Virus generation method

Abstract

The present invention addresses the problem of providing a virus production method with which it is possible to increase the production of viruses. According to the present invention, provided is a virus production method comprising: a nucleic acid introduction step for introducing a nucleic acid into a cell by electroporation to obtain a cell into which the nucleic acid has been introduced; and a culturing step for culturing the cells into which the nucleic acid has been introduced, the CNET product defined in the specification being 1 * 104 to 1 * 107 (inclusive) in the electroporation.

Description

Technical Field

[0001] This invention relates to a method for generating viruses by introducing nucleic acids into cells via electroporation. Background Technology

[0002] Gene therapy has been developed to treat intractable diseases such as genetic disorders or cancer, involving the administration of recombinant adeno-associated virus (AAV) vectors incorporating therapeutic genes. One method for generating AAV vectors involves neutralizing the charge of the plasmid DNA by forming a complex with a cationic polymer, polyethyleneimine (PEI). The plasmid DNA encoding the protein required for AAV production is then introduced into cells via endocytosis, where AAV is produced.

[0003] Furthermore, physical introduction methods are known as a way to introduce genes into cells without using viral vectors or chemical reagents (e.g., polyethyleneimine, lipid nanoparticles, etc.). Among these physical introduction methods is electroporation, which utilizes the principles of cell perforation and electrophoresis based on the application of an electric field. In electroporation, gene introduction into cells is completed instantaneously upon the application of the pulse (electrophoresis), thus eliminating the need for prolonged control of plasmid and cell agitation. Moreover, methods for large-scale processing via flow-through have also been investigated for electroporation.

[0004] Patent Document 1 describes the production of AAV by introducing recombinant AAV and REP mRNA into eukaryotic cells via electroporation. However, Patent Document 1 does not specify the exact conditions for electroporation and uses a viral vector to introduce the helper gene through viral infection for the purpose of introducing the helper function.

[0005] Existing technical documents

[0006] Patent documents

[0007] Patent Document 1: Japanese Patent Publication No. 2020-511153 Summary of the Invention

[0008] The technical problem to be solved by the invention

[0009] Because previous treatments were localized diseases, the need for large-scale AAV production was low. However, with the anticipated increase in demand for treating systemic diseases, improving AAV production efficiency has become a key challenge.

[0010] The problem to be solved by the present invention is to provide a method for generating viruses that can increase the production of viruses.

[0011] means for solving technical problems

[0012] The inventors, through in-depth research to solve the aforementioned problems, discovered that when obtaining cells with introduced nucleic acids by introducing nucleic acids into cells via electroporation, the CNET product defined in this specification in the electroporation process can be set to 1×10⁻⁶. 4 Above and 1×10 7 The following methods can increase the production of viruses. This invention is based on the above insights.

[0013] According to the present invention, the following invention is provided.

[0014] <1> A method for producing a virus, comprising:

[0015] The nucleic acid introduction process involves introducing nucleic acids into cells via electroporation to obtain cells infused with nucleic acids; and

[0016] The culturing process involves culturing cells infused with the aforementioned nucleic acids.

[0017] In the above methods of virus generation,

[0018] The CNET product in the aforementioned electroporation, expressed by the following formula, is 1 × 10⁻⁶. 4 Above and 1×10 7 the following.

[0019] [Formula 1]

[0020]

[0021] or

[0022] [Formula 2]

[0023]

[0024] In the formula, C represents the nucleic acid concentration in μg / mL.

[0025] N k N represents the number of pulses. k Represents integers greater than or equal to 1.

[0026] E k E represents the pulsed electric field in V / cm. k The voltage is above 500V / cm and below 2000V / cm.

[0027] T k Indicates pulse duration in milliseconds (ms).

[0028] N l N represents the number of pulses. l Represents integers greater than or equal to 1.

[0029] E l E represents the pulsed electric field in V / cm. lIt is above 50V / cm and less than 500V / cm.

[0030] T l Indicates pulse duration in milliseconds (ms).

[0031] k represents an integer from 1 to n.

[0032] l represents an integer from 1 to m.

[0033] n represents an integer greater than or equal to 1.

[0034] m represents an integer greater than or equal to 1.

[0035] <2> According to the virus generation method described in <1>, the introduction of nucleic acid is carried out only by electroporation.

[0036] <3> According to the virus generation method described in <1> or <2>, wherein the CNET product is 1×102. 5 Above and 1×10 6 the following.

[0037] <4> The virus generation method according to any one of <1> to <3>, wherein,

[0038] The above CNET product is represented by the following formula:

[0039] [Formula 3]

[0040]

[0041] And satisfy the following formula,

[0042] [Formula 4]

[0043]

[0044] <5> The method for generating a virus according to any one of <1> to <4>, wherein the nucleic acid concentration C is 10 to 500 μg / mL.

[0045] <6> The virus production method according to any one of <1> to <5>, wherein the ratio of the amount of capsid containing the complete gene to the total amount of capsid in the virus produced in the culture process is 30% or more.

