WO2022114170A1 - Method for producing non-enveloped virus utilizing bivalent positive ion - Google Patents

Abstract

The present invention provides a method for producing an adeno-associated virus, the method comprising (a) a step for freezing and thawing a culture medium containing cells capable of producing an adeno-associated virus and 5 mM or more of a bivalent positive ion and (b) a step for obtaining the adeno-associated virus from the culture medium obtained in step (a).

Description

Method for producing non-enveloped virus using divalent cations

The present invention relates to a method for producing an envelopeless virus (hereinafter, non-enveloped virus), preferably an adeno-associated virus (hereinafter, AAV).

Currently, in the field of gene recombination and medical field, as a method for introducing a gene into mammalian cells including humans, a physical method using electric perforation or metal fine particles, a chemical method using nucleic acid, polycation, or liposome, etc. In addition, as a biological method, there is a method using a vector for introducing a gene derived from a virus (hereinafter referred to as a virus vector). A viral vector is a vector obtained by modifying a naturally occurring virus so that a desired gene or the like can be transferred into a target cell, and technological development has been progressing in recent years. Generally, a virus vector prepared by using gene recombination technology is called a recombinant virus vector, and the virus from which such a recombinant virus vector is derived includes retrovirus, lentivirus, Sendai virus, herpes virus and the like. Encapsulated viruses, as well as adenoviruses, and non-enveloped viruses such as AAV are well known.

In particular, AAV can infect a wide variety of cells including humans, it also infects non-dividing cells that have completed differentiation such as blood cells, muscles, and nerve cells, and since it is not pathogenic to humans, there is little concern about side effects. Since the virus particles are physicochemically stable, it is useful as a vector for gene transfer used in gene therapy for the treatment of congenital gene diseases as well as cancer and infectious diseases. It has been attracting attention in recent years.

As a method for producing a recombinant virus vector, generally, among the elements essential for virus particle formation, those that require cis supply and those that can be trans-supplied are separated and introduced into cells that can be used as a host. Therefore, a method is taken to prevent the production of wild-type virus and the self-sustaining replication of the recombinant virus at the infected destination. Usually, the essential element for virus particle formation is introduced into a cell in the form of a nucleic acid construct to produce a cell having the ability to produce a virus (hereinafter referred to as a virus-producing cell). When the cells are cultured and all the essential elements for virus particle formation are expressed in the cells, a viral vector is generated.

The virus-producing cells in which virus production has been achieved are then collected and disrupted, and the cell disruption solution containing the obtained recombinant AAV (rAAV) vector is appropriately filtered, ultra-centrifuged, chromatographed, ultrafiltered, or the like. By subjecting to the process, the rAAV vector is purified and becomes the final product.

Currently, as the use of rAAV vectors spreads to the fields of basic research on gene therapy or clinical application, there is a need for a large-scale and easy method for obtaining rAAV vectors with higher titers and higher purity. The improvement method is disclosed. However, many of the disclosed improved methods are devised for the step of purifying the non-enveloped virus from the cell disruption solution, and the "method for treating virus-producing cells" carried out before the non-enveloped virus is subjected to purification. There are few improvements.

Examples of the method for treating virus-producing cells include freeze-thaw, ultrasonic treatment, mechanical crushing, crushing with a dairy pot / nipple, addition of a surfactant, etc., but in general, freeze-thawing may be performed. many. That is, a crude extract containing a virus vector is obtained by crushing the cells by freezing and thawing the solution containing the virus-producing cells, and then centrifuging or filtering the cells. For example, in Non-Patent Document 1, lysis buffer (50 mM Tris, 150 mM NaCl, pH 8.5) is added to pellets of Expi293F cells or BHK cells which are AAV-producing cells, freeze-thawed three times, and then subjected to Benzonase treatment. Then, the solution is clarified by centrifugation. Further, in Non-Patent Document 2, pellets of HEK293 cells or HeLaS3 cells, which are AAV-producing cells, are suspended in lysis buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 10 mM MgCl ₂ ), and freeze-thaw is performed three times. is doing.

As mentioned above, there are few improvements in the "method for treating virus-producing cells" that is carried out before the non-enveloped virus is subjected to purification, and there is still room for improvement.

As a result of diligent research for the purpose of providing a "method for treating virus-producing cells" capable of efficiently obtaining a non-enveloped virus without complicated operations, the present inventors have the ability to produce a non-enveloped virus. By freezing and thawing a solution containing divalent cations and obtaining a non-enveloped virus from the solution, it was found that the non-enveloped virus can be efficiently obtained without complicated operations, and the present invention has been completed.

That is, the present invention
[1] A method for producing an adeno-associated virus.
(A) a step of freezing and thawing a solution containing cells capable of producing an adeno-associated virus and a divalent cation of 5 mM or more, and (b) a step of obtaining an adeno-associated virus from the solution of (a).
Including, method,
[2] The method according to [1], wherein the divalent cation is a magnesium ion.
[3] The method according to [1] or [2], wherein the solution is a medium.
[4] The method according to [3], wherein the medium is a medium containing amino acids, sugars, and lipids.
[5] The method according to [4], wherein the medium is a medium selected from the group consisting of DMEM, IMDM, RPMI1640, Ham's F-12, EMEM and a mixture thereof.
[6] The method according to any one of [1] to [5], wherein the step (b) is carried out by solid-liquid separation.
[7] The method according to [6], wherein the solid-liquid separation is carried out by centrifugation.
Regarding.

INDUSTRIAL APPLICABILITY According to the present invention, a manufacturing method for efficiently obtaining a non-enveloped virus solution without complicated operations is provided. Further provided is a solution containing divalent cations used in the production method and a non-enveloped virus produced using the production method. The crude virus extract obtained by using the solution containing the divalent cation of the present invention can also be applied to a conventional non-enveloped virus purification method.

It is a figure which shows the viral load of each crude extract by the difference in the number of times of freezing and thawing using MgCl ₂ medium or PBS in Example 1. FIG. It is a figure which shows the viral load of each crude extract by freeze-thaw using _H2O containing magnesium chloride or sodium chloride in Example 2. FIG. It is a figure which shows the viral load of each crude extract by freeze-thaw using DMEM containing magnesium chloride or sodium chloride in Example 3. FIG. It is a figure which shows the viral load of each crude extract by freeze-thaw using the culture medium containing various divalent cations in Example 4. FIG. It is a figure which shows the viral load of each crude extract by freeze-thaw using various solvents containing magnesium chloride in Example 5. FIG. It is a figure which shows the viral load of each crude extract by freeze-thaw using _H2O containing magnesium chloride in Example 6. It is a figure which shows the viral load of the crude extract of various serotype AAV by freeze-thaw using the medium containing magnesium chloride in Example 7. FIG.

The present invention will be described in detail below.
<Definition>

In the present specification, the term "non-enveloped virus" refers to a virus other than the enveloped virus. Here, the enveloped virus refers to a virus having a lipid layer or a lipid bilayer on the surface of the virus. Typical non-enveloped viruses include adenovirus, parvovirus, papovavirus, human papillomavirus, etc. for viruses whose genome is DNA, and rotavirus, coxsackie virus, enterovirus, and sapovirus for viruses whose genome is RNA. , Norovirus, poliovirus, echovirus, hepatitis A virus, hepatitis E virus, rhinovirus, astrovirus and the like are exemplified.

