserted downstream of the NPTII gene. IRES derived from encephalomyocarditis virus (EMCV) and (iv) 3′-UTR, which directs translation from HCV protein NS2 or NS3 to NS5B. After transfection of Huh-7 cells, only cells supporting HCV RNA replication expressed NPTII protein and were resistant to drug 418. Cell lines obtained from such G418 resistant colonies is contained significant levels of replicon RNA and viral proteins, supported the HCV replication in a 10 ⁶ Huh-7 cells transfected There was only one.

Similar selectable HCV replicons were generated based on HCV-H genotype 1a infectious clones (Blight et al., Science 2000 290: 1972-74). The HCV-H derived replicon failed to establish efficient HCV replication. This suggests that the Lohmann (1999) replicon generated earlier depended on the specific genotype 1b consensus cDNA clone used in such experiments. The above-mentioned Blight et al. Reproduced the replicon structure produced by Lohmann (1999) by performing a gene assembly procedure based on PCR, and obtained a G418-resistant Huh-7 cell colony. By sequencing independent G418 resistant cell clones, it was determined whether replicon adaptation to host cells was required for high levels of HCV replication. Multiple independent matched mutations that cluster in the HCV nonstructural protein NS5A have been identified. The conferred mutations enhance replication capacity in vitro and transduction efficiencies are 0.2% to 10% of transfected cells, compared to those made earlier in the art. For example, the transduction efficiency of the I ₃₇₇ / NS3-3 ′ replicon was 0.0001%.

  In the present invention, since unique restriction enzyme sites are introduced at the 5 ′ and 3 ′ ends of the protease domain of the NS3 gene, the NS3 protease sequence obtained from a sample of an HCV-infected patient can be cloned into a shuttle vector. Characterized by the development of a novel HCV replicon shuttle vector so that the resulting replicon can be evaluated for replication fitness and sensitivity to HCV NS3 protease inhibitors. Because individual HCV-infected patients typically contain genetically diverse virus populations due to the high error frequency of NS5B RNA polymerase, the use of the shuttle vector of the present invention results in the The characteristics of NS3 protease variants and the sensitivity or resistance of these variants to drug treatment could be revealed.

  Accordingly, the present invention relates to a unique restriction enzyme sequence located between 5 'to 5' and 10 'to 3' of a polynucleotide sequence encoding NS3 protein; protease of NS3 protein; A polynucleotide sequence encoding the domain; a unique restriction enzyme sequence located between 10 nucleotides 5 'and 10 nucleotides 3' from the 3 'end of the polynucleotide sequence encoding the protease domain of NS3 protein; NS3 A polynucleotide sequence encoding the helicase domain of the protein; a polynucleotide sequence encoding the NS4A protein; a polynucleotide sequence encoding the NS4B protein; a polynucleotide sequence encoding the NS5A protein; Comprising a polynucleotide sequence encoding in this order to provide an HCV replicon shuttle vector comprising an HCV polynucleotide sequence. In one embodiment of the invention, the polynucleotide sequence encoding the protease domain of NS3 protein has been modified or deleted so that the protease domain of NS3 protein does not function.

  In another aspect of the invention, the unique restriction enzyme sequence at the 5 ′ end of the polynucleotide sequence encoding NS3 protein recognizes EcoRV and is a unique restriction at the 3 ′ end of the polynucleotide sequence encoding the protease domain of NS3 protein. The enzyme sequence recognizes AsiSI. In yet another aspect of the invention, the HCV replicon shuttle vector comprises an HCV polynucleotide sequence selected from SEQ ID NO: 3 or SEQ ID NO: 6.