[0046] <7> The virus production method according to any one of <1> to <6>, wherein the nucleic acid is introduced into the cell in the absence of an introduction reagent.

[0047] <8> The method for generating a virus according to any one of <1> to <7>, wherein, in the nucleic acid introduction step, the cells are in a suspension, the conductivity of which is 5 to 20 mS / cm.

[0048] <9> The method for generating a virus according to any one of <1> to <8>, wherein, in the nucleic acid introduction step and the culture step, the cells are in a suspension, wherein the suspension is a culture medium.

[0049] <10> The virus generation method according to any one of <1> to <9>, wherein no additional nucleic acid is introduced after the above-mentioned nucleic acid introduction step.

[0050] <11> The method for generating a virus according to any one of <1> to <10>, wherein the virus is an adeno-associated virus.

[0051] <12> The method for generating a virus according to any one of <1> to <11>, wherein the nucleic acid comprises one or more of an adeno-associated virus gene and a viral helper gene.

[0052] <13> The method for generating a virus according to any one of <1> to <12>, wherein the nucleic acid contains at least one or more viral auxiliary genes.

[0053] <14> A method for generating a virus according to any one of <1> to <13>, wherein the nucleic acid comprises four or more genes from the group consisting of a therapeutic or preventive gene, a Rep gene, a Cap gene, an E2 gene, an E4 gene, and a VA-RNA1 gene.

[0054] <15> A method for generating a virus according to any one of <1> to <14>, wherein nucleic acid is introduced into a cell using multiple plasmids.

[0055] <16> The method for generating a virus according to any one of <1> to <15>, wherein the electroporation is flow electroporation.

[0056] <17> The method for generating a virus according to any one of <1> to <16>, wherein the cell concentration during nucleic acid introduction is 10 × 102. 6 cells / mL ~200×10 6 cells / mL.

[0057] <18> The method for generating a virus according to any one of <1> to <17>, wherein liquid is aseptically transported in the nucleic acid introduction step and the culture step.

[0058] <19> The method for producing a virus according to any one of <1> to <18>, wherein the cell is an animal cell.

[0059] <20> The method for producing a virus according to any one of <1> to <19>, wherein the cell is an HEK cell.

[0060] <21> The method for generating a virus according to any one of <1> to <20> includes the step of recovering the generated virus.

[0061] Invention Effects

[0062] The virus production method according to the present invention can increase the amount of virus produced. Attached Figure Description

[0063] Figure 1 A top view of the electroporation device.

[0064] Figure 2 A cross-sectional view of an electroporation device.

[0065] Figure 3 Examples of pulse waveforms representing electroporation.

[0066] Figure 4 Examples of pulse waveforms representing electroporation.

[0067] Figure 5 Examples of pulse waveforms representing electroporation.

[0068] Figure 6 Examples of pulse waveforms representing electroporation.

[0069] Figure 7 A schematic diagram illustrating perfusion culture.

[0070] Figure 8 A schematic diagram showing pAAV-GFP, pAAV-RC5, and pHelper.

[0071] Figure 9 This is a schematic diagram of a continuous production unit for AAVs that connects a perfusion culture device and a flow electroporation device. Detailed Implementation

[0072] Hereinafter, an example of an embodiment of the present invention will be described. However, the present invention is not limited by the following embodiment, and appropriate modifications can be made to implement it within the scope of the object of the present invention. In this specification, the numerical range indicated by "~" represents the range in which the values ​​before and after "~" are respectively included as the minimum and maximum values.

[0073] This invention relates to a method for generating a virus, comprising: a nucleic acid introduction step, wherein nucleic acid is introduced into cells via electroporation to obtain cells infused with nucleic acid; and a culture step, wherein the cells infused with the nucleic acid are cultured. In the above method for generating a virus, the CNET product in the electroporation, expressed by the following formula, is 1 × 10⁻⁶. 4 Above and 1×10 7 the following.

[0074] [Formula 5]

[0075]

[0076] or

[0077] [Formula 6]

[0078]

[0079] In the formula, C represents the nucleic acid concentration in μg / mL.

[0080] N k N represents the number of pulses. k Represents integers greater than or equal to 1.

[0081] E k E represents the pulsed electric field in V / cm. k The voltage is above 500V / cm and below 2000V / cm.

[0082] T k Indicates pulse duration in milliseconds (ms).

[0083] N l N represents the number of pulses. l Represents integers greater than or equal to 1.

[0084] E l E represents the pulsed electric field in V / cm. l It is above 50V / cm and less than 500V / cm.

[0085] T l Indicates pulse duration in milliseconds (ms).

[0086] k represents an integer from 1 to n.

[0087] l represents an integer from 1 to m.

[0088] n represents an integer greater than or equal to 1.

[0089] m represents an integer greater than or equal to 1.

[0090] When applied to high-dose applications such as systemic diseases, improving the complete capsid rate is important from the perspective of reducing costs and patient side effects by increasing the production rate of gene therapy drugs. Therefore, improving the titer and the complete capsid rate in the early stages before purification have become research topics.

