WO2007037429A1 - 新規組換え型ヒトc型肝炎ウイルス様粒子とその産生方法 - Google Patents
新規組換え型ヒトc型肝炎ウイルス様粒子とその産生方法 Download PDFInfo
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Definitions
- Novel recombinant human hepatitis c virus-like particles and production method thereof Novel recombinant human hepatitis c virus-like particles and production method thereof
- the present invention relates to a human recombinant hepatitis C virus-like particle and a production method thereof.
- the physicochemical methods include calcium phosphate coprecipitation method, DEAE dextran method, lipofection method, microinjection method, and electoral position method.
- a biological method there is a method using a viral vector.
- the viral vector method is a method of introducing a gene by utilizing a cell entry mechanism possessed by a virus, that is, infectivity.
- Virus-derived structural proteins are present on the surface of recombinant virus particles produced using viral vectors, and receptors present on the cell surface. It has a mechanism for infecting cells through the cell and efficiently introducing genes. Therefore, recombinant virus particles prepared using such viral vectors are not only used for the purpose of introducing genes into animal cells to produce target gene-expressing cells, but also for gene therapy, transgenic animal production, etc. It can be used for any purpose.
- Virus vectors are classified into three categories: retrovirus vectors, DNA virus vectors, and RNA virus vectors.
- Retroviruses have plus-strand RNA as a genome.
- This RNA has typical eukaryotic mRNA properties, ie, methylated 5, end-capped structure 3, and poly A consisting of about 200 bases at the end! /.
- this RNA is converted to DNA by the reverse transcriptase of the virus.
- the integrated DNA is called a provirus.
- a repeat sequence (LTR) is generated at both ends of the provirus, and viral RNA is synthesized by the promoter in it. The synthesized RNA translates the viral protein, and the genome-size RNA is incorporated into the virus particle and becomes a child particle and released outside the cell.
- RNA structure necessary for producing virus particles is the LTR at both ends, the primer binding site surrounded by it, the knocking signal, and the polypurine signal. These are essential cis factors. On the other hand, if a viral protein is supplied in an infected cell where the gene encoding the viral protein is not essential as a cis factor, replication and particle production will be performed normally.
- a vector into which a gene of interest to be expressed is inserted instead of a gene such as gag, po 1, or env encoded by the retrovirus (referred to as a retrovirus vector)
- a retrovirus vector When this vector is introduced into a cell (usually called a packaging cell) supplied with a viral protein, a retroviral particle incorporating a foreign gene is produced (Non-patent Document 1).
- retroviruses examples include mouse leukemia virus, feline leukemia virus, baboon C-type oncovirus, human immunodeficiency virus, and adult T cell leukemia virus. Furthermore, those reported as recombinant retrovirus vectors include those based on murine leukemia virus (Non-patent document 1) and those based on human immunodeficiency virus (Non-patent document 2). Etc.
- a system for the production of recombinant retroviruses consists of two components: the genetic information to be introduced (target foreign gene) and the elements necessary for packaging and integration of the viral genome. It also works with retroviral vectors (recombinant retroviral DNA) that retain all of them in cis and retroviral packaging cells that provide viral proteins encoded by the gag, pol and env genes. Recombinant retroviral particles cannot be released only by packaging cells into which a recombinant vector expressing gag, pol and env genes has been introduced.
- the gag, pol and env proteins are trans Must be located in trans. Therefore, by introducing a retroviral vector into a packaging cell into which a recombinant vector that expresses the gag, pol, and env genes has been introduced, a recombinant retrovirus that retains the genetic information contained in the vector can be produced. . If these viruses are then used to infect cells, retroviral vectors will integrate into the cell's chromosomal genome according to the natural retroviral life cycle.
- the retroviral vector method is a system created for the purpose of efficiently integrating specific DNA into the host chromosome genome, and the insertion position of the target gene cannot be predicted. The possibility of damage, activation of nearby genes, overexpression of target foreign genes, or suppression of expression cannot be denied.
- the development of a transient expression system using a DNA virus vector that can be used as an extrachromosomal gene was advanced.
- a DNA virus vector is a vector derived from a DNA virus.
- a DNA virus retains DNA as genetic information in a virus particle, and its DNA replication is a self-form of DNA, and a host-derived DNA-dependent DNA replication enzyme is at least part of the catalyst. This is done by repeating the process of generating a complementary strand.
- DNA viral vectors that can be used as extrachromosomal genes include adenoviral vectors.
- Human adenovirus has approximately 36kb of linear double-stranded DNA as its genome, and the region contained in it is large in early genes El, E2, E3, E4 and late genes LI, L2, L3, L4, L5 Separated. The early genes are mainly involved in virus replication, and the late genes are involved in the synthesis of viral structural proteins such as force psid.
- Adenovirus vector used for gene transfer One is an early gene E1 region (divided into E1A and E1B, and all adenovirus promoters are activated by E1A) (target gene) ) Cells and proliferate in 293 cells, which are cell lines that can supply E1A to trans (293 cells express E1A).
- Non-patent Document 4 genes can be introduced into both non-proliferating cells and proliferating cells. Therefore, this method is suitable for in vivo gene transfer.
- One of the disadvantages of this vector is that the gene expression period is generally short (weekly). This is because the adenovirus genome exists only in the extrachromosomal region (episosome), and no replication or amplification is performed.
- the second disadvantage is that adenoviruses currently in common use cause non-specific inflammatory responses and enhance the cellular immune response against the vector itself. There are concerns about the difficulty of administration (Non-patent Document 5).
- RNA viruses are duplicated by repeating the process of generating complementary strands by using self RNA as a cage and catalyzing self-derived RNA-dependent RNA replication enzymes.
- RNA viruses are divided into (-) strand RNA viruses and (+) strand RNA viruses.
- a typical (-) strand RNA virus is influenza virus.
- the influenza virus genome is a virus consisting of eight segmented negative-strand RNAs.
- viral RNA polymerase cleaves host cell mRNA from the 5 'end cap structure at a portion of more than 10 nucleotides, and uses it as a primer to extend the RNA strand (plus strand).
- Viral proteins are translated from this plus-strand RNA.
- RNA that is completely complementary to the viral RNA is synthesized, and this is used as a cocoon to amplify the progeny viral RNA.
- the viral RNA is then packaged with viral proteins into viral particles.
- RNA polymerase II system promoter such as CMV or CAG promoter
- the viral RNA is expressed by a cap structure and polyA.
- the promoter RNA polymerase type I promoter such as rRNA gene If expressed in a child promoter, viral RNA is packaged together with viral proteins in the cell to form viral particles (Non-patent Document 6).
- the amount of virus produced is not shown, and the production power has been established as a technology that can be fully utilized.
- Sindbis virus and hepatitis C virus are viruses classified as (+) strand RNA viruses.
- the (+) strand RNA virus has a genomic RNA that simultaneously functions as a messenger RNA (hereinafter referred to as “mRNA”), and can produce proteins necessary for replication and particle formation depending on the translation function of the host cell. it can.
- mRNA messenger RNA
- the genomic RNA of the (+) strand RNA virus itself has the ability to propagate.
- a virus vector derived from Sindbis virus has a genomic RNA in which a structural gene region related to a viral structure is deleted, and a viral transcriptional replication protein gene group remains,
- the basic structure is RNA that has a desired foreign gene connected downstream of the transcription promoter.
- RNA or cDNA that can transcribe the RNA is introduced into the cell, autonomous replication of the RNA vector containing the foreign gene and transcription of the foreign gene downstream of the transcription promoter occur, and the desired foreign gene product Is expressed in cells.
- a cDNA unit (helper) that expresses the structural gene and a cDNA unit that expresses the above RNA vector coexist in the packaging cell to produce a complex that has infectivity but does not have the ability to propagate.
- Sindbis virus uses a 67-kilodalton high-affinity laminin receptor (LAMR) as a receptor and infects nerve cells with high efficiency. Is attracting attention as a system for gene-specific gene introduction (Non-patent Document 8). However, Sindbis virus infection has been shown to induce apoptosis in host cells (Non-patent Document 9), and there is concern about toxicity.
- LAMR high-affinity laminin receptor
- the genome of hepatitis C virus is a single-stranded RNA (+) chain consisting of about 9600 nucleotides.
- This genomic RNA consists of a 5 ′ untranslated region (also referred to as 5′NTR or 5′UTR), a translated region composed of a structural region and a nonstructural region, and a 3 ′ untranslated region (3′NTR or 3 ′ (Also referred to as UTR).
- the structural region encodes a structural protein of HCV
- the nonstructural region encodes a plurality of nonstructural proteins.
- HCV structural proteins Core, El, E2, and p7 and non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) are a series of polyproteins from the translation region. After being translated, they are released and produced by limited degradation by proteases.
- Core is the core protein and E1 and E2 are envelope proteins.
- Non-structural proteins are proteins involved in the virus's own replication.
