KR20010022420A - Compositions and methods for determining anti-viral drug susceptibility and resistance and anti-viral drug screening - Google Patents

Abstract

The present invention comprises the steps of (a) introducing a resistance test vector comprising a patient-derived fragment comprising a hepatitis C virus gene and an indicator gene into a host cell;

(b) culturing the host cell from (a);

(c) measuring the expression of the indicator gene in the target host cell;

(d) the expression of the indicator gene measured when (c) the test concentration of the HCV antiviral drug is present in steps (a)-(c); (b)-(c) or (c); A) A method for determining sensitivity to HCV antiviral drugs comprising comparing the indicator gene expression measured when step-(c) is performed in the absence of antiviral drugs. The invention also comprises the steps of (a) obtaining a patient-derived section from a patient near the time of sensitivity determination and determining the patient's initial antiviral drug sensitivity according to the method of claim 91;

(b) determining the late antiviral drug sensitivity of the patient; And,

(c) comparing the antiviral drug sensitivity determined in steps (a) and (b) when a decrease in late antiviral drug sensitivity in the early preparation indicates an increase or progress in the antiviral drug resistance of the patient. A method for determining HCMV antiviral drug resistance in a patient is provided. The present invention also provides a method for measuring the biological effect of an HCV or HCMV antiviral drug compound.

Description

COMPOSITIONS AND METHODS FOR DETERMINING ANTI-VIRAL DRUG SUSCEPTIBILITY AND RESISTANCE AND ANTI-VIRAL DRUG SCREENING}

Viral Drug Resistance

The use of antiviral compounds as chemotherapy and chemoprophylaxis of viral diseases is a relatively new development in the field of infectious diseases. This is especially true when compared to more than 50 years of experience with antimicrobial antibiotics.

The design of antiviral compounds is not easy because viruses create many unique problems. Viruses must replicate within cells and often use enzymes, macromolecules, and organelles from the host cell for the synthesis of viral particles. Therefore, a safe and effective antiviral compound must be able to very efficiently distinguish between cell function and virus specific function. Further, due to the replication properties of the virus, the in vitro sensitivity of the virus isolates to antiviral compounds should be assessed in complex culture systems made of living cells such as tissue cells. The results of the assay system vary greatly depending on the type of tissue culture cells applied and the assay conditions.

Viral drug resistance is a practical problem given the high rate of viral replication and mutation frequency. Drug-resistant mutations were initially identified as Poxviruses in Thiosemicarbazone (Appleyard and Way (1966) Brit. J. Exptl. Pathol. 47,144-51), and polioviruses in guanidine (Melnick). (1961) Science 134,557), influenza virus in amantadine (oxford et al. (1970) Nature 226, 8283; Cochran et al. (1965) Ann. NY Acad Sci 130, 423-429) and iodody Herpes simplexvirus (Jawetz et al. (1970) Ann. NY Acad sci 173, 282-291) in iododeoxyuridine was recognized. Approximately 140 HIV drug resistance mutations have been demonstrated to date for a variety of antiviral agents (Mellors et al. (1995) Intnl. Antiviral News, supplement and Condra, JH et al. (1996) J Virol. 70, 8270-8276). . Approximately 20 human cytomegalovirus (HCMV) drug resistance mutations have been demonstrated to date with approximately various antiviral agents. (Biron (1996) Antiviral Chemotherapy, 4, 135-143)

The small and efficient genome of the virus has helped intensive studies of the molecular genetics, structure and replication cycle of the most important human viral pathogens. As a result, sites and mechanisms were specified for activity and resistance to antiviral drugs more precisely than in other classes of drugs (Richman (1994) Trends Microbiol. 2. 401-407). The likelihood of resistant mutations is a function of at least four factors. First, the frequency of mutation of the virus; Second, essentially altering the target site of the virus for a particular antivirus; Third, selective pressure of antiviral drugs; Fourth, the scale and proportion of virus replication.

With respect to the first element, for single stranded RNA viruses that lack a proofreading mechanism for genome replication, the mutation frequency is approximately 3 × 10 ⁻⁵ per base pair of one replication cycle. Thus, a single 10-kilogram genome, such as human immunodeficiency virus (HIV) or hepatitis C virus (HCV), is expected to contain, on average, one mutation per three of the descendant virus genomes. . In a second factor, the intrinsic mutagenesis of the viral target in response to a particular antiviral agent can affect the probability of resistance mutations.

In one example, zidovudine (AZT) causes mutations in reverse transcriptase of HIV more easily in vitro or in vivo than other recognized thymidine analogs d4T (stavudine).

One of the consequent consequences of antiviral drug action is perhaps that it puts sufficient selective pressure on viral replication to screen for drug-resistant mutations (Herrmann et al. (1977) Ann NY Acad Sci 284. 6327).

With respect to the third factor, with increasing drug exposure, the selection pressure on the replicating viral population increases to promote the more rapid emergence of drug-resistant mutations. For example, higher doses of zidovudine tend to screen for drug resistant viruses faster than lower doses (Richman et al. (1990) J. AIDS.3.743-6). This selection pressure for resistant mutations increases the likelihood of mutations as long as an important stage of viral replication continues.

The fourth factor, the scale and proportion of viral populations, has a major influence on the likelihood of resistant mutations. Many viral infections are characterized by high levels of viral replication with a high rate of virus turnover (Perelson et al. (1996) Science, 271,1582-1586; Nowak et al. (1996), PNAS 93, 4398-4402). . This is especially true for chronic infections with HIV as well as hepatitis B and C. The likelihood of developing AZT resistance is increased in HIV infected patients with decreasing numbers of CD4 lymphocytes associated with an increase in HIV replication (Ibid).

Higher levels of the virus increase the likelihood of mutations previously present. The emergence of resistant populations has been known to be responsible for the survival and selective proliferation of previously existing sub-populations that appear irregular in the absence of selection pressure. All viruses have a basic mutation rate. When approximately 10 ¹⁰ new virions are produced daily during HIV infection, mutation rates of 10 ⁻⁴ to 10 ⁻⁵ per nucleotide indicate that most of the single point mutations are present at any point during the HIV infection. Evidence suggests that drug-resistant mutations are actually present in subpopulations of HIV-infected individuals (Najera et al. (1994) AIDS Res Hum Retroviruses 10,1479-88; Najera et al. (1995) J Virol. 69, 23). -31; Havlir et al. (1996) J. Virol. 70. 7894-7899). It is also well documented that drug-resistant picornavirus mutations pre-exist in approximately 10 ^-5 ratios.

Hepatitis C Virus (HIV)

Infection with hepatitis c virus occurs worldwide. And before its demonstration, it showed the major cause of blood transfusion-related hepatitis. Serum prevalence varies from 0.02% to 1.23% in recipients worldwide. HCV is also a major cause of hepatitis in individuals exposed to blood products. In the United States alone, there have been 150,000 new cases of HCV infection each year (Alter 1993, Infect, Agents Dis. 2.155-166; Houghton 1996. in Fields Virology, 3rd edition 1035-1058).

Hepatitis c virus (HCV) is an unsegmented, positive strand, RNA virus with a liquid epidermis and is a member of the flaviviridae virus species. Other members of the species are pestiviruses (eg bovine viral diarrheal virus, or BVDV, and classic swine fever virus, or CSFV) and flaviviruses ( flaviviruses (eg, yellow fever virus and Dengue virus). See Rice, 1996 in Fields Virology, 3rd edition, pp. 931-959. The understanding of the molecular dissection of HCV replication and the function of its encoded proteins has been greatly improved by the isolation of the virus and the continuity of the viral genome, but the development of an effective cell culture system for the production of natural or recombinant HCV from molecular clones. It has been limited to lack.

However, low levels of replication have been observed in several cell lines infected with HCV infected humans or chimpanzee viruses or transfected with RNA derived from cDNA clones of HCV.

HCV is closely associated with the endoplasmic reticulum and replicates in infected cells of the cytoplasm (FIG. 1).

The introduced positive sense RNA is freed and translation is initiated through an internal initiation mechanism. (Wang et al. 1993. J. Virol. 67.3338-3344; Tsukiyama-Kohara et al. 1992. J. Virol. 66.1476-1483) The internal initiation is a cis-acting RNA element at the fifth end of the genome. Is indicated by. Some reports indicate that the maximum activity of the internal ribosomal entry site (IRES) is determined by the first 700 nucleotides in which the first 123 amino acids of the 5 'untranslated region (UTR) and the open reading frame (ORF) lie. (Lu and Wimmer, PNAS 93,1412,1996). All of the protein products of HCV are produced by proteolytic cleavage of polyproteins according to large isolates (3010-3030 amino acids), which is performed by one of the following three proteases; Host signal peptidase, viral autologous metalloproteinase, NS2, or viral serine protease NS3 / 4A (FIG. 2). The enzymes combine to produce structural proteins (C, E1 and E2) and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) required for replication and packaging of viral genomic RNA. NS5B is a viral RNA-dependent RNA polymerase (RDRP) that is responsible for converting input genomic RNA into negative strand copies (complementary RNA, or cRNA). The cRNA then becomes the template for transcription by the positive sense genome / messenger RNA. Several associations and laboratories are attempting to identify and develop anti-HCV drugs. Currently the only effective treatment for HCV is alpha interferon, which regulates the amount of virus in the liver and blood in only a small number of infected patients (Houghton 1996, in Fields Virology, 3rd edition, pages 1035-1058). The molecular structure of the serine protease, NS3 / 4A (Love et al. 1996. Cell 87, 331-342; Kim et al. 1996. Cell 87. 343-355) is available and successful using protease inhibitors in the treatment of HIV-1 infection. If so, another useful countermeasure soon appears. In addition to HCV protease inhibitors, other inhibitors that may specifically interfere with HCV replication include virus specific activity targets such as IRES encoded by NS5B, RDRP activity or internal initiation directed by NS3 encoded RNA helicase activity. You can do

As a result of the high error rate of their RDRPs, RNA viruses can be particularly adapted to many new growth conditions. Most polymerases in this class have an error rate of 1 of 10,000 nucleotides copied. At least one nucleotide position in the HCV genome with a genome size of approximately 9.5 kb will maintain a mutation every time the genome is copied. Therefore, drug resistance tends to develop during continuous exposure to antiviral drugs. Given the choice of drug and a non-overlapping form of drug-resistant genotype, a fast and convenient analysis of drug-resistant HCV, as in the case of HIV, will enhance the likelihood of successful antiviral treatment.

Resistance-associated mutations can sometimes be quickly identified by culturing the virus in cell culture in the presence of the drug, with a significant success approach against HIV-1. However, to date there is a lack of convenient cell culture systems for HCV. Therefore, it is not possible to determine the exact nature of the genetic modification that gives drug resistance in vitro. Therefore, in the absence of a list of known resistance-associated mutations, a better resistance assay relies on the reading of the phenotype rather than the genotype.

There is no recently available drug resistance assay for HCV. However, some investigators have devised a chimeric-like viral system that includes HCV NS3 protease so that replication of chimera or reporter gene expression depends on NS3 activity. The system is designed to study the function of NS3 and its cofactors, NS4AD, and to serve as the basis for cell-based drug screening assays.

(Hiwartari et al .: Anal. Biochem. 225: 113.1995) synthesizes NS2-NS3-taxl fusion protein (taxl is a transcriptional trans-activator from HTLV-I retrovirus) suspended in endoplasmic reticulum. Expression vectors were prepared for this purpose. Upon cleavage by NS3 (or NS2), the taxl trans-activator is released and migrates to the nucleus. And there, it activates the expression of the reporter gene regulated by HTLV-1 LRT.

(Including Virology 226: 318, 1996) produced chimeric polioviruses comprising HCV NS3 upstream of a polyvirus polyprotein; Replication of the chimera depends on the activity of the NS3 protease. Because the release of the native N-terminus of the poliovirus polyprotein is essential for initiation of poliovirus replication. However, this chimera does not contain the NS4A protein of HCV, which is known to modify the activity of NS3.

(Philocamo et al .: J. Virol. 71: 1417,1997) produced a chimeric Sindbis virus containing HCV NS3 / 4A upstream of the Sindbis virus polyprotein: replication of the chimera was NS3. Depends on protease activity. This is because being free of the natural N-terminus of the Sindbis virus polyprotein is essential for initiating Sindbis virus replication.

In Vitro Expression Systems

Resistance test vectors rely on transfection, replication and expression of indicator genes. Some have described a system for transfection of complete HCV RNA into Huh7 cells synthesized in vitro from cDNA constructs. And it has been demonstrated that replication can occur (Yoo et al. (1995) J. Virol. 69, 32-38). In addition, some cell lines have demonstrated that HCV replication is helpful after being infected with viruses present in the serum or plasma of HCV-infected humans or chimpanzees (Valli et al. (1997) Res. Virol. 148,181-186; Shimizu (1992) PNAS 89,3477-5481; Shimizu et al. (1993) PNAS 90, 6037-6041; Shimizu and Yoshikura (1994) J. Virol. 68,8406-8408; Shimizu et al. (1994) J. Virol .68,1494-1500; Mizutani et al. (1996) J. Virol. 70,7219-7223)

Currently these systems are limited to systems where replication is detected in a sensitive manner such as RT / PCR. Therefore, it is not easy to consider efficient production of the indicator gene (IG). As soon as new cell lines or methods of infection have been discovered, improved methods will become viable. An example of a possible improved method is to infect RNA with an RNP complex prepared by in vitro transcription in the presence of purified NS5B so that transcription can be initiated as soon as it enters the cell. This strategy has been applied to negative strand RNA viruses such as influenza. Enami and Palese (1991) J. Virol. 65,2711-2713, rabies virus (Schnell et al. (1994) EMBO J. 13,4195-4203), and vesicular stomatitis virus (Lawson et al. (1995) PNAS 92, 4477-4481)

Since the genome length RNA of the flavivirus is infectious, the HCV vector may be in the form of a cRNA construct comprising a promoter of a 5-terminal T7 RNA polymerase and a 3-terminal T7 polymerase terminator sequence. Thus, RNA can be synthesized in large quantities in vitro and transfected into cells. Another approach is to infect DNA constructs, which include powerful promoters such as the CMV IE promoter. Possible benefits of RNA transfection over DNA include the following: RNA infection prevents specific, cell-specific limitations of promoter activity; Translation of genetically engineered proteins generally occurs within minutes to hours after transfection of biologically activated RNA, whereas translation after DNA transfection must be preceded by transcription and RNA processing before the maximum expression level is reached. There is a delay from hours to days.

Representation of pseudoviruses is easier when RNA is infected. This is because sufficient amounts of RNA can be synthesized from uncloned PCR products by including sequences of bacteriophage RNA polymerase in 5 'PCR primers. Such an approach is known to preserve the diversity of poliovirus-like species (Chumakov. J. Virol. 70: 7331-7334,1996). Generation of the correct 5 'and 3' ends of the RNA involves a promoter at the 5 'end and a restriction endonuclease recognition site at the 3' end (used for linearization of the DNA template prior to transcription) with respect to the viral sequence. Positioning makes it easier to obtain than in vitro transcription. However, the 3 'end can also be precisely generated by locating autologous RNA ribozyme sequences present in cDNA constructs (eg Chowrira et al. (1994), J. Biol. Chem. 269, 24856-25864). ).

The third transfection strategy, which has the advantage of RNA transfection, is that a construct containing a T7 RNA polymerase promoter at the 5 'end and a T7 RNA polymerase terminator at the 3' end is transfected into cells expressing the T7 RNA polymerase. It is infectious. Expression of the polymerase can be obtained in a variety of ways, of which it will be most effective to infect cells transfected with recombinant T7 polymerase / vaccinia virus.

Human Cytomegalovirus (HCMV)

Human Cytomegalovirus (HCMV) is endemic and infection rates appear to be constant throughout the year. Humans are the only known reservoir of HCMV, and natural transmission occurs through direct or indirect interpersonal contact. Between 0.2% and 2.2% of children born in the United States are hepatitis in the uterus. The other 8% to 60% are infected during the first six months of life as a result of infections born or suckled. The high incidence of HCMV infection reactivation in the breast may represent the world's most common mode of HCMV transmission, which is spread to breast milk. In most developed countries, 40% to 80% of children are infected before puberty. In other countries, 90% to 100% of the population is infected in childhood.

HCMV is a member of the herpesvirus family. A typical herpes virion contains an icosadeltahedral capsid, about 100 to 110 nm in diameter, containing linear double-stranded DNA and 162 capsomeres with holes along its long axis. It consists of a core, an epithelial "surface" surrounding the core, and an epidermis containing viral glycoproteins on its surface. The size of virions range from 120 to 130 nm due to the difference in outer layer thickness. The three subgroups of herpesviruses are as follows.

Alphaherpes virinae: HSV, VZV. Various host ranges, relatively short regeneration cycles, rapid proliferation in culture, effective destruction of infected cells, and potential infectivity in the ganglion.

2. Betaherpes virinae: HCMV. Limited host range, long regeneration cycles, slow infection progression in culture, infected cells expand and carrier cells are formed immediately. Viruses can be latent in the gland, lymph cells, kidneys and other tissues.

3. Gammaherpes virinae: EBV. Experimental hosts are extremely narrow in scope, replicate in lymphoblastoid cells, and cause lytic infection in some types of epithelial and fibroblasts.

Eight known human herpesviruses include human herpes simplex virus 1 (HSV-1), human herpes simplex virus 2 (HSV-2), and human herpes virus 3 (Varicella-zoster virus, VZV). , Human herpesvirus 4 (Epstein-Barr virus, EBV), human herpesvirus 5 (Human cytomegalovirus), human herpesvirus 6, human herpesvirus 7, human herpesvirus 8. The herpesvirus genome consists of linear double helical DNA in the virion that is circularized and concammerized while liberated from the viral capsid in the infected cell nucleus (FIG. 10). The herpesvirus genome ranges in size from 120 to 230 kilobase pairs (kbp); The genome is a polymorph within individual viral populations (up to size 10kbp difference). This diversity is due to the repeating sequences at the ends and inside. Herpesviruses can be classified into six groups from A to F based on their overall genomic organization. HSV and HCMV become the E group, in which the sequences at both ends are repeated in a reversed orientation and internally aligned, where the genome is divided into two components, L (Long) and S (Short). Each consists of unique sequences U _L and U _S flanked by the converted repeats (FIG. 10). In this virus, the two components can be converted to each other, and the extracted DNA is composed of four moles of the same number of moles having different relative orientations of the two components (FIG. 11).

HCMV is a beta herpesvirus and is the only one of the beta herpesviruses to be of the E group genome type. The genome of HCMV is approximately 230 kbps in length, fully sequenced (EMBL database accession # x17403) and contains 208 expected open reading frames that are greater than 100 amino acids in length (Mocarski, ES (1996) Ch. 76 In Fields Virology, Third Edition edited by BN Fields, DM Knipe, PMHowley, et al. 2447-2492; Chee et al. (1990) Curr top Microbiol Immunol 154: 125-170). ORFs are designated by their position in unique and repeated regions of the viral genome (TRL, UL, IRL, IRS and US) and numbered sequentially.

In a population of naturally occurring viruses, the genome exists as four isomers (FIG. 11). L-S conjugation can be deleted by freezing the genome of one of the four isomers without drastically affecting the ability of the virus to grow in cells cultured in HCMV as in HSV.

The HCMV genome comprises "a" and "a '" terminal repeat sequences that exist in various numbers while directly oriented at both ends of the linear genome. Various numbers of "a" repeats exist in the converted orientation in the LS junction. The number of "a" sequences at this position is from 1 to 10, of which one is dominant. The size of "a" in HCMV ranges from 700 to 900 bp. The "a" sequence conveys the sequence and the package signal. The package signal is a two highly conserved short sequence element located within the "a" sequence and designated pac-1 and pac-2. The 220-bp fragment carrying both the pac-1 and pac-2 elements is sufficient to deliver sites for cleavage / packages as well as conversion to recombinant CMV constructs. The ends of the linear genome are generated by cleavage, leaving a single 3 'overhang nucleotide at either end of the genome. The genome consists of "b" and "b '" (or TRL and IRL) and "c" and "c'" (or IRS and TRS) located at the unique sequences U _L and U _S that make up the L and S components of the genome. It is characterized by a largely switched repeat, called (Fig. 10).