The non-enveloped virus to which the production method of the present invention is applied is not particularly limited, and even a non-enveloped virus whose production method is already known is a non-enveloped virus newly obtained from nature or a gene recombination derived from them. It may be a virus vector. Preferred examples of the non-enveloped virus produced by the production method of the present invention include adenovirus, AAV of the Parvoviridae family, and bocavirus.

As used herein, AAV refers to a small virus belonging to the family Parvoviridae, Dependoparvovirus, which infects primate animals including humans and other vertebrates. AAV has an envelopeless icosahedron outer shell and a single single-stranded DNA inside it. As used herein, AAV includes wild-type viruses and derivatives thereof, including all serotypes and clades unless otherwise noted.

The production method of the present invention is applicable to any known serotype of AAV, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13. It can be used to produce at least one AAV selected from the group consisting of. The production method of the present invention is preferably applied to AAV1, AAV2, AAV5, and AAV6, and more preferably to AAV2. When describing the serotype of rAAV in the present specification, the serotype from which the capsid is derived is used as a reference. That is, the serotype of the rAAV shall be determined according to the origin of the cap gene used at the time of rAAV preparation, and shall not depend on the origin of the serotype of the AAV genome encapsulated in the rAAV particles. For example, if the capsid is derived from AAV6 and the ITR in the AAV genome encapsulated in the rAAV particles is derived from AAV2, the rAAV is referred to as serotype 6 herein. Furthermore, the production method of the present invention can also be applied to the production of AAV containing a variant of the capsid of AAV of each of the above serotypes.

In the present specification, a cell having an ability to produce a virus (hereinafter referred to as a virus-producing cell) refers to a cell that expresses an element necessary for virus production and produces virus particles. The virus-producing cells used in the production method of the present invention are not particularly limited, and may be virus-producing cells obtained in the environment or from clinical specimens of patients with infectious diseases, or may be artificially produced virus-producing cells. Preferably, artificially produced virus-producing cells are used in the present invention, for example, a nucleic acid that supplies essential elements for particle formation of a desired non-enveloped virus, and a nucleic acid encapsulated in particles of a non-enveloped virus. Virus-producing cells produced by introduction into arbitrary cells, and virus-producing cells in which the cells are artificially infected with a non-enveloped virus and, if necessary, a helper virus necessary for producing the virus are used. Virus.

The cells for producing virus-producing cells are not particularly limited, and mammalian cells such as humans, monkeys, and rodents can be used. Preferably, artificially established cell lines, such as 293 cells (ATCC CRL-1573), 293T cells (ATCC CRL-3216), 293T / 17 cells (ATCC CRL-11268), 293F cells, which have high transfection efficiency. Examples thereof include 293FT cells (all manufactured by Life Technologies), G3T-hi cells (International Publication No. 2006/035829), commercially available cell lines for virus production, and AAV293 cells (manufactured by Stratagene). Further, arthropod cells (insect cells) such as Sf9 cells (ATCC CRL-1711) are exemplified. For example, the 293 cells and the like constitutively express the adenovirus E1 protein, and one or some of the proteins necessary for rAAV production are modified to transiently or constitutively express. It may be a cell. The following elements necessary for virus formation can be introduced into these various cells using a known method or a commercially available kit to obtain virus-producing cells. In addition, the cells can be cultured under known culture conditions. For example, culture at a temperature of 30 to 37 ° C., a humidity of 95% RH, and a CO ₂ concentration of 5 to 10% (v / v) is exemplified, but the present invention is not limited to such conditions. If the desired proliferation of virus-producing cells and the production of virus can be achieved, the temperature, humidity, and CO ₂ concentration other than the above ranges may be used. The culture period is not particularly limited, and is, for example, 12 to 150 hours, preferably 48 to 120 hours.

Taking rAAV-producing cells as an example as virus-producing cells, as essential elements for rAAV formation, (A) nucleic acids encapsulated in rAAV particles, (B) AAV-derived elements such as Rep protein and Cap protein, and the like. In addition, (C) elements that exert a helper function, such as E1a protein, E1b protein, E2 protein, E4 protein, and VARNA derived from adenovirus can be mentioned. By introducing these elements into any cell, rAAV-producing cells can be produced.

The above-mentioned "(A) nucleic acid encapsulated in rAAV particles" is composed of an AAV-derived ITR sequence and a nucleic acid desired to be mounted on rAAV particles. Nucleic acids that are desired to be loaded on rAAV particles include arbitrary foreign genes such as polypeptides (enzymes, growth factors, cytokines, receptors, structural proteins, etc.), antisense RNA, ribozyme, decoy, RNA that causes RNA interference, and the like. Nucleic acid to supply is exemplified. In addition, one or more of the appropriate promoters, enhancers, terminators and other transcriptional regulatory elements may be inserted into the nucleic acid encapsulated in the rAAV particles to control the expression of foreign genes. The nucleic acid encapsulated in the rAAV particles can be introduced into cells in the form of a plasmid as a nucleic acid construct. The above-mentioned plasmid can be constructed, for example, by a known method using a commercially available or known plasmid. Examples of the plasmid include pAAV-ZsGreen1 Vector and pAAV-CMV Vector (both manufactured by Takara Bio Inc.).

The form of the above-mentioned "(B) AAV-derived element, for example, Rep protein and Cap protein" is not limited, and each element can be directly introduced into a cell as a protein, and each element can be supplied 1. Alternatively, it can be loaded into a plasmid or viral vector as a plurality of nucleic acid constructs and introduced into cells. Introduction of these nucleic acids into cells can be performed, for example, by a known method using a commercially available or known plasmid or viral vector. Examples of plasmids capable of supplying Rep protein and Cap protein include pRC1 Vector, pRC2-mi342 Vector, pRC5-Vector, and pRC6 Vector (all manufactured by Takara Bio Inc.).

The form of the above-mentioned "(C) element exhibiting helper function" is not limited, and each element can be directly introduced into a cell as a protein, or one or more nucleic acid constructs capable of supplying each element. It can be loaded into a plasmid or viral vector and introduced into cells. Introduction of these nucleic acids into cells can be performed, for example, by a known method using a commercially available or known plasmid or viral vector. An example of the plasmid is pHelper Vector (manufactured by Takara Bio Inc.). The above-mentioned object can also be achieved by directly infecting cells with a virus that exerts a helper function, such as adenovirus, instead of a plasmid or viral vector.

As a method for introducing a nucleic acid construct, a transient introduction method is exemplified.
The method for transient introduction is not particularly limited, and known transient introduction methods such as calcium phosphate method, lipofection method, DEAE dextran method, polyethyleneimine (PEI) method, electroporation method and the like can be used. .. Also, commercially available reagents such as TransIT (registered trademark) -293 Reagent, TransIT (registered trademark) -2020 (hereinafter, manufactured by Mirus), Lipofectamine 2000 Reagent, Lipofectamine 2000CD Reagent (hereinafter, manufactured by Life Technologies), FuGene (registered trademark). ) Transfection Reagent (manufactured by Promega) or the like may be used. Further, when insect cells are used as virus-producing cells, an introduction method using baculovirus can also be used.