- NS2 is metaprotease activity
- NS3 is serine protease activity (1/3 of the N-terminal side) and helicase activity (3 minutes of C-terminal side). 2).
- NS4A is a cofactor for NS3 protease activity
- NS5B has been reported to have RNA-dependent RNA polymerase activity.
- HCV is classified into a number of types according to genotype or serotype.
- HCV is genotype la, genotype lb, genotype 2a, genotype 2b, and gene type 3a. These are classified into 6 types, genotype 3b, and each type is further classified into several subtypes. And about the multiple genotypes of HCV, the base sequence of the full length of the genome is also determined (nonpatent literatures 10-13).
- HCV particles are captured by sulfate polysaccharides on the cell surface, bind to high-affinity receptors via envelope proteins, and are taken up into endosomes by endocytosis. Subsequently, the viral membrane and endosomal membrane fuse, and the nucleocapsid enters the cytoplasm. Translation of the naked viral genome is initiated by IRES. Translation and protein cleavage occur on the endoplasmic reticulum membrane. The core protein, E1 and E2 proteins, and viral RNA replicated in the endoplasmic reticulum assemble to form viral particles. After that, it buds into the endoplasmic reticulum cavity. It is believed that the budding particles pass through the Golgi apparatus and are released outside the cell.
- HCV subgenomic RNA replicons have been prepared as RNA having autonomous replication ability derived from HCV (Patent Documents 1 and 2 and Non-Patent Documents 14 to 16). It became possible to analyze the replication mechanism of HCV. These HCV subgenomic RNA replicons are downstream of HCV IRES in the 5 'untranslated region of HCV genomic RNA The structural protein was replaced with the neomycin resistance gene and EMCV-IRES linked downstream. This RNA replicon was proved to replicate autonomously in Huh7 cells when introduced into human hepatoma cells Huh7 and cultured in the presence of neomycin.
- Patent Document 3 proposes the production of HCV virus particles using the full-length HCV genome for the use of recombinant HCV as a gene therapy vector! No. 2002-171978
- Patent Document 2 Japanese Patent Laid-Open No. 2001-17187
- Patent Document 3 International Publication WO2004Z104198
- Non-patent literature l Mann, R. et al, Cell, 33 (1983) pl53-59
- Non-Patent Document 2 Simada, T et al., J Clin Invest. 88 (1991) pi 043-47
- Non-Patent Document 3 Betta, A et al "Proc. Nati. Acad. Sci. USA 91 (1994) p8802- 06
- Non-Patent Document 4 Burden, S & Yarden, Y., Neuron, 18 (1997) p847-55
- Non-Patent Document 5 Crystal, R.G Science, 270, (1995) p404-10
- Non-Patent Document 6 Neumann, G. & Kawaoka, Y., Virology 287 (2001) p243- 50
- Non-Patent Document 7 Berglund, P et al., Biotechnology, 11 (1993) p916-920
- Non-Patent Document 8 Wang, K.S et al., J. Virol. 66 (1992) p4992- 5001
- Non-Patent Document 9 Levine, B. et al., Nature, 361 (1993) p739-42
- Non-Patent Document 10 Simmonds, P. et al., Hepatology, 10 (1994) pl321-24
- Non-Patent Document ll Choo, Q.L et al "Science, 244 (1989) p359-362
- Non-Patent Document 12 Okamoto, H et al "J. Gen. Virol, 73 (1992) p673- 79
- Non-Patent Document 13 Mori, S. et al "Biochem. Biophis. Res. Commun. 183 (1992) p334-4
- Non-Patent Document 14 Blight et al., Science, 290 (2000) pl972-74
- Non-Patent Document 15 Friebe et al., J. Virol, 75 (2001) pl2047-57
- Non-Patent Document 16 Kato, T. et al., Gastroenterology, 125 (2003) pl808-17 Disclosure of the invention
- HCV has not actually been developed as a viral vector such as retrovirus, adenovirus, influenza virus, and Sindbis virus. If such H
- CV vectors are developed, it will be possible to introduce genes specifically into cells of tissues such as the liver. In that case, in order to ensure a higher degree of safety, it is desirable that the HCV vector infects cells but does not propagate.
- An object of the present invention is to develop recombinant hepatitis C virus (HCV) -like particles that can be used as vectors as described above. Another object is to provide a method for efficiently producing the HCV-like particles.
- HCV hepatitis C virus
- the present inventors tried to produce recombinant HCV-like particles in cultured cells that are considered industrially useful in terms of safety, convenience, and applicability.
- the HCV genome was divided into a vector that expresses HCV structural proteins and a vector that contains genes involved in replication.
- the latter vector can contain a desired foreign gene and / or an internal ribosome entry site (IRES).
- IRS internal ribosome entry site
- the present inventors constructed a vector in which a DNA comprising a desired foreign gene, IRES sequence, and a gene related to HCV replication is cloned downstream of the T7 promoter, and in vitro using T7 polymerase.
- An HCV subgenomic RNA replicon containing the foreign gene sequence was synthesized. This RNA replicon was introduced into cultured animal cells, and the HCV subgenomic RNA replicon was replicated to obtain a cell line.
- the present inventors have found that the recombinant HCV-like particles produced by the method of the present invention are infected, and that the cells infected with the recombinant HCV do not produce offspring virus particles, and are not easily transmitted. Na I confirmed. Due to these characteristics, the recombinant HCV particles of the present invention can be used as a vector for introducing a foreign gene or for gene therapy.
- the present invention is a method for producing recombinant hepatitis C virus particles
- Recombinant C-type produced by introducing a vector expressing Core protein, E1 protein, E2 protein and p7 protein derived from the same or different hepatitis C virus strain as above (0), culturing the cell, and The method is provided comprising recovering hepatitis virus particles.
- the (0 and GO hepatitis C virus strains are independently a group consisting of the virus strains of the gene types la, lb, 2a, 2b, 3a and 3b. Power At least one strain selected.
- the hepatitis C virus strains of (0 and (ii) are independently selected from at least one selected from the group strength of the virus strains of genotype lb and 2a. Is a stock.
- the (0 hepatitis C virus strain) is a virus strain of genotype lb.
- the genotype lb virus strain is a conl strain or a derivative thereof.
- the GO hepatitis C virus strain is a genotype 2a virus strain.
- the genotype 2a virus strain is a JFHI strain or a derivative thereof.
- the RNA levicon is at least one internal ribozo. It may further include a genome binding site (IRES) sequence.
- IFN genome binding site
- the RNA replicon can further comprise at least one foreign gene.
- the IRES and the foreign gene can be located between the 5 'untranslated region and the NS3.
- the cell is an animal cell.
- the animal cell is a mammalian cell.
- the expression vector is a viral vector.
- the viral vector is a vaccine virus vector.
- Recombinant hepatitis C virus particles produced and recovered by the above method of the present invention are further infected with HCV sensitive cells such as hepatocytes or lymphoid cells to propagate virus particles. Can do.
- HCV sensitive cells such as hepatocytes or lymphoid cells to propagate virus particles. Can do.
- the present invention includes such steps.
- the present invention is also produced in the second aspect by the method of the present invention described above.
- the present invention also provides recombinant hepatitis C virus particles that are infectious but have no propagation power.
- the recombinant hepatitis C virus particle is introduced with a foreign gene so that it can be expressed.
- the recombinant hepatitis C virus particle is a vector.
- a method for producing recombinant hepatitis C virus particles comprising:
- the method comprises the steps of: introducing a virus vector, culturing the cell, and recovering the produced recombinant hepatitis C virus particles.
- a suitable recombinant hepatitis C virus particle is produced by the above-mentioned preferred method.
- the present invention further includes the following [1] to [2].
- a method for producing recombinant hepatitis C virus-like particles comprising:
- the hepatitis C virus strain having the genotype lb of (i) is a conl strain.
- the (i) genotype 2a hepatitis C virus strain is JFH1 strain U.
- the hepatitis C virus strain of genotype la in (ii) is H77c strain, 1 strain, H strain and HC-J1 strain.
- the hepatitis C virus strain having the genotype lb of (ii) is a J1 strain, a conl strain, or a TH strain.
- J strain, JT strain, and BK strain are also preferable.
- the hepatitis C virus strain of genotype 2a of (ii) is a JFH1 strain, an HC-J6 strain,
- JCH1 strain and J6CF strain are also preferable.
- the genotype 3a hepatitis C virus strain of (ii) above is the NZL1 strain or the K3a / 650 strain.
- the genotype 3b hepatitis C virus strain of (ii) is a Tr strain.
- the vector of (ii) is a cuticular virus vector or an EF-1a promoter-containing vector.
- the RNA replicon further comprises at least one internal ribosome binding site (IRES) sequence and Z or at least one foreign gene.
- IRS internal ribosome binding site
- the IRES sequence and the Z or foreign gene are preferably located between the 5 'untranslated region and the NS3 protein coding sequence.
- the cell is preferably an animal cell.