  A further aspect of the invention is the presence in a sample using a step of obtaining a sample from a subject infected with HCV, a sense strand primer comprising a unique restriction enzyme sequence, and an antisense strand primer comprising a different unique restriction enzyme sequence. PCR amplification of a polynucleotide sequence encoding the protease domain of NS3 protein from a plurality of HCV pseudospecies, and cloning of the PCR amplified polynucleotide sequence into an HCV replicon shuttle vector to obtain a chimeric HCV replicon positivemid Creating a chimeric HCV replicon RNA by chaining the chimeric HCV replicon positivemid and subjecting the chained positivemid to in vitro transcription, and a Huh7 cell line as described above. HC Efficacy of an HCV NS3 protease inhibitor for controlling HCV infection in a subject comprising the step of transfecting with a replicon RNA and measuring the replication level of said HCV replicon RNA in the presence or absence of an HCV NS3 protease inhibitor Provides a method for evaluating

  Another further aspect of the present invention is to use a step of obtaining a sample from a subject infected with HCV, a sense strand primer comprising a unique restriction enzyme sequence, and an antisense strand primer comprising a different unique restriction enzyme sequence in the sample. PCR-amplifying a polynucleotide sequence encoding the protease domain of NS3 protein from a plurality of HCV pseudospecies present in HCV, a chimeric HCV replicon positive by cloning said PCR-amplified polynucleotide sequence into an HCV replicon shuttle vector A step of preparing a mid, a step of preparing a colony of a plurality of transformed cells by transforming the positive mid into the cell, a pool of the colonies, and a chimeric HCV replicon positive mid from the pooled colony. Isolate The step of preparing a chimeric HCV replicon RNA by chaining the chimeric HCV replicon positivemid and subjecting the chained positivemid to in vitro transcription, and the Huh7 cell line with the HCV replicon RNA Assessing the efficacy of an HCV NS3 protease inhibitor that controls HCV infection in a subject comprising measuring the replication level of said HCV replicon RNA in the presence or absence of HCV NS3 protease inhibitor Providing a method for

  The foregoing and other advantages and features of the present invention, as well as methods for achieving the same, are more readily considered in view of the following detailed description of the invention, in conjunction with the accompanying examples illustrating exemplary aspects. Will become apparent.

FIG. 1 is an illustration of the components of an HCV replicon shuttle vector. FIG. 2 shows a positive-mid diagram of NS3 protease replicon shuttle vector (A) pSC_1b_NS3 / protease_EcoRV_AsiSI (SEQ ID NO: 3); (B) pSC_1b_NS3 / protease / LacZ_EcoRV_AsiSI (SEQ ID NO: 6). FIG. 3 shows the replication ability of a replicon comprising pSC_1b_NS3 / protease_EcoRV_AsiSI and NS3 protease from a patient derived from HCV genotype 1a or genotype 1b. RLU indicates the level of firefly luciferase signal observed 96 hours after transfection.

  The terms used in the present invention are defined below.

  The term “HCV replicon” refers to a nucleic acid derived from hepatitis C virus that can direct the creation of a copy of itself. As used herein, the term “replicon” includes RNA and DNA, and hybrids thereof. For example, a double-stranded DNA form of the HCV genome can be used to generate a single-stranded RNA transcript that constitutes an HCV replicon. The HCV replicon can include a full length HCV genome, or an HCV subgenomic construct (also referred to as a “subgenomic replicon”). For example, the HCV subgenomic replicon described herein contains most of the genes for viral nonstructural proteins, but lacks most of the genes that encode structural proteins. The subgenomic replicon can direct the expression of all viral genes required for viral subgenomic replication and subgenomic replicon replication without producing viral particles.

  The basic HCV replicon is a subgenomic construct that includes an HCV 5 'untranslated (UTR) region, an HCV NS3-NS5B polyprotein coding region, and an HCV 3'-UTR. Other nucleic acid regions may also be present, such as HSC NS2, HCV structural proteins, and non-HCV sequences.

  The HCV 5'-UTR region provides an internal ribosome entry site (IRES) for protein translation and the elements required for replication. The HCV 5'-UTR region includes native HCV 5'-UTR and functional derivatives thereof that extend approximately 36 nucleotides within the HCV core coding region. The 5'-UTR region may be present at various positions, such as at sites downstream from sequences encoding a selection protein, reporter protein, or HCV polyprotein.

  In addition to the HCV 5'-UTR-PC region, non-HCV IRES elements may be present in the replicon. Non-HCV IRES elements can be present in various positions, including immediately upstream of the region encoding the HCV polyprotein. Examples of non-HCV IRES elements that can be used are EMCV IRES, poliovirus IRES, and bovine viral diarrhea virus IRES.