Although the rAAV-producing cells have been described above as an example, other non-enveloped virus-producing cells can also be produced by a known method. The virus-producing cells thus produced are cultured. The virus-producing cells can be cultured under known culture conditions. For example, culture at a temperature of 30 to 37 ° C., a humidity of 95% RH, and a CO ₂ concentration of 5 to 10% is exemplified, but the present invention is not limited to such conditions. If the desired proliferation of virus-producing cells and the production of non-enveloped virus can be achieved, the temperature, humidity, and CO ₂ concentration other than the above ranges may be used. The culture period is not particularly limited, and in the case of rAAV-producing cells, it is, for example, 12 to 150 hours, preferably 48 to 120 hours. The medium used for culturing virus-producing cells may contain components necessary for culturing cells, and for example, DMEM, IMDM, DMEM: F-12, RPMI1640, Ham's F-12, EMEM, etc. Basic synthetic medium (sold from Thermo Fisher, etc.), and if necessary, bovine fetal serum, growth factors, peptides are added to these basic synthetic media, and amino acids are increased. Can be mentioned.

<Manufacturing method of non-enveloped virus>

The virus-producing cells cultured as described above are brought into contact with a solution containing divalent cations. The contact is an operation in which the pellets of virus-producing cells collected by removing the culture medium by centrifugation or filtration after culturing are suspended in a solution containing divalent cations, or the culture medium of virus-producing cells (that is, that is, It is carried out by one of the operations of adding "a component capable of supplying divalent cations" to the medium after culturing the virus-producing cells). Therefore, in the present invention, the term "solution containing divalent cations" refers to a solution obtained by mixing a solvent described below with a "component capable of supplying divalent cations" and a solution, unless otherwise specified. It includes both solutions obtained by adding "a component capable of supplying divalent cations" to a culture solution of virus-producing cells. The virus-producing cells at the time of contact with the solution containing divalent cations are in a state where virus production has already been achieved, and are basically in a state of being in contact with the solution containing divalent cations. In some cases, virus production or cell proliferation may not be seen.

The "solution containing divalent cations" in the case of suspending the pellet of virus-producing cells in a solution containing divalent cations is higher than the concentration of divalent cations when the virus-producing cells are cultured. There is no limitation as long as it is a solution containing divalent cations having a concentration and which can obtain a crude extract containing a non-enveloped virus after freeze-thaw.

A solution containing divalent cations is prepared by appropriately mixing a "component capable of supplying divalent cations" and a "solvent".

The "divalent cation" used in the present invention is not particularly limited, and is, for example, magnesium ion, calcium ion, strontium ion, barium ion, cadmium ion, nickel (II) ion, zinc ion, copper (II). Examples thereof include an ion, a mercury (II) ion, an iron (II) ion, a cobalt (II) ion, a tin (II) ion, a lead (II) ion, and a manganese (II) ion. Although not particularly limited, magnesium ions are preferably used in the present invention.

The "component capable of supplying divalent cations" of the present invention is not particularly limited, and when magnesium ions are used, magnesium chloride (MgCl ₂ ) and magnesium sulfate (ו ₄ ) are exemplified. Although not particularly limited, magnesium chloride is preferably used in the present invention.

The "solvent" of the present invention is not particularly limited, and water, buffer solution, phosphate buffered saline (PBS), Tris buffered saline (TBS), and medium are exemplified and can be appropriately selected. Medium means a solution containing nutrients or components that nourish cells. Typically, nutrients include essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements that cells need for minimal growth and / or survival. These solutions include hormones and / or other growth factors, specific ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides, or nucleotides, inorganic compounds, amino acids, lipids, and / Or sugars (such as glucose), or additional nutrients or additional components that enhance growth and / or survival, including other energy sources. The medium is not limited as long as it can be used for culturing virus-producing cells, and media for culturing vertebrate (mammalian) cells, preferably DMEM, IMDM, RPMI1640, Ham's F-12 and DMEM: Basic synthetic media such as F-12 and EMEM are exemplified. Further, the medium may be a medium containing serum, a serum-free medium containing no serum, or a medium having a specified chemical composition. Although not particularly limited, DMEM is particularly preferably used in the present invention.

The concentration of the divalent cation contained in the "solution containing the divalent cation" is not particularly limited, but for example, 5 to 200 mM is exemplified. When the solvent is water, the concentration of the divalent cation is not particularly limited, but is 20 to 100 mM, preferably 30 to 80 mM, and more preferably 40 to 80 mM. On the other hand, when the solvent is a medium such as DMEM, the concentration of the divalent cation is not particularly limited, but is 5 to 100 mM, preferably 5 to 80 mM, and more preferably 10 to 40 mM. Particularly preferably, it is 15 to 20 mM. The concentration of the divalent cation is the final concentration of the divalent cation added to the solvent.

The concentration of divalent cations can be appropriately set according to the content of the nuclease treatment step described later. That is, it is preferable to use a solution containing divalent cations prepared at an appropriate concentration according to the nuclease. In the present invention, a high salt concentration resistant nuclease can be preferably used. For example, the cryobacterial Shewanella sp. The optimum concentration of magnesium ion of the derived endonuclease (Cryonase Cold-active Nuclease (manufactured by Takara Bio Inc.)) is higher than the optimum concentration of other nucleases such as DNaseI, DNaseII, and Benzonase®. .. That is, when treating with a high salt concentration resistance nuclease such as Cryonase, freeze-thaw using a solution containing a relatively high concentration magnesium ion, and then use the high salt concentration resistance nuclease as it is without diluting the solution. Can be processed.

Solutions containing divalent cations may contain other components, such as buffer components, sugars such as glucose and sucrose, and monovalent or plural cations or anions. good. The pH of the solution containing divalent cations is not limited, and examples thereof include pH 5.5 to 8.5, preferably pH 6.0 to 8.0, and more preferably pH 6.5 to 7.5. Will be done.

On the other hand, when performing an operation of adding a "component capable of supplying divalent cations" to the culture solution of virus-producing cells, the amount of the component added is a crude extract containing a non-enveloped virus after freezing and thawing. The amount is not particularly limited as long as it can be used, and examples thereof include an amount at which the concentration of divalent cations in the culture solution of virus-producing cells becomes the concentration in the above-mentioned "solution containing divalent cations".

There are no particular restrictions on the cell concentration and contact time in contact between the virus-producing cells and the solution containing divalent cations.

Freeze and thaw the solution containing the virus-producing cells and divalent cations obtained as described above.

Freezing is carried out using liquid nitrogen, dry ice, ultra-low temperature freezer, etc. The freezing temperature and freezing time are not particularly limited, and examples of the temperature are, for example, −200 to −20 ° C., preferably −90 to −70 ° C. The freezing time may be appropriately set according to the freezing temperature and the method, but may be selected in the range of, for example, 3 minutes to 1 hour. When ethanol dry ice is used, a freezing operation for 5 to 10 minutes is exemplified.

Melting is carried out using an incubator, a water bath, etc. The melting temperature and melting time are not particularly limited, and examples thereof include a temperature of, for example, 30 to 45 ° C., preferably 35 to 40 ° C., and a time of, for example, 3 minutes to 1 hour, preferably 5 to 10 minutes.

The number of times of freezing and thawing is not particularly limited, and examples thereof include 0 times, 1 time, 2 times, 3 times, 4 times, 5 times and more. In addition, when freeze-thawing is carried out multiple times, the length of the interval is not limited, and after the freeze-thaw is completed or while the frozen product is being thawed, it is re-used using an ultra-low temperature freezer at -80 ° C. Can be frozen. In the present invention, one freeze-thaw is sufficient. Performing freeze-thaw once by the method of the present invention is more efficient than performing freeze-thaw three times using a divalent cation-free lysis buffer, and / / cell disruption solution containing non-enveloped virus. Alternatively, a crude extract can be obtained.

