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WO2011074564A1 - Procédé de fabrication d'un vecteur d'adénovirus - Google Patents

Procédé de fabrication d'un vecteur d'adénovirus Download PDF

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Publication number
WO2011074564A1
WO2011074564A1 PCT/JP2010/072450 JP2010072450W WO2011074564A1 WO 2011074564 A1 WO2011074564 A1 WO 2011074564A1 JP 2010072450 W JP2010072450 W JP 2010072450W WO 2011074564 A1 WO2011074564 A1 WO 2011074564A1
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Prior art keywords
cells
adenovirus
vector
cell
gene
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English (en)
Japanese (ja)
Inventor
英人 蝶野
知恵 江頭
広文 吉岡
晃 高蔵
純一 峰野
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Takara Bio Inc
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Takara Bio Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10351—Methods of production or purification of viral material

Definitions

  • the present invention relates to an adenoviral vector with improved infection efficiency used for introducing a gene into a target cell in the fields of medicine, cell engineering, genetic engineering, developmental engineering, and the like, and a method for producing the same.
  • This application claims priority to Japanese Patent Application No. 2009-284475 filed on Dec. 15, 2009, and incorporates the entire contents of Japanese Patent Application No. 2009-284475. Is.
  • Adenovirus was isolated from pediatric tonsils and adenoid tissue cultures in 1953, and to date, the existence of more than 80 serotypes hosting humans, birds, cows, monkeys, dogs, mice or pigs has been revealed. Has been. About 50 types of serotypes have been discovered so far for adenoviruses having human hosts, among which types 2 and 5 have been used as gene therapy vectors.
  • Adenovirus is an icosahedral virus with a diameter of about 80 nm and has no envelope. Its genome is a linear double-stranded DNA.
  • the capsid wrapping the core is composed of 12 pentons (consisting of a penton base and fiber) located at each apex and 240 hexons.
  • Adenovirus infection is initiated by adenovirus fibers binding to coxackievirus and adenovirus receptor (CAR) on the host cell surface and adhering to the cells.
  • CAR coxackievirus and adenovirus receptor
  • endocytosis is induced by the binding of the penton base and the cell adhesion factor integrin, and is taken up into the cell.
  • the endosomal membrane is disrupted by the penton base and the adenovirus is released into the cytoplasm.
  • Adenovirus that reaches the nucleus sends its genomic DNA from the nuclear pore into the nucleus. Transcription, replication and packaging of adenoviral DNA occurs in the nucleus.
  • Non-patent Documents 1 and 2 There is a feature that the adhesion type cells generally have higher infection efficiency than the blood cell type cells. These reflect the feature that the vector is easily introduced into cells with many viral receptors. Although it can be introduced into a relatively large number of cell types, there are reports that the amount of receptors for adenoviral vectors is actually decreasing in many cancer cells for which gene therapy is expected. Development of vectors with mutations in viral fibers is also underway. However, problems such as low virus titer remain (for example, Non-Patent Documents 3 and 4).
  • Non-patent Document 5 Non-patent Document 5
  • An object of the present invention is to provide an adenoviral vector with improved infection efficiency, which is used when a gene is introduced into a target cell, and a method for producing the same.
  • the present inventors have found that a foreign gene can be introduced into a target cell with high efficiency by using an adenovirus vector treated with a protease, and the present invention has been completed. I let you.
  • the first aspect of the present invention relates to a method for producing an adenoviral vector, which comprises the following steps: (1) a step of culturing an adenovirus vector-producing cell to produce an adenovirus vector, and (2) a step of treating the adenovirus vector obtained in step (1) with a protease.
  • the adenovirus vector may contain a foreign gene.
  • Adenoviral vectors may also be treated with endo-type proteases.
  • the present invention provides an adenovirus vector with improved infection efficiency.
  • the second aspect of the present invention relates to an adenovirus vector obtained by the first aspect of the present invention.
  • the third aspect of the present invention relates to a method for introducing a gene into a cell, comprising the step of infecting a cell with an adenovirus vector containing a foreign gene obtained according to the first aspect of the present invention.