  HCV 3'-UTR assists in HCV replication. HCV 3'UTR includes native HCV 3'-UTR and functional derivatives thereof. The natural 3'-UTR contains a poly U tract and an additional region of about 100 nucleotides.

  The NS3-NS5B polyprotein coding region provides a polyprotein that can be processed into various proteins in the cell. Suitable NS3-NS5B polyprotein sequences (which may be part of the replicon) include sequences present in various HCV strains and functional groups such that NS3-NS5B processing results in a functional replication machine. Includes equivalents. Proper processing can be measured, for example, by assaying NS5B RNA-dependent RNA polymerase.

  A “vector” is a piece of DNA that allows another piece of DNA segment to attach to a piece of DNA, such as a positivemid, phage, or cosmid, thereby resulting in replication, expression, or integration of the attached DNA segment. It is a piece. “Shuttle vector” refers to a vector in which a DNA segment can be inserted into or excised from a specific restriction enzyme site in the vector. The DNA segment inserted into the shuttle vector generally encodes the polypeptide or RNA of interest, and this restriction enzyme site ensures that the DNA segment is inserted into the appropriate reading frame for transcription and translation. Designed to be

  A variety of vectors can be used to express a nucleic acid molecule. Such vectors include those derived from chromosomes, episomes and viruses, such as bacterial positivemids, bacteriophages, yeast episomes, yeast chromosome elements (including yeast artificial chromosomes), baculoviruses, SV40. And vectors derived from viruses such as papovavirus, vaccinia virus, adenovirus, poxvirus, pseudorabies virus, herpes virus, and retrovirus. Vectors can also be derived from combinations of these sources, such as vectors derived from positivemid and bacteriophage genetic elements, such as cosmids and phagemids. Suitable cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual. 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA. ing.

  A vector containing the appropriate nucleic acid molecule can be introduced into an appropriate host cell for propagation or expression using known techniques. Host cells include bacterial cells (including but not limited to E. coli, Streptomyces, and Salmonella typhimurium), eukaryotic cells ( This includes yeast, insect cells such as Drosophila, animal cells such as Huh-7, HeLa, COS, HEK293, MT-2T, CEM-SS, and CHO cells, and plant cells. But may be included).

  Vectors generally include a selectable marker that allows for selection of a subpopulation of cells containing the recombinant vector construct. The marker may be included on the same vector containing the nucleic acid molecules described herein or on a separate vector. Markers include tetracycline or ampicillin resistance genes in prokaryotic host cells and dihydrofolate reductase or neomycin resistance genes in eukaryotic host cells. However, any marker that provides selectivity for the phenotypic trait will be effective.

  “Polynucleotide” or “nucleic acid molecule” generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, but modified RNA or DNA. May be. “Polynucleotide” includes single-stranded and double-stranded DNA, mixed DNA of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and mixed of single-stranded and double-stranded regions. It can be RNA, single stranded or more typically double stranded, or includes, but is not limited to, a hybrid molecule comprising DNA and RNA mixed of single and double stranded regions. . Furthermore, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA, or both RNA and DNA. “Polynucleotide” also embraces relatively short polynucleotides, also referred to as oligonucleotides.

  Furthermore, the term “DNA molecule” refers only to the primary and secondary structure of the molecule and is not limited to any particular three-dimensional form. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (eg, restriction enzyme fragments), viruses, positive mids, and chromosomes. In discussing the structure of a particular double-stranded DNA molecule, only the 5 ′ to 3 ′ sequence is shown herein along the non-transcribed strand of DNA (ie, the strand having a sequence homologous to mRNA). The sequence can be described according to the usual convention.

  “RNA molecule” refers to a multimeric form of ribonucleotides in its single-stranded or double-stranded helix form. In discussing the structure of a particular RNA molecule, the sequences can be described herein according to the usual convention of indicating the sequence 5 'to 3'.