By the above freeze-thaw operation, virus-producing cells are crushed and non-enveloped virus is released extracellularly. The method of the present invention may or may not further include cell crushing methods such as ultrasonic crushing, enzyme treatment, and osmotic pressure treatment, which are generally used as conventional methods.

The cell disruption solution obtained through the above steps is used for obtaining a non-enveloped virus. For the cell disruption solution, perform solid-liquid separation, for example, physical separation method such as centrifugation or filter filtration to obtain a supernatant or filtrate to prepare a crude extract separated from cells and their residues. Allows the acquisition of non-enveloped viruses. Preferably, acquisition of the non-enveloped virus is performed by centrifugation. Although not particularly limited, after centrifuging the cell disruption solution at 9000 × g at 4 ° C. for 10 minutes, the supernatant can be collected to obtain a crude extract containing a non-enveloped virus.

The crude extract containing the non-enveloped virus thus obtained can be obtained by carrying out any "measurement method for the non-enveloped virus" described later, whereby the "amount of viral vector" and "capsid protein" in the sample can be obtained. Desirable values such as "amount", "ratio of viral vector particles to empty capsid of virus" can be measured.

According to the method of the present invention, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, and 8 times, as compared with the case of freeze-thawing using a thawing buffer containing no divalent cation. A crude extract containing a non-enveloped virus can be obtained with high efficiency of fold, 9 times, or 10 times or more.

In the method of the present invention, the crude extract is subsequently subjected to nuclease treatment, further subjected to ultracentrifugation, chromatography, ultrafiltration, precipitant treatment, and purification by other known methods to finalize the concentrated or purified non-enveloped virus. It can be obtained as a product. Commercially available reagents and kits can be used for purification. When purifying rAAV, for example, AAVpro (registered trademark) Purification Kit (manufactured by Takara Bio Inc.) can be used without particular limitation.

To explain more specifically the purification of AAV vector using AAVpro (registered trademark) Purification Kit, first, (1) 1/100 amount of Cryonase Cold-active Nuclearase (final concentration 200U) was added to the crude extract containing AAV vector. / Ml) Add and react at 37 ° C. for 1 hour. By this reaction, the contaminated cell genome, free plasmid, etc. are cleaved. Cryonase is a cryobacterial Shewanella sp. The derived endonuclease is expressed and purified in recombinant Escherichia coli, and can cleave any DNA and RNA substrate (single-stranded, double-stranded, linear, cyclic) at low temperature. Next, (2) 1/10 amount of Precipitator A is added to the above solution, mixed with vortex for 10 seconds, reacted at 37 ° C. for 30 minutes, and mixed again with vortex for 10 seconds. Further, 1/20 amount of Precipitator B is added to the solution (3), immediately mixed with vortex for 10 seconds, and centrifuged at 5000 to 9000 × g at 4 ° C. for 5 minutes. (4) The supernatant is filtered using Millex-HV 0.45 μm. (5) Add the filtered AAV vector solution to Amicon Ultra-15,100 kDa and centrifuge at 2000 × g at 15 ° C. for 5 minutes to confirm that the AAV vector solution is 1.5 ml or less. (6) After removing the filtrate, add 5 ml of Suspension Buffer into the cup, homogenize the solution by pipetting, and centrifuge at 2000 × g at 15 ° C. for 5 minutes. Confirm that the AAV vector solution is 1.5 ml or less. (7) The above operation 6 is repeated 4 times (5 times in total), and finally concentrated to an arbitrary volume. (8) After removing the filtrate, suspend it sufficiently with vortex for 30 seconds or by pipetting, and transfer the AAV vector solution in the Amicon Ultra-15,100 kDa cup to a tube.

The non-enveloped virus obtained by the method of the present invention can be stored for a long period of time in a suitable solution. For example, when the rAAV vector is replaced with phosphate buffered saline (pH 7.4), under the condition of -80 ° C, for example, 12 hours or more, 1 day or more, 3 days or more, 1 week or more, 2 weeks. It can be stored for 1 month or more, 2 months or more, 3 months or more, or 6 months or more.

<Measurement method for non-enveloped virus>
The method for measuring the non-enveloped virus is not particularly limited, but for example, (a) the amount of the non-enveloped virus vector in a certain amount of sample, for example, the amount of the virus genome, or (b) the protein constituting the non-enveloped virus. Examples include methods for measuring the amount, eg, the amount of capsid protein.

As the method (a) above, a method of measuring the number of copies of the virus genome in the sample by the PCR method is exemplified. Although not particularly limited, for example, the amount of AAV genome can be calculated by using the AAV qPCR rapid titer measurement kit (manufactured by Takara Bio Inc.) and the method described in the instruction manual.

Examples of the method (b) above include a method of analyzing the protein by SDS-PAGE or a method of quantifying the protein by an immunological method (ELISA method or the like).

By combining the above (a) and (b), the ratio of the virus vector particles contained in the sample to the empty capsid of the virus can be calculated. That is, the viral vector particles contain both the capsid protein and the viral genome, whereas the empty capsid of the virus contains the capsid protein but not the viral genome. Thus, for example, when a sample is measured, a small amount of viral genome per fixed amount of capsid protein indicates that the sample is high in empty capsids.

Another method for determining the ratio of the virus vector particles contained in the sample to the empty capsid of the virus is (c) a method of observing with an electron microscope.

In addition, as a method for measuring whether or not the viral vector particles contained in the sample are functional, that is, whether or not they have the ability to infect target cells, (d) the ability of the viral vector particles to infect cells experimentally. A method of measuring (infectious titer) can be mentioned. More specifically, for example, a method of infecting a suitable target cell such as HeLa cells with a serial diluted solution of a sample containing a virus vector particle to detect a change in cell shape (cell degeneration), and a method of detecting the expression of an introduced gene. Examples thereof include a method and a method for measuring the number of copies of a viral genome introduced into a cell.

<Other inventions>

INDUSTRIAL APPLICABILITY In addition to the method for producing a non-enveloped virus, the present invention also provides a solution containing a divalent cation used in the production method, and a non-enveloped virus produced by the production method. Further, a pharmaceutical composition containing a non-enveloped virus obtained by using the production method of the present invention as an active ingredient is also provided. The pharmaceutical composition can be appropriately prepared according to a technique for producing a viral vector preparation for gene therapy. For example, the non-enveloped virus obtained by further concentrating, purifying, and processing the non-enveloped virus from the crude extract obtained by the production method of the present invention by a known method can be used as a pharmaceutical composition. The pharmaceutical composition can be used in vitro to cells of patient origin or can be administered directly to the patient.

Further, as one aspect of the present invention, a kit for producing a non-enveloped virus is provided. The kit of the present invention contains a solution containing divalent cations or a component capable of supplying divalent cations as an essential component, but also contains a nucleic acid that supplies an essential component for particle formation of a non-enveloped virus. It may contain a vector, a vector containing nucleic acid encapsulated in non-enveloped virus particles, a nuclease, a filtration filter, and the like.

As yet another embodiment, the kit may include a solution containing divalent cations, a virus purification column, and various buffer solutions used in the column purification operation.

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

Example 1 Comparison with freeze-thaw using a lysis buffer containing no MgCl ₂ (1) Seeding of cells for rAAV vector preparation 100 mm culture dish (manufactured by Iwaki) for cell culture 8 sheets, 10% FBS (BioWest) DMEM High-glucose (manufactured by Thermo Fisher) containing 10 ml was put therein, and 293T cells were seeded therein in 1 × 10 ⁶ cells. Then, the cells were cultured in a CO ₂ incubator at 37 ° C. for 3 days.