  • a foreign gene can be introduced into a target cell with high efficiency.
  • the present invention provides a high-titer adenovirus vector suitable for introducing a foreign gene into a cell, and a method for producing the same.
  • the adenoviral vector obtained by the production method is extremely useful not only for the field of gene therapy but also for obtaining cells having various foreign genes in vitro.
  • Adenovirus is a linear double-stranded DNA virus whose genomic DNA is approximately 36 kb in size, and the end protein encoded by the virus is covalently bound to the 5 'ends of both ends of the genomic DNA, and the DNA-terminal protein.
  • a complex (DNA-terminal protein complex; DNA-TPC) is formed.
  • the adenovirus used in the present invention is not particularly limited.
  • human adenovirus type 2 or type 5 having high growth efficiency and slight pathogenicity can be used.
  • a recombinant adenovirus obtained by artificially modifying the viral genome is used as the vector.
  • a non-propagating adenovirus in which the E1 region involved in viral replication is deleted from the genome is used as the vector.
  • An adenovirus lacking the E1 region lacks the ability to proliferate and therefore is suitable for the present invention from the viewpoint of safety without causing virus propagation in infected cells, which may cause pathological situations.
  • Deletion of the viral genome is not particularly limited as long as it does not hinder the function of the vector.
  • those obtained by deleting a part or all of the E3 region that is not essential for virus growth in cultured cells can be used in the present invention.
  • An adenoviral vector prepared by deleting the E1 region, or an adenoviral vector prepared by deleting the E1 region and the E3 region is a cell that continuously expresses the gene of the E1 region, for example, human fetal kidney Origin cells 293 cells can be grown as a host.
  • a virus in which the E2 region or the E4 region is further deleted can also be used as a vector.
  • the gutless adenovirus vector [Proc. Natl. Acad. Sci. Bulletin of the American Academy of Sciences, which has been deleted except for the region necessary for replication and packaging of the viral genome (approximately 0.4 kb at the left end and approximately 0.2 kb at the right end). USA), volume 93, pages 13565-13570 (1996)] can also be used in the present invention.
  • adenoviral vectors can be propagated in cells that express the protein encoded by the deleted region or by co-infection with a helper virus that retains the deleted region.
  • the cells thus constructed are called virus producing cells.
  • Construction of the adenoviral vector, insertion of a foreign gene into the vector, and preparation of viral vector particles can all be performed by techniques known to those skilled in the art.
  • Adenoviral vector particles accumulate in the culture supernatant and production cells of virus producing cells.
  • An adenovirus vector can be obtained by subjecting a culture solution containing virus-producing cells to operations such as ultrasonic disruption or repeated freeze-thawing to disrupt cells contained in the culture solution. Although the obtained disrupted solution can be used as it is as an adenovirus vector, purified adenovirus vector particles can be obtained by means such as cesium chloride density gradient centrifugation according to the purpose.
  • the adenovirus vector used in the present invention may contain a foreign gene.
  • the foreign gene is not particularly limited, and any gene desired to be introduced into the cell can be selected.
  • a gene for example, a gene encoding an antisense nucleic acid, siRNA (small interfering RNA), ribozyme or decoy is used in addition to an enzyme, structural protein, cytokine, receptor or other protein encoding. Can do.
  • an appropriate marker gene that enables selection of the gene-introduced cell for example, a gene encoding a drug resistance gene or a fluorescent protein, a gene encoding an enzyme that can function as a reporter such as ⁇ -galactosidase, luciferase, etc.
  • a gene encoding a drug resistance gene or a fluorescent protein for example, a gene encoding an enzyme that can function as a reporter such as ⁇ -galactosidase, luciferase, etc.
  • the DNA molecule to be bound may be bound by known means such as ligation.
  • it may have a sequence in which a mutation is introduced into a natural sequence according to the purpose.
  • regulatory elements such as appropriate promoters and enhancers for controlling the expression of these genes may be added.
