JPWO2011074564A1 - Method for producing adenovirus vector - Google Patents

Abstract

The present invention includes (1) a step of culturing an adenoviral vector-producing cell to produce an adenoviral vector, and (2) a step of treating the adenoviral vector obtained in step (1) with a protease. The present invention relates to a method for producing an adenovirus vector. The production method provides an adenoviral vector with improved infection efficiency suitable for introducing a foreign gene into a cell.

Description

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 and adhering to the cell surface coxackievirus and adenovirus receptor (CAR) on the host cell surface. When adenovirus adheres to a cell, endocytosis is induced by the binding of the penton base and the cell adhesion factor integrin, and is taken up into the cell. After being 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.

  At the beginning of adenovirus vector development, it was considered optimal to administer the vector to the bronchus because of its pathogenicity. However, in reality, most vectors are transferred to the liver when the vector is administered to the mouse from the tail vein. (Non-patent Documents 1 and 2). In addition, 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).

  It has already been reported that pretreatment of cancer cells with protease increases the infection efficiency with recombinant adenovirus (Non-patent Document 5). However, there is no description or suggestion that treatment of an adenoviral vector with a protease increases the efficiency of infection of target cells.

Nature Genetics, Vol. 4, pp. 27-34 (1993) Journal of Clinical Investing (J. Clin. Invest.), 91, 225-234 (1993) Biochimika et Biophysica Actor (Biochim. Biophys. Acta), Volume 1568, pp. 13-20 (2001) Human Gene Therapy, Vol. 9, pp. 2503-2515 (1998) Human Gene Therapy, Vol. 11, pp. 2219-2230 (2000)

  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.

  As a result of diligent efforts to solve the above problems, 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.

That is, when the present invention is outlined, 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.

In the first aspect of the present invention, 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 adenoviral vector obtained by the first aspect of the present invention.

  A 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 adenoviral vector containing a foreign gene obtained according to the first aspect of the present invention. According to 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, the size of the genomic DNA is about 36 kb, and the end protein encoded by the virus is covalently bound to the 5 'ends of both ends of the genomic DNA. A complex (DNA-terminal protein complex; DNA-TPC) is formed.

  The adenovirus used in the present invention is not particularly limited. For example, human adenovirus type 2 or type 5 having high growth efficiency and slight pathogenicity can be used. Usually, a recombinant adenovirus obtained by artificially modifying the viral genome is used as the vector. Preferably, 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.

  The deletion of the viral genome is not particularly limited as long as it does not hinder the function as a vector. For example, in addition to the above-described E1 region, 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.

  From the viewpoint of reducing immunogen production that can cause side effects, in addition to the deletion of the E1 region and E3 region described above, a virus in which the E2 region or E4 region is further deleted can also be used as a vector. Furthermore, 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), 93, 13565-13570 (1996)] can also be used in the present invention.

These 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 adenoviral 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. As such a gene, for example, a gene encoding an antisense nucleic acid, siRNA (small interfering RNA), ribozyme or decoy is used in addition to a gene encoding an enzyme, structural protein, cytokine, receptor or other protein. Can do. In addition, 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 β-galactosidase, luciferase, etc.) May be introduced simultaneously. There are no particular limitations on the origin of these genes, and they may be derived from the same organism as the cell into which the gene is introduced, from different organisms, or genetically engineered, or from different origins. The DNA molecule to be bound may be bound by known means such as ligation. Furthermore, it may have a sequence in which a mutation is introduced into a natural sequence according to the purpose. In addition, 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, SRα promoter, EF1α 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 α1AT promoter that is a liver-specific promoter is used, a skeletal muscle-specific α-actin promoter can be used in a hepatocyte-specific manner. Specific to skeletal muscle cells, specific to nerve cells using a neuron-specific enolase promoter, specific to vascular endothelial cells using a vascular endothelial cell-specific tie promoter, specific to gastric cancer cells using a gastric cancer-specific CEA promoter Alternatively, if a liver cancer-specific AFP promoter is used, 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 prepared by a known method. In one embodiment, for example, 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. By culturing the cells under appropriate conditions, an adenovirus vector can be produced in the cells and / or from the culture solution.

  The production of the above virus-producing cells can be performed, for example, by a full-length virus genome introduction method [Human Gene Therapy, Vol. 9, pages 2577 to 2583 (1998)] or COS-TPC method [National Academy of Sciences]. Bulletin (Proc. Natl. Acad. Sci. USA), Vol. 93, pp. 1320-1324 (1996)] and the like are 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. Recombinant adenovirus production kits that can be used in both methods include, 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.