The term “restriction enzyme sequence” refers to a specific double-stranded DNA sequence that is recognized and cleaved by a bacterial enzyme, each of which has double-stranded DNA at or near a specific nucleotide sequence. Disconnect. The restriction enzyme “EcoRV” has the sequence

Recognizes the specified nucleotide position

Cleave the double-stranded DNA. The restriction enzyme “AsiSI” is a sequence

And cleaves after the T residue on the recognition sequence.

  The term “primer” as used herein is RNA or DNA, single or double stranded, derived from a biological system, made by digestion with a restriction enzyme, or synthetically. The oligonucleotides made in (1) above can function functionally as an initiator for template-dependent nucleic acid synthesis when placed in an appropriate environment. When presented with the appropriate nucleic acid template, the appropriate nucleic acid nucleoside triphosphate precursor, the polymerase enzyme, the appropriate cofactor, and conditions (eg, appropriate temperature and pH), the nucleotide of the nucleotide by the action of the polymerase or similar activity Addition extends the primer at its 3 'end, generating a primer extension product. Primers may vary in length depending on the specific conditions and requirements to be applied. For example, in a PCR reaction, the primer is typically 15-25 nucleotides in length or longer. The primer must have sufficient complementarity to the desired template to initiate synthesis of the desired extension product, i.e., to be used to initiate synthesis by a polymerase or similar enzyme. It must be able to fully anneal with the desired template strand so that the 3'-hydroxyl portion of the primer is properly juxtaposed. The primer sequence need not exhibit exact complementarity to the desired template. For example, a non-complementary nucleotide sequence (eg, a restriction enzyme recognition sequence) may be attached to the 5 'end of an otherwise complementary primer. Or if the primer sequence has sufficient complementarity to the sequence of the desired template strand so that the template-primer complex for synthesis of the extension product is functional, non-complementary Bases may be interspersed within the oligonucleotide primer sequences.

  The term “chimera” as used herein refers to a DNA molecule obtained from two or more different sources of DNA fused or spliced together.

  As used herein, “quasispecies” refers to a collection of microvariants of a dominant HCV genomic sequence (ie, genotype) that has a high mutation rate during HCV replication. As a result, it is formed in one infected subject or even in a single cell clone.

  As used herein, “subject” refers to a member of a vertebrate, particularly a mammalian species, including rodents, rabbits, shrews, and primates (including humans). However, this is not a limitation.

  The term “sample” refers to a tissue or fluid sample isolated from a subject, including, for example, plasma, serum, spinal fluid, lymph fluid, skin, respiratory organs, intestinal tract, and external sections of the genitourinary tract. , Tears, saliva, milk, blood cells, tumors, organs, and also samples of in vitro cell culture components (including conditioned media obtained from growth of cultured cells, putative virus-infected cells, recombinant cells, And cellular components), but not limited thereto.

  When exogenous or heterologous DNA or RNA is introduced into a cell, the cell is “transformed” or “transfected” by such exogenous or heterologous DNA or RNA. The DNA or RNA to be transformed or transfected may or may not be integrated (covalently linked) into the chromosomal DNA making up the genome of the cell. For example, in prokaryotic cells, yeast, and mammalian cells, the transforming DNA may be maintained on an episomal element such as a positive mid. With respect to eukaryotic cells, a stably transformed cell is one in which the transformed DNA is integrated into the chromosome so that the DNA is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of eukaryotic cells to establish cell lines or clones consisting of a collection of daughter cells containing transforming DNA. In the case of HCV replicons that transform mammalian cells as described in the present invention, RNA molecules, such as HCV RNA molecules, have the ability to replicate semi-autonomously. Huh-7 cells with an HCV replicon are detected by the presence of a selectable marker or reporter gene present on the replicon.

  A “clone” refers to a population of cells obtained from a single cell or consensus ancestor, generally by a mitotic process.

Examples The following preparations and examples are described to enable those skilled in the art to more clearly understand and to practice the present invention. They should not be construed as limiting the invention, but merely as descriptions and illustrations.