  • the size of the foreign gene introduced into the target cell is not particularly limited. From the viewpoint of packaging of the recombinant adenovirus vector, for example, in the case of a vector lacking the E1 region and the E3 region, it is about 7 kb. The following is desirable. Moreover, in the gutless adenovirus vector, the size of the foreign gene introduced into the target cell is desirably 37 kb or less.
  • the promoter used in the present invention is not particularly limited as long as it can express a foreign gene in a target cell, and examples thereof include CAG promoter, SR ⁇ promoter, EF1 ⁇ promoter, CMV promoter, PGK promoter, etc. Can be selected accordingly. If a tissue-specific promoter is used, a foreign gene can be expressed in a tissue-specific manner. For example, if an ⁇ 1AT promoter, which is a liver-specific promoter, is used, a skeletal muscle-specific ⁇ -actin promoter can be used in a hepatocyte-specific manner.
  • each foreign gene can be expressed specifically in the liver cancer cells.
  • Production of an adenovirus vector according to the method of the present invention is performed by a first step of culturing virus-producing cells to produce an adenovirus vector, and a second step of treating the adenovirus vector obtained in the first step with a protease. Is done.
  • virus-producing cells used in the first step can be produced by a known method.
  • virus-producing cells can be prepared by the following operation.
  • a cosmid vector containing an adenovirus genome from which a gene involved in replication is deleted is prepared, and a foreign gene is inserted if desired.
  • a suitable host cell for example, 293 cells, is transformed with digested cosmid DNA prepared by cleaving this cosmid vector with a restriction enzyme. The resulting transformants have acquired the ability to produce adenovirus.
  • an adenovirus vector can be produced in the cells and / or from the culture solution.
  • the above virus-producing cells can be prepared by, for example, the full-length virus genome introduction method [Human Gene Therapy, Vol. 9, pp. 2577-2583 (1998)] or the COS-TPC method [National Academy of Sciences]. Bulletin (Proc. Natl. Acad. Sci. USA), Volume 93, pp. 1320-1324 (1996)] etc. is carried out by preparing a recombinant adenoviral vector into which a desired foreign gene has been inserted. . According to this method, a recombinant adenovirus vector can be prepared with high efficiency without performing complicated operations.
  • a recombinant adenovirus production kit that can be used for both methods, for example, Adenovirus Expression Vector Kit (Dual Version) Ver. 2 (manufactured by Takara Bio Inc.) and the like, and a recombinant adenovirus vector can be prepared according to the instruction manual attached to the kit.
  • adenovirus vector particles recovered or purified from the inside of the virus-producing cells and the culture supernatant are treated with protease.
  • the protease to be used is not particularly limited.
  • an endo-type protease can be used.
  • the endo-type protease include trypsin, chymotrypsin, elastase, subtilisin, collagenase, dispase, and papain.
  • trypsin or subtilisin is used in the present invention.
  • the concentration at which trypsin is allowed to act on a solution containing adenovirus vector particles is not particularly limited, and may be set as appropriate using, for example, an increase in infectious titer against cells as an index. For example, 0.0010 to 0.030% by weight, particularly 0.0025 to 0.0125% by weight of trypsin is suitable. Further, the concentration at which subtilisin is allowed to act on a solution containing adenovirus vector particles is not particularly limited, and may be set as appropriate using, for example, an increase in infectious titer against cells as an index.
  • subtilisin 16KNPU / G, Sigma-Aldrich, Code P3111
  • the reaction conditions may be set as appropriate within the range suitable for the protease to be used so that the effects of the present invention are exhibited.
  • the treatment can be performed for 10 minutes to 3 hours in a temperature range of 30 to 40 ° C.
  • the adenoviral vector obtained in the first step is again infected with an appropriate host cell (for example, 293 cell), and the adenoviral vector secondary is infected from the infected cell and the culture supernatant.