[0091] In methods using polyethyleneimine, the control of the plasmid / polyethyleneimine complex (due to the decreasing efficiency of delivery over time due to aggregation) and the agitation control of cells and the plasmid / polyethyleneimine complex within the reactor are complex, making it difficult to scale up to high cell concentrations. Furthermore, there are issues with the cytotoxicity of polyethyleneimine itself. In electroporation-based gene delivery, it is necessary to control the amount of nucleic acid delivered into cells and reduce cell damage; therefore, there are no examples of electroporation being practically applied in the production of therapeutic viruses.

[0092] (1) Pre-culture of cells

[0093] There are no particular limitations on the cell type, but animal cells are preferred, more preferably mammalian or insect cells, and even more preferably mammalian cells. Examples of mammalian cells include human cells, mouse cells, rat cells, monkey cells, and hamster cells, but there are no particular limitations. Human cells are preferred. Examples of cells include mouse myeloma (NSO) cell lines, Chinese hamster ovary (CHO) cell lines, HT1080, H9, HepG2, MCF7, MDBKJurkat, NIH3T3, PC12, BHK (juvenile hamster kidney cells), VERO, SP2 / 0, YB2 / 0, Y0, C127, L cells, COS (e.g., COS1 and COS7), QC1-3, HEK293 (renal kidney cells derived from human fetuses), VERO, PER.C6, HeLa, EB1, EB2, EB3, oncolytic or hybridoma cell lines. HEK293 cells and CHO cells are preferred, with HEK293 cells being more preferred. HEK293 cells are cells that integrate the E1A and E2B genes derived from adenovirus. The cells can be either adhesion-type or suspension-type cells, but suspension-type cells are preferred for high-concentration / large-scale culture in the reactor.

[0094] As a cell line with high cell proliferation and high AAV production capacity per cell, the Thermofisher suspension cell line Viral Production Cells 2.0 (VPC s2.0) can be used, for example.

[0095] Furthermore, the genetic code set required for viral production can be pre-integrated into the cell's genome (encapsulating the cell). Examples of the genetic code set required for AAV production, as described later, include at least one selected from the target gene GoI (e.g., a therapeutic or preventative gene), REP, CAP, E2, E4, and VA-RNA.

[0096] The culture medium used in this process can be any medium commonly used for culturing animal cells. Various companies sell PBS-based media best suited for culturing cells such as HEK293. For example, Balan-CD HEK293 medium and Expi293 medium manufactured by FISI (FUJIFILM Irvine Scientific) can be used. TM Expression Medium (manufactured by ThermoFisher Scientific), Gibco ViralVector HEK Media Panel (manufactured by ThermoFisher Scientific), etc. Other media that can be used include CDOptiCHO (manufactured by ThermoFisher), Dulbecco modified Eagle medium (DMEM), Eagle minimum required medium (MEM), RPMI-1640 medium, RPMI-1641 medium, F-12K medium, Ham F12 medium, Isco b modified Dulbecco medium (IMDM), McCoy5A medium, Leibovitz L-15 medium, and EX-CELL (trademark) 300 series (JRH Biosciences), CHO-S-SFMII (Invitrogen), CHO-SF (Sigma-Aldrich), CD-CHO (Invitrogen), IS CHO-V (Irvine Scientific), PF-ACF-CHO (Sigma-Aldrich), etc. Alternatively, homemade media can be used. Additional components may be added to the culture medium as needed. Examples of such additional components include amino acids, salts, sugars (such as glucose), vitamins, hormones, growth factors, lipids, and trace elements, but there are no particular limitations. The pH of the culture medium is 6–8, preferably 6.8–7.6, and more preferably 7.2–7.6.

[0097] An antifoaming agent may also be added to the culture medium. Silicone-based antifoaming agents are preferred, especially polydimethylsiloxane. Antifoaming agents containing polydimethylsiloxane are preferred, and those containing micronized silica powder (polydimethylsiloxane) are more preferred. The rate at which silicone oil is added relative to the volume of the culture medium is not particularly limited, but is preferably 5 mg / hr / L or less, more preferably 2 mg / hr / L or less.

[0098] A block copolymer of polyoxypropylene and polyoxyethylene can be added to the culture medium. Poloxamer polymer is preferred as the copolymer of polyoxypropylene and polyoxyethylene, and poloxamer polymer-188 is more preferred. The content of the polyoxypropylene and polyoxyethylene block copolymer in the culture medium is not particularly limited, but is preferably 0.1% by mass or more and 2% by mass or less, more preferably 0.25% by mass or more and 2% by mass or less, and even more preferably 0.5% by mass or more and 2% by mass or less.