Example 1
NS3 protease replicon shuttle vector was obtained from the HCV replicon shuttle vector pSC_1b_NS3_EcoRV, an intermediate vector used to create the HCV NS3 replicon shuttle vector pSC_1b_NS3_EcoRV_XbaI, which was submitted on September 27, 2007. Entitled “HCV NS3 Replicon Shuttle Vector” in US patent application USSN 60 / 995,558 by Chua et al., Which is hereby incorporated by reference in its entirety. The components of the replicon shuttle vector are shown in FIG. 1 and include HCV polynucleotide sequences from 5′-UTR to NS3 to NS5B protein and 3′-UTR. The vector also contains a poliovirus internal ribosome entry site (IRES), which controls the translation of the firefly luciferase gene. IRES from encephalomyocarditis virus (EMCV), downstream of the firefly luciferase gene, controls the translation of HCV nonstructural genes (NS3, NS4A, NS4B, NS5A, and NS5B).

Mutations were introduced into pSC_1b_NS3_EcoRV_XbaI using the quick change site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions. In order to introduce the AsiSI restriction enzyme sequence at the 3 ′ end of the protease domain of NS3 gene (corresponding to amino acid position Ser181), the following primers were used:

These mutations did not change the amino acid coding sequence of the NS3 protein, thereby creating the NS3 protease shuttle vector pSC_1b_NS3 / protease_EcoRV_AsiSI (SEQ ID NO: 3, FIG. 2A). Another NS3 protease replicon shuttle vector was created by replacing the sequence encoding the protease domain of the NS3 gene with a β-galactosidase (lacZ) coding sequence from pUC19 (GenBank Accession No. M77789). An EcoRV restriction site at the 5 ′ end of the lacZ gene, an AsiSI restriction site at the 3 ′ end, and the following primers:

Was introduced by PCR amplification to prepare vector pSC_1b_NS3 / protease / LacZ_EcoRV_AsiSI (SEQ ID NO: 6, FIG. 2B).

  The replication ability of the NS3 protease replicon shuttle vector was assessed using the phenotypic assay described in Example 3 (and shown in FIG. 3).

Example 2
Cloning of NS3 Protease Domain PCR Sample Amplified from an Infected Patient into NS3 Protease Replicon Shuttle Vector The DNA sequence encoding the protease domain of the NS3 gene was obtained according to the manufacturer's protocol using the SuperScript III system (Invitrogen) according to the manufacturer's protocol. It was prepared by reverse transcription polymerase chain reaction (RT-PCR) of plasma-derived RNA obtained from patients infected with genotype 1a and genotype 1b. The primers used for this RT-PCR step were as follows:

First, annealing was performed at 50 ° C., followed by PCR cycles of denaturation at 94 ° C., annealing at 53 ° C., and extension at 68 ° C. The amplified product is then used with primers that introduce unique restriction enzyme sequences at the 5 ′ and 3 ′ ends of the protease domain of NS3 protein using the Phusion ™ high fidelity DNA polymerase system (New England Biolabs). A second round of PCR was performed. The sense primers used to introduce the EcoRV restriction enzyme sequence near the 5 'end of the patient's NS3 gene sequence were as follows:

Antisense primers used to introduce the AsiSI restriction enzyme sequence near the 3 ′ end of the protease domain of NS3 gene sequence (corresponding to amino acid position Ser181) were as follows:

  Amplification was performed by a PCR cycle including denaturation at 98 ° C, annealing at 53 ° C, and extension at 72 ° C. The amplified patient NS3 protease DNA was then purified using a Qiagen PCR purification column and digested with EcoRV and SgfI (AsiSI iso-restriction enzyme).

  NS3 protease shuttle replicon vectors pSC_1b_NS3 / protease_EcoRV_AsiSI or pSC_1b_NS3 / protease / LacZ_EcoRV_AsiSI were prepared by double digestion with restriction endonucleases EcoRV and SgfI. 25 ng of the shuttle vector was ligated with the digested patient amplicon using the MightMix ligation kit (Takara Bio Inc.) for 1.5 hours at 16 ° C. A ratio of 1: 2 to 1: 4 vector to insert was routinely used. 5 μl of the reaction was transformed into 50 μl of Top10 cells (Invitrogen) and seeded after 1 hour phenotypic expression at 37 ° C.