  • the virus is collected, the obtained secondary virus is again infected with the host cell, the adenovirus vector tertiary virus is collected from the infected cell and the culture supernatant, and the obtained tertiary virus is again infected with the host cell. It is possible to increase the titer of the adenoviral vector by repeating the operation of recovering the adenoviral vector quaternary virus from the infected cells and the culture supernatant.
  • the infection efficiency to the host cells is improved, virus production is enhanced, and a high titer adenovirus vector can be obtained.
  • the number of times the second step is performed is not particularly limited.
  • the second step may be performed for each series of operations, or only for the finally recovered adenovirus vector. .
  • the recovered adenoviral vector By subjecting the recovered adenoviral vector to the second step, it is possible to provide an adenoviral vector with increased infection efficiency that is effective for gene transfer into various target cells.
  • the adenovirus vector of the present invention obtained by the above method is included in the present invention.
  • the adenoviral vector of the present invention is a high-titer adenoviral vector with an increased number of infectious viral particles against various target cells, particularly cancer cells.
  • the present invention provides a method for introducing a foreign gene into a target cell.
  • the method for introducing the foreign gene of the present invention into the target cell is obtained as a first step, a step of culturing virus-producing cells to produce an adenovirus vector into which the foreign gene has been inserted, and a second step as a first step.
  • the step of treating the obtained adenoviral vector with a protease and the third step include a step of infecting the target cell with the adenoviral vector obtained in the second step.
  • the adenoviral vector obtained in the second step is brought into contact with the target cell, and the target cell is infected with the adenoviral vector.
  • the infection time is not particularly limited, but is preferably 15 minutes to 6 hours, and more preferably 1 to 3 hours.
  • the temperature at the time of infection may be a temperature suitable for the target cell.
  • the foreign gene introduction method of the present invention is useful in the field of gene therapy.
  • an ex vivo gene transfer method for infecting a target cell taken out of the body with the adenovirus of the present invention and then returning the infected cell to the living body, or an adeno of the present invention an ex vivo gene introduction method in which a viral vector is administered to a living body can be used.
  • a viral vector is administered to a living body can be used.
  • the disease targeted for gene therapy by this method can be used for the treatment of genetic diseases in which there is a genetic abnormality in nature, viral infections such as AIDS, and cancer.
  • a restricted-growth type oncolytic virus vector may be used.
  • the adenoviral vector inserted with a therapeutic gene produced by the method of the present invention can be used as a therapeutic agent for various diseases.
  • the adenovirus vector can be prepared as a preparation that can be administered by drip, injection, inhalation, etc. by mixing with a known organic or inorganic carrier, excipient, stabilizer, etc. suitable for parenteral administration.
  • the foreign gene introduction method of the present invention is useful not only for gene therapy but also for obtaining cells having various foreign genes in vitro. Useful for development.
  • Example 1 Preparation of ZsGreen-expressing adenoviral vector pZsGreen Vector (Clontech) was cleaved with restriction enzymes BamHI and EcoRI (Takara Bio), and subjected to agarose gel electrophoresis, which contains a sequence encoding green fluorescent protein ZsGreen A fragment of about 0.7 kbp was recovered. The recovered fragment was blunt-ended using DNA Blunting Kit (Takara Bio), and then swaI of pAxCAwtit2 (Takara Bio), a cosmid vector containing the full length of the adenovirus genome from which the E1 and E3 genes were deleted. Inserted into the site.
  • the thus obtained cosmid vector was transformed into Escherichia coli DH5 alpha (manufactured by Takara Bio Inc.) using Gigapack III XL Packaging Extract (manufactured by Agilent Technologies), and the obtained transformant was cultured in ampicillin-added LB medium. From the culture, pAxCAwtit2-ZsGreen cosmid DNA was prepared. Next, 293 cells were transformed using the digested cosmid DNA obtained by cleaving the obtained pAxCAwtit2-ZsGreen cosmid DNA with restriction enzyme BspT104I (Takara Bio) and TransIT-293 (Miras Bio). After that, culture was performed.