[0099] There are no particular limitations on the pre-culture method for cells; it can be any of the following: batch culture, fed-batch culture, perfusion culture, shaking culture, stirred culture, static culture, or adhesion culture. From the viewpoint of large-scale processing and high concentration, perfusion culture is preferred. In perfusion culture, by removing waste components based on filters and supplying fresh culture medium, the quality (component concentration) of the suspension is maintained at conditions suitable for cell proliferation, thereby enabling proliferation at high concentrations.

[0100] The preferred temperature for pre-culturing cells is 30–40°C, more preferably 32–37°C, and especially preferably 36–37°C. The CO2 concentration is 5–10%.

[0101] The optimal cell density for batch culture is 1×10⁻⁶. 6 ~20×10 6 The optimal cell / mL ratio for fed-batch culture is 1×10⁻⁶. 6 ~50×10 6 The optimal cell / mL ratio for perfusion culture (dynamic / continuous) is 1×10⁻⁶. 6 ~200×10 6 cells / mL.

[0102] Preferably, the pre-cultured cell density is 10 × 10⁻⁶. 6 cells / mL or higher and 200 × 10⁻⁶ 6 Cultured at a rate of less than 20 × 10⁶ cells / mL. More preferably, at a rate of 20 × 10⁶ cells / mL. 6 cells / mL or higher and 100×10 6 Below cells / mL, more preferably 40 × 10⁶ cells / mL 6 cells / mL or higher and 80 × 10 6 cells / mL.

[0103] The optimal culture time for pre-culture is 12 hours or more.

[0104] In perfusion culture, either a dynamic or continuous approach can be used. In a dynamic approach, cells are seeded at a low concentration, proliferated, and then the total volume of cell slurry is recovered after reaching a high concentration for gene transfer. In a continuous approach, cells are seeded at a low concentration, proliferated, and then a certain amount of cells is extracted (cell bleeding) after reaching a certain concentration for gene transfer. The same amount of fresh culture medium as the extracted amount is replenished to the reactor side to maintain a constant cell concentration within the reactor. Cell bleeding and culture medium supply can be continuous or intermittent (e.g., 10-50% once daily). During the next cell bleeding period, the concentration is restored through cell proliferation. This process can be repeated, for example, from 1 day to 3 months. The perfusion ratio is not particularly limited, but is typically 0.3 vvd to 5.0 vvd, preferably 0.5 vvd to 4.0 vvd. In the perfusion ratio, vvd represents the volume of culture medium aspirated / the volume of culture medium in the culture vessel / day.

[0105] Apparatus and methods for perfusion culture are well known in the art and are described in WO2018 / 159847, WO2019 / 049843, WO2019 / 181234, WO2019 / 239780, WO2020 / 003833, WO2020 / 162125, WO2021 / 187008, WO2022 / 196710 and WO2023 / 054556, and are incorporated herein by reference. Furthermore, perfusion culture, as described later, can also be used in pre-culture. For example, regarding the "membrane separation process for separating cell suspension extracted from a culture tank into cell-containing fluid and osmotic fluid by passing it through a separation membrane," as described later, and the... Figure 9 The instructions indicate that it can be used in pre-culture.

[0106] (2) Adjustment of cell concentration and replacement of culture medium

[0107] The optimal cell concentration for nucleic acid introduction is 10 × 10⁻⁶. 6 cells / mL ~200×10 6 cells / mL, more preferably 20 × 10⁻⁶ 6 cells / mL ~160×10 6 cells / mL, more preferably 40 × 10⁻⁶ 6 cells / mL ~120×10 6 cells / mL. The lower limit is preferably 10 × 10⁻⁶ cells / mL. 6 cells / mL or higher, more preferably 20 × 10⁻⁶ cells / mL 6 cells / mL, more preferably 40 × 10⁻⁶ 6cells / mL, more preferably 80 × 10⁻⁶ 6 cells / mL, more preferably 100 × 10⁻⁶ 6 Cells per mL. Higher cell concentrations result in more effective nucleic acid delivery.

[0108] In the polyethyleneimine (PEI) method, a negatively charged plasmid DNA complex is formed with PEI, a cationic polymer, to neutralize the charge. The PEI-plasmid complex can approach negatively charged cells and is introduced into the cell via endocytosis. After endosome expulsion and PEI release within the cell, gene transcription and translation occur in the nucleus. The optimal size for DNA / PEI complex introduction into cells is approximately several hundred nm to several μm. The DNA / PEI complex is prone to aggregation / precipitation in liquids over time. The PEI method is a complex reaction system influenced by factors such as the optimal state of the DNA / PEI complex, collision with cells, the concentration of free nucleic acids (derived from cells) in the culture medium, cell concentration, reactor size, and stirring speed. In the PEI method, the concentrations of added plasmid and PEI need to be optimized based on culture conditions, cell concentration, reactor size, and stirring method. Furthermore, the intracellular gene delivery process is complex, occurring within minutes to hours after the PEI complex is added to the cell culture medium. It also requires maintaining suitable culture conditions to preserve cell culture characteristics, typically at a concentration of 1×10⁻⁶. 6 Gene transfer was performed at a low cell concentration of approximately 100 cells / mL.