(2) plasmid transfection The cells obtained in Example 1- (1) were subjected to medium exchange in 10 ml of DMEM High-glucose (manufactured by Thermo Fisher). Then, three plasmids (pRC2-mi342 Vector (manufactured by Takara Bio Inc.), pHelper Vector (manufactured by Takara Bio Inc.), and pAAV-ZsGreen1 Vector (manufactured by Takara Bio Inc.)) were applied to 293T cells by the polyethyleneimine (PEI) method. So, it was transfected. After transfection, 293T cells were cultured in a CO ₂ incubator at 37 ° C. for 3 days.

(3) Recovery of rAAV vector-producing cells 125 μl of 0.5 mM EDTA was added to the 100 mm culture dish obtained in Example 1- (2), and the cells were detached by keeping the cells warm at room temperature for 10 minutes. Then, the cell suspension was collected, centrifuged at 1700 × g at 4 ° C. for 10 minutes, and the supernatant was removed to obtain cell pellets.

(4) Preparation of extraction solution MgCl ₂ was added to DMEM (manufactured by Thermo Fisher) so as to have a concentration of 20 mM to prepare "MgCl ₂ medium".

(5) Acquisition of crude extract of rAAV vector using "MgCl ₂ medium" When performing freeze-thaw once Example 1-The cell pellet obtained in (3) is loosened using a vortex mixer for about 15 seconds, and is an example. 500 μl of PBS was added as the MgCl ₂ medium prepared in 1- (4) or a dissolution buffer containing no MgCl ₂ , and the mixture was mixed using a vortex mixer. Then, the cells were allowed to stand in ethanol dry ice for about 5 to 10 minutes to freeze the cell suspension, and then thawed in a water bath at 37 ° C. for about 5 to 10 minutes. The cell disruption solution after thawing was mixed using a vortex mixer, centrifuged at 9000 × g at 4 ° C. for 10 minutes, and the supernatant was recovered to obtain a crude extract of rAAV vector (performed under each condition N = 2). rice field).
When freeze-thawing 3 times The cell pellet obtained in Example 1- (3) is loosened using a vortex mixer for about 15 seconds, and does not contain MgCl ₂ medium or MgCl ₂ prepared in Example 1- (4). 500 μl of PBS was added as a lysis buffer and mixed using a vortex mixer. Then, the cells were allowed to stand in ethanol dry ice for about 5 to 10 minutes to freeze the cell suspension, and then thawed in a water bath at 37 ° C. for about 5 to 10 minutes. The thawed cell disruption solution was mixed using a vortex mixer. This freeze-thaw cycle was performed 3 times. Then, the cell disruption solution was centrifuged at 9000 × g at 4 ° C. for 10 minutes, and the supernatant was collected to obtain a crude extract of rAAV vector (performed under each condition N = 2).

(6) Measurement of Genome Titer of Crude Extract The genomic titer of the crude extract of the rAAV vector prepared in Example 1- (5) was measured. An AAV qPCR rapid titer measurement kit (manufactured by Takara Bio Inc.) was used for genome titer measurement. According to the instructions, the crude extract was treated with DNase I to remove free genomic DNA and plasmid DNA.

The above reaction solution was kept warm at 37 ° C. for 30 minutes, and then heat-treated at 95 ° C. for 10 minutes to inactivate DNase I.
To 20 μl of this DNase I-treated solution, 20 μl of Lysis Buffer was added, and the mixture was kept warm at 70 ° C. for 10 minutes. After heat insulation, this solution was diluted 100-fold with EASY dilution, and 5 μl of this diluted solution was used for genomic titer measurement. After preparing a 50 × primer mix using the primers (AAV Forward Titter Primer, AAV Reverse Titter Primer) included in the kit, the reaction solution was prepared as follows according to the instructions (per reaction).

As real-time PCR, initial denaturation was performed at 95 ° C. for 2 minutes, and then a 2-step cycle of "95 ° C. for 5 seconds" and "60 ° C. for 30 seconds" was performed for 35 cycles. After that, melting curve analysis was performed. The standard product used the positive control included in the kit. The vector genome (vg) was determined from the obtained values and the amount of crude extract. The results are shown in Table 1 and FIG.

(7)
From Table 1 and FIG. 1, it was shown that the extraction efficiency of the rAAV vector by MgCl2 medium is higher _than that of PBS, and it can be efficiently extracted even by one freeze-thaw.

Example 2 About the effect of increasing AAV extraction efficiency by freezing and thawing using _H2O supplemented with MgCl ₂ (1) Seeding of cells for rAAV vector preparation T225 flask for cell culture (manufactured by Corning) 10% 40 ml of DMEM High-glucose (manufactured by Thermo Fisher) containing FBS (manufactured by BioWest) was added, and 293T cells were seeded in 4 × 10 ⁶ cells. Then, the cells were cultured in a CO ₂ incubator at 37 ° C. for 3 days.

(2) plasmid transfection The cells obtained in Example 2- (1) were subjected to medium exchange in 40 ml of DMEM High-glucose (manufactured by Thermo Fisher). Then, three plasmids (pRC2-mi342 Vector (manufactured by Takara Bio Inc.), pHelper Vector (manufactured by Takara Bio Inc.), and pAAV-ZsGreen1 Vector (manufactured by Takara Bio Inc.)) were applied to 293T cells by the polyethyleneimine (PEI) method. So, it was transfected. After transfection, 293T cells were cultured in a CO ₂ incubator at 37 ° C. for 3 days.

(3) Recovery of rAAV vector-producing cells To the T225 flask obtained in Example 2- (2), 500 μl of 0.5 mM EDTA was added, and the cells were detached by keeping the cells warm at room temperature for 10 minutes. Then, the cell suspension was divided into 26 equal parts and dispensed into tubes (1.5 ml / tube). Each cell suspension was centrifuged at 1700 × g at 4 ° C. for 10 minutes, and the supernatant was removed to obtain cell pellets.

(4) Preparation of Extraction Solution Water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added to 1M MgCl ₂ or 1M NaCl to prepare an "extraction solution" having the following salt concentration.

(5) Acquisition of crude extract of rAAV vector by various "extraction solutions" The cell pellet obtained in Example 2- (3) is loosened for about 15 seconds using a vortex mixer, and in Example 2- (4). 100 μl of the prepared various extraction solutions were added and mixed using a vortex mixer for about 15 seconds (performed under each condition N = 2). Then, the cell suspension was frozen at −80 ° C. for about 5 to 10 minutes using a freezer, and then thawed for about 5 to 10 minutes using an incubator at 37 ° C.
The thawed sample was mixed using a vortex mixer, centrifuged at 9000 × g at 4 ° C. for 10 minutes, and the supernatant was collected to obtain a crude extract of rAAV vector.

(6) Genome titer measurement of "crude extract" The genomic titer of the crude extract of the rAAV vector prepared in Example 2- (5) was measured. An AAV qPCR rapid titer measurement kit (manufactured by Takara Bio Inc.) was used for genome titer measurement. According to the instructions, the crude extract was treated with DNase I to remove free genomic DNA and plasmid DNA.