- Huh7 cells as animal cells
- H-marked G2 cells or cell lines derived from those cell forces are more preferred.
- a non-transmissible recombinant infectious HCV-like particle packaged with HCV subgenomic RNA containing a desired foreign gene and a method for producing the same.
- Such recombinant infectious HCV-like particles have the advantage of lack of transmissibility and are particularly used for gene transfer (eg, gene therapy) to liver or lymphoid cells or tissues. Or has the advantage that it can be used as a viral vector for the production of transgenic animals and even as an attenuated vaccine.
- FIG. 1 is a schematic view of an embodiment of the present invention, and shows a production process of recombinant HCV-like particles.
- FIG. 2 shows the structure of cDNA of HCV genomic RNA and HCV subgenomic RNA.
- the upper row is pFHl prepared from HCV genotype 2a, and the middle row is pSGR-JFHl.
- the lower row is I389 / NS3-3 ′ / wt prepared from HCV genotype lb.
- the symbols in the figure are as follows.
- T7 T7 RNA promoter. 5'UTR: 5 'untranslated region.
- Core Core protein.
- El, E2 Envelope protein.
- p7 p7 protein.
- NS2, NS3, NS4A, NS4B, NS5A, NS5B nonstructural proteins.
- 3'UTR 3 'untranslated region.
- Age I, Pme I, Xba I cleavage sites for restriction enzymes
- EMCV IRES Internal ribosome binding site of encephalomyocarditis virus.
- FIG. 3 shows a map of a vector for expressing the HCV structural protein of the present invention.
- the upper row is a plasmid clone pGAGC-p7JFHl created by inserting the JFH structural region gene downstream of the CAG promoter
- the lower row is the JFH structural region gene downstream of the Elon Gation Factor 1 ⁇ promoter sequence.
- 2 shows the structure of the plasmid clone ⁇ F4C-p7JFHl produced in the above manner. The symbols in the figure are as follows.
- CAG CAG promoter 1
- pA polyA-attached sequence
- EcoRI restriction enzyme EcoRI cleavage site
- EF-1 a Elongation factor 1 ⁇ promoter
- BGH pA ushi growth factor poly-attached sequence.
- Figure 4 shows the HCV structural gene inserted into the vectors pDIsHJFHst, pDIsH77st ⁇ pDIsJlst, pDIsJl (c) / JFH (El—p7) st and pDIsJFH (c) / Jl (El-p7) st! The map of is shown. Differences in the virus strains from which they are derived are shown in the shaded area.
- FIG. 5 shows that HCV replicon RNA in each fraction obtained by introducing pEF4C-p7JFHl into a replicon-retaining cell line IH4.1 and fractionating the cell culture supernatant (sup) with a sucrose density gradient. It is a graph (B) which shows the quantity (A) and the quantity of HCV Core protein. Mouth: Experiment 1, ⁇ : Experiment 2 0
- Fig. 6 shows that each of the fractions obtained by infecting the replicon-carrying cell line 5-15 with the viral virus vector DIsJFHst and fractionating the cell culture supernatant (sup) with a sucrose density gradient (horizontal It is a graph which shows the quantity (vertical axis) of HCV Core protein in (axis). The black circles show the results using the HCV Core (Core) protein, and the black squares show the results using the NP40-treated culture supernatant. Experiment 1 shows the results of untreated only ( Figure 6A), and Experiment 2 shows the results of untreated and NP40 treatment ( Figure 6B).
- RNA levicon refers to RNA having autonomous replication ability produced by modifying the HCV virus genome.
- Autonomous replication ability refers to autonomous reproduction of nucleic acid copies in cells like plasmid DNA.
- infectious refers to the ability to introduce a nucleic acid or the like inside a virus into a cell by retaining the ability to adhere to a cell and membrane fusion ability. Point to.
- Recombinant hepatitis C virus means a virus that has qualitatively / quantitatively altered the properties of the original HCV virus by genetic recombination technology. Viruses that have the ability to express foreign genes other than those that do not, viruses that lack the ability to replicate the virus genome, etc., in a broad sense, between the same virus types or subtypes. It also includes viruses that have been genetically modified.
- Propagation or “propagation force” refers to an infectious particle or a compound equivalent thereto after the nucleic acid is replicated after the nucleic acid is introduced into the cell by infection or an artificial technique. It means the ability to form the body and propagate to other cells.
- Core is the core structural protein of HVC.
- E1 and E2 are both envelope structural proteins.
- NS refers to a non-structural protein of HCV and is a protein involved in the replication of the virus itself.
- “NS2” has meta-oral protease activity.
- “NS3” has serine protease activity (1/3 on the N-terminal side) and helicase activity (2/3 on the C-terminal side).
- “NS4A” is a cofactor for the protease activity of NS3.
- the function of “NS4B” is not clear.
- NS5A is considered to have an activity to control the signal transduction of host cells.
- NS5B has RNA-dependent RNA polymerase activity.
- IRES sequence means an internal ribosome binding site capable of binding a ribosome inside RNA to initiate translation.
- Be able to express refers to a state in which a target gene can be transcribed and translated by a regulatory sequence such as a promoter or an enzyme.
- a regulatory sequence such as a promoter or an enzyme.
- the wild-type HCV genome consists of a single-stranded RNA of about 9.6 kb in length that encodes a precursor protein of about 3,000 amino acids.
- the HCV genome consists of 5 'untranslated region (5'UTR), Core, El, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B, 3' untranslated region (3'UTR). ing .
- the HCV subgenomic RNA replicon used in the method of the present invention includes a modified RNA composed in the order of 5 ′ untranslated region, NS3, NS4A, NS4B, NS5A, NS5B, 3 ′ untranslated region. This RNA replicon is introduced into a specific cell so that it can replicate under the action of a regulatory factor such as a promoter.
- the RNA replicon may further include a foreign gene and / or an IRES sequence.
- the foreign gene and the IRES sequence can preferably be located in the order of the foreign gene and the IRES sequence between the 5 ′ untranslated region and the NS3 coding sequence.
- IRES sequences include, but are not limited to: EMCV IRES (internal ribosome binding site of encephalomyocarditis virus), FMDV IRES, HCV IRES, etc. 1S EMCV IRES and HCV IRES force is preferred over S, and EMCV IRES is most preferred.
- a gene exhibiting drug resistance that is, this gene is a gene that enables selection of cells, and cells having the gene become resistant to the drug
- a reporter gene ie, this gene is a marker gene encoding a gene product that is an indicator of gene expression
- Genes encoding enzymes that catalyze luminescent or color reactions such as luciferase, green fluorescent protein (GFP), ⁇ -galactosidase
- target genes for gene therapy or therapeutic nucleic acids such as mammals including humans
- Nucleic acids for treatment of proteins such as enzymes, cytokines, chemokines, hormones, antibodies, immunoregulatory molecules, tumor suppressor proteins, growth factors, membrane proteins and vas
- HCV subgenomic RNA replicons include pSGR-JFHl (middle of Fig. 2), I / N
- HCV subgenomic RNA replicons are For example, it is produced using the method described in Kato, T. et al. Gastroenterology, 2003 125: 1808-1817, International Publication WO 2004/104198 (Patent Document 3), etc. be able to.
- HCV is classified into 6 types: genotype la, genotype lb, genotype 2a, genotype 2b, genotype 3a, and genotype 3b. Each of these types is classified into several subtypes.
- genotype la genotype la
- genotype lb genotype 2a
- genotype 2b genotype 3a
- genotype 3b genotype 3b
- each of these types is classified into several subtypes.
- the base sequence of the entire genome has also been determined (Simmonds, P. et al., Hepatology, 10 (1994) pl321-1324, and Choo, QL et al "Science, 244 (1989) p359-362, Okamoto, H et al "J. Gen. Virol, 73 (1992) p673-679, Mori, S. et al” Biochem. Biophys. Res. Commun. 183 (1992) p334- 342, International Publication WO 2004/104198) 0
- HCV strains of genotype la include H77c strain (H77 strain consensus sequence: GenBank accession number AF011751), 1 strain (GenBank accession number M62321), H strain (GenBank Cession number M67463) and HC-J1 strain (GenBank accession number D 10749) are known.
- the HCV strains of genotype lb include J1 strain (GenBank accession number D89815), conl strain (GenBank accession number AJ238799, sometimes referred to as Con-1 strain), TH strain (Wakita, T. et al. al "J. Biol.
- J strain (GenBank accession number D90208), JT strain (GenBank accession number D 01171), BK strain (GenBank The HCV strains of genotype 2a include JFH1 strain (GenBank accession number AB047639, sometimes referred to as JFH-1 strain), HC-J6 strain (GenBank (Accession number D00944), JCH1 strain (GenBank accession number AB047640), J6CF strain (GenBank accession number AF177036), etc.
- HC-J8 strain (genotype 2b HCV strain ( GenBank Accession No.