[0109] In electroporation (EP), intracellular gene delivery is achieved by applying an electrical pulse to create a perforation that, similar to the perforation formed by the cell, allows negatively charged plasmid DNA, which repels the cell, to approach / invade the cell (perforation). Therefore, unlike PEI, gene delivery in EP ends upon pulse application, eliminating the need for prolonged stirring / culture control as in PEI. Cell concentration can be freely adjusted temporarily during the EP process, as long as it maintains uniform dispersion of cells and plasmid DNA. Furthermore, since the delivery is based on electrophoresis, there is no need to individually optimize plasmid concentration based on cell concentration or culture medium conditions, allowing for the application of the same plasmid concentration conditions across a wide range of cell concentrations. Therefore, from the viewpoint of improving plasmid utilization efficiency or reducing throughput, it is preferable to set a higher cell concentration during EP.

[0110] Cell concentrations can also be increased compared to pre-culture levels through cell concentration methods such as centrifugation, continuous centrifugation, sonication, sonophoresis, and filtration (TF-F, ATF). Regarding concentration, a cell concentration of 80 × 10⁻⁶ is preferred. 6cells / ml or higher. More preferably 100 × 10⁻⁶. 6 cells / ml or higher, 120×10 6 cells / ml or higher.

[0111] Furthermore, in the same process, the culture medium for the suspension can be changed from the culture medium used during cultivation to fresh culture medium, electroporation-specific buffer, or the most suitable culture medium for subsequent cultivation.

[0112] As a buffer condition during EP, it is possible to use conditions suitable for typical cell culture (pH, pH buffering capacity, nutrients, salt concentration, etc.).

[0113] In the nucleic acid introduction process, the cells are contained in a suspension, the conductivity of which is preferably 5–20 mS / cm. However, a conductivity of 5–20 mS / cm is not strictly necessary as measures can be taken to reduce heat generation under electroporation conditions.

[0114] Membrane repair agents or nutrients can be added during electroporation.

[0115] Electroporation buffers and cell culture media share many similarities in function and composition. Sometimes, using a specialized medium best suited for each cell type, rather than a universal EP buffer, can improve cell viability and gene delivery / expression efficiency after EP. For HEK293 cell × EP plasmid delivery, Balan-CD-HEK293 medium is preferred.

[0116] Furthermore, when pre-culturing involves maintaining a certain concentration of waste / nutrients in the culture medium at a specific quality level, such as in perfusion culture, buffer exchange can be omitted, and the pre-culture suspension can be directly applied to EP. By eliminating the concentration / culture medium replacement steps, it is possible to simplify the process, reduce the risk of contamination, improve process stability / reproducibility, and reduce costs.

[0117] (3) Addition and mixing of nucleic acids

[0118] As the nucleic acid, the nucleic acid encoding the target virus is used, preferably the exogenous recombinant nucleic acid encoding the virus.

[0119] A target virus is a virus created by introducing exogenous nucleic acid into a cell. Examples of target viruses include non-enveloped viruses. More specifically, examples include adeno-associated viruses, adenoviruses, lentiviruses, baculoviruses, and retroviruses, with adeno-associated viruses being the preferred choice.

[0120] Non-enveloped viruses are well known in the art and are described in WO2015 / 005430, which are incorporated herein by reference.

[0121] Adeno-associated virus (AAV) refers to a family of viruses in the Parvoviridae family and the Parvovirus genus, consisting of small, non-enveloped viruses with a single-stranded DNA of approximately 4700 bases and incomplete replication capacity. It is known that there are over 100 serotypes of AAV, and the host range or viral characteristics vary depending on the serotype. Serotype 2 (AAV2) is one of the most extensively studied serotypes, with a very broad host range. Serotypes 1 (AAV1), 5 (AAV5), and 6 (AAV6) are serotypes with higher tissue specificity. Specifically, AAV1 has high gene delivery efficiency for muscles, liver, respiratory tract, central nervous system, etc.; AAV5 has high gene delivery efficiency for the central nervous system, liver, retina, etc.; and AAV6 has high gene delivery efficiency for the heart, muscles, liver, etc. In this invention, serotype 2 or serotype 5 is preferred. Serotype 5 is particularly preferred.

[0122] Adeno-associated virus (AAV) genes are genes composed of one or more nucleic acid sequences derived from one or more serotypes of accompanying adenoviruses. Preferably, AAV genes are those involved in AAV replication and packaging, and those encoding AAV constituent proteins.

[0123] AAVs are non-enveloped viruses that replicate in the presence of helper viruses such as adenoviruses and herpesviruses. In the production of AAVs for gene therapy or nucleic acid delivery, the classic approach is to co-infect host cells with adenoviruses to replicate the AAVs. Furthermore, the gene responsible for the helper role of the adenovirus has been identified, and plasmids integrating this gene are used. For example, recombinant AAVs (rAAVs) can be packaged by simultaneously transfecting cells with plasmids containing the Rep and Cap genes, an adenovirus helper plasmid, and a plasmid containing genes for treatment or prophylaxis.