  Ninety-six independent colonies were picked and inoculated into 200 μl terrific medium (TB) supplemented with 50 μg / ml ampicillin. This “stock” 96-well plate was incubated at 37 ° C. overnight. The next day, a replica plate was prepared by using this plate. A 48 pin stamp was used to replica plate on two LB plates supplemented with 100 μg / ml carbenicillin. 96 individual 200 μl cultures were also transferred to 1.5 ml TB broth supplemented with 50 μg / ml ampicillin and used for DNA preparation. After overnight incubation at 37 ° C., the replica plate was spread with 6 ml of LB to obtain a heterogeneous pool of 96 clones, which was used for mini-preparation of DNA. This DNA patient pool was then used for subsequent replicon phenotype assays. After shaking overnight at 37 ° C., 96 individual 1.5 ml TB cultures were precipitated in a centrifuge, decanted, and positive-mid DNA was extracted using Qiagen's Qiaprep 96 Turbo Mini-DNA kit did. These individual molecular clones corresponding to the complex 96 pool used in the replicon phenotype assay were used for sequencing reactions.

  Heterogeneous clone pool positivemid DNA or individual molecular clones were sequenced to confirm the identity of the patient sample and to examine the sensitivity to inhibitors in the phenotypic replicon assay.

Example 3
Phenotypic replicon assay Preparation of RNA transcribed in vitro 5 μg of DNA positivemid was chained with ScaI restriction enzyme (Roche). After digesting overnight at 37 ° C., the DNA was purified using a Qiagen PCR purification kit. 1 μg of strand DNA was used for in vitro transcription using T7 RiboMAX Express (Promega) according to the manufacturer's protocol. After incubating at 37 ° C. for 2 hours, the DNA template was removed by treatment with DNase at 37 ° C. for 30 minutes. The in vitro transcribed RNA was then purified using an RNeasy spin column (Qiagen) according to the manufacturer's protocol.

B. Hepatocytoma cell line Hepatocytoma Lunet Huh-7 cell line was conditioned in a humidified atmosphere containing 5% CO ₂ in Dulbecco's modified Eagle medium (DMEM) supplemented with Glutamax ™ and 100 mg / ml sodium pyruvate. Incubated at 37 ° C. The medium was further supplemented with 10% (v / v) FBS and 1% (v / v) penicillin / streptomycin. All reagents were from Invitrogen / Gibco.

C. Measurement of transient replicon replication levels 4 million Lunet Huh7 cells were transfected with 5 μg of in vitro transcribed RNA using electroporation. Cells were then resuspended in 7.2 ml DMEM containing 5% FBS and seeded in 96-well plates at 50000 cells / well (final volume 90 μl). Inhibitors (if used) were diluted 3 fold and added 24 hours after transfection at a final DMSO concentration of 1%, and the firefly luciferase reporter signal was added using the luciferase assay system (Promega). 72 hours later. The IC ₅₀ value, the concentration of inhibitor at which a 50% reduction in the level of firefly luciferase reporter was observed, compared to the level of firefly luciferase signal when no compound was added was evaluated.

The replication ability of the replicon shuttle vector pSC_1b_NS3 / protease_EcoRV_AsiSI and the replication ability of the replicon containing NS3 protease domain derived from HCV genotype 1a and genotype 1b patients were tested using the luciferase signal as a decoding value, and the results are shown in FIG. . The inhibitory action of HCV protease inhibitors BILN2061, VX-950, and NM107 were also tested in these replicons and the results are shown in Table 1.

Claims (34)