The HCMV replication cycle is relatively slow compared to other herpesviruses. Viral replication involves the ordered expression of a contiguous set of viral genes. These sets are expressed at different times after infection, ?? (early intermediate), ?? 1 and ?? 2 (early delayed), and ?? 1 and ?? 2 (late) based on the time post-infection transcription accumulated. It includes a set. DNA replication, genome maturation and generation of virions are arranged in order through time control of the appropriate gene product required for each step. ?? Gene products express rapidly. Late genes are delayed and expressed for 24 to 36 hours. Late virions begin to accumulate late infections for 48 hours and reach peak stages at 72 to 96 hours. DNA replication in any fibroblast can be detected early, such as 14-16 hours of late infection. HCMV stimulates host DNA, RNA and protein synthesis. HCMV replicates faster in actively detached cells, and HCMV replication is inhibited by pretreatment of cells with agents that reduce host cell metabolism. The HCMV genome will be round soon after infection. Circles give rise to concatamers and genome conversion takes place within the concatameric form of DNA. The majority of the cloned DNA is longer than the base length and lacks terminal segments based on southern blot analysis.

Target for drug resistance

Drugs currently used to treat HCMV (ganciclovir (GCV), foscarnet, cidofovir) are known to select UL97 phosphotransferase and UL54 virus DNV polymerase for mutation of two viral genes.

UL97: phosphotransferase 707 amino acid (aa) (2121 bp). Mutations associated with GCV tolerance include aa #, ie: 460,520,590,591,592,593,594,595,596,600,603,607,659 and 665. Phosphotransferase proteins have two functional domains. 1) amino terminal 300 aa code for regulatory domain; 2) The carboxyl end 400 aa refers to a catalytic domain. All known drug resistance mutations are found in the catalytic domain (approximately 1.2 kb of sequence). The thymidine kinase gene product (TK) in HSV causes phosphate addition of GCV in cells, and resistance to GCV in HSV is associated with mutations in the thymidine kinase gene. HCMV is homologous with the HSV thymidine kinase gene. Genes that are homologues with UL97 in HSV (UL13) are protein kinases.

UL54: Viral DNA Polymerase, 1242 a.a. (3726 bp). Mutations in this gene end in resistance to foscarnet as well as GCV and other nucleoside analogues (including cidofovir). Mutations associated with foscarnet resistance include aa #, i.e. 700 and 715. Mutations associated with GCV resistance include aa #, ie 301,412,501,503 and 987. The mature protein has four recognized domains: 1) 5'-3 'exoRNase H, 3'-5'exonuclease, a domain that acts as the intended catalyst and a subunit protein that binds the domain.

New therapies being developed include drugs targeted by CMV protease (UL80) and DNA maturase ("terminase").

GCV-resistant HCMV was recovered from the central nervous system of patients with neurological diseases associated with HCMV who had undergone long-term GCV maintenance therapy. Resistant strains of HCMV can be selected and preferentially placed in the CNS. It is not possible to culture the virus from cerebral spinal fluid, but it is possible to amplify HCMV DNA using PCR.

Primary isolates of CMV will replicate slowly. There is also a significant delay in the growth rate of drug resistant clinical isolates. In mixed virus populations the resistant virus population may be covered with sensitive. Therefore, the results of analysis based on the growth of the virus are unreliable.

Most virus culture assays use blood or urine. This is because the blood or urine is easy to obtain. However, viruses obtained from these do not represent viruses of specific tissues from which the disease develops (especially vitreous fluids and Csf). Although there are some amino acid residues that are relatively frequently modified among drug-resistant strains of herpesviruses recovered from patients, a wide distribution of mutations makes the rapid genetic screening method impractical. Importantly, the biological sensitivity to the antiviral susceptibility of HCMV will be more beneficial because of the complexity and diversity of drug-sensitive phenotypes resulting from individual genetic modifications.

Current virus resistance analysis

The definition of viral drug sensitivity is generally understood as the concentration of antiviral drug when viral replication is suppressed at a given percentage (eg IC ₅₀ for antiviral drugs is when 50% of viral replication is inhibited). Concentration.)

Thus, the decrease in viral drug sensitivity is evidence that the antivirus has selected a mutant virus that is resistant to the antiviral drug. Viral drug resistance is generally defined as a decrease in viral drug sensitivity in a given patient. Clinically, viral drug resistance proves that antiviral drugs are less effective or no longer effective for patients.

Several types of assays are useful for monitoring and measuring the sensitivity of HCMV antiviral drugs. The two most commonly used methods are plaque reduction assays and DNA hybridization assays. Plaque reduction analysis is currently considered the standard. Both assays require passage in HCMV isolation and cell culture. In general, it takes four to six weeks to obtain analytical results.

Plaque reduction assays with increased sensitivity can be performed immediately on clinical samples, including blood, urine, bronchoalveolar lavage and spinal fluid. Two assays modified from standard plaque reduction assays detect early or late CMV antigens. The process is essentially the same as the standard plaque reduction assay, except that the virus is tested immediately without preferential passage and the incubation time is reduced to 96 hours (Gerna et al. (1995) J. Clin. Microbiol. 33.738-741). The limitation of these analyzes is that they can only be performed in patients with high levels of viremia. Viral culture remains an essential step in detecting drug resistant isolates.

Another approach is to detect specific viral DNA mutations related to drug resistance. In this assay, PCR primers are used to amplify viral DNA, and restriction sites present in mutant viral DNA other than wild-type DNA are used to determine the genotype of viral DNA. Analysis of two PCR products using a total of three or four restriction digests ranged from 78% to tolerance to ganciclovir (Chou et al. (1995) J. Infect. Dis. 172,239-242). It is known to be suitable for detecting 83% of UL97 (some mutation of UL97 that specifies genetic information for phosphotransferase results in resistance to ganciclovir). The main limitation of this assay is that infrequent mutations or new resistance mutations are not identified. In addition, DNA polymerase mutations (UL54), which show the high degree of ganciclovir resistance and the possibility of high multi-drug resistance, are not detected.

It is an object of the present invention to provide a drug sensitivity and resistance test that can show whether a patient's viral population is resistant to a prescribed drug. Another object is to provide a test that allows a physician to substitute one or more drugs in a therapeutic regimen for patients who are resistant to a given drug (s) after a previous course of treatment. Yet another object of the present invention is to provide a test that allows the selection of an effective drug diet in treating viral infections. It is yet another object of the present invention to provide a safe, standardized, fast, accurate and reliable analysis of drug sensitivity and tolerance for clinical and research applications. It is also an object of the present invention to assess the biological efficacy of candidate drug compounds that act on specific viral genes and / or viral proteins, particularly related to viral drug resistance and cross resistance. It is also an object of the present invention to provide a means and composition for assessing viral drug resistance and sensitivity. The above objects of the present invention will become clear throughout this specification.

Figure 1: HCV Replication

Schematic of the HCV replication cycle. The virion binds to the cell surface by a specific interaction between the viral surface glycoprotein and the cell surface receptor (1). Nucleocapsid cores are released into the cytoplasm 4 following membrane fusion with receptor-mediated endocytosis 2 and low pH. The virion RNA is translated in close association with the endoplasmic reticulum, and the polyprotein is a specific endoproteolitic mediated by either a host signal peptidase or one of two viral proteases (5) in the ER. It is processed by endoproteolytic cleavage. After sufficient production of the nonstructural protein, viral RNA is replicated through the negative strand intermediate to generate more positive sense RNA for translation and packaging into a new virion (6). Structural proteins and RNA are secreted via cell pathways 8, 9 to assemble and form new viral particles that grow into ER and release later virions.

Figure 2: HCV genome construct

Schematic of 9.5 kb HCV RNA. Said individual HCV protein size is indicated using the putative function associated with that indicated below in HCV polyprotein. Cleavage sites for the proteolytic process are indicated by triangles (open triangles for host signal peptidase, black triangles for NS2 / 3, and gray triangles for NS3 / 4A). The internal ribosomal entry (IRES) is located at the 5 'end of the RNA and comprises several sequences and a region (UTR) that is not fully translated at the beginning of the C ORF. The 3 'end of the RNA includes a poly (A) or poly (U) bottom, depending on the type of HCV.

Figure 3: Resistance Test Vectors (Luciferase Fusion Proteins)

A, Schematic of a resistance test vector (pXHCVluc with X being CMV or T7) with patient sequence receptor sites for carrying sections derived from patients indicated by arrows below polyprotein (PSAS). Promoter and terminator sequences are generally shown in this figure as well as in the following figures, as several different regulatory element types may be used (as described below). The luciferase reporter gene is expressed as a fusion protein using HCV polyprotein and then divided by the activity of NS3 / 4A.

B. Transfection method using DNA transfection of a resistance test vector (pCMVHCV-luc) comprising a CMV IE promoter and an SV40 polyadenylation signal. RNA is transcribed in the transfected cell nucleus by the RNA polymerase of the cell and then transported to the cytoplasm where translation and replication can occur.

C. Transfection method using DNA transfection of a resistance test vector (pT7HCV-luc1) comprising a T7 RNA polymerase promoter and a T7 RNA polymerase terminator. DNA is transfected with cells expressing T7 RNA polymerase (e.g. after infection with recombinant vaccine virus or by cotransfection with T7 RNA polymerase expressing plasmid), and RNA is transfected by T7 polymerase. Transcribed in the cytoplasm.

D. Transfection Method Using RNA Transfection of RNA Derived from Resistance Test Vector (pT7HCV-luc2) The resistance test vector is positioned at the 3 'end for linearization of DNA prior to ex vivo transcription with the T7 RNA polymerase promoter. Included restriction sites. Synthetic RNA can then be directly transfected into cells and translation and replication can occur.

Figure 4: Resistance test vector (bicistronic luciferase expression).

Structure of the resistance test vector (pXHC-IRESluc) containing the IRES element for luciferase translation. IRES may be native HCV IRES or may be derived from other viruses using such elements for internal initiation of mRNA. Expression of luciferase occurs by translational initiation from bicistronic RNA in the cytoplasm of transfected cells.

Figure 5: Resistance test vector (positive sense small genome).

Schematic of resistance test vectors (pXHCV and pXIRESluc) containing positive sense luciferase RNA small genome. Two constructs are cotransfected into cells and HCV nonstructural proteins expressed from pXHCV act on both RNAs that replicate and package them. The cloned RNA is packaged into a later virion and the virion can be used for infection of a new subject cell, and the subject cell is either transfected with pXHCV or infected with HCV, and the luciferase minigenome (now Positive sense RNA) is expressed.

7: Resistance test vector (defective genome).

Schematic of resistance test vectors (pXluc-NSHCV and pXSHCV) expressing binding genomic RNA. Two constructs are cotransfected into cells, nonstructural proteins expressed from pXluc-NSHCV act to replicate luc-NSHCV RNA, and the newly cloned RNA uses structural proteins (C, E1 and E2) from pXSHCV. Packaged in virion. Subsequent virions are then used for infected new cells.

Figure 8: Resistance test vector (BVDV NS3 / 4A chimeras, luc fusion protein)

The genomic schematic of BVDV is shown at the top. The HCV protease cleavage site is indicated by a gray triangle and the BVDV protease cleavage site is represented by a reticulated diamond (single peptide degrading enzyme and NS2 / 3 protease cleavage site not shown). Resistance test vector pXBVDV (HCVNS3) luc includes BVDV structural protein gene, BVDV NS2, HCV NS3 / 4A protease, and BVDV NS4B and NS5, and the cleavage site of the nonstructural protein region is altered so that they are directed to HCV NS3 / 4A protease Is recognized by. The luciferase reporter gene is expressed by fusion with chimeric polyprotein and released by cleavage of HCV NS3 / 4A.

Figure 9: Resistance test vector (BVDV NS5B chimera, luc fusion protein)

Is located in the amino terminal regions of the resistance test vectors pXBVDV (HCVNS5B) luc, including the BVDV structural protein gene, BVDV NS2, NS3 / 4 protease and NS4B and NS5A, and HCV NS5B, and 3 'UTR and 5'UTR and C ORF The cis-activity regulatory element recognized by NS5B polymerase is derived from HCV. The luciferase reporter gene is expressed in fusion with chimeric polyprotein and is released by cleavage of BVDV NS3 / 4A.

Figure 10:

A. Schematic of the HCMV Genome. The genome has direct terminal repeats referred to as "a" which exist from 1 to 10 copies per genome. The "a" sequence is also present in the opposite orientation at the LS junction (a '). The converted repeats "b" and "c" are referred to as blocks and "b" and "c" are used to refer to "b" and "c" repeats in the antisense orientation. U _L and U _S refer to the unique regions of the genome L and S components. Blocks of ORFs are shown below the genome. Three genes encoding for the antiviral agent, UL54, US80 and UL97 subjects are indicated by arrows. OriLYT refers to the HCMV soluble source of replication.

B. Circulation of the Monomer HCMV Genome After Infection

Figure 11:

A. Schematic of the HCMC Genome

B. Circulation of the Monomer HCMV Genome After Infection

C. Four isomers of the HCMV genome following conversion of the genome during replication.

The arrows below the U _L and U _S fragments highlight the conversion of genomic L and S fragments to each other.

Figure 12:

Schematic of HCMV Genome. Β _2.7 transcripts present in the genomic “b” region are shown to be modified in that present in wild type HCMV (A) and to HCMV-β _2.7 -F-IG (B).

13:

Schematic of amplicon plasmid lacking function indicator genes. ORF gene X refers to an antiviral subject (eg UL54, UL80, UL97). Large black arrows represent promoters and circles (pA +) represent polyadenylation signals. The promoter and polyadenylation signal may be appropriately derived from the HCMV genome to the viral gene / drug subject (gene X) or may be a heterogeneous regulatory element described in the text. PSAS represents the sequence receiving site of the patient.

14:

Schematic of an amplicon plasmid with a nonfunctional indicator gene comprising an altered promoter. ORF gene X refers to an antiviral subject (eg, UL54, UL80, UL97). Large black arrows indicate promoters and round circles (pA +) indicate polyadenylation signals. Promoters and polyadenylation signals may be appropriately derived for viral genes / drug subjects (gene X) from the HCMV genome or may be heterogeneous regulatory elements described in the text. PSAS represents the sequence receiving site of the patient. Such amplicons include altered promoter cassettes as described in the text.

Figure 15:

Schematic of an amplicon plasmid containing an altered coding region and a nonfunctional indicator gene. ORF gene X refers to an antiviral subject (eg UL54. UL80. UL97). Large black arrows represent promoters and circles (pA +) represent polyadenylation signals. Promoters and polyadenylation signals may be appropriately derived from the HCMV genome for viral genes / pharmaceutical subjects (gene X) or may be heterogeneous regulatory elements described in the text. PSAS represents the sequence receiving site of the patient. This amplicon comprises a modified coding region cassette as described in the text.

Figure 16:

Schematic of the four isomers of the HCMV genome present after viral replication and the relative position of the altered coding region cassette after rearrangement. Note that in panel C, two half of the cassettes are properly oriented for direct expression of the reporter gene. The arrangement shown in panel B will also properly juxtapose two half of the cassettes following concammerization of the rearranged genome.

Figure 17:

Schematic of amplicon plasmid containing function indicator genes. ORF gene X refers to an antiviral subject (eg UL54, UL80, UL97). Large black arrows indicate promoters and round circles (pA +) indicate polyadenylation signals. Promoters and polyadenylation signals may be appropriately derived for viral genes / drug subjects (gene X) from the HCMV genome or may be heterogeneous regulatory elements described in the text. PSAS represents the sequence receiving site of the patient.

Such amplicons comprise a functional indicator gene cassette as described in the text.

The following description makes the invention described herein more fully understood. The following flow chart illustrates various vectors and host cells that can be used in the present invention. But not everything is included.

vector

Indicator Cassette + Virus Vector

(Functional / Non-Function Indicative Genes)

↓

Directing gene virus vector

(Functional / non-functional indicator genes)

+ Patient sequence receiving site

+ Section derived from patient

Immunity test vector

(Section derived from patient + indicating gene)

Host cell

Host Cell Packaging-Transfected by Packaging Expression Vector

Resistance Test Vector Host Cells-Packaged Host Cells Transfected with the Resistance Test Vector

Subject host cell-resistance test vector A host cell that is infected with a resistance test vector virus particle produced by a host cell. Components of the resistance test vector system comprising the indicator gene will either be delivered to the subject host cell upon infection or stably integrated into the subject host cell chromosomal DNA.

Immunity test vector

"Endurance test vector" means one or more vectors comprising both DNA or RNA comprising a patient-derived fragment and an indicator gene. If the resistance test vector contains more than one vector, the patient-derived fragments may be included in one vector and the indicator gene may be included in another vector. Immunity test vectors containing one or more such vectors are referred to herein as immunity test vector systems for clarity, but are nevertheless understood as immunity test vectors. Thus, the RNA or DNA of the resistance test vector is contained within one or more DNA or RNA molecules. In one embodiment, the resistance test vector is obtained by inserting a patient-derived fragment into a directed gene viral vector. In another embodiment, a resistance test vector is obtained by inserting a segment derived from a patient into a package vector while the indicator gene is included in a second vector (eg, the indicator viral vector). As used herein, “patient derived fragment” refers to one or more viral sections obtained directly from a patient using various means. Viral DNA or complementary DNA (cDNA) prepared from viral RNA present in other physical fluids, serum or cells (e.g., peripheral blood mononuclear cells, PBMCs) of an infected patient by such means. There is a polymerase chain reaction (PCR) amplification, or molecular cloning, of the group of patient-derived sections used. Virus fragments are obtained "directly" from the patient without the virus passing through the culture or, if the virus is cultured, by minimal passage to remove the mutation selection from the culture. A “viral segment” refers to a viral gene or any functional viral sequence that encodes a functional viral sequence or gene product (eg a protein) that is the subject of an antiviral drug. As used herein, "functional viral sequence" means a functional such as an enhancer, a promoter, a polyanimation site, an active site of a trans activator, an internal ribosomal entry site, a translation frameshift site, a packaging sequence, an integration sequence, a splicing sequence Refers to any nucleic acid sequence (DNA or RNA) having activity. If the drug targets more than one functional viral sequence or viral gene product, the patient-derived fragments corresponding to each viral gene will be inserted into the resistance test vector. For combination therapies in which two or more antivirals of two different functional viral sequences or viral gene products are evaluated, the patient-derived segments corresponding to each functional viral sequence or viral gene product are subjected to a resistance test vector. Will be inserted. Patient-derived fragments are inserted into a specific restriction site or designated location, called a patient sequence receiving site, in an indicator viral vector or, for example, a packaging vector that depends on the particular fabrication described herein.

As used herein, “patient-derived fragments” encompass fragments derived from humans and various animal species. Such animal species are not limited to chimpanzees, and other primates, horses, cattle, cats and dogs are also included.

Patient-derived sections can also be incorporated into resistance test vectors using several different cloning techniques. Such cloning includes, for example, cloning by introducing a class II restriction site into both the plasmid backbone and the patient-derived fragment, uracil DNA glycosylase primary cloning, or site specific recombination, or exonuclease overhang cloning. .

Patient-derived fragments are obtained by introducing a patient sequence receiving site at the end of a patient-derived fragment introduced into the resistance test vector described below by methods such as molecular cloning, gene amplification or modifications thereof. For example, in gene amplification methods such as PCR, restriction sites corresponding to patient sequence receiving sites can be incorporated at the primer ends used in the PCR reaction. Similarly, in molecular cloning, such as cDNA cloning, the restriction sites can be combined at the ends of the primers used for the first or second strand cDNA synthesis, in which DNA has been cloned or not, or in methods such as primer repair of DNA. The restriction site may be incorporated into the primer used for the recovery reaction. Patient sequence receptor sites and primers are designed to enhance the display of patient-derived fragments. The set of resistance test vectors that devised the patient sequence receiving site alone provide an indication of the patient-derived segment that exhibits a lower degree than actually present in one resistance test vector.