[0124] The Rep and Cap genes encode proteins involved in virion replication and packaging. In the wild type, the Rep gene is expressed by the p5 and p19 promoters. The Cap region expresses VP1, VP2, and VP3. The p40 promoter is a naturally retained promoter of the Cap gene.

[0125] Adeno-associated virus genes (such as Rep and Cap genes) can be wild-type genes, but as long as the gene performs its original function, it can be a wild-type gene that has been modified by base substitution, deletion, insertion, or addition.

[0126] When modifying wild-type adeno-associated virus (AAV) genes (such as the Rep gene and Cap gene) by base substitution, deletion, insertion, or addition, the number of modified bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. The modified AAV gene sequence preferably shows more than 85% sequence homology with the wild-type AAV gene sequence, more preferably more than 90% sequence homology, even more preferably more than 95% sequence homology, and even more preferably more than 98% sequence homology.

[0127] When introducing the Rep and Cap genes into cells, a vector containing the Rep and Cap genes can be introduced into the cells. There are no particular restrictions on the configuration of the Rep and Cap genes in the vector; the Rep gene can be located upstream or downstream of the Cap gene, but it is preferred that the Rep gene be located upstream of the Cap gene.

[0128] The Rep gene refers to a region of the AAV genome that encodes viral replication proteins required for replication of viral genomes known to those skilled in the art, or, for example, the Rep gene of human herpesvirus 6 (HHV-6) (known for mediating AAV-2 DNA replication) and its functional homologs. Therefore, the coding region of the Rep gene at least includes genes encoding AAV's REP78 and REP68 (long forms of REP proteins) and REP52 and REP40 (short forms of REP proteins), or their functional homologs. The coding region of the Rep gene used in this invention can be derived from any AAV serotype, but is preferably derived from AAV2. Examples of viruses derived from AAV2 include REP78 and REP68, as well as REP52 and REP40, and ITR.

[0129] The Cap gene refers to the region in the AAV genome that encodes viral capsid proteins known to those skilled in the art. Examples of these capsid proteins are AAV capsid proteins VP1, VP2, and VP3. The Cap gene used in this invention can be derived from any AAV serotype, but is preferably derived from AAV2 or AAV5. AAV2 is particularly preferred.

[0130] As a vector containing the Rep gene and the Cap gene, for example, plasmids, viral nucleic acid sequences, artificially synthesized nucleic acids, etc., are preferred.

[0131] In this invention, therapeutic or preventative genes can be introduced into cells. Preferably, the therapeutic or preventative genes are introduced while being held in place by an ITR.

[0132] Genes used for treatment or prevention can be genes that are incomplete or missing in the genome of target cells, or genes that encode non-natural proteins with the desired biological or therapeutic effect (e.g., antiviral function), but there are no particular limitations. Specific examples of genes used for treatment or prevention include genes used for the treatment or prevention of infectious diseases, inflammatory diseases, autoimmune diseases, chronic and infectious diseases (including acquired immunodeficiency syndrome (AIDS), cancer, nervous system diseases, cardiovascular diseases, hypercholesterolemia, etc.), various blood diseases such as anemia and hemophilia, and gene defects (e.g., cystic fibrosis, Gaucher disease, adenosine deaminase (ADA) deficiency, emphysema, etc.).

[0133] Genes for therapeutic or preventative purposes may also be useful in antisense therapies for cancer and viral diseases, such as short oligonucleotides (e.g., short oligonucleotides complementary to the sequence surrounding the translation initiation site (AUG codon) of mRNA).

[0134] The therapeutic or preventative gene can be linked to a promoter used to express the therapeutic or preventative gene. There are no particular limitations on the promoter used to express the therapeutic or preventative gene, but examples include promoters derived from cytomegalovirus (including promoters as needed), SV40 early promoters, human elongation factor-1α (EF-1α) promoters, human ubiquitin C promoters, the Rous sarcoma virus LTR promoter (a retrovirus), dihydrofolate reductase promoters, β-actin promoters, and phosphoglycerate kinase (PGK) promoters. Preferably, the therapeutic or preventative gene and the promoter used to express the gene are sandwiched between the ITR sequence.

[0135] As a vector containing genes for treatment or prevention, for example, plasmids, viral sequences, artificially synthesized nucleic acids, etc., are preferred.

[0136] In this invention, it is preferable to introduce a viral helper gene derived from adenovirus into the cell. A viral helper gene refers to a non-adeno-associated virus gene used for the replication and packaging of adeno-associated virus. As a viral helper gene, genes derived from viruses other than adeno-associated virus can be used. Specific examples of viral helper genes include those derived from adenovirus or herpesvirus, with adenovirus being the preferred source.