  • DMEM Dulbecco's modified Eagle medium
  • FBS 10% fetal bovine serum
  • DMEM Dulbecco's modified Eagle medium
  • FBS 10% fetal bovine serum
  • the culture solution containing the cells was sonicated, and then centrifuged to collect the supernatant, thereby obtaining a primary adenovirus solution (ZsGreen expression adenovirus vector).
  • a primary adenovirus solution ZsGreen expression adenovirus vector
  • 293 cells infected with the primary adenovirus solution were cultured, the culture solution containing the cells was sonicated, centrifuged, and the supernatant was collected to obtain a secondary adenovirus solution.
  • this quaternary adenovirus solution was used as a ZsGreen expression adenovirus vector in the following experiments.
  • Example 2 Trypsinization of Adenovirus Vector-1 To 20 ⁇ L of the ZsGreen-expressing adenovirus vector prepared in Example 1, 4 ⁇ L of 0.25% trypsin-EDTA aqueous solution (manufactured by Invitrogen) for cell detachment and 376 ⁇ L of PBS were prepared to prepare a reaction solution having a final trypsin concentration of 0.0025%.
  • a reaction solution prepared by adding 380 ⁇ L of PBS to 20 ⁇ L of ZsGreen-expressing adenovirus vector and diluting 20 times was prepared. Each of the prepared reaction solutions was incubated in a 37 ° C. water bath for 1 hour, and then cooled with ice to stop the reaction.
  • Example 3 Cell Infection Experiment-1 1 ⁇ 10 5 HeLa cells [ATCC CCL-2] were added to each well of a 12-well plate and cultured for 24 hours.
  • As the medium 1 mL of Dulbecco's modified Eagle medium (DMEM, Sigma) containing 10% fetal bovine serum (FBS, manufactured by GIBCO) was used.
  • DMEM Dulbecco's modified Eagle medium
  • FBS fetal bovine serum
  • each adenovirus solution prepared in Example 2 was serially diluted with DMEM medium containing 10% FBS and added to cells in each well, and the cells were infected at 37 ° C. for 1 hour. After completion of the infection, the virus supernatant was removed, and 1 mL of DMEM medium containing 10% FBS was added per well, followed by further culturing for 2 days.
  • the detached and recovered cells are suspended in 4% formaldehyde solution and fixed, and then analyzed with a flow cytometer (FACSCant, manufactured by Becton Dickinson). The ratio of the green fluorescent protein ZsGreen expressed is determined. Analysis and gene transfer efficiency was calculated. The number of infectious virus particles (IVP / mL) was calculated from the value obtained by multiplying the obtained gene transfer efficiency by the virus dilution rate. The number of infectious virus particles is shown in Table 1. As shown in Table 1, the number of infectious virus particles increased in the group in which the adenovirus vector was trypsinized compared to the group in which the protease treatment was not performed.
  • Example 4 Trypsinization of Adenovirus Vector-2 20 ⁇ L of a 0.25% trypsin-EDTA aqueous solution for cell detachment and 360 ⁇ L of PBS were added to 20 ⁇ L of the ZsGreen-expressing adenovirus vector prepared in Example 1 to prepare a reaction solution having a final trypsin concentration of 0.0125%.
  • a reaction solution was prepared by adding 380 ⁇ L of PBS to 20 ⁇ L of adenovirus vector and diluting 20 times. The prepared reaction solution was reacted in a 37 ° C. water bath for 1 hour and then cooled with ice to stop the reaction.
  • the virus supernatant was removed, 1 mL of each culture medium was added per well, and the cells were further cultured for 2 days. After culturing, the cells detached and recovered are suspended in 4% formaldehyde solution and fixed, then analyzed with a flow cytometer (FACSCant), and the proportion of green fluorescent protein ZsGreen expressed is analyzed to determine the efficiency of gene transfer. Calculated.
  • the number of infectious virus particles (IVP / mL) was calculated from the value obtained by multiplying the obtained gene transfer efficiency by the virus dilution rate. The number of infectious virus particles is shown in Table 2. As shown in Table 2, by treating the adenovirus vector with trypsin, the number of infectious virus particles for various cells increased.