The resistance test vector system introduces a patient sequence receptive site, modifies or clones a patient-derived fragment, and modifies the amplified or cloned sequence into a patient by modifying the indicator gene viral vector (described below) or packaging the vector. It is prepared by precise insertion into a viral vector or packaging vector at the sequence receiving site. Resistance test vector systems constructed from directed gene viral vectors are alternately constructed from viral vectors or subgenomic viral vectors and indicator gene cassettes, each of which is described below. Resistance test vector systems constructed from packaging instruction vectors are alternately constructed from packaging vectors of genome or packaging vectors of subgenomics, and directed gene cassettes, each of which is described below. Resistance test vectors are then inserted into host cells. Or a resistance test vector (also referred to as a tolerance test vector system) introduces a package vector into the patient sequence receiving site, amplifies or replicates the patient-derived fragment and inserts the amplified or replicated sequence into the packaging vector exactly at the patient sequence receiving site And the package vector is transfected with the indicated gene viral vector.

In one preferred embodiment, the resistance test vector is introduced into a packaging host cell with a packaging expression vector as defined below and the resistance test vector virus particles used in drug resistance and sensitivity tests referred to as "particle-based tests". Can produce In another preferred embodiment, the resistance test vector can be introduced into a host cell in the absence of a packaging expression vector to perform drug resistance and sensitivity tests referred to herein as "particle-based tests".

As used herein, a "package expression vector" provides a gene that is required by a packaging protein (e.g., a structural protein such as a core polypeptide of the core) transactive element, or a replication defective virus. In this situation, the viral genome, which is capable of replication, is slightly weakened so that it cannot replicate on its own. This means that although the package expression vector can produce the transactivity or lacking genes required to rescue defective viral genomes in cells containing the weakened genome, the weakened genome cannot be rescued on its own.

Indicator or Gene

An “indicator or indicator” is a nucleic acid, or indicator, that encodes a protein, DNA, or RNA structure that can be measured or recognized in a modifiable or recognizable manner, such as a color or light of a measurable wavelength. In the case of DNA or RNA used, it refers to the change or occurrence of a particular DNA or RNA structure. Preferred examples of indicator genes are the E. coli lacZ gene, which encodes beta galactosidase, and the luciferase from Photonis pyralis or Renillaniformis. Luc gene, E.coli phoA gene encoding alkaline phosphatase, green fluorescent protein, bacterial CAT gene encoding chloramphenicol acetyltransferase, and bacterial β-lactamase ) Genes. Preferred examples of indicator genes also include radioimmunoassay (RIA), including, for example, growth factors, cytokines and cell surface antigens (e.g. growth hormone, I1-2 or CD4, respectively). Or secreted proteins or cell surface proteins that are readily measured by assays such as fluorescence active cell sorting (FACS). An “indicator” is also understood to include a selection gene and is considered as an indicator capable of selection. Examples of appropriately selectable indicators of mammalian cells include dihydrofolate reductase, thymidine kinase, E. coli gpt, antibiotic hygromycin, neomycin, It is a gene encoding resistance to puromycin and zeocin. In the above example for the indicator gene, the indicator gene and the patient-derived segment are separate, distinct and divided genes. In some cases, patient-derived fragments may also be used as indicator genes. In one embodiment where the patient-derived segment corresponds to one or more viral genes that are targets of an antiviral, one of the viral genes described above, for example the HCV protease gene, alternates the ability to cleave a chromosomal substrate or chromosomal substrate Its ability to activate an inactive enzyme source that cleaves can also act as an indicator gene, in each case causing a color reaction. In the second embodiment, the HCMV phosphotransferase gene functions as an indicator gene by its ability to upregulate and downregulate its activity by phosphorylating a substrate. All of the indicator genes above are either "functional" or "non-functional" but in each case the expression of the indicator gene of the target cell ultimately depends on the action of the patient-derived fragment.

Functional indicator genes.

For “functional indicator genes” the indicator genes may be expressed in “packaging host cells / resistant test vector host cells”, regardless of patient-derived fragments, as described below. However, functional indicators cannot be expressed in the target host cells described below without the production of functional resistance test vector particles and effective infection of the target host cells with them. In one embodiment of a functional indicator, the indicator cassette comprising a regulatory element and a gene encoding the indicator protein is a viral viral vector or packaging virus in the same or opposite transcriptional orientation as the natural or foreign enhancer / promoter of the viral vector. Inserted as a vector. For HCV, examples of functional indicator genes are the same as or opposite to the T7 phage RNA polymerase promoter (herein referred to as the T7 promoter) associated with the indicator gene and its promoter (CMV IE enhancer / promoter), respectively, with the HCV enhancer / promoter or viral vector. Place in the transfer orientation.

Nonfunctional indicator genes.

On the other hand, an indicator gene is a "non-functional" that is not effectively expressed in a package host cell transfected with a resistance test vector until it is converted into a functional indicator gene through one or more actions of the patient-derived fragment product. "It can be. Hereinafter, the host cell is referred to as a resistance test vector host cell. The indicator gene is made nonfunctional through genetic manipulation according to the present invention.

1.Replaced promoter

In a first embodiment the indicator gene becomes nonfunctional because of the position of the promoter. This is because although the promoter is in the same transcription direction as the indicator gene, it follows rather than precedes the indicator coding sequence. Such a misplaced promoter is called a "substituted promoter". Non-functional indicator genes and substituted promoters thereof become functional by one or more actions of the viral protein. In the case of HCMV one embodiment of a non-functional indicator gene with a substituted promoter places the promoter in the "b" region and the IRES and encodes and terminates the region of the indicator gene in the c '/ and / or adjacent U _S region. In a resistance test vector, the non-functional indicator is converted to a functional indicator inversion of the U _L / U _S junction obtained by relocating the CMV IE promoter associated with the indicator gene coding region upon target cell infection. After the inversion, appropriately arranged indicator genes are expressed in target cells.

Substituted promoters are eukaryotic or prokaryotic promoters capable of being transcribed in target host cells. In the first embodiment, the CMV IE promoter / enhancer region may be used. In the second embodiment the promoter should be small enough to be inserted into the viral genome without interfering with viral replication. More preferably, a promoter, such as a bacteriophage promoter, which is small in size and capable of being transcribed by a single subunit RNA polymerase introduced into the target host cell, will be used. Examples of such bacteriophage promoters and RNA polymerases homologous to them include those of phage T7, T3 and Sp6. Nuclear localization sequences (NLSs) can be attached to RNA polymerases to position expression of RNA polymerase in the nucleus where they are needed to transcribe repaired indicator genes. The NLS will be obtained from a nuclear-transfer protein such as SV40 T antigen. If phage RNA polymerase is used, an internal ribosomal entry site (IRES), such as the EMC virus 5 'untranslated region (UTR), can be added before the indicator gene for translation of transcripts that are generally not capped. . In the case of HCMV, a substituted promoter can be introduced at any position that does not destroy the cis-acting elements required for HCMV DNA replication. Blocking sequences can be added to the ends of resistance test vectors with inappropriate expression of nonfunctional indicators due to transfection artifacts (DNA linkages). In the embodiment of the substitutional T7 promoter described above, such block sequences also consist of T7 transcription terminators, positioned to interfere with transcriptional read through resulting from the DNA chain.

2. Substituted encryption domain

In a second embodiment the indicator gene becomes nonfunctional because of the relative positions of the 5 'and 3' coding regions of the pointing gene, because the 3 'coding region precedes the 5' coding region. The incorrectly designated encryption region is referred to as a "permuted coding region". The orientation of the nonfunctional indicator gene may be the same as or opposite to that of the viral vector's natural or foreign promoter / enhancer, as mRNA encoding the functional gene is produced in either orientation. Nonfunctional indicator genes and substituted coding regions thereof become functional by one or more actions of the patient-derived fragment product. In the case of HCMV, an embodiment of a non-functional indicator gene having a substituted coding region may include a 5 'directed gene encoding region having an associated promoter in b region and a 3' pointing gene coding region in which the coding region has a reverse transcription orientation. place in the c 'region and / or in the adjacent U _S region of the HCMV genome. In both embodiments the 5 'and 3' coding regions may also be associated with splice donor and receptor sequences, respectively, which may be heterologous or artificial splicing signals. The indicator gene cannot be functionally transcribed by the associated promoter or viral promoter because the substituted coding region interferes with the formation of the functional transcript. Nonfunctional indicator genes of the resistance test vector are converted to functional indicator genes by inversion of the U _L / U _S junction obtained by rearranging the interrelated 5 'and 3' indicator gene coding regions upon infection of the target cell. After transcription by a promoter associated with the 5 'coding region, RNA with the 5' and 3 'coding regions properly arranged produces a functional indicator gene.

3. Negative Strand RNA Coding Region—In the third embodiment, the indicator gene becomes nonfunctional due to the fact that it is expressed from RNA that is negative sense with respect to virally encoded gene products. Expression of luciferase from mini-genomic RNA containing the luc gene in the opposite orientation requires negative strand RNA made during viral replication (FIG. 5).

Pointer Gene Virus Vector-Preparation

As used herein, "director viral vector" refers to a vector comprising an indicator gene, its regulatory elements, and one or more viral genes. The indicator viral vector is assembled from the pointer cassette and a "virus vector" as defined below. The indicator viral vector may additionally include an enhancer, a splicing signal, a polyadenylation sequence, a transcription terminator or other regulatory sequence. In addition, the indicator viral vector may be functional or non-functional. If the target viral segments of the antiviral drug (patient-derived for drug sensitivity and resistance testing) are not included in the indicator viral vector, they are supplied from a second vector, which may be a packaged viral vector. An "directive cassette" includes an indicator gene and a regulatory element. A "viral vector" refers to a vector comprising some or all of the following: viral genes, regulatory sequences, viral package sequences encoding gene products. In addition, the viral vector comprises one or more viral segments, one or more of which may be the target of an antiviral drug. Two embodiments of viral vectors comprising viral genes are referred to herein as "genomic virus vectors" and "subgenomic virus vectors". A "genomic virus vector" is a vector that includes the deletion of one or more viral genes which renders viral replication impossible, while preserving the mRNA expression and processing characteristics of the complete virus. In one embodiment for HCV drug sensitivity and resistance testing, genomic viral vectors include C, E1, E2, NS2, NS3, NS4 and NS5 (see infra, pages 52-53). In one specific example of HCMV drug sensitivity and resistance testing, genomic viral vectors include viruses deleted from one or several genes, such as JL54, UL80, UL97. "Subgenomic viral vector" refers to a vector comprising a coding region of one or more viral genes capable of encoding a protein targeted by an antiviral drug. For HCV, preferred embodiments are subgenomic viral vectors comprising the HCV NS2, NS3, NS4, NS5 genes. (FIG. 6) For HCMV, preferred embodiments are one or several viral genes such as UL54, UL80, UL90. It is a subgenomic virus vector comprising the HCMV amplicon plasmid containing. Examples of viral clones used for viral vector production are Toune, Toledo and AD169. Viral coding genes may be under the control of a natural enhancer / promoter or foreign virus or cellular enhancer / promoter. A preferred embodiment of the HCV drug sensitivity and resistance test is that the viral coding region of the genome or subgenomic is subjected to control of the T7 promoter. A preferred embodiment of the HCMV drug sensitivity and resistance test is that the viral coding region of the genome or subgenomic is subjected to control of the endogenous HCMV promoter. In the case of an indicator viral vector comprising one or more viral genes which are targets or which encode a protein of interest for an antiviral drug, said vector comprises a patient sequence receptor site. The patient-derived fragment is inserted at the patient sequence receptor site of the indicator viral vector called the resistance test vector as described above.

A "patient sequence receptor site" is a site in a vector for insertion of patient-derived fragments. Such sites include 1) intrinsic restriction sites introduced into the vector by site specific mutations; 2) intrinsic restriction sites that occur naturally in the vector; 3) selected sites where patient-derived fragments are inserted in alternative cloning methods (eg, UDG cloning, exonuclease overhang cloning); 4) site specific recombination target sites. In one embodiment the patient sequence receiving site is introduced into an indicator gene viral vector. The patient sequence receiving site is preferably located in or near the coding region of the viral protein that is the target of the antiviral drug. The viral sequence used for introduction of the patient sequence receiving site is preferably selected such that no change or conservative change occurs in the amino acid coding sequence found at that position. Preferably the patient sequence receiving site is preferably located within a relatively conserved region of the viral genome to facilitate the introduction of patient-derived fragments. Or the patient sequence receiving site is located between functionally important genes or regulatory sequences. The patient sequence receiving site may be located at or near an area within the virus genome that is relatively conserved to allow priming by primers used to introduce the corresponding restriction site into the patient-derived fragment. To further enhance the description of patient-derived fragments, the primers may be designed into degenerate pools to accommodate viral sequence heterogeneity or may include residues such as deoxyinosine I with many base pair possibilities. A set of resistance test vectors with patient sequence receiving sites that clarify the same or overlapping restriction site intervals may be used to describe drug resistance and sensitivity to account for patient-derived fragments that contain the same internal restriction site as a given patient sequence receiving site. It will be used together in the test and therefore devalued in only one immunity test vector.

Host cell

Resistance test vectors are introduced into host cells. Suitable host cells are mammalian cells. Suitable host cells are obtained from human tissues and cells that are the principal targets of viral infection. In the case of HCV, host cells include human cells such as hepatocytes, hepatoma cell lines and other cells. For HCMV, suitable host cells include MRC5, HF, human epidermal fibroblasts and other cells. Using human-derived host cells, the antiviral drug enters the cell effectively and is transformed into a form suitable for the metabolism of the antiviral inhibitor by cellular enzyme machinery. Host cells are referred to herein as "packaging host cells," "resistant test vector host cells," or "target host cells." "Packaging host cell" means a host cell that provides the trans-acting factors and viral packaging proteins required by the replication defective viral vectors used herein, such as the resistance test vector, to produce a resistance test vector virus particle. . The package protein can be supplied by expression of the viral gene contained in the resistance test vector itself, in the package expression vector (s) or both. A package host cell is a host cell transfected with one or more package expression vectors and when infected with a resistance test vector is referred to herein as a "tolerance test vector host cell", sometimes referred to as a package host cell / resistance test vector host cell. do. Preferred host cells used as package host cells for HCV include huh7, HepG2. Preferred host cells used as package host cells for HCMV include MRC5 and HF. "Target host cell" means a cell infected with a resistance test vector virus particle produced by a resistance test vector host cell in which expression or inhibition of an indicator gene occurs. Preferred host cells used as target host cells for HCV are HepG2 (Hiramatsu et al. (1997), J. Viral Hepatol. 4 (suppl. 1), 61-67), Huh7 (Yoo et al. (1995), J Virol. 69, 32-38), Vero (Valli et al. (1997) Res. Virol. 148, 181-186), Molt4Ma (Shimizu et al. (1992) .PNAS 89, 5477-5481), HPBMa (Shimizu et al. (1993), PNAS 90, 6037-6041; Shimizu and Yoshikura (1994), J. Virol. 68, 8406-8408; Shimizu et al. 1994. Virol. 68. 1494-1500), MT-2 (Mizutani et al. (1996). J. Virol. 70, 7219-7223). Preferred host cells used as target host cells for HCMV include MRC5 and HF.

Drug Sensitivity and Tolerance Testing

Drug sensitivity and resistance testing of the present invention can be performed in one or more host cells. Viral drug sensitivity is determined by the concentration of the antiviral agent when the indicator expression is inhibited at a predetermined rate (e.g., the IC50 of the antiviral agent is the concentration when 50% of the indicator expression is inhibited). . The standard curve for drug sensitivity of a given antiviral drug is a virus test from a standard experimental virus fragment or a patient who has never received drug administration using the methods of the present invention (eg, a patient who has never received any antiviral drug). Can be deployed. Correspondingly, viral drug resistance is measured by increased suppression of indicator expression, ie, reduced indicator expression, such that the subject patient is compared with drug sensitivity as described above, or the same patient is continuously measured over time. Of viral drugs is a decrease in sensitivity.

In the first type of drug sensitivity and resistance test, resistance test vector virus particles are produced by the first host cell (resistant test vector host cell) prepared by transfecting the package host cell with the resistance test vector and packaging with an expression vector. Resistance test vector virus particles are then used to infect a second host cell (target host cell) whose indicator expression is measured. The two cell systems which have been transfected with the resistance test vector and then called the resistance test vector host cell and the resistance test vector host cell are used in the case of functional or non-functional indicator genes. The functional indicator genes are effectively expressed upon transfection of the packaged host cell and it is necessary to infect the target host cell with the resistance test vector host cell supernatant to perform the test of the present invention. Non-functional indicator genes with substituted promoters, substituted coding regions, or negative sense strand directed RNA are not effectively expressed upon infection of the package host cell. Therefore, infection of the target host cell can be obtained through co-cultivation of the resistance test vector host cell and the target host cell or infection of the target host cell using the supernatant of the resistance test vector host cell. In the second type of drug sensitivity and resistance test, a single host cell (resistant test vector host cell) also functions as a target host cell. Package host cells are transfected to produce resistance test vector virus particles, and some package host cells are also targeted for infection by resistance test vector particles. Drug sensitivity and resistance testing using a single host cell type is possible with a viral resistance test vector containing a non-functional indicator gene with a substituted promoter, a substituted coding region, and a negative sense strand directed RNA. Such indicator genes are not effectively expressed in the infection of the first cell but are effectively expressed in the second infection. This provides an opportunity to measure the effectiveness of antiviral drugs. In the case of drug sensitivity and resistance tests using resistance test vectors consisting of functional indicator genes, neither co-cultivation nor resistance and sensitivity tests using one cell type can be used to infect target cells. Resistance test vectors composed of functional indicator genes require two cellular systems using supernatants filtered from the resistance test vector host cells to infect target host cells. In one embodiment of the invention in the case of HCV, the particle-based resistance test comprises a resistance test vector derived from a viral vector of the genome, eg, pXHCV-luc; pXHCV-IRESluc; pXHCV / pxIRESluc; pXHCV / pXASIRESluc; pXluc-NSHCV / pXsHCV; pXBVDV (HCVNS3) luc; pXBVDV (HCVNS5B) luc. In a preferred embodiment of the invention in the case of HCMV, the particle-based resistance test comprises a resistance test vector obtained from a viral vector of the genome, ie pA-CMV-VS-gene X; pA-CMV-CS-gene X; pA-CMV-VS-gene X- (NF-IG) PP / pA-CMV-CS-gene X- (NF-IG) PP; pA-CMV-VS-gene X- (NF-IG) PCR / pA-CMV-CS-gene X- (NF-IG) PCR; Performed with pA-CMV-VS-gene XF-IG / pA-CMV-CS-gene XF-IG and they were infected together by package expression vector HCMVβ _2,7 FIG / ?? gene X or HCMV ?? gene X.

For particle-based sensitivity and resistance tests, resistance test vector virus particles are produced by first host cells (resistant test vector host cells) prepared by transfecting a package host cell with a resistance test vector and a package expression vector. Resistance test vector virus particles are then used to infect a second host cell (target host cell) whose expression of the indicator gene is measured. In sensitivity and resistance tests based on the second type of particle, a single host cell type (resistance test vector host cell) serves two purposes. That is, in a given culture some package host cells are transfected to produce resistance test vector virus particles, and in the same culture some host cells are targets of infection by the resistance test vector particles produced. Resistance tests using a single host cell type have been altered because the indicator gene is effectively expressed in the infection of acceptable host cells, but not effectively expressed in host cells of the same type, providing an opportunity to measure the effectiveness of antiviral agents. This is possible by resistance test vectors containing nonfunctional indicator genes with promoters. For similar reasons, resistance tests using two cell types can be performed by co-culturing the two cell types, such as by infecting the second cell type with viral particles obtained from the supernatant of the first cell type.

Package host cells are transfected with the resistance test vector and the appropriate package expression vector to produce a resistance test vector host cell. Each antiviral agent for HCV, including protease inhibitors, IRES inhibitors, combinations thereof, as well as polymerase inhibitors, is added to each package host cell plate at the time of infection in the appropriate concentration range. 24 to 48 hours after transfection, the target host cell is infected by co-culture with resistance test vector virus particles obtained from the supernatant of the resistance test vector host cell or resistance test vector host cell. Each antiviral agent or combination thereof is added to target cells prior to or upon infection to achieve the same final concentration of a given agent (s) present during transfection.