- HCV strains with genotype 3a are NZL1 strains (GenBank Accession No. No. D17763), K3a / 650 strain (GenBank accession number D28917), 452 strain (GenBank accession number DQ437509), E-bl strain (Chan, S. et al., J. Gen. Virol, 73 pl l31-1141 (1992)) and Tr strains (Chayama, K. et al., J. Gen. Virol, 75 p3623-3628 (1994)) are known as genotype 3b HCV strains.
- a list of GenBank accession numbers has already been reported for other strains (Tokita, T. et al., J. Gen. Vir ol. 79, 1847—1857,1998, Columbia J. & Colina R. Virolgy Journal, 3, 1-8, 2006).
- the elements constituting the HCV subgenomic RNA replicon used in the present invention can be replicated in cells.
- an HCV subgenomic RNA replicon can be constructed, it may be derived from any of the above genotypes or strains of those subtypes.
- the present invention is a method for producing recombinant hepatitis C virus-like particles that are infectious but not transmissible.
- the RNA of the present invention Preferably, the replicon does not contain genes encoding each of the HCV structural proteins (Core, El, E2 and p7).
- Each of the above elements may be derived from the same HCV strain or may take the form of a hybrid (chimera) derived from two or more different HCV strains.
- Preferred HCV strains are at least one strain selected for the strength of HCV strains of genotype lb and 2a, more preferably conl strains that are HCV strains of genotype lb, or HCV strains of genotype 2a JFHI stock.
- the HCV strain in the present invention is an isolated strain obtained by spontaneously or artificially mutating a conl strain or JFHI strain as a parent strain, for example, and at least the genotype is changed from that of the parent strain. Even if it is a stock (derived stock). Even if the phenotypic trait is the same as the parent strain, it is different! / Take it! /, But prefer the same strain traits U ,.
- HCV subgenomic RNA replicons include sequences encoding 5 'untranslated region, NS3 protein, NS4A protein, NS4B protein, NS5A protein derived from HCV strain genomes other than JFH1 strain (genotype 2a) and HCV subgenomic RNA replicon consisting of NS5B protein coding sequence and 3 'untranslated region of JFH1 strain; 5' untranslated region of JFH1 strain, NS3 protein, NS4A protein, NS4B protein, NS5A protein, NS5B protein HCV subgenomic RNA replicon consisting of an untranslated region and 3, an HCV-conl strain (genotype lb, GenBank accession number AJ238799, Lohmann.V.
- HCV subgenomic RNA consisting of 5 'untranslated region, NS3 protein, NS4A protein, NS4B protein, NS5A protein and NS5B protein coding sequence and 3' untranslated region Silicon, and the like.
- the desired foreign gene and the IRES distribution are located between the 5 'untranslated region of these HCV subgenomic RNA replicons and the NS3 protein coding sequence. It may contain columns.
- the 5 'untranslated region that can be used as a constituent element of the HCV subgenomic RNA replicon in the present invention is represented by nucleotide numbers 1 to 340 in the nucleotide sequence of SEQ ID NO: 10. Yes, and the region coding for Core protein strength p7 protein (Core, El, E2, p7) is base number 341 to 2779, from NS3 protein to 3 'untranslated region (NS3, The region encoding NS4A, NS4B, NS5A, NS5B, 3'UTR) has base numbers 3431 to 9678. By comparing with the sequence of each region derived from this JFH1 strain, each region can be identified even in genomic cDNA derived from other HCV strains.
- HCV subgenomic RNA replicon synthesis was performed by DNA-dependent RNA polymerase using a vector in which DNA complementary to the HCV subgenomic RNA was cloned downstream of a promoter that transcribes RNA from the DNA sequence. can do.
- the T7 promoter which includes T7, T3, SP6, etc., is suitable as a promoter that transcribes RNA from a DNA sequence, and HCV subgenomic RNA can be synthesized by T7 polymerase.
- the cell is most preferably an animal cell, for example, a mammalian cell preferred by vertebrate cells such as fish, reptiles, amphibians, birds and mammalian cells.
- the cells include liver, cervix, fetal kidney-derived normal cells, tumor cells, and established cell lines thereof, such as cells such as Huh7, HepG2, IMY-N9, HeLa, and HEK293 ( Date, T. et al "J. Biol. Chem., 279, p.22371-22376, (2004), Ito, T. et al, Hepatology 34, p.56 6-572, (2001)) preferably Huh7 HepG2 or clones derived from these cells.
- Another method for replicating the HCV subgenomic RNA in cultured cells is a system using HCV cDNA without using an RNA replicon.
- HCV cDNA When HCV cDNA is expressed with an RNA polymerase type II promoter, a CAP structure is added to the 5 'end of the transcribed RNA, and a poly A chain is added to the 3' end.
- HCV genomic RNA replication does not occur.
- Heller et al. Ligated ribozyme sequences to the 5 'and 3' ends of the HCV genome, transcribed with RNA polymerase II in the cell, and then cleaved with ribozyme.
- a DNA vector was prepared that could synthesize HCV RNA intracellularly with a cap and poly A (Heller T et al. Proc. Natl. Acad. Sci. USA., 102, p. 2579-2583, 2005). By introducing this vector into a cell, a cell that replicates the HCV subgenomic RNA replicon can be obtained.
- the complementary DNA of the HCV subgenomic RNA is cloned into an RNA polymerase I promoter / terminator-type vector and the vector is introduced into a cell that allows HCV growth, Cells that replicate the HCV subgenomic RNA replicon can be obtained.
- pHH21 Neuronal Biolistics et al., Proc. Natl. Acad. Sci. USA, 96 (1999) p9345-9350
- pHH21 is a vector comprising a human RNA polymerase ⁇ promoter as a promoter and a mouse RNA polymerase I terminator as a terminator.
- the restriction enzyme BsmBI recognition sequence was added to the 5 'end and 3' end of the cDNA for the HCV subgenomic replicon RNA using PCR, digested with BsmBI, and the HCV genome was inserted into the BsmBI site of pHH21.
- the terminator can be linked to the HCV genome without any extra nucleotide sequence.
- Intracellular introduction of a vector such as an HCV subgenomic RNA replicon or a vector that expresses an HCV subgenomic RNA replicon can be performed using any technique known to those skilled in the art. Examples of such introduction methods include an electoral position method, a particle gun method, a lipofusion method, a calcium phosphate method, a microinjection method, a DEAE dextran method, and the like.
- a cell in which the HCV subgenomic RNA replicon that is useful in the present invention is replicated in the cell can be produced.
- Such RNA levicons are Since it autonomously replicates continuously in the cell, it is maintained in a certain amount even in the cell undergoing RNA degradation. Therefore, a cell into which a HCV subgenomic RNA replicon or a vector that expresses an HCV subgenomic RNA replicon according to the present invention as described above is introduced into the cell can retain the RNA replicon.
- the “cell retaining an RNA replicon” means that the RNA replicon is present in the cell continuously in a significant amount, not transiently, due to its autonomous replication ability.
- a virus-like particle in which the HCV subgenomic RNA replicon is packaged by introducing and expressing the HCV structural protein expression vector described below into a cell carrying the HCV subgenomic RNA replicon of the present invention. Can be produced.
- an HCV structural protein by expressing an HCV structural protein gene in a cell that holds an HCV subgenomic RNA replicon.
- the HCV structural protein consists of Core, El, E2, and p7.
- the genes encoding these proteins for supplying HCV structural proteins are not limited to HCV genotypes, and each of the genes may be derived from the same HCV strain, or two or more A hybrid (chimera) form may be derived from different HCV strains.
- the origin of the HCV structural protein gene may be at least one virus strain selected from la, lb, 2a, 2b, 3a and 3b HCV strains, but preferred HCV strains are la, 2a, It is at least one virus strain selected from the group consisting of 3a and 3b, and a more preferred HCV strain is at least one virus strain whose genotype 1b and 2a HCV strain power is also selected. .
- H77c strain, 1 strain, H strain and HC-J1 strain of genotype la, J1 strain, conl strain, TH strain, J strain, JT strain and BK strain of genotype lb, or genotype 2a are at least one type of virus strain that can be selected for its strength, such as JFH1 strain, HC-J6 strain, JCH1 strain and J6CF strain. More preferably, H77 strain of genotype la, J1 strain of genotype lb or JFH1 strain of genotype 2a, most preferably JFH1 strain (GenBank accession number AB047639, Kato, T. et al., Gastroenterology, 125 (2003 ) pl808-1817).
- cells preferably animal cells
- any method can be used as long as it can be expressed in mammalian cells.
- the preferred V ⁇ method is a method using an expression vector incorporating the above gene.
- an HCV structural protein expression vector (preferably an expression vector containing an HCV structural protein gene in a form that can be expressed under the control of a promoter) is used to supply the HCV structural protein. Then, the HCV subgenomic RNA replicon described in 2 above is introduced and expressed.