The above reaction solution was kept warm at 37 ° C. for 30 minutes, and then heat-treated at 95 ° C. for 10 minutes to inactivate DNase I.
To 20 μl of this DNase I-treated solution, 20 μl of Lysis Buffer was added, and the mixture was kept warm at 70 ° C. for 10 minutes. After the heat insulation was completed, this solution was diluted 100-fold with EASY dilution, and 5 μl of this diluted solution was used for genomic titer measurement. After preparing a 50 × primer mix using the primers (AAV Forward Titter Primer, AAV Reverse Titter Primer) included in the kit, the reaction solution was prepared as follows according to the instructions (per reaction).

As real-time PCR, initial denaturation was performed at 95 ° C. for 2 minutes, and then a 2-step cycle of "95 ° C. for 5 seconds" and "60 ° C. for 30 seconds" was performed for 35 cycles. After that, melting curve analysis was performed. The standard product used the positive control included in the kit. The vector genome (vg) was determined from the obtained values and the amount of crude extract. The results are shown in Table 2 and FIG.

(7)
From Table 2 and FIG. 2, it was shown that when the salt concentration of MgCl ₂ was 40 mM or 80 mM, the rAAV vector was extracted with high efficiency as compared with other conditions.

Example 3 Effect of increasing AAV extraction efficiency by freezing and thawing using DMEM supplemented with MgCl ₂ (1) Seeding of cells for rAAV vector preparation T225 flask for cell culture (manufactured by Corning) 10% FBS (1) 40 ml of DMEM High-glucose (manufactured by Thermo Fisher) containing (manufactured by BioWest) was put therein, and 293T cells were seeded therein with 4 × 10 ⁶ cells. Then, the cells were cultured in a CO ₂ incubator at 37 ° C. for 3 days.

(2) plasmid transfection The cells obtained in Example 3- (1) were subjected to medium exchange in 40 ml of DMEM High-glucose (manufactured by Thermo Fisher). Then, three plasmids (pRC2-mi342 Vector (manufactured by Takara Bio Inc.), pHelper Vector (manufactured by Takara Bio Inc.), and pAAV-ZsGreen1 Vector (manufactured by Takara Bio Inc.)) were applied to 293T cells by the polyethyleneimine (PEI) method. So, it was transfected. After transfection, 293T cells were cultured in a CO ₂ incubator at 37 ° C. for 3 days.

(3) Recovery of rAAV vector-producing cells 500 μl of 0.5 mM EDTA was added to the T225 flask obtained in Example 3- (2), and the cells were detached by keeping the cells warm at room temperature for 10 minutes. Then, the cell suspension was divided into 26 equal parts and dispensed into tubes (1.5 ml / tube). Each cell suspension was centrifuged at 1700 × g at 4 ° C. for 10 minutes, and the supernatant was removed to obtain cell pellets.

(4) Preparation of extraction solution DMEM (manufactured by Thermo Fisher) was added to 1M MgCl ₂ or 1M NaCl to prepare an "extraction solution" having the following salt concentration.

(5) Acquisition of crude extract of rAAV vector by various "extraction solutions" The cell pellet obtained in Example 3- (3) is loosened using a vortex mixer for about 15 seconds, and in Example 3- (4). 100 μl of the prepared various extraction solutions were added and mixed using a vortex mixer for about 15 seconds (performed under each condition N = 2). Then, the cell suspension was frozen at −80 ° C. for about 5 to 10 minutes using a freezer, and then thawed for about 5 to 10 minutes using an incubator at 37 ° C.
The thawed sample was mixed using a vortex mixer, centrifuged at 9000 × g at 4 ° C. for 10 minutes, and the supernatant was collected to obtain a crude extract of rAAV vector.

(6) Genome titer measurement of "crude extract" The genomic titer of the crude extract of the rAAV vector prepared in Example 3- (5) was measured. An AAV qPCR rapid titer measurement kit (manufactured by Takara Bio Inc.) was used for genome titer measurement. According to the instructions, the crude extract was treated with DNase I to remove free genomic DNA and plasmid DNA.

The above reaction solution was kept warm at 37 ° C. for 30 minutes, and then heat-treated at 95 ° C. for 10 minutes to inactivate DNase I.
To 20 μl of this DNase I-treated solution, 20 μl of Lysis Buffer was added, and the mixture was kept warm at 70 ° C. for 10 minutes. After the heat insulation was completed, this solution was diluted 100-fold with EASY dilution, and 5 μl of this diluted solution was used for genomic titer measurement. After preparing a 50 × primer mix using the primers (AAV Forward Titter Primer, AAV Reverse Titter Primer) included in the kit, the reaction solution was prepared as follows according to the instructions (per reaction).

As real-time PCR, initial denaturation was performed at 95 ° C. for 2 minutes, and then a 2-step cycle of "95 ° C. for 5 seconds" and "60 ° C. for 30 seconds" was performed for 35 cycles. After that, melting curve analysis was performed. The standard product used the positive control included in the kit. The vector genome (vg) was determined from the obtained values and the amount of crude extract. The results are shown in Table 3 and FIG.

(7)
From Table 3 and FIG. 3, it was shown that under the condition of low ionic strength (15 to 60 mM), the condition to which MgCl ₂ was added was more efficient than the condition to which NaCl was added.

Example 4 AAV extraction efficiency using divalent cations other than magnesium ion (1) Seeding of cells for rAAV vector preparation 10% FBS (BioWest) per well in 6-well plate (manufactured by Corning) for cell culture 2 ml of DMEM High-glucose (manufactured by Thermo Fisher) containing (manufactured by The Company) was put therein, and 293T cells were seeded therein with 1.5 × 10 ⁵ cells. Then, the cells were cultured in a CO ₂ incubator at 37 ° C. for 3 days.

(2) plasmid transfection The cells obtained in Example 4- (1) were subjected to medium exchange in 2 ml of DMEM High-glucose (manufactured by Thermo Fisher). Then, three plasmids (pRC2-mi342 Vector (manufactured by Takara Bio Inc.), pHelper Vector (manufactured by Takara Bio Inc.), and pAAV-ZsGreen1 Vector (manufactured by Takara Bio Inc.)) were applied to 293T cells by the polyethyleneimine (PEI) method. So, it was transfected. After transfection, 293T cells were cultured in a CO ₂ incubator at 37 ° C. for 3 days.

(3) Recovery of rAAV vector-producing cells The cells obtained in Example 4- (2) were stripped by pipetting. Then, the cell suspension was collected, centrifuged at 1700 × g at 4 ° C. for 10 minutes, and the supernatant was removed to obtain cell pellets.

(4) Preparation of extraction solution Add MgCl ₂ , MnCl ₂ , or CaCl ₂ to DMEM (manufactured by Thermo Fisher) so as to be 20 mM, and add "MgCl ₂ medium", "MnCl ₂ medium", and "CaCl ₂ medium". Was produced. Moreover, DMEM to which nothing was added was defined as "medium (-)".

(5) Acquisition of crude extract of rAAV vector by various "extraction solutions" The cell pellet obtained in Example 4- (3) was loosened for about 15 seconds using a vortex mixer, and in Example 4- (4). 100 μl of the prepared various extraction solutions were added and mixed using a vortex mixer for about 15 seconds (performed under each condition N = 2). Then, the cell suspension was frozen at −80 ° C. for about 5 to 10 minutes using a freezer, and then thawed for about 5 to 10 minutes using an incubator at 37 ° C.
The thawed sample was mixed using a vortex mixer, centrifuged at 9000 × g at 4 ° C. for 10 minutes, and the supernatant was collected to obtain a crude extract of rAAV vector.