[0137] Examples of viral helper genes derived from adenovirus include E1A, E1B, E2A, E4, and VA-RNA. In host cells possessing all or part of the E1 region, the AAV genome is replicated and packaged into the capsid. The regions of the adenovirus genome required for the formation of the viral virion are the E2A, E4, and VA-RNA regions. Regarding the function based on the E4 region, AAV replication requires the E4 34kDa protein encoded by the open reading frame 6 (E4ORF6) of the E4 region. Preferred viral helper genes are the E2, E4, and VA-RNA genes. The VA-RNA gene is preferably the VA-RNAI gene.

[0138] Viral helper genes derived from adenovirus (such as E1A, E1B, E2A, E4, and VA-RNA) can be wild-type genes. However, as long as the gene performs its original function, it can be a gene that has been modified by base substitution, deletion, insertion, or addition to the wild-type gene.

[0139] When modifying the wild-type viral helper gene by base substitution, deletion, insertion, or addition, the number of modified bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. The base sequence of the modified viral helper gene preferably shows more than 85% sequence homology with the base sequence of the wild-type viral helper gene, more preferably more than 90% sequence homology, even more preferably more than 95% sequence homology, and even more preferably more than 98% sequence homology.

[0140] When introducing viral helper genes into cells, it is possible to introduce vectors containing viral helper genes into cells.

[0141] As a vector containing viral helper genes, for example, plasmids, viral sequences, artificially synthesized nucleic acids, etc., plasmids are preferred.

[0142] Viral helper genes are preferably controlled by promoters, but can also be controlled by promoters whose expression can be regulated.

[0143] There are no specific limitations on the specific examples of promoters, but examples that can be cited include promoters derived from cytomegalovirus (CMV promoters) (which may include enhancers as needed), SV40 early promoters, human elongation factor-1α (EF-1α) promoters, human ubiquitin C promoters, Rous sarcoma virus (RSV) LTR promoters, dihydrofolate reductase promoters, β-actin promoters, and phosphoglycerate kinase (PGK) promoters.

[0144] As described above, the nucleic acid preferably contains one or more of the adeno-associated virus gene and viral helper genes, and preferably contains at least one viral helper gene. More preferably, the nucleic acid contains four or more of the following groups: therapeutic or prophylactic gene, Rep gene, Cap gene, E2 gene, E4 gene, and VA-RNA1 gene.

[0145] As described above, in this invention, nucleic acids can be introduced into cells using multiple plasmids. However, the morphology of the introduced nucleic acid is not limited as long as the purpose of introducing the nucleic acid encoding the gene required for virus production is achieved. For example, a giant plasmid carrying the aforementioned gene onto a single plasmid can be used. However, in electroporation, the larger the plasmid size, the greater the cell damage tends to be during electroporation; therefore, the plasmid size is preferably 15 kbp or less.

[0146] The method for manufacturing the virus can be either the TT (Triple Transfection) method or the packaging cell method.

[0147] The TT method refers to a method of packaging recombinant AAV (rAAV) by simultaneously transfecting cells with a plasmid containing the Rep and Cap genes, an adenovirus helper plasmid, and a plasmid containing the introduced gene (e.g., the desired therapeutic or preventative gene).

[0148] The packaging cell method refers to a method of producing AAV by transfecting cells into which a portion of the genes from the TT method have been pre-integrated into the chromosome.

[0149] The volume, cell concentration, and nucleic acid stock solution of the cell suspension before nucleic acid mixing can be appropriately adjusted and changed to obtain the desired cell and nucleic acid concentrations. The mixing method is not particularly limited as long as shear damage to cells can be minimized and uniform dispersion of cells and nucleic acids can be achieved. For small-scale processing, nucleic acids can be mixed by pipetting and directly added and mixed within the electroporation device. From the perspective of reducing contamination risk, for large-scale processing, stirring / mixing or continuous flow mixing in a sterile, dedicated container is preferred.

[0150] (4) Electroporation (gene transfer)

[0151] If the applied voltage (necessary electric field E × gap) and power capacity (pulse current, pulse time, pulse interval) meet the necessary requirements, then the type of power supply is irrelevant. A function generator has a voltage of ~tens of V and tens of μm; a function generator + power amplifier has a voltage of ~hundreds of V and a gap of ~2 mm. A dedicated pulse power supply (DC power supply + capacitor + power switch circuit) has a voltage of ~several kV and a gap of ~1 cm.

[0152] As a pump and method for delivering liquid, it can be any of the following: syringe pump, hose pump, magnetic pump, diaphragm pump, etc., or it can be pressurized air delivery.

[0153] Electroporation can be performed using any of the following methods: intermittent electroporation (single-use cuvette (approximately tens of μL to 1 mL, pipette operation), continuous intermittent electroporation (several mL to hundreds of mL), or flow electroporation (several mL to tens of L). Flow electroporation is preferred when processing large volumes (culture medium volumes). In the case of flow electroporation, a liquid delivery method with minimal pulsation is preferred.

[0154] There is no particular limitation on the apparatus used for electroporation. Apparatus is well known in the art and described in WO2022 / 224803, WO2023 / 157673 and WO2023 / 223931, which are incorporated herein by reference.