Expression or inhibition of indicator genes in target host cells infected with the supernatant of the co-infected or filtered virus is determined by indicator expression analysis, for example, if the pointer gene is the firefly luc gene, luciferase. It is determined by measuring its activity. The decrease in luciferase activity observed for target host cells infected with a given preparation of resistance test vector virus particles in the presence of a given antiviral agent (s) compared to a control advanced in the absence of antiviral agents. A sigmoidal curve is related to the log concentration of the antiviral agent. This inhibition curve is used to calculate the exact inhibitory concentration (IC) of the agent or drug combination for the viral target product encoded by the patient-derived fragments present in the resistance test vector.

In the case of one cell sensitivity and resistance test, the host cell is transfected with the resistance test vector and the appropriate package expression vector (s) to produce the resistance test vector host cell. Each antiviral agent or combination thereof is added to each plate of transfected cells when they are transfected in the appropriate concentration range. At appropriate times after transfection, cells are collected and analyzed for the luciferase activity of fireflies. Since the transfected cells in the culture do not effectively express the indicator gene, not only the infected cells but also the transfected cells in the culture can act as target host cells for the expression of the indicator gene. Luciferase activity observed on cells transfected in the presence of a given antiviral (s) is generally compared with the log concentration of the antiviral agent as a sigmoidal curve when compared to the control in the absence of the antiviral agent. Related. This inhibition curve is used to calculate the exact inhibitory concentration of the drug or drug combination against the viral target product encoded by the patient-derived fragments present in the resistance test vector.

Antiviral Drugs / Drug Candidates

The antiviral drug added to the test system is added at a selected time depending on the target of the antiviral drug. For example, HCV protease inhibitors are added to each plate of package host cells when they are transfected with a resistance test vector at an appropriate concentration range. HCV protease inhibitors are added to target host cells upon infection to achieve the same final concentration added during transfection. In HCMV, protein inhibitors including phosphotransferase, GCV, cidofovir, and foscarnet, DNA polymerase and phosphatase were transfected with resistance test vector virus particles at test concentrations. It is added to each of the target host cells upon infection. Optionally, antiviral agents may be present during the analysis. Test concentrations are selected within a range of concentrations designed to provide a satisfactory inhibition profile for resistant or sensitive isolates.

In another specific embodiment of the invention, candidate antiviral compounds are tested in the drug sensitivity and resistance test of the invention. Candidate antiviral compounds are added to the test system at appropriate concentrations and selected times depending on the protein target of the candidate antiviral. Optionally, one or more candidate antiviral compounds will be tested or one candidate antiviral compound will be tested in combination with an approved antiviral drug such as GCV for HCMV or a compound in clinical trials. The effect of the candidate antiviral will be assessed by measuring the expression or inhibition of the indicator gene. In another aspect of this embodiment, drug sensitivity and resistance tests can be used to screen for viral mutations. After identifying mutations that are resistant to a known antiviral or candidate antiviral, the resistant mutation is isolated and the DNA is analyzed. Thus, a library of viral resistant mutations can be assembled to allow for the screening of candidate antiviruses. This will allow one general technique to identify effective antivirals and to devise effective treatments.

Common Materials and Methods

Most of the techniques used to construct vectors, transfect and infect cells are widely practiced in the art, and most practitioners are familiar with standard materials describing particular conditions and processes. Conveniently, however, the following provide guidelines.

"Plasmids" and "vectors" are designed by the lowercase case p followed by letters and / or numbers. The plasmids disclosed herein can be used commercially, publicly available, or made from available plasmids consistent with known methods. Plasmid equivalents to those described are also known in the art and will generally be apparent to those skilled in the art.

The preparation of the vectors of the present invention uses standard ligation and restriction techniques that are well understood in the art (Ausubel et al. (1987) Current Protocol in Molecular Biology, Wiley-Interscience or Maniatis et al. (1992) in Molecular Cloning : A laboratory Manual, Cold Spring Harbor Laboratory, NY). Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, aligned, and degraded in the desired form. All DNA preparation sequences that make synthetic DNA are confirmed by DNA sequencing (Sanger et al. (1977) Proc. Natl. Acad. Sci. 74,5463-5467).

"Digestion" of DNA refers to the catalytic cleavage of DNA by restriction enzymes that act only at restriction sites in a particular sequence of DNA. Various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements are known to those of ordinary skill in the art. For analytical methods, 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer. Optionally, excess restriction enzymes are used to ensure complete digestion of the DNA substrate. Parameters may vary, but incubation times of 1 to 2 hours are available at temperatures of about 37 °. After each incubation the protein is extracted and removed with phenol / chloroform followed by ether extraction and the nucleic acid is recovered from the water soluble portion upon precipitation by ethanol. Hopefully the size of the cleaved sections can be separated by polyacrylamide gels, agarose gels or electrophoresis using standard techniques. A description of general size separation is found in Methods of Enzymology 65: 499-560 (1980).

Restricted cleaved sections were incubated at about 15-25 minutes at 200 ° C. in 50 mM Tris (pH7.6) 50 mM NaCl, 6 mM MgCl ₂ , 6 mM MgCl ₆ , 6 mM DTT and 5-10 micromole dNTPs. While blunted by treatment with large fragments of E. coli DNA polymerase I (Klenow) in the presence of four deoxynucleotide triphosphates. The Klenow fragment chews back a single 3 'strand that fills the 5' sticky end, although there are four dNTPs. Hopefully the selective repair can be performed by feeding one dNTP or selected dNTPs at the limit determined by the nature of the tacky end. After treatment with Cleno, the mixture is extracted with precipitated ethanol and phenol / chloroform. Hydrolyze any single-stranded portion by treatment with S1 nuclease or Bal-31 under appropriate conditions.

Ligations are run in 15-50 μl volumes at the following standard conditions and temperatures. Ie 20 mM Tris-Cl pH 7.5, 10 mM MgCl ₂ , 10 mM DTT, 33 mg / ml BSA, 10 mM to 50 mM NaCl, and 40 μM ATP, 0.01-0.02 at 0 ° C. (for “sticky end) (Weiss) units T4 DNA ligase or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14 ° C. (for “blunt end). The "sticky end" ligation inside the molecule usually performs well at 33-100 μg / ml total DNA concentration (5-100 mM total tip concentration).

“Blunt end” ligation inside the molecule (typically applying a molar excess of 10 to 30 times the linker) is performed at 1 μM total end concentration.

"Temporary expression" refers to expression that has not been amplified during transfection of about a day or two. The optimal time for transient expression of particularly preferred heterologous proteins will vary depending on any processing factor used, translational control mechanisms, and various factors, including host cells. Transient expression occurs when the particular plasmid transformed functions. That is, when it is transcribed and translated. During this time, the plasmid DNA that enters the cell is transferred to the nucleus. DNA is free in the nucleus without coalescing. Transcription of the plasmid by cells occurs during this period. After transfection, plasmid DNA is degraded or attenuated by cell division. Irregular integration occurs in cellular chromatin.

In general, vectors comprising promoters and regulatory sequences derived from species that can coexist with a host cell are used with a particular host cell. Suitable promoters for use with prokaryotic hosts include beta-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan promoter systems, and tac promoters. Hybrid promoters. However, other functional bacterial promoters are suitable. In addition to prokaryotes, eukaryotic microorganisms such as yeast culture may also be used. Saccharomyces cerevisiae or yeast of common bread is the most commonly used eukaryotic microorganism, although many other strains are commonly available.

Promoters that regulate transcription from vectors in mammalian host cells include a variety of sources, such as Polyoma, simianvirus40, adenovirus, retrovirus, hepatitis B virus, cytomegalovirus, Obtainable from heterologous mammalian promoters such as beta-actin promoters. Early and late promoters of the SV40 virus are readily obtained as SV40 restriction fragments containing the SV40 virus origin of replication. Adjacent early promoters of human cytomegalovirus are readily obtained as HindIII restriction fragments. Of course, promoters from host cells or related species are also useful herein.

As used herein, a vector may comprise a selection gene, which is called a selectable indicator. The selection gene encodes a protein necessary for the survival or growth of host cells modified by the vector. Examples of suitable selectable markers of mammalian cells include the dihydrofolate reductase gene (DHFR), the onnitine decarboxylase gene, the multi-drug resistance gene (mdr), and the adenosine diamine Adenosine deaminase genes, glutamine synthase genes, and the like. When such a selectable indicator is successfully delivered into a mammalian host cell, the modified mammalian host cell can survive if it is under selective pressure. There are two unusual categories of regimes to choose from. The first category is based on the metabolism of cells and the use of mutant cell systems that lack the ability to grow independently of the media supplied. The second category is called dominant, which refers to the screening scheme used in all cell types and does not require the use of mutant cell lines. Such schemes typically use drugs to arrest host cell growth. Cells with new genes will express proteins that carry drug resistance and survive selection. Examples of such dominant selection include the drug neomycin (Southern and Berg (1982) J. Molec. Appl. Genet. 1.327), mycophenolec acid (Mulligan and Berg (1980) Science 209,1422), Or hygromycin (Sugden et al. (1985) Mol. Cell. Biol. 5,410-413). The three examples use bacterial genes under eukaryotic control to impart resistance to the appropriate drug neomycin (G418 or genticin), xgpt (mycophenolic acid), hygromycin, respectively.

"Transformation" refers to the introduction of DNA into a host cell where the DNA is functionally or nonfunctionally expressed, and the DNA can also replicate as a non-chromosomal element or by chromosomal binding. Unless otherwise stated, the methods used for the modification of host cells herein are calcium phosphate precipitation methods of Graham and van der Eb (1973) Virology 52,456-457. Other methods of transfection are electroporation, DEAE-dextran method, lipofection, and biolistics (Kriegler (1990) Gene Transfer and Expression: A Laboratory Manual, Stockton Press).

Host cells are transfected with the expression vector of the present invention and cultured in a conventional nutrient medium modified to be suitable for an induction promoter, selection transformant, or amplification gene. Host cells are cultured in F12: DMEM (Gibco) 50:50 without antibiotics and with glutamine. Culture conditions such as temperature, pH, etc. are previously used for the host cell selected for expression, and can be known to those skilled in the art.

The following examples represent the best mode presently known for carrying out the invention but should not be construed as limiting the invention. References in this application are expressly incorporated by reference.

Example 1

Resistance test and sensitivity of HCV drug using resistance test vectors comprising functional indicator genes fused to HCV polyprotein and segment (s) derived from the patient.

Direct Gene Gene Vector-

Indicator Gene Viral Vector (IGVV) pXHCV-luc was designed using an HCV genomic virus vector containing a functional indicator gene fused to HCV polyprotein. IGVV is produced by injecting an open translation frame for the indicator gene into a cDNA construct that contains the complete HCV genome that produces in-frame fusion proteins. The IGVV also includes all cis-activity regulatory elements within the 5 'and 3' untranslated regions (UTRs) required for replication, transcription and translation of HCV RNA.

In one embodiment, the luciferase transcriptional translation frame lies downstream of the NS5 coding region and has a spacer region containing the recognition sequence for the NS3 / 4A protease (FIG. 3A). An example of such a cleavage site is TEDVVCC-SMSYTWT, which shows a junction between NS5A and NS5B (Grakoui et al 1993, J. Virol. 67: 2832; Steinkuler et al., 1996, J. Virol. 70: 6694). Expected cleavage products are HCV NS5B with an extended C-terminus (eg TEDVVCC) and luciferase with an extended N-terminus (eg SMSYTWT). In a second embodiment, the luciferase open reading frame lies between NS5A and NS5B open reading frames and the NS5A-5B cleavage sequence has an N-terminal NS5A-luc and a C-terminal luc-NS5B junction. Luciferase proteins generated from this construct include N-terminal SMSYTWT and C-terminal TEDVVCC extension. In a third embodiment, the luciferase open reading frame is located between NS4B and NS5A open reading frames and the NS4B-5A cleavage sequence (SECTTPC-SGSWLRD) is the N-terminal NS4B-luc and the C-terminal luc-NS5A junction. Has Luciferase proteins prepared from such constructs include an SGSN-terminal SGSWLRD and a C-terminal SECTTPC extension. In a fourth embodiment, the luciferase open reading frame is located between NS4A and NS4B open reading frames and the NS4A-4B cleavage sequence (FDEMEEC-SQHLPYI) is N-terminal NS4A-luc and C-terminal luc-NS4B It has a junction. Luciferase proteins prepared from such constructs include N-terminal SQHLPYI and C-terminal FDEMEEC extensions. Short extension of the N- and C-terminus of luciferase or the C-terminus of NS5B does not dramatically affect activity.

The viral vector is made of a full length cDNA construct of HCV and the construct consists of a 5 'UTR, an open reading frame for 3010 amino acid polyprotein, and a 3' UTR (in the 5 'to 3' orientation). The polyprotein has a capsid (C) open reading frame, envelope glycoprotein genes (E1 and E2), NS2 (cis-active auto-protease that cleaves polyprotein at specific sites of NS2-NS3 junctions), NS3 ( Helicase and serine proteases), NS4A (required as cofactor for NS3 activity), NS4B, NS5A and NS5B (RNA-dependent RNA polymerase) open reading frames. The luciferase open reading frame is also included in the polyprotein open reading frame and positioned in various ways as described above.

In one embodiment, the IGVV has a eukaryotic promoter at the 5 'end of the HCV sequence and a terminator at the 3' end to produce RNA in the transfected cells. Examples of transcriptional promoters include, but are not limited to, CMV proximal-initial promoters, or SV40 promoters. And examples of transcription terminators include, but are not limited to, transcription terminator / polyadenylation signals found in the SV40 or human β-globin genes (FIG. 3B). In a second embodiment, the promoter is a promoter for a bacteriophage RNA polymerase such as T7, T3 or SP6 and the terminator is a sequence or self cleaving ribozyme that signals the transcription termination recognized by the polymerase (eg Chowrira et al. al. 1994, J. Biol. Chem. 269: 25864). IGVV is transfected into cells expressing RNA polymerase in the cytoplasm as DNA. Such expression can be transfected together by a polymerase expression vector, or by various methods such as infection with recombinant vaccinia virus expressing polymerase (Fuerst et al. 1986, PNAS 83: 8122), or permanent expression of polymerase. This is done by introducing a cell system in advance (FIG. 3C). The IGVV additionally comprises a poly-A or poly-U sequence immediately after the HCV 3 'end so that the transcribed RNA comprises a poly-A or poly-U bottom at the 3' end. In a third embodiment IGVV having a bacteriophage RNA polymerase promoter at the 5 'end and a transcription terminator sequence at the 3' end is transduced in vitro and the nucleic acid representing the IGVV is transfected as RNA. Transcription terminators can be specific sequences recognized by bacterial phage RNA polymerase or self-cleaving ribozymes as termination sites (see chowrira et al. (1994) J. Biol. Chem. 269, 25856-25864). In addition, the terminator is a restriction endonuclease site that can linearize the DNA template prior to transcription (FIG. 3A). Also in this case the vector comprises poly-A or poly-U at the 3 'end.

In transfected cells, RNA is translated using an internal initiation mechanism in the body by the internal ribosomal introduction sequence (IRES) to obtain an HCV polyprotein-luc fusion protein. Depending on the activity of NS3 / 4A expressed from genomic RNA, active luciferases are released from HCV polyprotein fusions. When genomic RNA is replicated and amplified in transfected cells, high levels of expression occur depending on the activity of viral proteases NS2 and NS3 / 4A as well as viral polymerase NS5B. When luciferase is inactive as part of a large HCV polyprotein, the activity can be measured directly in the transfected cells since active luciferase is released upon HCV RNA replication (one cell assay). If luciferase has important activity as a fusion moon protein, progeny virions will be collected and used to infect new subject cells (two cell assays). IGVV RNA migrates from transfected cells to infected subject cells following the replication and encapsulation of RNA in the transfected cells, in turn depending on the correct expression, processing and activity of proteins of the HCV viral structure and non-structural proteins. To increase metastasis efficiency (ie IGVV RNA package into a new virion) the subject cells can be infected simultaneously with wild type HCV virus or can be transfected with wild type HCV RNA or cDNA expression constructs. To further increase replication and package of IGVV RNA, the input RNA can be transfected together by purified NS5B protein (ie RNP complex) to initiate transcription to the cell. Such methods or variations of methods include influenza virus (Enami and Palese 1991, J. Virol. 65: 2711-2713), rabies virus (Schnell et al. 1994, EMBO J. 13: 4195-4203), and vesicular oralitis virus ( vesicular stomatitis (Lawson et al. 1995, PNAS 92: 4477-4481).

Immunity test vector-production

Resistance test vectors (RTVs) are blood and / or blood of a patient (a segment or PDS derived from a patient) infected with an HCV genomic region corresponding to a protein that is an antiviral drug target (e.g., NS3 / 4A, NS5B, or IRES). It is constructed from a directed gene viral vector by replacing with a corresponding region derived from virus and / or RNA present in the cell. In one embodiment, for NS3 / 4A protease inhibitors, IGVV is modified by introducing a unique restriction site called the patient sequence receiving site (PSAS) into or near the NS3 / 4A gene (nucleotides 3418-5473 of the H strain of HCV). do. Sections obtained from the virus derived from the patient then metastasize to PSAS of IGVV (FIG. 3A). The wild type NS3 / 4A region is digested with restriction endonucleases that recognize the patient sequence receiving site and removed from IGVV. That is, such sequences are replaced with DNA fragments, where the DNA fragments are generated by PT / PCR from viral RNA derived from patients obtained from plasma, serum or cells. PCR products are generated using primers, wherein the primers contain restriction endonuclease sites required for the generation of compatible aggregation ends for cloning into digested IGVV. RT and PCR primer binding sites are selected and the primer sequences are designed to allow for the amplification of as many different HCV subtypes as possible. In a second embodiment, for the inhibitor of NS5B RDRP, the sequence spanning the NS5B open reading frame (nucleotides 7601-9373 of the H strain of HCV) is removed from the IGVV at the only patient sequence receiving site and then such sequence is plasma, serum or It is replaced by the corresponding PDS generated by RT / PCR from patient viral RNA obtained from the cells. In a third embodiment, for an IRES inhibitor, the sequence spanning the IRES (nucleotides 1-709 of the H strain of HCV) is removed from the IGVV at a unique patient sequence receiving site and then the sequence is obtained from the patient viral RNA obtained from plasma, serum or cells. From the corresponding PDS generated by RT / PCR. The method described above can be applied to other subjects of anti-HCV drugs by identifying genes encoding drug subjects in IGVV, introducing unique patient sequence receptors into IGVV, and replacing subject genes with PDS.

Drug Sensitivity and Tolerance Testing

Drug resistance and sensitivity tests are performed using one cell assay or two cell assays with resistance test vectors (DNA or RNA) prepared as described above by transfection. Transfection of the host cell with the resistance test vector results in HCV virus particles comprising the directed gene RNA encapsidated.

Repeat for package host cell culture series maintained in the presence of antiviral drug or at elevated concentrations of anti HCV drug (e.g., HCV NS3 / 4A protease inhibitor, NS5B polymerase inhibitor, or IRES inhibitor) Transfection takes place. After holding the package host cells for several days in the presence or absence of the anti-HCV agent, directing them from the host package cell filtrate, or directing the isolated HCV particles obtained by obtaining the host package cell culture medium. Gene expression can be measured to assess drug sensitivity or resistance. An alternative approach can be used to assess drug sensitivity and resistance in HCV particles isolated from the cell filtrate.

In one embodiment referred to as one cell assay, drug sensitivity or resistance is assessed by measuring luciferase expression or activity in packaged host cells transfected with or without an antiviral drug. Cells transfected with a given antiviral drug or combination drug are observed to have a decreased luciferase activity when compared to a control without the antiviral drug (s) and such luciferase activity. The reduction of is used to calculate the inhibition constant (Ki) of the drug or to generate a log concentration-related sigmoidal curve of the antiviral drug for luciferase activity.