- CDM8 pEFl / Myc-Hisl, 2,3, pEF4 / Myc-Hisl, 2,3, pc DNA3.1, pREP4, pCEP (all from Invitrogen), pC neo (Promega) ).
- Pc DNA5 / TO Invitrogen containing a promoter whose expression can be regulated by tetracycline can also be used.
- the promoter can be expressed in animal cells, the cytomegalovirus IE (immediate early) promoter, SV40 early or late promoter, metamouthone promoter, retrovirus promoter, heat shock promoter SR a promoter, Elon Gation Factor 1a promoter, albumin promoter and the like.
- examples of usable vectors include virus vectors. Any viral vector can be used as long as it can infect animal cells and express a desired foreign gene. Preferred examples include a retrovirus vector, an adenovirus vector, a Sindbis virus vector, and a cuticle virus vector. In particular, vaccine vectors can express large amounts of gene products (Elroy-Stein, 0., et al, Pro Natl. Acad. Sci. USA, 86 (1989) p6126- 6130). A virus vector is preferred.
- the HCV structural protein expression vector used in the method of the present invention contains, as an HCV structural protein gene, a Core protein gene, an E1 protein gene, an E2 protein gene, and a p7 protein gene in a state that can be expressed in a host cell. I prefer it.
- HCV structural protein expression vectors that are relevant to the present invention include the Core protein gene, the E1 protein gene, the E2 protein gene, and the p7 protein gene under the control of a promoter that allows expression of the inserted gene. It is a vector.
- the Core protein gene, E1 protein gene, E2 protein gene, and p7 It is possible to use a vector containing an elongation factor 1 ⁇ promoter in which a protein gene is inserted under the control of the elongation factor 1a (EF-1 ⁇ ) promoter as a particularly suitable HCV structural protein expression vector.
- the vector containing an elongation factor 1 ⁇ promoter refers to the promoter arrangement of the elongation gene 1 ⁇ gene IJ (EF-1 ⁇ promoter; Mizushima et. Al, Nucleic Acids Res., 1990, 18, 5322) in such an arrangement that the gene under its control can be expressed in the host cell. Examples are pEFl / Myc-Hisl, 2,3, pEF4 / Myc-Hisl, 2,3 (V, both are Invitrogen).
- HCV structural protein expression vector useful for the present invention includes a core protein gene, an E1 protein gene, an E2 protein gene, and a p7 protein gene in an expressible state. It is a viral vector (a recombinant vector virus vector).
- a viral vector a recombinant vector virus vector
- a recombinant virus vector such as DIs strain, WR strain, IBTd strain (Meis, RJ & Condit, RC. Virol. 182, 442-454, (1992)
- a strain can be preferably used.
- the method for producing the recombinant viral vector is also described in detail in the examples below.
- the above HCV structural protein gene is cloned under the control of the ⁇ -quinoless promoter such as p.7.5 in the ⁇ -quinoline 'transfer vector, and the transfer vector is further infected with ⁇ -virus.
- the target recombinant vector virus vector is introduced by electroporation or the like, cultured to produce virus particles, and more preferably, the virus is selected and purified. Can be manufactured.
- Such a virus vector can be prepared in the form of recombinant virus-like particles.
- HCV The structural proteins of HCV (Core, El, E2, p7) and nonstructural proteins (NS3, NS4A, NS4B, NS5A, and NS5B) are translated as a continuous polyprotein, and then converted by protease. As these proteins are released and produced upon limited degradation, it is desirable to express these HCV structural proteins as a series of polyproproteins of Core, El, E2, and p7. Can be expressed in separate expression vectors.
- Confirmation can be detected by reacting a protein extracted from a cell culture medium or cell shell with an antibody against a structural protein (International Publication WO 2004/104198) o [0110] Specifically, for example, cell force is also detected.
- the extracted protein sample is fractionated by SDS-polyamide gel electrophoresis and blotted on a nitrocellulose membrane, and anti-HCV protein antibody (for example, anti-core specific antibody or hepatitis C patient force is also collected. The antiserum) is reacted, and the antibody is detected (Western plot method).
- cells expressing HCV protein can be immunostained, and the expression and subcellular localization of these proteins can be confirmed.
- a virus in which an HCV subgenomic RNA replicon is packaged with a structural protein in the cell by supplying a vector that expresses the HCV structural protein into the cell holding the HCV subgenomic RNA replicon.
- HCV subgenomic RNA replicon To knock an HCV subgenomic RNA replicon from a cell replicating the HCV subgenomic RNA replicon into a viral particle, a structural protein (Core, El, E2, p7) expression vector is introduced into the cell and expressed. That's fine.
- HCV subgenomic RNA may be introduced into cells in which structural proteins (Core, El, E2, p7) are stably expressed.
- Examples of such an introduction method include known methods such as an electopore position method, a particle gun method, a lipofusion method, a calcium phosphate method, a microinjection method, and a DEAE dextran method.
- HCV-like particle production Cells can produce recombinant virus-like particles.
- the produced recombinant HCV-like particles have infectivity and the ability to replicate HCV subgenomic RNA.
- the infected cells cannot produce offspring virus particles, they do not have propagating properties. Therefore, recombinant HCV-like particles can be produced in a cell culture system by culturing the recombinant HCV particle-producing cells of the present invention.
- HCV-like particles can be obtained by culturing recombinant HCV-like particle-producing cells and recovering virus-like particles produced in the culture (preferably culture medium).
- the virus-like particles can be recovered, for example, by a technique such as the above-described culture fluid sucrose density gradient centrifugation.
- the ability of the recombinant HCV-like particle producing cell of the present invention to produce virus particles may be confirmed according to any virus detection method known to those skilled in the art. For example, fractionating a cell culture medium that seems to be producing virus-like particles using a sucrose density gradient, and measuring the density of each fraction and the HCV core protein concentration or HCV replicon RNA concentration of each fraction Therefore, it can be judged by the power that matches the specific gravity of HCV that has been conventionally known. In addition, the density power of the fraction in which the peak of the core protein is detected is 0.25% NP40 (Polyoxyethylene (9) Octylphenyl Ether)). If the density of the fraction is light, it can be determined that the cell has the ability to produce virus-like particles.
- NP40 Polyoxyethylene (9) Octylphenyl Ether
- virus-like particle of the recombinant HCV-like particle-producing cell has infectivity is detected by detecting the phenotype of the foreign gene present in the HCV subgenomic RNA packaged in the virus particle. That's fine.
- the foreign gene is a drug resistance gene
- HCV-permissive cells are inoculated with the virus particles, and are usually cultured for 2 to 3 weeks in the presence of the drug, and drug resistant clones are counted. Can be evaluated.
- an extract of the infected cells preferably a sample in the supernatant of the infected cell culture Whether or not HCV structural protein is present can be determined by the Western blot method described above.
- the recombinant HCV-like virus particles produced by the method of the present invention have the ability to infect cells (preferably HCV-permissive cells).
- Recombinant including culturing recombinant HCV-like particle producing cells and infecting other cells (preferably HCV-permissive cells) with virus-like particles in the resulting culture (preferably culture medium)
- a method for producing hepatitis C virus-infected cells is also provided.
- HCV-permissive cells are sensitive to HCV genomic RNA replication and / or HCV. It is a cell having the ability to stain, but is not limited thereto.
- hepatocytes include primary liver cells, Huh7 cells, HepG2 cells, IMY-N9 cells, HeLa cells, etc., and lymphoid cells include Molt4 cells, HPB-Ma cells, and Daudi cells.
- the powers that can be mentioned are not limited to these.
- the recombinant HCV-like particle of the present invention produced by the method of the present invention has the above HCV strain (genotype la, lb, 2a, 2b, 3a and 3b, preferably lb and 2a force selected) on its RNA genome.
- HCV strain gene la, lb, 2a, 2b, 3a and 3b, preferably lb and 2a force selected
- It has a feature of including a base sequence including a base sequence to be encoded and a 3 ′ untranslated region.
- HCV-like particles produced in recombinant HCV-like particle-producing cells by the method of the present invention are infected with cells (for example, the HCV-permissive cells exemplified above), the infected cells Among them, there is an interesting feature that no force virions that replicate the HCV subgenomic RNA as described above are formed.
- the recombinant HCV-like particle of the present invention has an HCV subgenome R packaged therein.
- HC V-like virus particle of the present invention By inserting a desired foreign gene into the NA levicon, it can be used as a vector for gene transfer Z expression.
- the recombinant HC V-like virus particle of the present invention containing such a foreign gene produces an HC V subgenomic RNA replicon in which the foreign gene is inserted between the 5 ′ untranslated region and the IRES sequence, It can be produced by packaging by the method of the present invention. Since HCV particles produced in recombinant HCV particle-producing cells have no propagation power, they can also be used as vectors for gene transfer targeting, for example, liver or lymphoid cells or tissues.