  • Example 6 Protease treatment of adenovirus vector Bacillus sp.
  • the derived protease (Subtilisin, 16.9 KNPU / G, manufactured by Sigma-Aldrich) was diluted 50-fold, 250-fold, 1250-fold or 6250-fold with PBS.
  • To a 1.5 mL microtube 85 ⁇ L of PBS and 5 ⁇ L of the ZsGreen-expressing adenovirus vector prepared in Example 1 were added, and 10 ⁇ L of the previously diluted protease solution was added (the final protease concentration was 33.8 NPU, respectively).
  • 10 ⁇ L of PBS without protease was added.
  • the prepared reaction solution was reacted in a 37 ° C. water bath for 1 hour and then cooled with ice to stop the reaction.
  • Example 7 Cell Infection Experiment The day before adenovirus infection, 1 ⁇ 10 5 HeLa cells were seeded in each well of a 12-well plate and cultured in DMEM medium containing 10% FBS for 24 hours. Next, the adenovirus solution reacted in Example 6 and the unreacted adenovirus solution were serially diluted with 10% FBS-containing DMEM medium and infected at 37 ° C. for 2 hours. After completion of the infection, the virus supernatant was removed, 1 mL of each culture medium was added per well, and the cells were further cultured for 2 days.
  • the cells detached and recovered are suspended in 4% formaldehyde solution and fixed, then analyzed with a flow cytometer (FACSCant), the proportion of green fluorescent protein ZsGreen expressed is analyzed, and the gene transfer efficiency is determined. Calculated.
  • the number of infectious virus particles (IVP / mL) was calculated from the value obtained by multiplying the obtained gene transfer efficiency by the virus dilution rate.
  • the number of infectious virus particles is shown in Table 3. As shown in Table 3, the number of infectious virus particles against HeLa cells increased in the group treated with the protease of the adenovirus vector compared to the group not treated with the protease.
  • the present invention provides a high-titer adenovirus vector suitable for introducing a foreign gene into a cell, and a method for producing the same.
  • the adenoviral vector obtained by the production method is extremely useful not only for the field of gene therapy but also for obtaining cells having various foreign genes in vitro.

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Abstract

L'invention concerne un procédé de fabrication d'un vecteur d'adénovirus, caractérisé en ce qu'il comporte (1) une étape dans laquelle des cellules productrices du vecteur d'adénovirus sont mises en culture et le vecteur d'adénovirus est produit, et (2) une étape dans laquelle le vecteur d'adénovirus obtenu dans l'étape (1) est traité avec une protéase. Grâce à ce procédé de fabrication, on obtient un vecteur d'adénovirus adapté à l'introduction de gènes exogènes dans des cellules et pour lequel l'efficacité de transmission est augmentée.
PCT/JP2010/072450 2009-12-15 2010-12-14 Procédé de fabrication d'un vecteur d'adénovirus Ceased WO2011074564A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015005430A1 (fr) * 2013-07-11 2015-01-15 タカラバイオ株式会社 Procédé de fabrication d'un virus non enveloppé
US10023846B2 (en) 2014-07-10 2018-07-17 Takara Bio Inc. Production method for non-enveloped virus particles
US10415020B2 (en) 2015-01-09 2019-09-17 Takara Bio Inc. Method for producing non-enveloped viral particles

Citations (5)

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Publication number Priority date Publication date Assignee Title
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JPWO2015005430A1 (ja) * 2013-07-11 2017-03-02 タカラバイオ株式会社 非エンベロープウイルスの製造方法
US10072250B2 (en) 2013-07-11 2018-09-11 Takara Bio Inc. Method for manufacturing non-enveloped virus
KR102069561B1 (ko) 2013-07-11 2020-01-23 다카라 바이오 가부시키가이샤 외피 비보유 바이러스의 제조 방법
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US10415020B2 (en) 2015-01-09 2019-09-17 Takara Bio Inc. Method for producing non-enveloped viral particles

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