In the second embodiment, referred to as two cell assays, drug sensitivity or resistance is assessed by measuring luciferase gene expression and / or activity in a subject host cell infected with HCV particles obtained by transfecting the host cell. At the time of transfection or infection, an appropriate concentration of antiviral drug is added to the host or subject cell culture depending on the drug subject. Several days after infection, host cells are lysed and luciferase expression is measured. Lucy on infected cells by inhibiting the protease (NS3 / 4A) or RDRP (NS5B) activity of HCV in the presence of a drug that inhibits HCV replication, for example compared to a control without the drug. It is observed that reduced ferase expression.

In a third embodiment where RNA structural change is used as an indicator, drug sensitivity or resistance is assessed by measuring the extent of HCV RNA replication that occurs in the transfected host cell. In host cells transfected with the DNA of an HCV viral vector, RNA is transcribed as positive (mRNA) sense RNA (or RNA is transcribed in vitro and directly transfected) and the positive (mRNA) sense RNA is then NS5B polymerase. It can be used as a template for the generation of negative sense cRNA by activity. Positive sense RNAs are also transformed which translate and serve as templates for negative sense cRNA synthesis. To measure HCV RNA replication, RNA is isolated from transfected cells and treated with DNAases to remove residue input DNA. (If a cell is transfected with positive sense RNA, DNAase treatment will not be absolutely required.) will be). RT primers are designed to hybridize specifically for negative sense HCV RNA to prepare for synthesis of positive sense cDNA and cDNA after digesting with RNA degrading enzymes to prevent reverse transcription of positive sense RNA using PCR primers. Amplified by PCR.

Following initiation of HCV RNA replication to form a negative sense RNA, cDNA to be amplified for positive sense is formed in the transfected cells. Antiviral agents that inhibit HCV RNA replication (RNA polymerase activity according to RNA), or the production of active forms of the polymerase (by NS3 / 4A protease) limit the formation of RNA target sequences and Formation is measured by the reduction of amplified DNA products using one of a number of quantitative amplification assays.

In other embodiments where modifications of the RNA construct are used as indicators, rather than measuring the production of amplified DNA, the 5 'exonuclease activity of the amplifying enzyme (eg Taq polymerase) is measured (Held et al., 1996 , Genome research 6: 986-994). The 5 'exonuclease activity is measured by monitoring the nucleolytic cleavage of the fluorescence-tagged oligonucleotide probe. In this case, the oligonucleotide probe may bind to an amplified DNA template region flanked by a PCR primer binding site. This assay is performed in the immediate vicinity of the upstream primer 3 'end to the oligonucleotide probe 5' end. The extension of the primer replaces the 5 'end of the oligonucleotide probe, so the 5' exonuclease activity of the polymerase cleaves the oligonucleotide probe.

Drug Screening

Drug screening is performed using an indicator gene viral vector comprising a function indicator gene fused with HCV polyprotein. In a transfected host cell, the indicator gene viral vector produces an RNA transcript comprising the indicator gene (or optionally RNA is transcribed ex vivo and directly transfected). Translation of such RNA, or mRNA resulting from replication and transcription by viral RDRP (NS5B), provides the essential structural enzymatic viral function for viral RNA replication and particle formation. The transfected cells give rise to HCV virus particles comprising the directed gene viral vector RNA surrounded by a membrane. In this case, the indicator gene viral vector RNA includes a function indicator gene fused to the HCV polyprotein gene.

Drug screening is performed as follows. Directed Gene Viral Vector DNA or RNA is used to transfect host cells. Repeat transfection is performed on a series of packaged host cell cultures maintained in the presence or absence of potential antiviral compounds (eg, candidate HCV NS3 / 4A protease or NS5B polymerase inhibitors). After maintaining the transfected host cell for several days in the presence or absence of the candidate antiviral drug, the degree of inhibition of RNA replication was directly obtained from the host cell filtrate, or harvested from the host transfected cell culture medium. It is assessed by measuring the directed gene expression in isolated HCV particles obtained by or in subject cells infected with the isolated HCV particles. The RNA detection or indicator gene activity methods described above can be used to assess potential anti HCV drug candidates.

Example 2

HCV Drug Sensitivity and Tolerance Testing Using Resistance Test Vectors Including Functional Indices Expressed from Internal Ribosome Initiation Sequences and Patient-Derived Sections

Direct Gene Gene Vector-

Translational initiation of HCV polyprotein is caused by an internal initiation mechanism with a cap independent. The 5 'end of the viral RNA comprising the untranslated region (UTR) and the first 369 nucleotides of the C open reading frame contains sequences and / or constructs in which the caps direct independent translation initiation (Tsukiyama-Kohara et al, J Virol. 66: 1476, 1992; Wang et al, J Virol. 67: 3338, 1993; Lu and Wimmer, PNAS 93: 1412, 1996). HCV is closely related to pestiviruses, novel death virus (BVDV) (Poole et al. (1995) Virology, 189, 285-292), as well as poliovirus (PV) (Pelletier and Sonenberg (1998), Nature, 334, 320 325), encephalomyocarditis virus (EMCV) (Jang et al. (1989), J. Virol. 63, 1651-1660), common rhinovirus (RV), (Rohll et al. 1994), J. Virol. 68, 4384-4391), Hepatitis A virus (HAV) (Brown et al. (1994), J. Virol. 68, 1066-1074; Glass et al. (1993) Virol. 193 Other viruses, such as 842-852, use a similar mechanism for initiating translation, although the sequence acting as the internal lysosomal entry (IRES) is different for each virus. Some cellular mRNAs are known to initiate translation internally by IRES (Macejak and Sarnow (1991), Nature, 353, 90-94). These RNA elements are shown to be able to direct translation initiation when located between the 5 'end of RNA as well as between two open reading frames. Such bicistronic RNAs can be used to direct the translation of two open reading frames to obtain two protein expressions from the same RNA.

The indicator gene viral vector comprising the functional IG expressed from the internal ribosome initiation sequence is opened to cDNA preparations containing the complete HCV genome as a second cistron element preceded by IRES, such as open translation for indicator genes, for example luciferase. It is made by injecting a mold. IRES (from native HCV 5 ′ UTRs or other viruses) and HCV polyproteins are injected under HCV polyproteins to provide luciferase gene expression independent of HCV protein expression (FIG. 4). When testing the resistance of drugs that inhibit the function of HCV IRES, note that the IRES used to express luciferase is derived from HCV and other viruses, and these viruses are not affected by the drug. IGVV thus comprises the following elements in the 5 'to 3' orientation: promoter sequence, HCV 5'UTR, complete HCV polyprotein coding sequence, IRES, luciferase coding region, HCV 3'UTR, and transcription terminator .

In one embodiment, the IGVV comprises a eukaryotic promoter at the 5 'end of the HCV sequence and a transcription terminator at the 3' end to produce RNA in the transfected cells. Examples of transcriptional promoters include, but are not limited to, CMV proximal-early promoters, or SV40 promoters, and examples of transcriptional terminators include, but are not limited to, transcriptional terminator / polyadenylation signals found in SV40 or human β-globin genes. (See Figure 3b). In a second embodiment, the promoter is a promoter for a bacterial phage RNA polymerase such as T7, T3, or SP6 and the transcription terminator is a sequence that signals the transcription termination recognized by the polymerase, or a self-cleaving ribozyme. IGVV is transfected into cells expressing RNA polymerase in the cytoplasm as DNA. Such expression is obtained in a number of ways, including transfection together with a polymerase expression vector, infection with recombinant vaccinia virus expressing polymerase, or by permanently establishing a cell system expressing polymerase ( 3c). Since IGVV additionally contains a poly-A or poly-U sequence immediately following the HCV 3 'end, the transcribed RNA comprises a poly-A or poly-U underneath. In a third embodiment, an IGVV having a bacteriophage RNA polymerase promoter at the 5 'end and a terminator sequence at the 3' end is transduced in vitro and the nucleic acid representing the IGVV is transfected as RNA. The terminator may be a specific sequence recognized by bacteriophage RNA polymerase as a terminator or self-cleaving ribozyme. In addition, the terminator is a restriction endonuclease site that allows linearization of the DNA template prior to transcription (see FIG. 3D). Also in this case the vector has poly-A or poly-U at the 3 'end.

Immunity test vector-production

Resistance test vectors with function indicator genes expressed from internal ribosome initiation sequences are constructed from IGVV described above and HCV sequences derived from patients as described in Example 1. The IGVV is modified to include PSAS for injecting IRES, NS5B, or NS3 / 4A containing PDS (described in Example 1, see FIG. 3A).

Drug Sensitivity and Tolerance Testing

Drug resistance and sensitivity tests are performed using resistance test vectors prepared as described above by transfection using one cell or two cell assays. Host cells are transfected with the resistance test vector to generate HCV virus particles comprising the directed gene RNA surrounded by the membrane. Drug resistance and sensitivity tests are performed as described in Example 1.

Drug Screening

Drug screening is performed using IGVV comprising a functional indicator gene expressed from an internal ribosomal initiation sequence and essentially as described in Example 1 above.

Example 3

HCV drug susceptibility and resistance testing using a resistance test vector comprising a functional indicator gene expressed from a replication defective small genome and the segment (s) derived from the patient.

Direct Gene Gene Vector-

Expression dependent HCV replication of the indicator gene can be achieved by using an artificial HCV genome virus vector, or the HCV 5 ′ UTR and, if necessary, the amino-terminus of the C open reading frame (required as part of the IRS), IG, eg Lucifera. And a small genome composed of HCV 3 ′ UTRs (see FIG. 5). Luciferases are produced with an N-terminal extension derived from the C open reading frame and ATG is mutated at the beginning of the C open reading frame so that translation begins at ATG of luciferase. Along with the N-terminus of C and 3 'UTR, the 5' UTR contains all cis-activation signals required for translation, replication and package of RNA. The luciferase small genome is transfected together as DNA or RNA (see Example 1, Figures 3b-3d) by a full-length helper HCV genomic construct, and a small genome as well as a package of cis-activity regulatory elements Package and replication of genomic RNA into later viruses depends on HCV replication devices, including NS3 / 4A protease and NS5B RDRP.

An indicator gene viral vector comprising a function indicator gene expressed from a replication binding small genome and a helper HCV genomic construct is constructed as follows. The small genome has the following elements in the 5 'to 3' orientation: promoter sequence, HCV 5 'UTR, first 24 or 369 nucleotides of C open reading frame, luciferase open reading frame, HCV 3' UTR, and transcription termination Contains the arguments. Helper HCV genomic constructs include a promoter, complete HCV cDNA, and terminator.

In one embodiment, the IGVV comprises a circular promoter at the 5 'end of the HCV sequence and a transcription terminator at the 3' end to generate RNA in the transfected cells. Examples of transcriptional promoters include, but are not limited to, CMV proximal-early promoters, or SV40 promoters, and examples of transcriptional terminators include, but are not limited to, transcriptional terminator / polyadenylation signals found in the SV40 or human β-globin genes. (See Figure 3b). In a second embodiment, the promoter is a promoter for bacteriophage RNA polymerase, such as T7, T3, or SP6 and the transcription terminator is a sequence or self-cleaving ribozyme that signals the transcription termination recognized by the polymerase. IGVV is transfected into cells expressing RNA polymerase in the cytoplasm as DNA. Such expression is obtained in a number of ways, including transfection together with a polymerase expression vector, infection with recombinant vaccinia virus expressing polymerase, or by pre-establishing a cell system that permanently expresses polymerase ( 3c). Since IGVV concomitantly contains a poly-A or poly-U sequence directly at the HCV 3 'end, the transcribed RNA includes a poly-A or poly-U bottom at the 3' end at the 3 'end. In a third embodiment, an IGVV having a bacteriophage RNA polymerase promoter at the 5 'end and a terminator sequence at the 3' end is transduced in vitro and the nucleic acid representing the IGVV is transfected as RNA. The terminator may be a specific sequence or self-cleaving ribozyme recognized by the bacteriophage RNA polymerase as terminator. In addition, the terminator is a restriction endonuclease site that allows linearization of the DNA template prior to transcription (see FIG. 3D). Also in this case the vector has a poly-A or poly-U sequence at the 3 'end.

Immunity test vector-production

Resistance test vectors comprising a function binding gene expressed from a replication binding small genome and a helper HCV genomic construct are constructed from a patient-derived HCV sequence and a helper HCV genomic construct as described in Example 1. The helper HCV genomic construct is modified to include PSAS for injecting PDS or NS3 / 4A containing NS5B (described in Example 1, see FIG. 3A). In the case of drugs targeting the function of the IRES, the PSAS is introduced into the small genome structure.

Drug Sensitivity and Tolerance Testing

Drug resistance and sensitivity testing was performed as described above by transfection using an IGVV system containing a functional indicator gene expressed from a small genome and helper HCV genome constructs and using one cell or two cell assays. This is done using a vector. HCV viral particles comprising membrane-enclosed luciferase gene RNA and / or membrane-enclosed HCV genomic RNA by transfection of host cells with a resistance test vector (luciferase small genome plus helper HCV genome construct comprising PDS) Create Drug resistance and sensitivity tests are performed as described in Example 1.

Drug Screening

Drug screening is performed using an IGVV system comprising a functional indicator gene expressed from a small genome and helper HCV genome constructs and essentially as described in Example 1 above.

Example 4

HCV drug susceptibility and resistance testing using resistance test vectors comprising non-functional indicator genes expressed from antisense replication-binding small genomes and segment (s) derived from the patient.

Direct Gene Gene Vector-

Directed Gene Viral Vectors contain nonfunctional directed genes expressed from replication defective small genomes and helper HCV genomic constructs and are constructed as follows. Small genomes have the following elements in the 5 'to 3' orientation: promoter sequence, HCV 3 'UTR (in antisense orientation), luciferase open translation frame (antisense), C open translation frame first 24 or 369 nucleotides (antisense ), HCV 5 'UTR (antisense), and transcription terminator. Helper HCV genomic constructs include a promoter, complete HCV cDNA (in sense orientation), and terminator. In one embodiment, the small genome is introduced into the cell as negative stranded RNA, ie, the replication intermediate RNA replica of the small genome described above (see FIG. 6).

Expression in transfected cells depends on the production and activation of NS5B, which in turn depends on the activity of cis-activating regulatory elements such as IRES and NS3 / 4A. Thus, the indicator gene does not function until activated by the viral replication device.

In one embodiment, the IGVV comprises a circular promoter at the 5 'end of the HCV sequence and a transcription terminator at the 3' end to generate RNA in the transfected cells. Examples of transcriptional promoters include, but are not limited to, CMV proximal-early promoters, or SV40 promoters, and examples of transcriptional terminators include, but are not limited to, transcriptional terminator / polyadenylation signals found in SV40 or human β-globin genes. (See Figure 3b). In a second embodiment, the promoter is a promoter for bacteriophage RNA polymerase, such as T7, T3, or SP6 and the terminator is a sequence, or self-cleaving ribozyme, that signals transcription termination recognized by the polymerase. IGVV is transfected into cells expressing RNA polymerase in the cytoplasm as DNA. Such expression is obtained in a number of ways, including transfection together with a polymerase expression vector, infection with recombinant vaccinia virus expressing polymerase, or by pre-establishing a cell system that permanently expresses polymerase ( 3c). IGVV concomitantly comprises a poly-A or poly-U sequence immediately following the HCV 3 'end, so the transcribed RNA comprises a poly-A or poly-U bottom at the 3' end. In a third embodiment, an IGVV having a bacteriophage RNA polymerase promoter at the 5 'end and a terminator sequence at the 3' end is transduced in vitro and the nucleic acid representing the IGVV is transfected as RNA. The terminator may be a specific sequence or self-cleaving ribozyme recognized by the bacteriophage RNA polymerase as terminator. In addition, the terminator is a restriction endonuclease site that allows linearization of the DNA template prior to transcription (see FIG. 3D). Also in this case the vector has a poly-A or poly-U sequence at the 3 'end.

Immunity test vector-production

Resistance test vectors comprising a function binding gene expressed from a replication binding small genome and a helper HCV genomic construct are constructed from a patient-derived HCV sequence and a helper HCV genomic construct as described in Example 1. The helper HCV genomic construct is modified to include PSAS for injecting PDS or NS3 / 4A containing NS5B (described in Example 1, see FIG. 3A). In the case of drugs intended for the functioning of the IRES, PSAS is also introduced in small genome structures.

Drug Sensitivity and Tolerance Testing

Drug resistance and sensitivity tests were prepared as described above by transfection using resistance test vectors containing nonfunctional indicator genes expressed from small genomes and helper HCV genome constructs and using one cell or two cell assays. It is performed using an immunity test vector. HCV viral particles comprising membrane-enclosed luciferase gene RNA and / or membrane-enclosed HCV genomic RNA by transfection of host cells with a resistance test vector (luciferase small genome plus helper HCV genome construct comprising PDS) Create Drug resistance and sensitivity tests are performed as described in Example 1.

Drug Screening

Drug screening is performed using resistance test vectors comprising non-functional indicator genes expressed from small genomes and helper HCV genome constructs and essentially as described in Example 1 above.

Example 5

HCV drug sensitivity and resistance testing using a resistance test vector comprising a functional indicator gene expressed as a replication binding genome and fragment (s) derived from the patient.

Direct Gene Gene Vector-

Directed Gene Viral Vectors contain a functional directed gene expressed from a replication defective genome and a package vector construct and are constructed as follows. IGVV is the NS2 through the following elements in the 5 'to 3' orientation: promoter sequence, HCV 5 'UTR, the first 24 or 369 nucleotides of the C open reading frame, luciferase open reading frame, NS5B portion of HCV genome Nucleotides 2768 to 9373 of the H strain of HCV), HCV 3 ′ UTR, and transcription terminators. The package vector comprises a promoter, C, E1 and E2 open reading frames of HCV (nucleotides 342-2578 of H strain of HCV) and terminators. In case the indicator gene is luciferase or other cytoplasmic protein, the IG-NS2 junction will be modified to be properly processed by the host signal peptide degrading enzyme of the endoplasmic reticulum. If the secreted indicator gene is, for example, alkaline phosphate, no modification is required.

Infectious recombinant virions are generated from cells transfected with two vectors: IGVV, which contains IG and viral non-structural proteins, and second, package vector, which contains proteins of viral structure (C / E1 / E2: 7). To generate infectious particles, IGVV DNA (or corresponding RNA, see FIGS. 3A-3D) is transfected together as a packaging vector. The particles are also pseudotyped as the envelope glycoprotein gene from related flaviviruses, such as BVDV or the classical swine fever virus (CSFV). The false positive virus is used to establish a cell culture system for single cycle infection assays. The virus produced by this method can then be used to infect the cells of the subject and the luciferase expression was subsequently determined. This approach has the added advantage of minimizing the manipulations performed with infectious agents suitable for replication.

In one embodiment, the IGVV comprises a circular promoter at the 5 'end of the HCV sequence and a transcription terminator at the 3' end to generate RNA in the transfected cells. Examples of transcriptional promoters include, but are not limited to, CMV proximal-early promoters, or SV40 promoters, and examples of transcriptional terminators include, but are not limited to, transcriptional terminator / polyadenylation signals found in SV40 or human β-globin genes. (See Figure 3b). In a second embodiment, the promoter is a promoter for a bacteriophage RNA polymerase, such as T7, T3, or SP6 and the terminator is a sequence, or self-cleaving ribozyme, that signals the transcription termination recognized by the polymerase. IGVV is transfected into cells expressing RNA polymerase in the cytoplasm as DNA. Such expression is obtained in a number of ways, including transfection together with a polymerase expression vector, infection with recombinant vaccinia virus expressing polymerase, or by pre-establishing a cell system that permanently expresses polymerase ( 3c). Since IGVV concomitantly comprises a poly-A or poly-U sequence immediately following the HCV 3 'end, the transcribed RNA comprises a poly-A or poly-U bottom at the 3' end. In a third embodiment, an IGVV having a bacteriophage RNA polymerase promoter at the 5 'end and a terminator sequence at the 3' end is transduced in vitro and the nucleic acid representing the IGVV is transfected as RNA. The terminator may be a specific sequence or self-cleaving ribozyme recognized by the bacteriophage RNA polymerase as terminator. In addition, the terminator is a restriction endonuclease site that allows linearization of the DNA template prior to transcription (see FIG. 3D). Also in this case the vector has a poly-A or poly-U sequence at the 3 'end.