- the HCV subgenomic RNA packaged in the virus particle by the virus particle production method of the present invention is not integrated into the chromosome genome in HCV-permissive cells infected by the virus-like particle of the present invention. So normal gene is lost by gene insertion There is an advantage in that it is not possible to activate a wounded or inserted gene in the vicinity.
- the vector of the present invention can be used for introduction of a foreign gene, for example, for gene therapy or production of a transgenic animal.
- Foreign genes (or foreign nucleic acids) introduced into HCV subgenomic RNA replicons and packaged into virus particles include proteins derived from mammals including humans, such as various proteins related to diseases, such as enzymes and sites. Blocks or suppresses the translation of proteins, polypeptides or peptides encoding proteins, proteins such as force-in, chemokines, hormones, antibodies, immunoregulatory molecules, tumor suppressor proteins, growth factors, membrane proteins and vasoactive proteins
- the ability to include therapeutic nucleic acids such as antisense RNA and siRNA is not particularly limited.
- Target HCV sensitive cells or tissues are mammalian cells or tissues, preferably human cells or tissues, eg, human liver and lymphoid cells or tissues.
- the vector of the present invention is allowed to act on target cells or tissues under in vivo, in vitro or ex vivo conditions.
- the vector of the present invention can be used for human therapy such as gene therapy and cancer (eg liver cancer, lymphoma, etc.).
- plasmid pJFHl was cleaved with restriction enzymes Agel and Clal, and a sequence extending from the 5'NTR to the Core region derived from pJFH1 and a neomycin resistance gene derived from pRSV5NEO were obtained by PCR amplification.
- a fragment that had been ligated and cleaved with restriction enzymes Agel and Pmel, and a fragment that had been ligated by PCR amplification of the sequence from EMC V IRES to NS3 region and cleaved with restriction enzymes Pmel and Clal were inserted and ligated.
- pSGR-JFHl was cleaved with the restriction enzyme Xbal. Then, 10-20 ⁇ g of each of these Xbal cleaved fragments was further processed by incubation with 30 units of Mung Bean Nuclease (total reaction volume 50 1) at 30 ° C. for 30 minutes.
- Mung Bean Nuclease is an enzyme that catalyzes a reaction that selectively degrades a single-stranded portion in double-stranded DNA.
- RNA synthesis is performed using the above Xbal cleaved fragment as it is as a cage, a replicon RNA in which 4 bases of CTGA, which is part of the Xbal recognition sequence, are added to the 3 'end is synthesized. . Therefore, in this example, the Xbal cleavage fragment was treated with Mung Bean Nuclease to remove the 4 bases of CTGA from the Xbal cleavage fragment force. After this, the Mbean Bean Nuclease-treated solution containing the Xbal cleaved fragment is purified by purifying the Xbal cleaved fragment from which CTGA's 4 bases have been removed by protein removal according to the usual method. DNA.
- RNA was synthesized in vitro from this caged DNA using T7 RNA polymerase.
- Ambion MEGAscript was used for this RNA synthesis.
- a reaction solution (20 ⁇ l) containing 0.5 to 1.0 g of the vertical DNA was reacted according to the manufacturer's instructions.
- RNA extraction with acidic phenol was performed to remove the cage DNA.
- RNA RNA
- replicon RNA total cellular RNA extracted from Huh7 cells
- the mixed RNA was introduced into Huh7 cells by the electroporation method.
- Huh7 cells that had been subjected to electopore position treatment were seeded in a culture dish and cultured for 16 to 24 hours, and then G418 (neomycin) was added to the culture dish at various concentrations. Then change the culture medium twice a week. The culture was continued. A colony of viable cells was cloned from the culture dish after 21 days of the culture and the culture was continued. Multiple clones of cell clones could be established by such colony cloning.
- One cell line carrying the HCV subgenomic RNA replicon was named 1H4.1.
- HCV subgenomic RNA replicon (GenBank accession number: AJ242654) prepared from the full-length genomic cDNA derived from the HCV genotype lb Con-1 strain by the same method as above (GenBank accession number: AJ242654; I / NS3 in the lower part of Fig. 2) -3 '/ wt) was introduced into the Huh7 cell line,
- Structural region gene of JFH1 strain (GenBank accession number AB047639) (Kato T. et al., J. Med. Viol. 2001, 64: 334-339) isolated from fulminant hepatitis (base sequence numbers 249 to 2781) The region containing was amplified by PCR. A fragment containing the structural gene obtained by digesting this DNA fragment with Nhel and EcoRI was purified by agarose gel electrophoresis, and blunt-ended with DNA polymerase. This blunt-ended cDNA was inserted downstream of the CAG promoter sequence (CAG) in the plasmid vector.
- CAG promoter sequence CAG
- a cDNA containing a structural region gene obtained by digestion with Nhel and EcoRI was used as an elongation factor 1 ⁇ gene promoter sequence (EF-1 ⁇ promoter; Mizushima et. Al., Nucleic Acids Res. , 1990, 18, 5322), pEF4 / Myc-His (Invitrogen) Spel and EcoRI.
- the resulting plasmids were named pCAGC-p7JFHl and pEF4C-p7JFHl (Fig. 3).
- pEF4C-p7JFHl shown in the upper part of Fig. 3 is digested with restriction enzymes BamHI and EcoRI, and Core protein, E1 protein The regions encoding E2 protein and p7 protein were separated by agarose gel electrophoresis. Next, this fragment is the target alien site. It was ligated to pDIsgptmH5 designed to insert the XGPRT gene together with the gene (Ohnishi, K. et al "Jap. J. Infect. Dis. 58 (2005) p88-94, Ishii, K.
- This pDIsgptmH5 incorporates the Escherichia coli xanthine-guanine phosphoribosyltransferase (XGPRT) gene under the control of the cuticular virus p7.5 promoter inserted into the cloning site of the pUc / DIs vector. It is a transfer vector (Ishii, K. et al., Virology 302 (2002) p.433-444). The resulting vector was named pDIsJFHst.
- XGPRT Escherichia coli xanthine-guanine phosphoribosyltransferase
- a vector containing the structural gene of the H77c strain and capable of expressing the protein encoded by it was produced by the following method. First, using a clone of the H77c strain HCV genomic cDNA (GenBank Accession No. AF011751) as a saddle, add 10 X buffer 5 ⁇ 1, 2.5 mM to the LA-PCR kit (Takara Bio Inc.). 1 ⁇ l each of 5 ⁇ ⁇ , 10 ⁇ row number 1) and ⁇ 77 reverse (AAGAGCTCTCATAACCCGACAAGAACAACGCCGC C: SEQ ID NO: 2) was added to the dNTP mixture, and finally deionized water was added to make the total amount 49 ⁇ 1.
- PCR reaction was performed under the conditions of 25 cycles, with one cycle consisting of 98 ° C for 20 seconds and 68 ° C for 5 minutes. When a portion of this PCR product was electrophoresed on an agarose gel, an amplification product of about 2.5 kb was confirmed.
- a PCR reaction 1 was used to perform a ligation reaction in which the amplified product was ligated into a plasmid vector.
- E. coli was transformed according to a conventional method, and plasmid DNA was prepared from the resulting transformant.
- the DNA fragment inserted into this plasmid DNA was digested with a restriction enzyme that can be excised, and subjected to agarose gel electrophoresis to confirm that a DNA fragment of about 2.5 kb was inserted into the plasmid DNA.
- the nucleotide sequence of the inserted DNA fragment was determined by a conventional method.
- the determined base sequence was consistent with the sequences from 271 to 2819 of the base sequence of GenBank accession number AF011751.
- This plasmid DNA was then digested with Bglll and Sacl, subjected to agarose electrophoresis, and a DNA fragment containing the structural gene region of the H77c strain was isolated, and the cuticular virus transfer vector pDIsgptmH5 (Ohnishi, K. et al., Jap. J. Inf ect. Dis. 58 (2005) p88-94, Ishii, K. et al., Virology 302 (2002) p433-444). The resulting vector was named pDIsH77st.
- J1 strain HCV genomic cDNA (GenBank accession number D89815) cloned as a template, attached to LA-PCR kit (Takara Bio Inc.), and 10 X buffer solution 5 ⁇ 1, 2.5 mM dNTP mix 5 1, 10 M primer
- this PCR product 21 was used to perform a ligation reaction in which the amplified product was ligated into a plasmid vector.
- E. coli was transformed according to a conventional method, and plasmid DNA was prepared from the obtained transformant.
- the DNA fragment inserted into the plasmid DNA was digested with a restriction enzyme that can be excised, and subjected to agarose gel electrophoresis, confirming that a DNA fragment of about 2.5 kb was inserted into the plasmid DNA! .
- the nucleotide sequence of the inserted DNA fragment was determined by a conventional method.
- the determined base sequence was identical to the base sequence of GenBank accession number D89815 up to 271st to 2819th.