Less than:
(A) a unique restriction enzyme sequence located between 10 nucleotides 5 'and 10 nucleotides 3' from the 5 'end of the polynucleotide sequence encoding NS3 protein;
(B) a polynucleotide sequence encoding the protease domain of NS3 protein;
(C) a unique restriction enzyme sequence located between the 3 ′ end of the polynucleotide sequence encoding the protease domain of the NS3 protein and 5 ′ to 10 nucleotides and 3 ′ to 10 nucleotides;
(D) a polynucleotide sequence encoding a helicase domain of NS3 protein;
(E) a polynucleotide sequence encoding an NS4A protein;
(F) a polynucleotide sequence encoding NS4B protein;
An HCV replicon shuttle vector comprising an HCV polynucleotide sequence comprising (g) a polynucleotide sequence encoding an NS5A protein; and (h) a polynucleotide sequence encoding an NS5B protein in this order.   The HCV replicon shuttle vector according to claim 1, wherein the polynucleotide sequence encoding the protease domain of NS3 protein is modified or deleted so that the protease domain of NS3 protein does not function.   A unique restriction enzyme sequence at the 5 ′ end of the polynucleotide sequence encoding NS3 protein recognizes EcoRV, and a unique restriction enzyme sequence at the 3 ′ end of the polynucleotide sequence encoding the protease domain of NS3 protein recognizes AsiSI, The HCV replicon shuttle vector according to claim 1 or 2.   An HCV replicon shuttle vector comprising an HCV polynucleotide sequence selected from SEQ ID NO: 3 or SEQ ID NO: 6. A method for assessing the efficacy of an HCV NS3 protease inhibitor that controls HCV infection in a subject comprising the following steps:
(A) providing a sample from a subject infected with HCV;
(B) a polynucleotide encoding a protease domain of NS3 protein from a plurality of HCV pseudospecies present in a sample using a sense strand primer containing a unique restriction enzyme sequence and an antisense strand primer containing a different unique restriction enzyme sequence PCR amplification of the sequence:
(C) producing a chimeric HCV replicon positiveimide by cloning the PCR amplified polynucleotide sequence into an HCV replicon shuttle vector;
(D) producing the chimeric HCV replicon RNA by chaining the chimeric HCV replicon positivemid and subjecting the chained positivemid to in vitro transcription;
(E) Transfecting a Huh7 cell line with the HCV replicon RNA and measuring the replication level of the HCV replicon RNA in the presence or absence of an HCV NS3 protease inhibitor.   6. The method of claim 5, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 1.   6. The method of claim 5, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 2.   6. The method of claim 5, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 3.   6. The method of claim 5, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 4. A method for assessing the efficacy of an HCV NS3 protease inhibitor that controls HCV infection in a subject comprising the following steps:
(A) providing a sample from a subject infected with HCV;
(B) NS3 protein protease domain from multiple HCV pseudospecies present in a sample using a sense strand primer containing a restriction enzyme sequence recognizing EcoRV and an antisense strand primer containing a restriction enzyme sequence recognizing AsiSI PCR amplification of a polynucleotide sequence encoding
(C) producing a chimeric HCV replicon positiveimide by cloning the PCR amplified polynucleotide sequence into an HCV replicon shuttle vector;
(D) producing the chimeric HCV replicon RNA by chaining the chimeric HCV replicon positivemid and subjecting the chained positivemid to in vitro transcription;
(E) Transfecting a Huh7 cell line with the HCV replicon RNA and measuring the replication level of the HCV replicon RNA in the presence or absence of an HCV NS3 protease inhibitor.   11. The method of claim 10, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 1.   11. The method of claim 10, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 2.   6. The method of claim 5, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 3.   6. The method of claim 5, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 4. A method for assessing the efficacy of an HCV NS3 protease inhibitor that controls HCV infection in a subject comprising the following steps:
(A) providing a sample from a subject infected with HCV;
(B) present in a sample using a sense strand primer comprising a nucleotide sequence selected from SEQ ID NO: 11 or SEQ ID NO: 12 and an antisense strand primer comprising a nucleotide selected from SEQ ID NO: 13 or SEQ ID NO: 14 PCR amplification of a polynucleotide sequence encoding the protease domain of NS3 protein from a plurality of HCV pseudospecies:
(C) producing a chimeric HCV replicon positiveimide by cloning the PCR amplified polynucleotide sequence into an HCV replicon shuttle vector;
(D) producing the chimeric HCV replicon RNA by chaining the chimeric HCV replicon positivemid and subjecting the chained positivemid to in vitro transcription;