Immunity test vector-production

Resistance test vectors comprising the functional binding genes expressed from the replication binding genome and the package vector construct are constructed from the IGVV described above and HCV sequences derived from patients as described in Example 1. The IGVV is modified to include PSAS for injecting IRES, NS5B or NS3 / 4A containing PDS (described in Example 1, see FIG. 3A).

Drug Sensitivity and Tolerance Testing

Drug resistance and sensitivity tests are performed using resistance test vectors (DNA or RNA) prepared as described above by transfection using one cell or two cell assays. Host cells are transfected with the resistance test vector to produce HCV virus particles containing the directed gene RNA surrounded by the membrane. Drug resistance and sensitivity tests are performed as described in Example 1.

Drug Screening

Drug screening is performed using IGVV comprising a functioning gene expressed from a replication defective genome and a package vector construct and essentially as described in Example 1 above.

Example 6

HCV protease inhibitor sensitivity and resistance testing using a resistance test vector comprising a functional indicator gene of the NS3 / 4A BVDV chimeric virus vector and the segment (s) derived from the patient.

Direct Gene Gene Vector-

Appropriate site (s) of chimeric IGVV and HCV (eg, NS3 / 4A protease domains) containing function indicator genes have been designed using viral backbones involved in replication in culture. Complete cDNA to the genome of BVDV is shown to be assembled to generate infectious RNA by in vitro transcription (Vassilev et al. 1997, J. Virol. 71: 471-478). BVDV polyproteins are treated in much the same way as for HCV using both the host (signal peptide degrading enzyme) and the virus encoded protease. Chimeric IGVVs based on the BVDV backbone include the complete NS3 / 4A open reading frame or NS3 proteaane domain of HCV, replacing the corresponding region of BVDV (FIG. 8). By mutating the cleavage site normally recognized by the BVDV NS3 protease to the site recognized by HCV NS3 / 4A, replication of BVDV chimeric RNA and expression of IG will be dependent on HCV NS3 / 4A activity.

The chimeric indicator gene viral vector contains a function indicator gene in the NS3 / 4A BVDV chimeric virus vector and is constructed as follows. IGVV has the following elements in the 5 'to 3' orientation: promoter sequence, BVDV 5 'UTR, C through NS2 region of BVDV, NS3 / 4A region of HCV, HCV NS4A / 4B cleavage site, BVDV NS4B open reading frame, HCV NS4B / 5A cleavage site, BVDV NS5A open frame, HCV NS5A / 5B cleavage site, BVDV NS5B open reading frame, luciferase open reading frame, BVDV 3 ′ UTR and transcription terminator. In a second embodiment, the IGVV comprises a luciferase open reading frame following IRES in a configuration similar to that described in Example 2. In a third embodiment, the luciferase gene is expressed from a small genome similar to that described in Example 3 or 4.

In one embodiment, IGVV comprises a circular promoter at the 5 'end of the HCV sequence and a transcription terminator at the 3' end to produce RNA in the transfected cells. Examples of transcriptional promoters include, but are not limited to, CMV intermediate-initial promoters, or SV40 promoters, but examples of transcriptional terminators include, but are not limited to, transcriptional terminator / polyadenylation signals found in SV40 or human β-globin genes. It is not limited (see FIG. 3B). In a second embodiment, the promoter is a promoter for bacteriophage RNA polymerase, such as T7, T3, or SP6 and the terminator is a sequence, or self-cleaving ribozyme, that signals transcription termination recognized by the polymerase. IGVV is transfected into cells expressing RNA polymerase in the cytoplasm as DNA. Such expression is obtained in a number of ways, including transfection together with a polymerase expression vector, infection with recombinant vaccinia virus expressing polymerase, or by pre-establishing a cell system that permanently expresses polymerase ( 3c). Since IGVV concomitantly comprises a poly-A or poly-U sequence immediately following the HCV 3 'end, the transcribed RNA comprises a poly-A or poly-U bottom at the 3' end. In a third embodiment, an IGVV having a bacteriophage RNA polymerase promoter at the 5 'end and a terminator sequence at the 3' end is transduced in vitro and the nucleic acid representing the IGVV is transfected as RNA. The terminator may be a specific sequence or self-cleaving ribozyme recognized by the bacteriophage RNA polymerase as terminator. In addition, the terminator is a restriction endonuclease site that allows linearization of the DNA template prior to transcription (see FIG. 3D). Also in this case the vector has a poly-A or poly-U sequence at the 3 'end.

Immunity test vector-production

Resistance test vectors comprising a function indicator gene in the NS3 / 4A BVDV chimeric virus vector are constructed from the IGVV described above and HCV NS3 / 4A sequences derived from the patient as described in Example 1. The IGVV is modified to include a PSAS for injecting a PDS containing NS3 / 4A (described in Example 1, see FIG. 3A).

Drug Sensitivity and Tolerance Testing

Drug resistance and sensitivity tests are performed using resistance test vectors (DNA or RNA) prepared as described above by transfection using one cell or two cell assays. The host cell is transfected with the resistance test vector to produce HCV virus particles containing the membrane-directed gene RNAA. Drug resistance and sensitivity tests are performed as described in Example 1.

Drug Screening

Drug screening is performed using IGVV comprising a functional indicator gene expressed from an internal ribosomal initiation sequence and essentially as described in Example 1 above.

Example 7

HCV drug sensitivity and resistance testing using resistance test vectors comprising segment (s) derived from the patient and functional indicator genes in the NS5B BVDV chimeric virus vector.

Direct Gene Gene Vector-

Chimeric IGVVs, including BVDV structural and nonstructural proteins, except NS5B derived from HCV, are designed using the backbone of BVDV. In addition, BVDV 5 'and 3' UTR are replaced by the corresponding regions from HCV to ensure recognition by the same polymerase (Figure 9).

An indicator gene viral vector comprising a function indicator gene in an NS5B BVDV chimeric virus vector is constructed as follows. IGVV is the next element in the 5 'to 3' orientation: promoter sequence, HCV 5 'UTR, sequence from the N-terminus of the HCV C open reading frame required for IRES function, Npro (NADL) through the NS5A region of BVDV Strain), NS5B region of HCV, luciferase open reading frame, HCV 3 ′ UTR, and transcription terminator. In a second embodiment, the IGVV comprises a luciferase open reading frame following the IRES in a configuration similar to that described in Example 2. In a third embodiment, the luciferase gene is expressed from a small genome similar to that described in Example 3 or 4.

In one embodiment, the IGVV comprises a circular promoter at the 5 'end of the HCV sequence and a transcription terminator at the 3' end to generate RNA in the transfected cells. Examples of transcriptional promoters include, but are not limited to, CMV proximal-early promoters, or SV40 promoters, and examples of transcriptional terminators include, but are not limited to, transcriptional terminator / polyadenylation signals found in SV40 or human β-globin genes. (See Figure 3b). In a second embodiment, the promoter is a promoter for bacteriophage RNA polymerase, such as T7, T3, or SP6 and the terminator is a sequence, or self-cleaving ribozyme, that signals transcription termination recognized by the polymerase. IGVV is transfected into cells expressing RNA polymerase in the cytoplasm as DNA. Such expression is obtained in a number of ways, including transfection together with a polymerase expression vector, infection with recombinant vaccinia virus expressing polymerase, or by pre-establishing a cell system that permanently expresses polymerase ( 3c). Since IGVV concomitantly comprises a poly-A or poly-U sequence immediately following the HCV 3 'end, the transcribed RNA comprises a poly-A or poly-U bottom at the 3' end. In a third embodiment, an IGVV having a bacteriophage RNA polymerase promoter at the 5 'end and a terminator sequence at the 3' end is transduced in vitro and the nucleic acid representing the IGVV is transfected as RNA. The terminator may be a specific sequence or self-cleaving ribozyme recognized by the bacteriophage RNA polymerase as terminator. In addition, the terminator is a restriction endonuclease site that allows linearization of the DNA template prior to transcription (see FIG. 3D). Also in this case the vector has a poly-A or poly-U sequence at the 3 'end.

Immunity test vector-production

Resistance test vectors comprising a function indicator gene in the NS5B BVDV chimeric virus vector are constructed from the IGVV described above and HCV NS5B sequences derived from patients as described in Example 1. The IGVV is modified to include a PSAS for injecting a PDS containing NS5B (described in Example 1, see FIG. 3A).

Drug Sensitivity and Tolerance Testing

Drug resistance and sensitivity tests are performed using resistance test vectors (DNA or RNA) prepared as described above by transfection using one cell or two cell assays. The host cell is transfected with the resistance test vector to produce HCV virus particles containing the directed gene RNA surrounded by the membrane. Drug resistance and sensitivity tests are performed as described in Example 1.

Drug Screening

Drug screening is performed using IGVV comprising a functional indicator gene expressed from an internal ribosomal initiation sequence and essentially as described in Example 1 above.

Example 8

HCV drug sensitivity and resistance testing using a resistance test vector system comprising a segment (s) derived from a patient, a transcriptional transactivator, and a function indicator gene.

Direct Gene Gene Vector-

Indicator gene viral vector systems have been designed with respect to HCV dependent expression and release of transcriptional transactivators that activate expression of indicator genes. Indicator genes such as luciferase are introduced into the host cell by transient or stable transfection as expression vectors. Genes encoding transactivator proteins such as the genes of HIV-1, tat are fused to HCV polyprotein (ie, by a NS3 / 4A cleavage site linker in a manner similar to the fusion of luciferase described in Example 1) At the C-terminus or anywhere). Depending on the expression of the polyprotein and the activity of the NS3 / 4A protease, tat activates the transcription of reporter genes such as luciferase cleaved from the polyprotein and under the control of the HIV-1 terminal repeat sequence (LTR).

Directed Gene The viral vector system includes a functional directed gene including a segment derived from a patient, a transcriptional activator, and a functional directed gene and is constructed as follows. The viral vector is directed to the 3010 amino acid HCV polyprotein, which is located in the 5 'to 3' orientation with the following elements: promoter, HCV 5 'UTR, variously positioned as described in Example 1 and including an open reading frame for tat. Open reading frame, 3 ′ UTR, and transcription terminator. Indicator gene constructs include HIV-1 LTR, luciferase open reading frame, and transcription terminator. The indicator gene construct may be transfected together by a viral vector or is preferably present as a DNA fragment that is stably integrated into the host cell DNA.

In one embodiment, the viral vector comprises a circular promoter at the 5 'end of the HCV sequence and a transcription terminator at the 3' end to generate RNA in the transfected cells. Examples of transcriptional promoters include, but are not limited to, CMV proximal-early promoters, or SV40 promoters, and examples of transcriptional terminators include, but are not limited to, transcriptional terminator / polyadenylation signals found in SV40 or human β-globin genes. (See Figure 3b). In a second embodiment, the promoter is a promoter for bacteriophage RNA polymerase such as T7, T3, or SP6 and the terminator is a sequence, or self-cleaving ribozyme, that signals the termination of transcription recognized by the polymerase. The viral vector is transfected into cells expressing RNA polymerase in the cytoplasm as DNA. Such expression is obtained in a number of ways, including transfection together with a polymerase expression vector, infection with recombinant vaccinia virus expressing polymerase, or by pre-establishing a cell system that permanently expresses polymerase ( 3c). Since IGVV concomitantly comprises a poly-A or poly-U sequence immediately following the HCV 3 'end, the transcribed RNA comprises a poly-A or poly-U bottom at the 3' end. In a third embodiment, a viral vector having a bacteriophage RNA polymerase promoter at the 5 'end and a terminator sequence at the 3' end is transduced in vitro and the nucleic acid representing the viral vector is transfected as RNA. The terminator may be a specific sequence or self-cleaving ribozyme recognized by the bacteriophage RNA polymerase as terminator. In addition, the terminator is a restriction endonuclease site that allows linearization of the DNA template prior to transcription (see FIG. 3D). Also in this case the vector has a poly-A or poly-U sequence at the 3 'end.

Immunity test vector-production

Resistance test vectors comprising fragments derived from the patient, transcriptional activators, and functional indicator genes including the functional indicator genes are constructed from the viral vectors described above and HCV sequences derived from the patient as described in Example 1. The viral vector is modified to include PSAS for injecting PDS containing the appropriate site of the HCV genome (described in Example 1, see FIG. 3A).

Drug Sensitivity and Tolerance Testing

Drug resistance and sensitivity tests are performed using resistance test vectors prepared as described above by transfection with a host cell containing the indicator construct. Drug resistance and sensitivity tests are performed as described in Example 1.

Drug Screening

Drug screening uses an indicator gene viral vector system comprising a function indicator gene, wherein the function indicator gene comprises a segment (s), a transcriptional transactivator, and a function indicator gene derived from a patient and described in Example 1 above. It is performed essentially the same as that and the function indicator gene includes a segment derived from a patient, a transcriptional activator and a function indicator gene.

Example 9

Cytomegalovirus Drug Sensitivity and Tolerance Testing Using Resistance Test Vectors Comprising a Functional Indicating Gene Inserted into a Defective Helper Virus and Section (s) Derived from the Patient.

Direct Gene Gene Vector-

Directed gene viral vectors are functional directed genes injected into the ORF of HCMV under the control of heterologous viral promoters that regulate the expression of RNA in wild-type viruses, for example β _2.7 located in TR _L or "b" repeats (Figure 12). It was designed to include. The directed gene viral vector (HCMV-β _2.7 F-IG / Δgene X) is further modified to be defective for replication by deleting genomic fragments containing viral gene (s) that are the subject of antiviral drug (s). Viral genes that are the subject of antiviral drugs are referred to herein as gene X. The gene X product is the cis-active function required for trans-complementation of the indicator gene viral vector and the patient sequence receiving site (PSAS) (in detail, this indicates the HCMV origin HCMV genome cleavage and package of replication HCMV). amplicon plasmid (pA-CMV-VS-gene X) comprising the “a” sequence) (FIG. 13). The PSAS is designed to receive PDS in a cassette containing appropriate regulatory signals for individual viral genes / drug subjects and is derived from the context of the viral gene / drug subjects in wild type HCMV. The binding indicator gene viral vector and the amplicon / gene X plasmid constitute the resistance test vector system. The defect indicating gene viral vector is propagated to the package host cell / subject host cell system as a viral colony, in which a function mimic of viral gene X is provided in trans. Virus colonies from such package host cells / subject host cell lines are prepared and used to infect cells and / or DNA from such viral colonies is isolated to make package host cells / subject host cells as part of a resistance test vector system, HCMV. It is used for transfection with an amplicon / gene X plasmid that is transfected into an acceptable cell type for infection. The trans complementarity of the deleted gene by the amplicon / gene X plasmid results in its own persistent virus population that increases the expression of the reporter gene. At this time, the reporter gene increases in expression with the activity of the viral gene encoded by the segment derived from the patient introduced into the amplicon / gene X plasmid.

In another embodiment, the amplicon / gene X plasmid (pA-CMV-CS-gene X) can be used to express PSAS that accepts PDS by expressing a gene X sequence derived from a patient under the control of a heterologous promoter and polyadenylation signal. Include. In one embodiment, the expression cassette comprising the CMV IE enhancer promoter region, PSAS, and SV40 polyadenylation (pA) signal is a cis-active function required for trans-complementarity of the directed gene viral vector (in detail this is replication In addition to the HCMV “a” sequence indicating the HCMV genome cleavage and package of the HCMV origin of replication and amplification / gene X plasmid (pA-CMV-CS-gene X).

In another embodiment, helper virus vectors are supplied as a series of overlapping cosmids that are recombinant for transfection into cells and expressed in a complete array of helper functions. Such modifications can be used to supply helper virus sequences in all other examples in the same manner. In various embodiments of the invention, the viral gene / drug subject (gene X) comprises 1) HCMV DNA polymerase (UL54), 2) phosphotransferase (UL97), 3) viral serine protease (UL80), 4 ) Can be any viral gene that encodes a real or potential subject for drug sensitivity testing or drug screening tests. Such viral genes include, but are not limited to, UL44, UL57, UL105, UL102, UL70, UL114, UL98, or UL84.

The plasmids described for CMV resistance test vectors are named using the following rules. That is, the lowercase letter case p represents the production of plasmid DNA molecules that can be replicated in the laboratory strain of E. coli, and "A" performs the cis-active function required for propagation by helper viruses since the plasmid is an amplicon. And in detail these amplicon plasmids are meant to include the viral origin of replication and the "a" sequence indicates genomic mutations, cleavage and packages and to produce a genome capable of inversion. And CMV indicates that the amplicon sequence is specific for HCMV (optionally HSV-1 may indicate that a homologous signal of HSV-1 is present in the amplicon) and V regulates expression of the viral gene / drug subject Wherein the regulatory region is derived from the HCMV genome and is the regulatory region used for expression of the viral gene / drug subject in the overall viral context, C denotes that the regulatory element used to regulate expression of the viral gene / drug subject is heterologous. In the example it is shown that it contains a CMV IE promoter / enhancer and SV40 polyadenylation signal, S is the construct is a genomic construct and gene X is the subject (s) of the antiviral drug (s) in the examples given herein. Viral oil, which can be UL54, UL80, or UL97 to indicate polymerase, serine protease or phosphatase Indicates that the identified characters. Helper virus or directed gene helper virus vectors are named as follows. HCVM represents the strain of HCMV, β _2.7 F-IG represents the functional indicator gene inserted into β _2.7 ORF in the appropriate reading frame under the control of the β _2.7 regulatory region and Δgene X deletes the viral gene / drug subject from the virus In the examples given, it can be ΔUL54, ΔUL80 or ΔUL97, respectively, to indicate deletion of DNA polymerase, serine protease, or phosphatase.

Immunity test vector-production

Resistance test vectors are: 1) amplicon / gene X plasmid (pA-CMV-VS-gene X or pA-CMV-CS-gene X) by introducing a unique site called the patient sequence receiving site (PSAS) into the gene X coding region. 2) amplify the viral DNA present in the tissue or blood of the infected patient to amplify the fragment (PDG) derived from the patient corresponding to the CMV drug target (geneX), and 3) the amplified fragments Prepared by inserting into an amplicon / gene X plasmid in PSAS. Another embodiment includes using reverse transcription to isolate viral RNA from tissue and convert the RNA to DNA simulation prior to amplification of PDS.

Drug Sensitivity and Tolerance Testing

Drug susceptibility and resistance testing is performed by the corresponding directed gene viruses such as amplicon / gene X plasmid (pA-CMV-VS-gene X or pA-CMV-CS-gene X) and HCMV-β _2.7 F-IG / Δgene X. This is done using a two-part immunity test vector system containing a vector. In one embodiment, the amplicon / gene X plasmid is transfected into a package host cell / subject cell and then the cells are infected with a defect indicating gene viral vector. In another embodiment the amplicon / gene X plasmid and the defect indicator gene viral vector DNA are simultaneously transfected together into the package host cell / subject host cell. The package host cell / subject host cell can be any cell allowed for wild-type HCMV infection. The trans complementarity of the deleted gene by the amplicon / gene X plasmid results in its own persistent virus population that increases the expression of the reporter gene. The reporter gene is then increased in expression with the activity of the viral gene / drug subject encoded by the segment derived from the patient introduced into the amplicon / gene X plasmid. Some reporter gene expression will be observed due to the degree of underlying expression from the defect directing viral vector, replication and thus trans complementarity by the amplicon / gene X plasmid, resulting in the amplification of the fault indicating gene viral vector and the reporter gene in the target cell. Will significantly increase its expression. Antiviral drugs that inhibit HCMV replication through inhibition of viral genes / drug subjects will limit the propagation and expansion of the defect indication gene viral vector, which can be measured as a decrease in expression of the reporter gene product.