- This plasmid DNA was then extinguished with Bglll and Sad, subjected to agarose electrophoresis, a DNA fragment containing the structural gene region of J1 strain was isolated, and the Kuchi virus transfer vector pDIsgptmH5 (Ohnishi, K. et al , Jap. J. Infect. Dis. 58 (2005) p88-94, Ishii, K. et al., Virology 302 (2002) p.433-444). The resulting vector was named pDIsJlst.
- a vector containing a chimeric structural gene sequence consisting of the Core gene derived from the J1 strain and the El, E2, and p7 genes derived from the JFH1 strain and capable of expressing the protein encoded by them is obtained by the following method. It was made with.
- a vector obtained by cloning the HCV genomic cDNA (GenBank accession number D89815) of the J1 strain is Primer H77 / J1 forward (SEQ ID NO: 1) and J1 / JFH1 that were supplied with the A-PCR kit (Takara Bio Inc.) revers
- AACAGGACAG SEQ ID NO: 4
- deionized water was added to make the total amount to 49 ⁇ 1.
- 1 ⁇ l of Takara LA Taq Takara LA Taq (Takara Bio Inc.) was added, and PCR was performed. The PCR reaction was performed under 25 cycles under the condition of 98 ° C for 20 seconds and 68 ° C for 5 minutes.
- JFH1 reverse (AAGAGCTCTCAATCAATATCAACAAACCCACGCCT: SEQ ID NO: 6) each was added in an amount of 1 ⁇ l, and finally deionized water was added to make the total amount 49 ⁇ l.
- 1 ⁇ l of Takara LA Taq (Takara Bio Inc.) was added and PCR was performed. The PCR reaction was performed under the conditions of 25 cycles, with one cycle consisting of 98 ° C for 20 seconds and 68 ° C for 5 minutes. Purify each amplified fragment, dissolve in 1 H 0, and dilute 1 ⁇ 1 each 100-fold.
- Each 1 ⁇ 1 was mixed into one. Using this mixed solution as a template, LA-PCR was performed for 5 cycles under the above conditions without adding primers. Thereafter, primers H77 / J1 forward (SEQ ID NO: 1) and JFH1 reverse (SEQ ID NO: 6) were added, and LA-PCR was further performed for 10 cycles to purify the amplified chimeric DNA fragment. This fragment was cloned into a plasmid vector, and the base sequence of the DNA fragment was determined. As a result, it was confirmed that the DNA fragment was a chimeric structural gene sequence consisting of the Core gene derived from the J1 strain and the El, E2, and p7 genes derived from the JFH1 strain.
- this plasmid was digested with Bglll and Sad and the fragment obtained was digested with the viral virus transfer vector pDIsgptmH5 (Ohnishi, K. et al, Jap. J. Infect. Dis. 58
- a vector containing a chimeric structural gene sequence consisting of the Core gene derived from the JFH1 strain and the El, E2, and p7 genes derived from the J1 strain and capable of expressing the proteins encoded by these genes is described below. It was produced by the following method. First, in order to amplify the Core gene of JFH1 strain, the HCV genomic cDNA (GenBank accession number AB047639) of JFH1 strain was used as a template and attached to the LA-PCR kit (Takara Bio Inc.).
- a vector obtained by cloning the HCV genomic cDNA (GenBank accession number D89815) of the J1 strain is used as a cocoon-type kit, and the LA-PCR kit (Takara Bio Inc.) is used. Attached, 10X buffer 5 ⁇ 1, 2.5 mM dNTP mix 5 1, 10 M primer
- CAACGTGTCCGGGATATACC: SEQ ID NO: 9) and Jl reverse (AAGAGCTCTCATA GACCTACAAAAACCCCGCCTCC: SEQ ID NO: 3) were each added at 1 ⁇ 1, and finally deionized water was added to make the total amount at 49 ⁇ 1.
- 1 ⁇ l of Takara LA Taq was added and PCR was performed. The PCR reaction was performed under conditions of 25 cycles, with one cycle consisting of 98 ° C for 20 seconds and 68 ° C for 5 minutes. Purify each amplified fragment, dissolve in 50 1 H 0,
- Each 1 ⁇ 1 was diluted 100 times and each 1 ⁇ 1 was mixed together. Using this mixed solution as a template, 5 cycles of LA-PCR were carried out under the above-mentioned conditions without preparing primers. Thereafter, primers JFH1 forward (SEQ ID NO: 7) and J 1 reverse (SEQ ID NO: 3) were added, and LA-PCR was performed for 10 cycles to purify the amplified chimeric DNA fragment. This fragment was cloned into a plasmid vector and the base sequence of the DNA fragment was determined. As a result, it was confirmed that the DNA fragment was a chimeric structural gene sequence consisting of the Core gene derived from the JFH1 strain and the El, E2, and p7 genes derived from the J1 strain.
- this plasmid was digested with Bglll and Sad and the fragment obtained was digested with the cuticle virus transfer vector pDIsgptmH5 (Ohnishi, K. et al, Jap. J. Infect. Dis. 58 (2005) p88-94, Ishii, K. et al., Virology 302 (2002) p433-444).
- the resulting vector was named pDIsJFH (c) / Jl (El-p7) st.
- Recombinant viral virus vector DIs strains having the above vectors were prepared and selected as follows, for example.
- the cell suspension was centrifuged at 200 Orpm for 3 minutes, and the cells were collected and suspended in 1 PBS. 10 g of the above transfer vector was dissolved in this cell suspension, and electroporation was performed by applying a voltage once at 250 v, 500 FD in a 0.4 cm cuvette using Gene Pulser II (Bio Rad). Cells are suspended in 2 ml of DMEM containing 10% FCS, seeded in a 35 mm dish, and cultured for 7 days at 37 ° C and 5% CO.
- Infected cells were collected together with the medium, freeze-dried 3 times and sonicated for 2 minutes, then diluted 10, 100, and 1000 times with the same medium. 10 6 cells are seeded in a 35 mm dish, and MPA, xantine and hypoxantin are added to the medium (DMEM containing 10% FCS) at 25 ⁇ g / ml, 250 ⁇ g / ml, 15 After addition of ⁇ g / ml and incubation, the above diluted cell solution was inoculated.
- DMEM containing 10% FCS medium
- the formed plaque was picked up with a Pasteur pipette, suspended in DMEM containing 200 ⁇ 1 10% FCS, and sonicated for 2 minutes to release the virus from the agar.
- This culture solution was diluted 10, 100, and 1000 times with the same medium, and the plaque passage procedure similar to the above was repeated two more times to purify the recombinant virus, and then scaled up by infecting CEF cells. .
- pDIsJFHst pDIsH77st
- pDIsJlst pDIsJl (c) / JFH (El—p7) st
- pDIsJ The virus vectors (recombinant virus-like particles) prepared from FH (c) / Jl (El-p7) st were used as DIsJFHst, DIsH77st, DIsJlst, DIsJl (c) / JFH (El—p7) st ⁇ DIsJFH (c ) / Jl (El—p7) st.
- HCV structural protein expression vector into levulin-bearing cells and production of structural proteins in cows.
- ribofetasion of HCV structural protein expression vector pCAGC-p7JFHl or pEF4C-p7JFHl prepared in Example 2 was used. It was introduced into levulin-contained cells by the method or the like.
- the viral vectors DIsJFHst, DIsH77st, DIsJlst, DIsJl (c) / JFH (El-p7) st or DIsJFH (c) / Jl (El-p7) st that express the HCV structural protein prepared in Example 2 are replicon-carrying cells. Infected with.
- pEF4C-p7JFHl was introduced into replicon-retaining cells IH4.1 by the ribofusion method.
- HCV Core protein could be detected in the culture supernatant by Western blotting. Therefore, after culturing the replicon-retaining cell IH4.1 into which pEF4C-p7JFHl was introduced for 4 days, the culture solution (8 ml) was collected, centrifuged at 8,000 g for 60 minutes at 4 ° C, and the culture supernatant was collected. It was. Next, the supernatant was centrifuged at 25,000 rpm for 4 hours at 4 ° C.
- sucrose density gradient is 60% (weight / weight) sucrose solution (dissolved in 50 mM Tris pH7.5 / 0.1M NaCl / lmM EDTA) 2ml, 50% sucrose solution lml, 40% sucrose
- the solution was prepared by overlaying 1 ml of a solution, 1 ml of a 30% sucrose solution, 1 ml of a 20% sucrose solution, and 1 ml of a 10% sucrose solution on a centrifuge tube.
- HCV Core protein was measured using the ortho HCV antigen IRMA test (Aoyagi et al., J. Clin. Microbiol., 37 (1999) pl 802-1808). Quantification of HCV replicon RNA was according to Takeuchi (Takeuchi et al., Gastroenterol ogy 116 (1999) p.636-642). As shown in Figure 5, in both experiments, the replicon RNA and Core protein peaks were both in fraction 8 and were consistent. The density of this fraction was approximately 1.17 g / ml, consistent with the reported density of HCV particles. This showed that virus particles were produced.