  In the second step, the adenovirus vector particles recovered or purified from the inside of the virus-producing cells and the culture supernatant are treated with protease. In the second step, the protease to be used is not particularly limited. For example, an endo-type protease can be used. Examples of the endo-type protease include trypsin, chymotrypsin, elastase, subtilisin, collagenase, dispase, and papain. Preferably, 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. When using a commercially available subtilisin (16KNPU / G, Sigma-Aldrich, Code P3111), for example, 0.016 to 80 NPU / mL, particularly 0.25 to 35 NPU / mL is suitable. Similarly, 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. For example, it can process for 10 minutes-3 hours in the temperature range of 30-40 degreeC.

Although the present invention is not particularly limited, 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. By performing the second step as a pretreatment when infecting host cells, 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. For example, the second step may be performed for each series of operations, or only for the finally recovered adenovirus 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 adenoviral vector of the present invention obtained by the above method is included in the present invention. As described in Examples 3, 5 and 7, 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 first and second steps are as described above. In the third 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. As a means for carrying out the gene transfer method of the present invention for therapeutic purposes, 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 in vivo gene introduction method in which a viral vector is administered to a living body can be used. There are no particular limitations on the disease targeted for gene therapy by this method, and it 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. When targeting 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. For example, 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.

  Further, 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.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the scope of the examples.

Example 1 Preparation of ZsGreen-expressing adenovirus 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 (manufactured by Takara Bio Inc.) and then swaI of pAxCAwtit 2 (manufactured by Takara Bio Inc.), 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 with the digested cosmid DNA obtained by cleaving the obtained pAxCAwtit2-ZsGreen cosmid DNA with restriction enzyme BspT104I (Takara Bio Inc.) and TransIT-293 (Miras Bio Inc.). After that, culture was performed. Dulbecco's modified Eagle medium (DMEM, manufactured by BioWhittaker) containing 10% fetal bovine serum (FBS, manufactured by Nargenunk International) was used as the medium. After culturing, 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). After 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. Further, the same operation was performed to prepare a tertiary adenovirus solution and a quaternary adenovirus solution. Hereinafter, this quaternary adenovirus solution was used as the ZsGreen expression adenovirus vector in the following experiments.

Example 2 Trypsinization of Adenovirus Vector-1
To 20 μL of the ZsGreen-expressing adenovirus vector prepared in Example 1, 4 μL of 0.25% trypsin-EDTA aqueous solution (manufactured by Invitrogen) for cell detachment and 376 μL of PBS were added to prepare a reaction solution having a final trypsin concentration of 0.0025%. Further, 20 μL of a 0.25% trypsin-EDTA aqueous solution for cell detachment (manufactured by Invitrogen) and 360 μL of PBS were added to 20 μL of a ZsGreen-expressing adenovirus vector to prepare a reaction solution having a final trypsin concentration of 0.0125%. As a comparative control, a reaction solution prepared by adding 380 μL of PBS to 20 μL 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 a medium, 1 mL of Dulbecco's modified Eagle medium (DMEM, Sigma) containing 10% fetal calf serum (FBS, manufactured by GIBCO) was used. Next, 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. After 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 treated with the trypsin of the adenovirus vector compared to the group not treated with the protease.

Example 4 Trypsinization of Adenovirus Vector-2
20 μL of a 0.25% trypsin-EDTA aqueous solution for cell detachment and 360 μL of PBS were added to 20 μL of the ZsGreen-expressing adenovirus vector prepared in Example 1 to prepare a reaction solution having a final trypsin concentration of 0.0125%. As a comparative control, a reaction solution was prepared by adding 380 μL of PBS to 20 μL 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.

Example 5 Cell Infection Experiment-2
HeLa cells and 293 cells [ATCC CRL-1573] were cultured in DMEM medium containing 10% FBS, and MKN1 cells [RIKEN cell bank RCB1003] and MKN45 cells [RIKEN cell bank RCB1001] were cultured in RPMI1640 medium containing 10% FBS. The day before virus infection, 1 × 10 ⁵ cells were seeded in each well of a 12-well plate and cultured for 24 hours. Next, the adenovirus solution reacted in Example 4 and the unreacted adenovirus solution were serially diluted with DMEM medium containing 10% FBS and added to the cells of each well and infected at 37 ° C. for 3 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. After completion of the culture, 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 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 μL of PBS and 5 μL of the ZsGreen-expressing adenovirus vector prepared in Example 1 were added, and 10 μL of the previously diluted protease solution was added (the final protease concentration was 33.8 NPU, respectively). / ML, 6.76 NPU / mL, 1.35 NPU / mL or 0.27 NPU / mL). As a comparative control, 10 μL 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. After completion of the culture, 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.

Claims (5)

A method for producing an adenoviral vector comprising 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 method according to claim 1, wherein the adenoviral vector contains a foreign gene.   2. The method of claim 1, wherein the adenoviral vector is treated with an endoprotease.   The adenovirus vector manufactured by the method of any one of Claims 1-3.   A method for introducing a gene into a cell, comprising the step of infecting a cell with an adenoviral vector produced by the method according to claim 2.