Drug Screening

Drug screening is tolerated with a directed gene viral vector such as an amplicon / gene X plasmid (pA-CMV-VS-gene X or pA-CMV-CS-gene X) and HCMV-β _2.7 F-IG / Δgene X plasmid It is performed using a test vector system. The PDS may be derived from a genome of a laboratory strain of HCMV or from a patient obtained from a patient and may comprise a wild type sequence or a sequence that supplies a viral gene / drug subject resistance to a known antiviral drug.

Drug screening is performed as follows. Directing gene viral vectors such as amplicon / gene X plasmids (pA-CMV-VS-gene X or pA-CMV-CS-gene X) and HCMV-β _2.7 F-IG / Δgene X plasmids are potentially antiviral The compound is introduced into the cell in the presence or absence of the compound. After maintaining the culture for a suitable period to spread the defect indication gene viral vector through the culture, the degree of amplicon expression of the reporter gene is measured and the degree of inhibition in the presence of the drug is calculated.

Example 10

Cytomegalovirus Drug Sensitivity and Tolerance Testing Using Resistance Test Vectors Containing a Function Directing Gene and Patient-Derived Section (s) Under the Control of a Viral Promoter.

Direct Gene Gene Vector-

Subject host cell systems that express function indicator genes under the control of an HCMV viral promoter active according to viral replication are constructed. Defects in replication are due to the fact that a defective helper viral vector (HCMV / Δgene X) has been constructed so that fragments of the genome containing the viral gene / drug subject (geneX) are deleted from the virus. The viral gene / drug subject (gene X) is required for trans-complementarity of the directed gene viral vector and cis-active function (PSAS) (in detail this indicates the HCMV origin of replication and HCMV genome cleavage and package). Is provided on an amplicon plasmid (pA-CMV-VS-gene X) with an HCMV “a” sequence). The PSAS of pA-CMV-VS-gene X is designed to receive PDS in a cassette containing regulatory signals appropriate for the individual viral gene / drug subject and is derived from the context of the viral gene / drug subject in wild type HCMV. The defect package / helper virus vector (HCMV / Δgene X) and the amplicon / gene X plasmid (pA-CMV-VS-gene X) and the indicator cell line constitute the resistance test vector system. The defective package / helper viral vector can only be propagated as a virus colony to the package host cell / subject host cell system and the viral gene / drug subject deleted from such system is provided in trans. Virus colonies may be prepared from such package host cells / subject host cell lines and used to infect package host cells / subject host cells, or DNA from such viral colonies may be isolated to render the package host cells / subject host cells of a resistance test vector system. As part, it can be used for transfection with an amplicon / gene X plasmid by transfection with an acceptable cell type for HCMV infection. The trans complementarity of the deleted gene by the amplicon / gene X plasmid results in its own persistent virus population that increases the expression of the reporter gene. In this case, the reporter gene is increased in expression according to the activity of the viral gene / drug subject encoded by a segment derived from a patient introduced into the amplicon / gene X plasmid.

In another embodiment, the amplification / gene X plasmid (pA-CMV-CS-gene X) receives PDS by expressing a gene drug subject (gene X) derived from a patient under the control of a heterologous promoter and polyadenylation signal. It includes PSAS. In one embodiment of this embodiment, an expression cassette comprising a CMV IE enhancer promoter region, PSAS, and an SV40 polyadenylation (pA) signal comprises a cis-active function required for trans-complementarity of the directed gene viral vector (detailed). Preferably it is included on an amplification / gene X plasmid (pA-CMV-CS-gene X) in addition to the HCMV “a” sequence that directs HCMV genome cleavage and package of replication and HCMV origin of replication.

In another embodiment of the invention, the viral gene / drug subject is 1) HCMV DNA polymerase (UL54), 2) phosphotransferase (UL97), 3) viral serine protease (UL80), 4) drug sensitivity test or drug screening It can be any viral gene that encodes a real or potential subject for the test. Such viral genes include, but are not limited to, UL44, UL57, UL105, UL102, UL70, UL114, UL98, or UL84.

Immunity test vector-production

Resistance test vectors include: 1) introducing a unique site called the patient sequence receiving site (PSAS) into the viral gene / drug subject (gene X), which is an amplicon / gene X plasmid (pA-CMV-gene X or pA-CMV-CS- Modifying gene X), 2) amplifying a segment (PDG) derived from a patient corresponding to the CMV drug subject (geneX) from viral DNA present in the tissue or blood of an infected patient, and 3) the amplified fragment Are prepared by inserting into an amplicon / gene X plasmid in PSAS. Another embodiment includes using reverse transcription to isolate viral RNA from tissue and convert the RNA to DNA simulation prior to amplification of PDS.

Drug Sensitivity and Tolerance Testing

Drug susceptibility and resistance testing is performed according to amplicon / gene X plasmid (pA-CMV-VS-gene X or pA-CMV-CS-gene X), defective viral vectors such as HCMV / Δgene X, and virus replication. It is performed using a resistance test vector system comprising a subject cell line containing an indicator gene under the control of an HCMV promoter. In one embodiment, the amplicon / gene X plasmid is transfected into a package host cell / subject cell and then the cells are infected with a defective helper virus vector. In another embodiment the amplicon / gene X plasmid and the defective helper virus vector DNA are simultaneously transfected together into the package host cell / subject host cell. The trans complementarity of the deleted gene by the amplicon / gene X plasmid results in its own persistent virus population that increases the expression of the reporter gene. The reporter gene is then increased in expression with the activity of the viral gene / drug subject encoded by the segment derived from the patient introduced into the amplicon / gene X plasmid. Antiviral drugs that inhibit HCMV replication through inhibition of viral genes / drug subjects will limit the propagation and expansion of the defective helper viral vector, which in turn is measured as a decrease in expression of the reporter gene product.

Drug Screening

Drug screening is performed using a resistance test vector system consisting of helper virus vectors such as amplicon / gene X plasmid (pA-CMV-VS-gene X or pA-CMV-CS-gene X) and HCMV / Δgene X. . The PDS may be derived from a sample derived from the genome or patient of a laboratory strain of HCMV and may comprise a wild type sequence or a sequence that supplies a viral gene / drug subject resistance to a known antiviral drug.

Drug screening is performed as follows. Helper viral vectors, such as amplicon / gene X plasmid (pA-CMV-VS-gene X or pA-CMV-CS-gene X) and HCMV / Δgene X, are present or present in the presence of latent antiviral compounds It is introduced into the indicator cell in a state where it is not. After maintaining the culture for a suitable period to spread the defective helper virus vector through the culture, the expression level of the reporter gene is measured and the degree of inhibition in the presence of the drug is calculated.

Example 11

Cytomegalovirus Drug Sensitivity and Tolerance Testing Using Resistance Test Vectors Including Nonfunctional Indicative Genes with Modified Promoter and Patient-Derived Section (s).

Direct Gene Gene Vector-

Directed Gene Viral Vectors Including Nonfunctional Directed Genes with Modified Promoters Designed using HCMV amplicon plasmids containing viral genes of interest for antiviral drug (s) (gene X). Directed gene viral vector (pA-CMV-VS-gene X- (NF-IG) PP) is a nonfunctional indicator gene with a modified promoter, all cis-activity regulatory elements required for HCMV replication (ie, the "a" sequence). And HCMV origin of replication), and viral gene / drug subject expression cassette with PSAS. PSAS is designed to receive PDSs in cassettes containing regulatory signals appropriate for individual viral genes / drug subjects and are derived from the context of viral genes / drug subjects in wild type HCMV. The non-functional indicator gene cassette is assembled so that the promoter region is positioned in the wrong orientation, ie antisense, or in the wrong position, ie, below the indicator gene ORF relative to the indicator gene ORF (FIG. 14). The promoter and indicator gene ORF are located in genomic regions that are separated and switch relative to each other during replication of the genome (FIG. 11). This conversion allows the two parts of the modified promoter indicator gene cassette to be appropriately cultured to enable expression of the indicator gene product. Defect helper viral vectors (HCMV / Δgene X) are defective for replication because genomic sections comprising the viral gene / drug subject (gene X) are deleted from the virus. The indicator gene viral vector and the binding helper / package viral vector constitute a tolerance test vector system. The defective helper / package viral vector (HCMV / Δgene X) may be propagated only in cellular systems in which the deleted viral gene / drug subject is provided in trans. Virus colonies may be prepared from such package host cells / subject host cell lines and used to infect package host cells / subject host cells, or DNA from such viral colonies may be isolated to render the package host cells / subject host cells of a resistance test vector system. As part it may be used to transfect with cell types that are acceptable for HCMV infection with the introduction of the indicator gene viral vector. The trans complementarity of deleted genes by the directed gene viral vector results in its own persistent virus population. These two fragments are formed because during the replication of the directed viral viral vector, the directed viral viral vector concatamer is formed and the conversion takes place as part of the normal replication cycle of the HCMV (FIG. 11) so that the two fragments of the modified promoter cassette are rearranged. An orientation is suitable to direct the transcription of the RNA that enables expression of the reporter gene.

In another embodiment, the directed gene viral vector comprises a PSAS that receives PDS by expressing a gene X sequence derived from a patient under the control of a heterologous promoter and a polyadenylation signal. In one embodiment of this embodiment, an expression cassette comprising a CMV IE enhancer promoter region, PSAS, and an SV40 polyadenylation (pA) signal comprises a cis-active function required for trans-complementation of the directed gene viral vector. Specifically, it includes an HCMV "a" sequence that indicates HCMV genome cleavage and package of replication and HCMV origin of replication, and a modified promoter cassette fragment, in addition to the directed gene viral vector (pA-CMV-CS-gene- (NF- IG) PP).

In various embodiments of the invention, the viral gene / drug subject is 1) HCMV DNA polymerase (UL54), 2) phosphotransferase (UL97), 3) viral serine protease (UL80), 4) drug sensitivity test or drug screening It can be any viral gene that encodes a real or potential subject for the test. Such viral genes include, but are not limited to, UL44, UL57, UL105, UL102, UL70, UL114, UL98, or UL84.

Immunity test vector-production

Resistance test vectors: 1) introduce a unique restriction site called the patient sequence receiving site (PSAS) into the viral gene / drug subject (gene X) coding region to direct the directed gene viral vector (pA-CMV-VS-gene X- (NF- IG) modifying PP or pA-CMV-CS-gene X- (NF-IG) PP), and 2) from the patient corresponding to the CMV drug subject (gene X) from viral DNA present in the tissue or blood of the infected patient. Amplified induced fragments (PDS), and 3) the correctly amplified fragments were prepared by inserting them into amplicon / gene X in PSAS. Another embodiment includes using reverse transcription to isolate viral RNA from tissue and convert the RNA to DNA simulation prior to amplification of PDS.

Drug Sensitivity and Tolerance Testing

Drug susceptibility and resistance tests were performed with the directed gene viral vectors (pA-CMV-VS-gene X- (NF-IG) PP or pA-CMV-CS-gene X- (NF-IG) PP) and HCMV / Δgene X It is performed using an immunity test vector system that includes a defective helper virus vector. In one embodiment, the indicator gene viral vector (pA-CMV-VS-gene X- (NF-IG) PP or pA-CMV-CS-gene X- (NF-IG) PP) is transfected with appropriate cells and The cells are then infected with the defective helper virus vector (HCMV / Δgene X). In another embodiment the indicator gene viral vector and the defective helper / package virus vector DNA are simultaneously transfected together into the package host cell / subject host cell. The trans complementarity of deleted genes by the directed gene viral vector results in its own persistent virus population. During replication of the indicator viral vector, the two fragments allow expression of the indicator gene because contamers of the indicator viral vector are formed and conversion occurs as part of the normal replication cycle of HCMV, resulting in rearrangement of the two fragments of the modified promoter cassette. In a suitable orientation to direct transcription of the RNA. The indicator gene is expressed according to the activity of the viral gene / drug subject encoded by the segment introduced from the patient introduced into the indicator gene viral vector. Antiviral drugs that inhibit HCMV replication through inhibition of viral genes / drug subjects limit replication of the directed gene viral vector, which in turn can limit the number of genomes that can be reversed and decrease the expression of reporter gene products. .

Drug Screening

Drug screening involves defects such as directed gene viral vectors (pA-CMV-VS-gene X- (NF-IG)) PP or pA-CMV-CS-gene X- (NF-IG) PP) and HCMV / Δgene X It is performed using an immunity test vector system consisting of a package / helper virus vector. The PDS may be derived from a sample derived from the genome or patient of a laboratory strain of HCMV and may comprise a wild type sequence or a sequence that supplies a viral gene / drug subject resistance to a known antiviral drug.

Drug screening is performed as follows. Defective helper viral vectors such as the directed gene viral vectors (pA-CMV-VS-gene X- (NF-IG) PP or pA-CMV-CS-gene X- (NF-IG) PP) and HCMV / Δgene X The latent antiviral compound is introduced into the cell in the presence or absence of it. After maintaining the culture for a suitable period of time that the directed gene viral vector can replicate, the expression level of the reporter gene is measured and the degree of inhibition in the presence of the drug is calculated.

Example 12

Cytomegalovirus Drug Sensitivity and Tolerance Testing Using Resistance Test Vectors Including Nonfunctional Indicating Genes with Modified Coding Regions and Section (s) Derived from Patients.

Direct Gene Gene Vector-

Directed Gene Viral Vectors Including Non-Function Directed Genes with Modified Coding Regions Designed using HCMV amplicon plasmids containing viral genes that are the subject of antiviral drug (s) (gene X). Indicator gene viral vectors (pA-CMV-VS-gene X- (NF-IG) PCR) are nonfunctional indicator genes with modified coding regions, all cis-activity regulators required for HCMV replication (ie, "a" HCMV origin of sequence and replication), and viral gene / drug subject expression cassette with PSAS. PSAS is designed to receive PDSs in cassettes containing regulatory signals appropriate for individual viral genes / drug subjects and are derived from the context of viral genes / drug subjects in wild type HCMV. The non-functional indication gene cassette is assembled so that the 5 'portion of the coding region and the promoter region are positioned in the wrong orientation, ie antisense, or wrong position, ie the remaining 3' portion of the coding region, relative to the remaining 3 'portion of the coding region ( 15). The 5 'portion of the coding zero and the promoter are located in genomic regions that are separated from the 3' portion of the coding region and switch to each other during replication of the genome (Figure 11). This inversion allows the two parts of the modified coding region indicator gene cassette to be appropriately cultured to allow expression of the indicator gene product (FIG. 16). Defect helper viral vectors (HCMV / Δgene X) are defective for replication because genomic sections comprising the viral gene / drug subject (gene X) are deleted from the virus. The indicator gene viral vector and the binding helper / package viral vector constitute a tolerance test vector system. The defective helper / package viral vector (HCMV / Δgene X) is propagated only in packaged host cells / subject host cell systems where deleted viral genes are provided in trans. Virus colonies may be prepared from such package host cell / subject host cell lines and used to infect cells or DNA from such viral colonies may be isolated to allow package host cells / subject host cells as part of a resistance test vector system for HCMV infection. It can be used to transfect with an acceptable cell type while introducing the indicator gene viral vector. The trans complementarity of deleted genes by the directed gene viral vector results in its own persistent virus population. These two fragments allow expression of the reporter gene because during the replication of the indicator viral vector the indicator viral vector concatamer is formed and the conversion takes place as part of the normal replication cycle of the HCMV so that the two fragments of the modified coding region cassette are rearranged. In a suitable orientation to direct transcription of the RNA.

In another embodiment, the directed gene viral vector comprises a PSAS that accepts PDS by a method of expressing a viral gene sequence (gene X) derived from a patient under the control of a heterologous promoter and a polyadenylation signal. In one embodiment, the expression cassette comprising the CMV IE enhancer promoter region, PSAS, and SV40 polyadenylation (pA) signal is a cis-active function required for trans-complementarity of the directed gene viral vector (in detail this is replication In addition to the HCMV "a" sequence that indicates HCMV genome cleavage and package of HCMV and the HCMV origin of replication) and the modified promoter cassette fragment, the directed gene viral vector (pA-CMV-CS-gene- (NF-IG) PCR) Included in the phase.

In various embodiments of the invention, the viral gene / drug subject is 1) HCMV DNA polymerase (UL54), 2) phosphotransferase (UL97), 3) viral serine protease (UL80), 4) drug sensitivity test or drug screening It can be any viral gene that encodes a real or potential subject for the test. Such viral genes include, but are not limited to, UL44, UL57, UL105, UL102, UL70, UL114, UL98, or UL84.

Immunity test vector-production

Resistance test vectors can be used to: 1) introduce a unique restriction site called a patient sequence receiving site (PSAS) into a viral gene / drug subject (gene X) coding region to direct a directed viral viral vector (pA-CMV-VS-gene X- (NF- IG) PCR or pA-CMV-CS-gene X- (NF-IG) PCR), and 2) from the patient corresponding to the CMV drug subject (gene X) from viral DNA present in the tissue or blood of the infected patient. Amplified induced fragments (PDS), and 3) the correctly amplified fragments were prepared by inserting them into amplicon / gene X in PSAS. Another embodiment includes using reverse transcription to isolate viral RNA from tissue and convert the RNA to DNA simulation prior to amplification of PDS.

Drug Sensitivity and Tolerance Testing

Drug susceptibility and resistance tests were performed with the directed gene viral vector (pA-CMV-VS-gene X- (NF-IG) PCR or pA-CMV-CS-gene X- (NF-IG) PCR) and HCMV / Δgene X It is performed using an immunity test vector system that includes a defective helper virus vector. In one embodiment, the indicator gene viral vector (pA-CMV-VS-gene X- (NF-IG) PCR or pA-CMV-CS-gene X- (NF-IG) PCR) is transformed into a suitable subject host cell. After infection, the cells are infected by a defective helper virus vector (HCMV / Δgene X). In another embodiment the indicator gene viral vector and the defective helper viral vector DNA are transfected together into the cell simultaneously. The trans complementarity of deleted genes by the directed gene viral vector results in its own persistent virus population. The two fragments are capable of expression of the indicator gene because concatemers of the indicator gene viral vector are formed during replication of the indicator gene viral vector and conversion occurs as part of the normal replication cycle of HCMV, resulting in rearrangement of the two fragments of the modified coding region cassette. In a suitable orientation to direct the transcription of the RNA to cause (FIG. 16). The reporter gene is expressed according to the activity of the viral gene / drug subject encoded by the segment introduced from the patient which is introduced into the indicator gene viral vector. Antiviral drugs that inhibit HCMV replication through inhibition of viral gene / drug subjects limit replication of the directed gene viral vector, which in turn limits the number of genomes in which reversal can occur and is measured as a decrease in expression of reporter gene products. Can be.

Drug Screening

Drug screening involves defects such as directed gene viral vectors (pA-CMV-VS-gene X- (NF-IG)) PCR or pA-CMV-CS-gene X- (NF-IG) PCR and HCMV / Δgene X It is performed using a resistance test vector system consisting of helper virus vectors. The PDS may be derived from a sample derived from the genome or patient of a laboratory strain of HCMV and may comprise a wild type sequence or a sequence that supplies a viral gene / drug subject resistance to a known antiviral drug.

Drug screening is performed as follows. Defective helper viral vectors such as the directed gene viral vector (pA-CMV-VS-gene X- (NF-IG) PCR or pA-CMV-CS-gene X- (NF-IG) PCR) and HCMV / Δgene X The latent antiviral compound is introduced into the cell in the presence or absence of it. After maintaining the culture for a suitable period of time that the directed gene viral vector can replicate, the expression level of the reporter gene is measured and the degree of inhibition in the presence of the drug is calculated.

Example 13

Cytomegalovirus Drug Sensitivity and Tolerance Testing Using Resistance Test Vectors Containing Functional Indicating Genes and Patient-Derived Section (s).