- the supernatant was centrifuged at 25,000 rpm for 4 hours at 4 ° C in a SW20 rotor (Beckman), and 8 ml of cell culture broth was added to a buffer containing 1 ml of 0.2% NP40 and a buffer containing no NP40. Suspended in one. After incubation at 4 ° C for 20 minutes, the sample was overlaid on a 10-60% sucrose density gradient prepared in a tube for SW41E rotor (Beckman), and centrifuged at 35,000 rpm for 16 hours at 4 ° C.
- sucrose density gradient is 60% (weight / weight) sucrose solution (dissolved in 50mM Tris pH7.5 / 0.1M NaCl / lmM EDTA) 2ml, 50% sucrose solution lml, 40% sucrose It was prepared by layering sugar solution lml, 30% sucrose solution lml, 20% sucrose solution lml, 10% sucrose solution lml on a centrifuge tube.
- HCV Core protein was measured using the ortho HCV antigen IRMA test (Aoyagi et al., J. Clin. Microbiol., 37 (1999) p. 1802-1808).
- the replicon-retaining cell line 5-15 was added to viral vectors DIsJlst, DIsJl (c) / JFH (El- p7) st or DIsJFH (c) / Jl (El-p7) st was infected in the same manner as described above. After infection, 8 ml of the supernatant cultured for 4 days was concentrated with an ultrafiltration membrane and fractionated by the sucrose density gradient centrifugation method described above. The density of each fraction and the HCV Core protein concentration were quantified.
- HCV Core protein From the density distribution pattern of HCV Core protein, it was produced in culture supernatants infected with DIsJlst, DIsJl (c) / JFH (El-p7) st, or DIs JFH (c) / Jl (El-p7) st It was shown to contain virus-like particles.
- the recombinant HCV particles produced in the above examples have a neo gene as a drug resistance marker. Therefore, in order to confirm whether or not the particles obtained in Example 3 are infectious, it is necessary to infect Huh7 cells with these particles and examine whether G418 (neomycin) resistant colonies can be obtained.
- the culture supernatant obtained by infecting DIsJFHst with a levulin-contained cell line 5-15 and culturing for 4 days was concentrated 30-fold with an ultrafiltration membrane (cut off 1 X 10 5 Da), and Huh7 cells Infected with. After infection, G418 was added to the culture dish at lmg / ml. Thereafter, the culture medium was changed twice a week, and the culture was continued with the force S. After culturing for 21 days from the time of seeding, viable cells were stained with crystal violet. As a result, colony formation was confirmed.
- a non-transmissible recombinant infectious HCV-like particle packaged with an HCV subgenomic RNA containing a desired foreign gene and a method for producing the same.
- Such recombinant infectious HCV-like particles have the advantage of lack of transmissibility, so they can be used in vivo in mammals, particularly human liver and lymphoid cells or yarns and tissues.
- it can be used for gene therapy via ex vivo gene transfer, or it can be used as a viral vector for producing a transgenic animal, and further as an attenuated vaccine.
- sequences of SEQ ID Nos: 1 to 9 represent primers.
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EP06810937A EP1930416B1 (en) | 2005-09-30 | 2006-09-29 | Novel recombinant human hepatitis c virus-like particle and method for producing the same |
KR1020087008304A KR101416143B1 (ko) | 2005-09-30 | 2006-09-29 | 신규 재조합형 인간 c형 간염 바이러스 유사 입자와 그산생 방법 |
CA2624130A CA2624130C (en) | 2005-09-30 | 2006-09-29 | Novel recombinant human hepatitis c virus-like particle and method for producing the same |
AU2006295716A AU2006295716B2 (en) | 2005-09-30 | 2006-09-29 | Novel recombinant human hepatitis C virus-like particle and method for producing the same |
PL06810937T PL1930416T3 (pl) | 2005-09-30 | 2006-09-29 | Nowa rekombinowana cząstka podobna do ludzkiego wirusa zapalenia wątroby typu C i sposób jej wytwarzania |
US11/992,646 US8183044B2 (en) | 2005-09-30 | 2006-09-29 | Recombinant human hepatitis C virus-like particle and method for producing the same |
CN2006800360034A CN101278042B (zh) | 2005-09-30 | 2006-09-29 | 新型重组人丙型肝炎病毒样颗粒及其生产方法 |
ES06810937T ES2376354T3 (es) | 2005-09-30 | 2006-09-29 | Nueva part�?cula recombinante similar al virus de la hepatitis c humano y procedimiento para producir la misma. |
JP2007537739A JP5035985B2 (ja) | 2005-09-30 | 2006-09-29 | 新規組換え型ヒトc型肝炎ウイルス様粒子とその産生方法 |
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CA (1) | CA2624130C (ja) |
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Cited By (8)
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WO2009080053A1 (en) * | 2007-12-20 | 2009-07-02 | Hvidovre Hospital | Efficient cell culture system for hepatitis c virus genotype 1a and 1b |
US8454974B2 (en) | 2007-04-13 | 2013-06-04 | Hvidovre Hospital | Adaptive mutations allow establishment of JFH1-based cell culture systems for hepatitis C virus genotype 4A |
US8506969B2 (en) | 2008-08-15 | 2013-08-13 | Hvidovre Hospital | Efficient cell culture system for hepatitis C virus genotype 7a |
US8569472B2 (en) | 2007-12-20 | 2013-10-29 | Hvidovre Hospital | Efficient cell culture system for hepatitis C virus genotype 6A |
US8618275B2 (en) | 2007-05-18 | 2013-12-31 | Hvidovre Hospital | Efficient cell culture system for hepatitis C virus genotype 5A |
US8663653B2 (en) | 2008-08-15 | 2014-03-04 | Hvidovre Hospital | Efficient cell culture system for hepatitis C virus genotype 2B |
US8772022B2 (en) | 2008-10-03 | 2014-07-08 | Hvidovre Hospital | Hepatitis C virus expressing reporter tagged NS5A protein |
US8945584B2 (en) | 2007-04-13 | 2015-02-03 | Hvidovre Hospital | Cell culture system of a hepatitis C genotype 3a and 2a chimera |
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WO2015123592A2 (en) | 2014-02-13 | 2015-08-20 | Synthetic Genomics, Inc. | Recombinant rna particles and methods of use |
CN110346239B (zh) * | 2019-07-10 | 2022-02-11 | 国家纳米科学中心 | 一种纳米材料密度的检测方法 |
AU2022208435A1 (en) | 2021-01-18 | 2023-08-10 | Conserv Bioscience Limited | Coronavirus immunogenic compositions, methods and uses thereof |
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WO2004024904A2 (en) | 2002-09-13 | 2004-03-25 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Infectious hepacivirus pseudo-particles containing functional e1, e2 envelope proteins |
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US8454974B2 (en) | 2007-04-13 | 2013-06-04 | Hvidovre Hospital | Adaptive mutations allow establishment of JFH1-based cell culture systems for hepatitis C virus genotype 4A |
US8945584B2 (en) | 2007-04-13 | 2015-02-03 | Hvidovre Hospital | Cell culture system of a hepatitis C genotype 3a and 2a chimera |
US8618275B2 (en) | 2007-05-18 | 2013-12-31 | Hvidovre Hospital | Efficient cell culture system for hepatitis C virus genotype 5A |
WO2009080053A1 (en) * | 2007-12-20 | 2009-07-02 | Hvidovre Hospital | Efficient cell culture system for hepatitis c virus genotype 1a and 1b |
US8563706B2 (en) | 2007-12-20 | 2013-10-22 | Hvidovre Hospital | Efficient cell culture system for hepatitis C virus genotype 1A and 1B |
US8569472B2 (en) | 2007-12-20 | 2013-10-29 | Hvidovre Hospital | Efficient cell culture system for hepatitis C virus genotype 6A |
US8506969B2 (en) | 2008-08-15 | 2013-08-13 | Hvidovre Hospital | Efficient cell culture system for hepatitis C virus genotype 7a |
US8663653B2 (en) | 2008-08-15 | 2014-03-04 | Hvidovre Hospital | Efficient cell culture system for hepatitis C virus genotype 2B |
US8772022B2 (en) | 2008-10-03 | 2014-07-08 | Hvidovre Hospital | Hepatitis C virus expressing reporter tagged NS5A protein |
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AU2006295716A1 (en) | 2007-04-05 |
CA2624130A1 (en) | 2007-04-05 |
EP1930416B1 (en) | 2011-12-14 |
US20090221028A1 (en) | 2009-09-03 |
PT1930416E (pt) | 2012-02-02 |
US8183044B2 (en) | 2012-05-22 |
AU2006295716B2 (en) | 2011-11-03 |
KR101416143B1 (ko) | 2014-07-08 |
ES2376354T3 (es) | 2012-03-13 |
JPWO2007037429A1 (ja) | 2009-04-16 |
CN101278042B (zh) | 2011-12-14 |
CN101278042A (zh) | 2008-10-01 |
JP5035985B2 (ja) | 2012-09-26 |
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