(E) Transfecting a Huh7 cell line with the HCV replicon RNA and measuring the replication level of the HCV replicon RNA in the presence or absence of an HCV NS3 protease inhibitor.   16. The method of claim 15, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 1.   16. The method of claim 15, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 2.   16. The method of claim 15, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 3.   16. The method of claim 15, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 4. A method for assessing the efficacy of an HCV NS3 protease inhibitor that controls HCV infection in a subject comprising the following steps:
(A) providing a sample from a subject infected with HCV;
(B) a polynucleotide encoding a protease domain of NS3 protein from a plurality of HCV pseudospecies present in a sample using a sense strand primer containing a unique restriction enzyme sequence and an antisense strand primer containing a different unique restriction enzyme sequence PCR amplification of the sequence:
(C) producing a chimeric HCV replicon positiveimide by cloning the PCR amplified polynucleotide sequence into an HCV replicon shuttle vector;
(D) a step of preparing colonies of a plurality of transformed cells by transforming the positivemid into cells;
(E) pooling said colonies and isolating chimeric HCV replicon positivemids from the pooled colonies;
(F) producing the chimeric HCV replicon RNA by concatenating the chimeric HCV replicon positivemid of step (e) and subjecting the chained positivemid to in vitro transcription;
(G) A method comprising transfecting a Huh7 cell line with the HCV replicon RNA and measuring the replication level of the HCV replicon RNA in the presence or absence of an HCV NS3 protease inhibitor.   21. The method of claim 20, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 1.   21. The method of claim 20, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 2.   21. The method of claim 20, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 3.   21. The method of claim 20, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 4. A method for assessing the efficacy of an HCV NS3 protease inhibitor that controls HCV infection in a subject comprising the following steps:
(A) providing a sample from a subject infected with HCV;
(B) NS3 protein protease domain from multiple HCV pseudospecies present in a sample using a sense strand primer containing a restriction enzyme sequence recognizing EcoRV and an antisense strand primer containing a restriction enzyme sequence recognizing AsiSI PCR amplification of a polynucleotide sequence encoding
(C) producing a chimeric HCV replicon positiveimide by cloning the PCR amplified polynucleotide sequence into an HCV replicon shuttle vector;
(D) a step of preparing colonies of a plurality of transformed cells by transforming the positivemid into cells;
(E) pooling said colonies and isolating chimeric HCV replicon positivemids from the pooled colonies;
(F) producing the chimeric HCV replicon RNA by concatenating the chimeric HCV replicon positivemid of step (e) and subjecting the chained positivemid to in vitro transcription;
(G) A method comprising transfecting a Huh7 cell line with the HCV replicon RNA and measuring the replication level of the HCV replicon RNA in the presence or absence of an HCV NS3 protease inhibitor.   26. The method of claim 25, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 1.   26. The method of claim 25, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 2.   26. The method of claim 25, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 3.   26. The method of claim 25, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 4. A method for assessing the efficacy of an HCV NS3 protease inhibitor that controls HCV infection in a subject comprising the following steps:
(A) providing a sample from a subject infected with HCV;
(B) present in a sample using a sense strand primer comprising a nucleotide sequence selected from SEQ ID NO: 11 or SEQ ID NO: 12 and an antisense strand primer comprising a nucleotide selected from SEQ ID NO: 13 or SEQ ID NO: 14 PCR amplification of a polynucleotide sequence encoding the protease domain of NS3 protein from a plurality of HCV pseudospecies:
(C) producing a chimeric HCV replicon positiveimide by cloning the PCR amplified polynucleotide sequence into an HCV replicon shuttle vector;
(D) a step of preparing colonies of a plurality of transformed cells by transforming the positivemid into cells;
(E) pooling said colonies and isolating chimeric HCV replicon positivemids from the pooled colonies;
(F) producing the chimeric HCV replicon RNA by concatenating the chimeric HCV replicon positivemid of step (e) and subjecting the chained positivemid to in vitro transcription;
(G) A method comprising transfecting a Huh7 cell line with the HCV replicon RNA and measuring the replication level of the HCV replicon RNA in the presence or absence of an HCV NS3 protease inhibitor.   32. The method of claim 30, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 1.   32. The method of claim 30, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 2.   32. The method of claim 30, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 3.   31. The method of claim 30, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 4.