Direct Gene Gene Vector-

Indicator genes comprising function indicator genes under the control of an endogeneous viral promoter The viral vector is based on an HCMV amplicon plasmid containing viral genes that are the subject of antiviral drug (s) (gene X). The indicator gene viral vector (pA-CMV-VS-gene XF-IG) is a function indicator gene under the control of the viral promoter according to viral replication, all cis-activity regulatory elements required for HCMV replication (ie, the "a" sequence and HCMV origin of replication), and viral gene / drug subject (gene X) expression cassette with PSAS (FIG. 17). PSAS is designed to receive PDS in a cassette containing regulatory signals appropriate for individual viral genes and are derived from the context of the viral gene / drug subject (gene X) in wild type HCMV. Defect helper viral vectors (HCMV / Δgene X) are defective for replication since genomic segments comprising viral genes / drug subjects are deleted from the virus. The directed gene viral vector and defective helper viral vector constitute a tolerance test vector system. The defective helper virus vector (HCMV / Δgene X) is propagated only in packaged host cells / subject host cell systems where deleted viral genes are provided in trans. Virus colonies may be prepared from such cell lines and used to infect package host cells / subject host cells or DNA from such viral colonies may be isolated to render package host cells / subject host cells as part of a resistance test vector system for HCMV infection. It can be used to transfect with an acceptable cell type while introducing the indicator gene viral vector. The trans complementarity of deleted genes by the directed gene viral vector results in its own persistent virus population. The indicator gene viral vector replicates according to trans complementarity between the indicator gene viral vector pA-CMV-VS-gene X-F-IG and the defective helper virus vector HCMV / Δgene X.

In another embodiment, the directed gene viral vector comprises a PSAS that receives PDS by way of expressing a viral gene / drug subject (gene X) derived from a patient under the control of a heterologous promoter and a polyadenylation signal. In one embodiment, the expression cassette comprising the CMV IE enhancer promoter region, PSAS, and SV40 polyadenylation (pA) signal is a cis-active function required for trans-complementarity of the directed gene viral vector (in detail this is replication In addition to the HCMV "a" sequence, which indicates the HCMV genome cleavage and package of HCMV, and the HCMV origin of replication) and modified coding region cassette fragments, on a directed gene viral vector (pA-CMV-CS-gene XF-IG) do.

In various embodiments of the invention, the viral gene / drug subject is 1) HCMV DNA polymerase (UL54), 2) phosphotransferase (UL97), 3) virus serine protease (UL80), 4) drug sensitivity test or drug screening It can be any viral gene that encodes a real or potential subject for the test. Such viral genes include, but are not limited to, UL44, UL57, UL105, UL102, UL70, UL114, UL98, or UL84.

Immunity test vector-production

Resistance test vectors: 1) introduce a unique restriction site called the patient sequence receiving site (PSAS) into the viral gene / drug subject (gene X) coding region to direct the directed viral viral vector (pA-CMV-VS-gene XF-IG or pA). -CMV-CS-gene XF-IG), and 2) amplify fragments derived from patients corresponding to the CMV drug subject (gene X) from viral DNA present in the tissue or blood of the infected patient, 3) The correctly amplified fragments are prepared by inserting them into an amplicon / gene X plasmid in PSAS. Another embodiment includes using reverse transcription to isolate viral RNA from tissue and convert the RNA to DNA simulation prior to amplification of PDS.

Drug Sensitivity and Tolerance Testing

Drug susceptibility and resistance tests include resistance including a directed gene viral vector (pA-CMV-VS-gene XF-IG or pA-CMV-CS-gene XF-IG) and a defective helper virus vector such as HCMV / Δgene X. It is implemented using a test vector system. In one embodiment, the indicator gene viral vector (pA-CMV-VS-gene XF-IG or pA-CMV-CS-gene XF-IG) is transfected into a suitable package host cell / subject host cell and thereafter. Is strongly infected by a defective helper virus vector (HCMV / Δgene X). In another embodiment the indicator gene viral vector and the defective helper viral vector DNA are simultaneously transfected together into the package host cell / subject host cell. The trans complementarity of deleted genes by the directed gene viral vector results in its own persistent virus population that increases the expression of the reporter gene. The reporter gene is then directly increased in expression with the activity of the viral gene / drug subject encoded by the segment derived from the patient being introduced into the indicator gene viral vector. Antiviral drugs that inhibit HCMV replication through inhibition of viral genes / drug subjects will limit the propagation and expansion of the defective helper viral vector, which in turn is measured as a decrease in expression of the reporter gene product.

Drug Screening

Drug screening uses a resistance test vector system consisting of a defective helper virus vector such as a directed gene viral vector (pA-CMV-VS-gene XF-IG or pA-CMV-CS-gene XF-IG) and HCMV / Δgene X. Is performed. The PDS may be derived from a sample derived from the genome or patient of a laboratory strain of HCMV and may comprise a wild type sequence or a sequence that supplies a viral gene / drug subject resistance to a known antiviral drug.

Drug screening is performed as follows. Defective helper viral vectors, such as the directed gene viral vector (pA-CMV-VS-gene XF-IG or pA-CMV-CS-gene XF-IG) and HCMV / Δgene X, have potential antiviral compounds Is introduced into the cell in or without it. After maintaining the culture for a suitable period of time that the directed gene viral vector can replicate, the expression level of the reporter gene is measured and the degree of inhibition in the presence of the drug is calculated.

It is an object of the present invention to provide a drug sensitivity and resistance test that can show whether a virus population of a patient is resistant to a prescribed drug. Another object is to provide a test that allows a physician to substitute one or more drugs in a therapeutic regimen for patients who are resistant to a given drug (s) after a previous course of treatment. Yet another object of the present invention is to provide a test that allows the selection of an effective drug diet in treating viral infections. It is yet another object of the present invention to provide a safe, standardized, fast, accurate and reliable analysis of drug sensitivity and tolerance for clinical and research applications. It is also an object of the present invention to assess the biological efficacy of candidate drug compounds that act on specific viral genes and / or viral proteins, particularly related to viral drug resistance and cross resistance. It is also an object of the present invention to provide a means and composition for assessing viral drug resistance and sensitivity.

Claims (111)

(a) introducing into a host cell a resistance test vector comprising a patient-derived fragment comprising a hepatitis C virus gene and an indicator gene; (b) culturing the host cell from (a); (c) measuring the expression of the indicator gene in the target host cell; (d) the expression of the indicator gene measured when (c) the test concentration of the HCV antiviral drug is present in steps (a)-(c); (b)-(c) or (c); A) A method of determining sensitivity to an HCV antiviral drug comprising comparing the indicator gene expression measured when step-(c) is performed in the absence of the antiviral drug. The method of claim 1, The resistance test vector comprises DNA of a genomic viral vector. The method of claim 2, The resistance test vector comprises RNA of a genomic viral vector. The method of claim 1, The resistance test vector is characterized in that it comprises a gene encoding C, E1, E2, NS2, NS3, NS4 or NS5. The method of claim 1 Wherein said patient-derived segment comprises a functional viral sequence. The method of claim 1 Wherein said patient-derived segment encodes one protein that is a target of an antiviral drug. The method of claim 1 Wherein said patient-derived segment encodes two or more proteins that are targets of an antiviral drug. The method of claim 5 Wherein said functional viral sequence comprises an IRES. The method of claim 1 The indicator gene is a functional indicator gene and the host cell is a resistance test vector host cell comprising an imposing step of infecting a target host cell using the supernatant filtered from the resistance test vector host cell with resistance test vector virus particles. Characterized in that the method. The method of claim 1 Wherein said indicator gene is a non-functional indicator gene. The method of claim 10 The host cell is characterized in that the package host cell / resistance test host cell. The method of claim 11, The culture is characterized in that by co-culture. The method of claim 11, The target host cell is infected with resistance test vector virus particles using supernatant filtered from the package host cell / resistant test vector host cell. The method of claim 1 The indicator gene is a luciferase gene. The method of claim 1 The indicator gene is characterized in that the β-lactamase gene. The method of claim 11, Wherein said package host cell / resistance test vector host cell is a human cell. The method of claim 11, The package host cell / resistance test vector host cell is characterized in that the human liver cells. The method of claim 11, Wherein said package host cell / resistance test vector host cell is a Huh7 cell. The method of claim 1 The target host cell is characterized in that the HepG2 cells. A resistance test vector comprising a patient-derived fragment comprising a Flaviviridae gene and an indicator gene. The method of claim 20, The patient-derived segment comprises a flavivirus gene. The method of claim 20, And said patient-derived segment is one gene. The method of claim 20, The patient-derived segment is two or more genes. The method of claim 20, The patient-derived segment comprises an HCV gene. The method of claim 20, The patient-derived segment comprises a NS3 / 4A protease gene. The method of claim 20, The patient-derived segment comprises a NS5B RDRP gene. The method of claim 20, The patient-derived segment comprises an IRES. The method of claim 20, The indicator gene is a resistance test vector, characterized in that the functional indicator gene. The method of claim 20, The indicator gene is a resistance test vector, characterized in that the non-functional indicator gene. The method of claim 20, The indicator gene is a resistance test vector, characterized in that the luciferase gene. A packaging host cell transfected with the resistance test vector of claim 20. The method of claim 31, wherein The packaging host cell is a packaging host cell, characterized in that the mammalian host cell. The method of claim 31, wherein The packaging host cell is a packaging host cell, characterized in that the human host cell. The method of claim 31, wherein The packaging host cell is a packaging host cell, characterized in that the human liver cells. The method of claim 31, wherein The packaging host cell is a packaging host cell, characterized in that HepG2. The method of claim 31, wherein The packaging host cell is a packaging host cell, characterized in that Huh7. (a) introducing into a host cell a resistance test vector comprising a patient-derived fragment comprising hepatitis C virus gene and a nonfunctional indicator gene; (b) culturing the host cell from (a); (c) measuring the expression of the indicator gene in the target host cell; (d) the expression of the indicator gene measured when (c) the test concentration of the HCV antiviral drug is present in steps (a)-(c); (b)-(c) or (c); A) A method of determining sensitivity to an HCV antiviral drug comprising comparing the indicator gene expression measured when step-(c) is performed in the absence of the HCV antiviral drug. The method of claim 37, The resistance test vector comprises DNA of a genomic viral vector. The method of claim 37, The resistance test vector comprises RNA of a genomic viral vector. The method of claim 37, The resistance test vector is characterized in that it comprises a gene encoding C, E1, E2, NS2, NS3, NS4 or NS5. The method of claim 37, Wherein said patient-derived segment encodes one protein. The method of claim 37, And said patient-derived segment encodes two or more proteins. The method of claim 37, Wherein said patient-derived segment comprises a functional viral sequence. The method of claim 37, The indicator gene is a luciferase gene. The method of claim 37, The host cell is characterized in that the package host cell. The method of claim 37, The package host cell is characterized in that the human cell. The method of claim 37, The package host cell is characterized in that the human liver cells. The method of claim 37, Package host cell characterized in that the Huh7 cells. The method of claim 37, The package host cell is characterized in that HepG2 cells. The method of claim 37, Wherein said nonfunctional indicator gene comprises a negative sense sequence. The method of claim 37, The host cell and the target cell is characterized in that the same cell. The method of claim 37, The target cell is characterized in that the human cell. The method of claim 37, And said target host cell is a target cell infected with resistance test vector virus particles using supernatant filtered from said package host cell / resistant test vector host cell. The method of claim 37, The culture is characterized in that the co-culture. (a) developing a standard curve of drug sensitivity to HCV antiviral drugs; (b) determining the patient's HCV antiviral drug sensitivity according to the method of claim 1; And (c) comparing the HCV antiviral drug sensitivity of step (b) with the standard curve determined in step (a) when a decrease in HCV antiviral sensitivity indicates an increase in HCV antiviral drug resistance in the patient. How to determine the HCV antiviral drug resistance in a patient. (a) developing a standard curve of drug sensitivity to HCV antiviral drugs; (b) determining the patient's HCV antiviral drug sensitivity according to the method of claim 37; And (c) comparing the HCV antiviral drug sensitivity of step (b) with the standard curve determined in step (a) when a decrease in HCV antiviral sensitivity indicates an increase in HCV antiviral drug resistance in the patient. How to determine the HCV antiviral drug resistance in a patient. (a) obtaining a patient-derived section from the patient near the time of sensitivity determination and determining the patient's initial HCV antiviral drug sensitivity according to claim 1; (b) determining the late HCV antiviral drug sensitivity of the patient; (c) comparing the HCV antiviral drug sensitivity determined in steps (a) and (b) above, when a decrease in late antiviral drug sensitivity at initial preparation indicates an increase or progress in HCV antiviral drug resistance in the patient. How to determine the HCV antiviral drug resistance of a patient comprising a. (a) obtaining the patient-derived section from the patient near the time of sensitivity determination and determining the patient's initial HCV antiviral drug sensitivity according to claim 37. (b) determining the initial HCV antiviral drug sensitivity of the patient; (c) comparing the HCV antiviral drug sensitivity determined in steps (a) and (b) above, when a decrease in the sensitivity of late HCV antiviral drug resistance to initial preparation indicates an increase or advancement of HCV antiviral drug resistance in the patient. A method of determining HCV antiviral drug resistance in a patient comprising a step. (a) introducing a patient-derived fragment resistance test vector comprising a HCMV gene and an indicator gene into a host cell; (b) culturing the host cell from (a); (c) measuring the expression of the indicator gene in the target host cell; (d) From (c), the test concentration of the HCMV antiviral drug is (a)-(c); (b)-(c); comparing the expression of the indicator gene measured when present in step (c) with the expression of the indicator gene measured when steps (a)-(c) are performed in the absence of an antiviral drug. How to determine the sensitivity to. The method of claim 59, The resistance test vector comprises DNA of a genomic viral vector. The method of claim 59, The resistance test vector comprises DNA of a subgenomic virus vector. The method of claim 59, The resistance test vector comprises DNA encoding phosphotransferase (UL97), DNA polymerase (UL54), protease (UL80), UL54, UL44, UL57, UL105, UL102, UL70, UL114, UL98 or UL84. Characterized in that. The method of claim 59, Wherein said patient-derived segment comprises a functional viral sequence. The method of claim 59, Wherein said patient-derived segment encodes one protein that is a target of an antiviral drug. The method of claim 59, Wherein said patient-derived segment encodes two or more proteins that are targets of an antiviral drug. The method of claim 59, The indicator gene is a functional indicator gene and the host cell is a resistance test vector host cell comprising an imposing step of infecting the target host cell with resistance test vector virus particles. The method of claim 59, Wherein said indicator gene is a non-functional indicator gene. The method of claim 59, Wherein said host cell is a packaged host cell / resistant test vector host cell. The method of claim 68, The culture is characterized in that the co-culture. The method of claim 69, wherein And said target host cell is a target host cell infected with resistance test vector virus particles from said package host cell / resistant test vector host cell. The method of claim 59, The indicator gene is a luciferase gene. The method of claim 59, The indicator gene is characterized in that the β-lactamase gene. The method of claim 68, The package host cell / resistance test vector host cell is characterized in that the human cell. The method of claim 68, The package host cell / resistance test vector host cell is characterized in that the fibroblasts of human skin. The method of claim 68, The package host cell / resistance test vector host cell is characterized in that the MRC5 cells. The method of claim 59, The target host cell is characterized in that the human fetal lung cells. Patient-derived segment resistance test vector comprising a herpesviridae gene and an indicator gene. 78. The method of claim 77 wherein The patient-derived segment comprises alpha herpesvirinae. 78. The method of claim 77 wherein And said patient-derived segment is one gene. 78. The method of claim 77 wherein The patient-derived segment is two or more genes. 78. The method of claim 77 wherein The patient-derived segment comprises an HCMV gene. 78. The method of claim 77 wherein The indicator gene is a resistance test vector, characterized in that the functional indicator gene. 78. The method of claim 77 wherein The indicator gene is a resistance test vector, characterized in that the non-functional indicator gene. 78. The method of claim 77 wherein The indicator gene is a resistance test vector, characterized in that the luciferase gene. Package host cell transfected with the resistance test vector of claim 77 86. The method of claim 85, The packaging host cell is a packaging host cell, characterized in that the mammalian host cell. 86. The method of claim 85, The packaging host cell is a packaging host cell, characterized in that the human host cell. 86. The method of claim 85, The packaging host cell is a packaging host cell, characterized in that the human fetal lung cells. 86. The method of claim 85, The packaging host cell is a packaging host cell, characterized in that the MRC5 cell. 86. The method of claim 85, The packaging host cell is a packaging host cell, characterized in that the fibroblast line of human skin. (a) introducing into a host cell a resistance test vector comprising a patient-derived fragment comprising a hepatitis C virus gene and a nonfunctional indicator gene; (b) culturing the host cell from (a); (c) measuring the expression of the indicator gene in the target host cell; (d) the expression of the indicator gene measured when (c) the test concentration of the HCV antiviral drug is present in steps (a)-(c); (b)-(c) or (c); A) A method of determining sensitivity to an HCV antiviral drug comprising comparing the indicator gene expression measured when step-(c) is performed in the absence of the HCMV antiviral drug. 92. The method of claim 91, The resistance test vector comprises DNA of a genomic viral vector. 92. The method of claim 91, The resistance test vector is characterized in that it comprises the DNA of the subgenomic virus vector. 92. The method of claim 91, The resistance test vector comprises DNA encoding phosphotransferase (UL97), DNA polymerase (UL54), protease (UL80), UL54, UL44, UL57, UL105, UL102, UL70, UL114, UL98 or UL84. Characterized in that. 92. The method of claim 91, Wherein said patient-derived segment encodes one protein. 92. The method of claim 91, And said patient-derived segment encodes two or more proteins. 92. The method of claim 91, The indicator gene is a luciferase gene. 92. The method of claim 91, The host cell is characterized in that the package host cell. 92. The method of claim 91, The package host cell is characterized in that the human cell. 92. The method of claim 91, The package host cell is characterized in that the human fetal lung cells. 92. The method of claim 91, The package host cell is characterized in that the fibroblasts of human skin. 92. The method of claim 91, Wherein said non-functional indicator gene comprises a substituted promoter. 92. The method of claim 91, Wherein said non-functional indicator cell comprises a substituted coding region. 92. The method of claim 91, The host cell and the target cell is characterized in that the same cell. 92. The method of claim 91, The target cell is characterized in that the human cell. 92. The method of claim 91, Wherein said target host cell is a target host cell infected with resistance test vector virus particles from a package host cell / resistant test vector host cell. 107. The method of claim 106, The culture is characterized in that the co-culture. (a) developing a standard curve of drug sensitivity for HCMV antiviral drugs; (b) determining the patient's HCMV antiviral drug sensitivity according to the method of claim 59; And, (c) comparing the HCMV antiviral drug sensitivity of step (b) with the standard curve determined in step (a) when a decrease in HCMV antiviral sensitivity indicates an increase in patient HCMV antiviral drug resistance. How to determine the HCMV antiviral drug resistance of a patient comprising a. (a) developing a standard curve of drug sensitivity for HCMV antiviral drugs; (b) determining the patient's HCMV antiviral drug sensitivity according to the method of claim 91; And (c) comparing the HCMV antiviral drug sensitivity of step (b) with the standard curve determined in step (a) when a decrease in HCMV antiviral sensitivity indicates an increase in patient HCMV antiviral drug resistance. How to determine the HCMV antiviral drug resistance of a patient comprising a. (a) obtaining a patient-derived section from the patient near the time of sensitivity determination and determining the patient's initial HCMV antiviral drug sensitivity according to the method of claim 59; (b) determining late HCMV antiviral drug sensitivity of the patient; And, (c) comparing the HCMV antiviral drug susceptibility determined in steps (a) and (b) above, when a decrease in late antiviral drug susceptibility at initial preparation indicates an increase or progress in the antiviral drug resistance of the patient. How to determine the HCMV antiviral drug resistance of a patient comprising a. (a) obtaining a patient-derived section from the patient near the time of sensitivity determination and determining the patient's initial HCMV antiviral drug sensitivity according to the method of claim 91; (b) determining late HCMV antiviral drug sensitivity of the patient; And, (c) comparing the HCMV antiviral drug susceptibility determined in steps (a) and (b) above, when a decrease in late HCMV antiviral drug susceptibility at initial preparation indicates an increase or progress in the patient's HCMV antiviral drug resistance. step How to determine the HCMV antiviral drug resistance of a patient comprising a.