(6) Genome titer measurement of "crude extract" The genomic titer of the crude extract of the rAAV vector prepared in Example 4- (5) was measured. An AAV qPCR rapid titer measurement kit (manufactured by Takara Bio Inc.) was used for genome titer measurement. According to the instructions, the crude extract was treated with DNase I to remove free genomic DNA and plasmid DNA.

The above reaction solution was kept warm at 37 ° C. for 30 minutes, and then heat-treated at 95 ° C. for 10 minutes to inactivate DNase I.
To 20 μl of this DNase I-treated solution, 20 μl of Lysis Buffer was added, and the mixture was kept warm at 70 ° C. for 10 minutes. After the heat insulation was completed, this solution was diluted 100-fold with EASY dilution, and 5 μl of this diluted solution was used for genomic titer measurement. After preparing a 50 × primer mix using the primers (AAV Forward Titter Primer, AAV Reverse Titter Primer) included in the kit, the reaction solution was prepared as follows according to the instructions (per reaction).

As real-time PCR, initial denaturation was performed at 95 ° C. for 2 minutes, and then a 2-step cycle of "95 ° C. for 5 seconds" and "60 ° C. for 30 seconds" was performed for 35 cycles. After that, melting curve analysis was performed. The standard product used the positive control included in the kit. The vector genome (vg) was determined from the obtained values and the amount of crude extract. The results are shown in Table 4 and FIG.

(7)
From Table 4 and FIG. 4, it was shown that the rAAV vector can be efficiently extracted even when a divalent cation other than magnesium ion is used.

Example 5 AAV extraction efficiency using a solvent other than DMEM (1) Dissemination of cells for rAAV vector preparation Cell culture 6-well plate (manufactured by Corning) contains 10% FBS (manufactured by BioWest) per well. 2 ml of DMEM High-glucose (manufactured by Thermo Fisher) was put therein, and 293T cells were seeded in 1.5 × 10 ⁵ cells. Then, the cells were cultured in a CO ₂ incubator at 37 ° C. for 3 days.

(2) plasmid transfection The cells obtained in Example 5- (1) were subjected to medium exchange in 2 ml of DMEM High-glucose (manufactured by Thermo Fisher). Then, three plasmids (pRC2-mi342 Vector (manufactured by Takara Bio Inc.), pHelper Vector (manufactured by Takara Bio Inc.), and pAAV-ZsGreen1 Vector (manufactured by Takara Bio Inc.)) were applied to 293T cells by the polyethyleneimine (PEI) method. So, it was transfected. After transfection, 293T cells were cultured in a CO ₂ incubator at 37 ° C. for 3 days.

(3) Recovery of rAAV vector-producing cells The cells obtained in Example 5- (2) were stripped by pipetting. Then, the cell suspension was collected, centrifuged at 1700 × g at 4 ° C. for 10 minutes, and the supernatant was removed to obtain cell pellets.

(4) Preparation of extraction solution DMEM (manufactured by Thermo Fisher), RPMI-1640 (manufactured by Thermo Fisher), DMEM / F-12 (manufactured by Thermo Fisher), EMEM (manufactured by Fujifilm Wako Junyaku Co., Ltd.), IMDM MgCl ₂ was added to TBS (manufactured by Nippon Gene) or PBS (manufactured by PromoCell) to 20 mM to prepare various extraction solutions.

(5) Acquisition of crude extract of rAAV vector by various "extraction solutions" The cell pellet obtained in Example 5- (3) was loosened for about 15 seconds using a vortex mixer, and in Example 5- (4). 100 μl of the prepared various extraction solutions were added and mixed using a vortex mixer for about 15 seconds (performed under each condition N = 2). Then, the cell suspension was frozen at −80 ° C. for about 5 to 10 minutes using a freezer, and then thawed for about 5 to 10 minutes using an incubator at 37 ° C.
The thawed sample was mixed using a vortex mixer, centrifuged at 9000 × g at 4 ° C. for 10 minutes, and the supernatant was collected to obtain a crude extract of rAAV vector.

(6) Genome titer measurement of "crude extract" The genomic titer of the crude extract of the rAAV vector prepared in Example 5- (5) was measured. An AAV qPCR rapid titer measurement kit (manufactured by Takara Bio Inc.) was used for genome titer measurement. According to the instructions, the crude extract was treated with DNase I to remove free genomic DNA and plasmid DNA.

The above reaction solution was kept warm at 37 ° C. for 30 minutes, and then heat-treated at 95 ° C. for 10 minutes to inactivate DNase I.
To 20 μl of this DNase I-treated solution, 20 μl of Lysis Buffer was added, and the mixture was kept warm at 70 ° C. for 10 minutes. After the heat insulation was completed, this solution was diluted 100-fold with EASY dilution, and 5 μl of this diluted solution was used for genomic titer measurement. After preparing a 50 × primer mix using the primers (AAV Forward Titter Primer, AAV Reverse Titter Primer) included in the kit, the reaction solution was prepared as follows according to the instructions (per reaction).

As real-time PCR, initial denaturation was performed at 95 ° C. for 2 minutes, and then a 2-step cycle of "95 ° C. for 5 seconds" and "60 ° C. for 30 seconds" was performed for 35 cycles. After that, melting curve analysis was performed. The standard product used the positive control included in the kit. The vector genome (vg) was determined from the obtained values and the amount of crude extract. The results are shown in Table 5 and FIG.

(7)
From Table 5 and FIG. 5, it was shown that the rAAV vector can be efficiently extracted even in a solvent other than DMEM when magnesium ion is added.

Example 6 About the effect of increasing AAV extraction efficiency by freezing and thawing using _H2O to which MgCl ₂ is added (1) Seeding of cells for rAAV vector production 6 well plate (manufactured by Corning) for cell culture per well 2 ml of DMEM High-glucose (manufactured by Thermo Fisher) containing 10% FBS (manufactured by BioWest) was put therein, and 293T cells were seeded therein with 1.5 × 10 ⁵ cells. Then, the cells were cultured in a CO ₂ incubator at 37 ° C. for 3 days.

(2) plasmid transfection The cells obtained in Example 6- (1) were subjected to medium exchange in 2 ml of DMEM High-glucose (manufactured by Thermo Fisher). Then, three plasmids (pRC2-mi342 Vector (manufactured by Takara Bio Inc.), pHelper Vector (manufactured by Takara Bio Inc.), and pAAV-ZsGreen1 Vector (manufactured by Takara Bio Inc.)) were applied to 293T cells by the polyethyleneimine (PEI) method. So, it was transfected. After transfection, 293T cells were cultured in a CO ₂ incubator at 37 ° C. for 3 days.

(3) Recovery of rAAV vector-producing cells The cells obtained in Example 6- (2) were stripped by pipetting. Then, the cell suspension was collected, centrifuged at 1700 × g at 4 ° C. for 10 minutes, and the supernatant was removed to obtain cell pellets.

(4) Preparation of Extraction Solution Water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added to 1M MgCl ₂ to prepare an "extraction solution" having the following salt concentration.

(5) Acquisition of crude extract of rAAV vector by various "extraction solutions" The cell pellet obtained in Example 6- (3) was loosened for about 15 seconds using a vortex mixer, and in Example 6- (4). 100 μl of the prepared various extraction solutions were added and mixed using a vortex mixer for about 15 seconds (performed under each condition N = 2). Then, the cell suspension was frozen at −80 ° C. for about 5 to 10 minutes using a freezer, and then thawed for about 5 to 10 minutes using an incubator at 37 ° C.
The thawed sample was mixed using a vortex mixer, centrifuged at 9000 × g at 4 ° C. for 10 minutes, and the supernatant was collected to obtain a crude extract of rAAV vector.

(6) Genome titer measurement of "crude extract" The genomic titer of the crude extract of the rAAV vector prepared in Example 6- (5) was measured. An AAV qPCR rapid titer measurement kit (manufactured by Takara Bio Inc.) was used for genome titer measurement. According to the instructions, the crude extract was treated with DNase I to remove free genomic DNA and plasmid DNA.

The above reaction solution was kept warm at 37 ° C. for 30 minutes, and then heat-treated at 95 ° C. for 10 minutes to inactivate DNase I.
To 20 μl of this DNase I-treated solution, 20 μl of Lysis Buffer was added, and the mixture was kept warm at 70 ° C. for 10 minutes. After the heat insulation was completed, this solution was diluted 100-fold with EASY dilution, and 5 μl of this diluted solution was used for genomic titer measurement. After preparing a 50 × primer mix using the primers (AAV Forward Titter Primer, AAV Reverse Titter Primer) included in the kit, the reaction solution was prepared as follows according to the instructions (per reaction).

As real-time PCR, initial denaturation was performed at 95 ° C. for 2 minutes, and then a 2-step cycle of "95 ° C. for 5 seconds" and "60 ° C. for 30 seconds" was performed for 35 cycles. After that, melting curve analysis was performed. The standard product used the positive control included in the kit. The vector genome (vg) was determined from the obtained values and the amount of crude extract. The results are shown in Table 6 and FIG.

(7)
From Table 6 and FIG. 6, it was shown that when MgCl ₂ was added to H ₂ O, the rAAV vector was extracted with high efficiency at a salt concentration of 30 mM or more.

Example 7 Extraction efficiency of AAV of serotypes other than AAV2 (1) DMEM High containing 10% FBS (manufactured by BioWest) in one T225 flask (manufactured by Corning) for seeding cells for producing rAAV vector. -Glucose (manufactured by Thermo Fisher) 40 ml was put therein, and 293T cells were seeded in 4 × 10 ⁶ cells. Then, the cells were cultured in a CO ₂ incubator at 37 ° C. for 3 days.

(2) plasmid transfection The cells obtained in Example 7- (1) were subjected to medium exchange in 40 ml of DMEM High-glucose (manufactured by Thermo Fisher). Then, in 293T cells, AAV1 is pRC1 Vector (manufactured by Takara Bio), pHelper Vector (manufactured by Takara Bio), and pAAV-ZsGreen1 Vector (manufactured by Takara Bio), AAV2 is pRC2-mi342 Vector (manufactured by Takara Bio). , PHelper Vector (manufactured by Takara Bio), and pAAV-ZsGreen1 Vector (manufactured by Takara Bio), AAV5 is pRC5 Vector (manufactured by Takara Bio), pHelper Vector (manufactured by Takara Bio), and pAAV-ZsGreen1 Vector (manufactured by Takara Bio). , AAV6 was transfected with pRC6 Vector (manufactured by Takara Bio) and pHelper Vector (manufactured by Takara Bio and pAAV-ZsGreen1 Vector (manufactured by Takara Bio)) by the polyethyleneimin (PEI) method. After transfection, 293T cells were cultured in a CO ₂ incubator at 37 ° C. for 3 days.

(3) Recovery of rAAV vector-producing cells 500 μl of 0.5 mM EDTA was added to the T225 flask obtained in Example 7- (2), and the cells were detached by keeping the cells warm at room temperature for 10 minutes. Then, the cell suspension was collected, centrifuged at 1700 × g at 4 ° C. for 10 minutes, and the supernatant was removed to obtain cell pellets.

(4) Preparation of extraction solution MgCl ₂ was added to DMEM (manufactured by Thermo Fisher) so as to be 20 mM to prepare "MgCl ₂ medium".

(5) Acquisition of crude extract of rAAV vector using extraction solution The cell pellet obtained in Example 7- (3) was loosened using a vortex mixer for about 15 seconds, and MgCl produced in Example 7- (4) was used. ₂ ml of PBS (manufactured by PromoCell) was added as a solvent containing no medium or MgCl ₂ , and the mixture was mixed using a vortex mixer for about 15 seconds (performed under each condition N = 2). Then, the cells were allowed to stand in ethanol dry ice for about 5 to 10 minutes to freeze the cell suspension, and then thawed for about 5 to 10 minutes using a water bath at 37 ° C.
The thawed sample was mixed using a vortex mixer, centrifuged at 9000 × g at 4 ° C. for 10 minutes, and the supernatant was collected to obtain a crude extract of rAAV vector.

(6) Genome titer measurement of "crude extract" The genomic titer of the crude extract of the rAAV vector prepared in Example 7- (5) was measured. An AAV qPCR rapid titer measurement kit (manufactured by Takara Bio Inc.) was used for genome titer measurement. According to the instructions, the crude extract was treated with DNase I to remove free genomic DNA and plasmid DNA.

The above reaction solution was kept warm at 37 ° C. for 30 minutes, and then heat-treated at 95 ° C. for 10 minutes to inactivate DNase I.
To 20 μl of this DNase I-treated solution, 20 μl of Lysis Buffer was added, and the mixture was kept warm at 70 ° C. for 10 minutes. After the heat insulation was completed, this solution was diluted 100-fold with EASY dilution, and 5 μl of this diluted solution was used for genomic titer measurement. After preparing a 50 × primer mix using the primers (AAV Forward Titter Primer, AAV Reverse Titter Primer) included in the kit, the reaction solution was prepared as follows according to the instructions (per reaction).

As real-time PCR, initial denaturation was performed at 95 ° C. for 2 minutes, and then a 2-step cycle of "95 ° C. for 5 seconds" and "60 ° C. for 30 seconds" was performed for 35 cycles. After that, melting curve analysis was performed. The standard product used the positive control included in the kit. The vector genome (vg) was determined from the obtained values and the amount of crude extract. The results are shown in Table 7 and FIG.

(7)
From Table 7 and FIG. 7, it was shown that extraction with MgCl ₂ medium can be efficiently extracted even with serotypes of AAV other than AAV2.

According to the method for producing a non-enveloped virus of the present invention, a non-enveloped virus solution can be efficiently obtained without complicated operations. The non-enveloped virus produced by the method of the present invention or a composition containing the non-enveloped virus as an active ingredient is very useful as a gene transfer method in the field of basic research or clinical application of gene therapy.

Claims (6)

1. A method for producing adeno-associated virus
   (A) a step of freezing and thawing a medium containing cells capable of producing an adeno-associated virus and a divalent cation of 5 mM or more, and (b) a step of obtaining an adeno-associated virus from the medium of (a).
   Including the method.
2. The method according to claim 1, wherein the divalent cation is a magnesium ion.
3. The method according to claim 1 or 2, wherein the medium is a medium containing amino acids, sugars, and lipids.
4. The method according to claim 3, wherein the medium is a medium selected from the group consisting of DMEM, IMDM, RPMI1640, Ham's F-12, EMEM and a mixture thereof.
5. The method according to any one of claims 1 to 4, wherein the step (b) is carried out by solid-liquid separation.
6. The method according to claim 5, wherein the solid-liquid separation is carried out by centrifugation.

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