[0155] As an example of a device for performing flow electroporation, it is possible to use Figure 1 and Figure 2 The electroporation device shown. Figure 1 This is a top view of the electroporation device 100. Figure 2 This is a cross-sectional view of the electroporation device 100.

[0156] In the electroporation device 100, an electrode pair 41 is formed by arranging an upper electrode 41a held on an upper electrode holding plate 10 and a lower electrode 41b held on a lower electrode holding plate 20 opposite each other. A power supply (not shown) is connected to the upper electrode 41a and the lower electrode 41b. D represents the distance (gap) between the electrodes. Figure 1 In this context, L represents the electrode length, and W represents the electrode width (flow path width). Figure 2 In the diagram, a1 represents the surface portion of the upper electrode, a2 represents the back portion of the upper electrode, b1 represents the surface portion of the lower electrode, and b2 represents the back portion of the lower electrode. A flow path plate 30 is provided between the upper electrode holding plate 10 and the lower electrode holding plate 20. Fluid introduced from the inlet 50 is discharged from the outlet 52 through the opening (flow path) 32.

[0157] Preferably, the liquid reservoir, delivery tubing, electroporation device, and recovery reservoir are aseptically connected. That is, in this invention, liquid delivery is preferably performed aseptically in the nucleic acid introduction and culture processes. The pre-culture process (+concentration / culture medium replacement + mixing system) and the post-culture process can be connected continuously or separately.

[0158] In this invention, nucleic acid introduction is preferably performed solely by electroporation. In this invention, nucleic acid is preferably introduced into cells in the absence of an introduction reagent. In this invention, it is preferable that no additional nucleic acid introduction is performed after the electroporation-based nucleic acid introduction step.

[0159] In this invention, the CNET product in the electroporation, expressed by the following formula, is 1 × 10⁻⁶. 4 Above and 1×10 7 the following.

[0160] [Formula 7]

[0161]

[0162] or

[0163] [Formula 8]

[0164]

[0165] In the formula, C represents the nucleic acid concentration in μg / mL.

[0166] N k N represents the number of pulses. k Represents integers greater than or equal to 1.

[0167] E k E represents the pulsed electric field in V / cm. k The voltage is above 500V / cm and below 2000V / cm.

[0168] T k Indicates pulse duration in milliseconds (ms).

[0169] N l N represents the number of pulses. l Represents integers greater than or equal to 1.

[0170] E l E represents the pulsed electric field in V / cm. k It is above 50V / cm and less than 500V / cm.

[0171] T l Indicates pulse duration in milliseconds (ms).

[0172] k represents an integer from 1 to n.

[0173] l represents an integer from 1 to m.

[0174] n represents an integer greater than or equal to 1.

[0175] m represents an integer greater than or equal to 1.

[0176] The concentration of nucleic acid (C, μg / mL) is preferably 10–500 μg / mL, more preferably 30–450 μg / mL, even more preferably 50–400 μg / mL, and especially preferably 60–375 μg / mL.

[0177] and,

[0178] [Formula 9]

[0179]

[0180] In this case, the preferred nucleic acid concentration is 100–300 μg / mL.

[0181] [Formula 10]

[0182]

[0183] In this case, the preferred nucleic acid concentration is 10–100 μg / mL.

[0184] Cell-nucleic acid suspensions are prepared by adding (mixing) plasmid stock solution to a cell suspension, and therefore must be diluted to some extent by the concentration of the plasmid stock solution. The upper limit of the plasmid stock solution concentration is around several mg / mL (above this concentration, it cannot dissolve and will precipitate / aggregate), making it difficult to adjust the plasmid concentration to above 500 μg / mL. Furthermore, at low nucleic acid concentrations (CNET), in order to ensure the desired CNET product (to obtain the required amount of plasmid to be introduced into the cells), it is necessary to increase the NET to further increase the electrophoretic distance. A trade-off exists between increased cell damage or decreased efficiency due to increased NT.

[0185] N k N represents the number of pulses. k It represents an integer greater than or equal to 1, preferably an integer greater than or equal to 1 and less than 10, more preferably an integer greater than or equal to 1 and less than 5, and even more preferably an integer greater than or equal to 1 and less than 3.

[0186] N l N represents the number of pulses. l It represents an integer greater than or equal to 1, preferably an integer greater than or equal to 1 and less than 10, more preferably an integer greater than or equal to 1 and less than 5, and even more preferably an integer greater than or equal to 1 and less than 3.

[0187] N k +N l Preferably, it represents an integer greater than or equal to 1 and less than or equal to 10; more preferably, it represents an integer greater than or equal to 1 and less than or equal to 5.

[0188] When performing multiple pulses, the interval is preferably less than 500ms.

[0189] n represents an integer greater than or equal to 1, preferably an integer greater than or equal to 1 and less than 10, more preferably an integer greater than or equal to 1 and less than 5, and even more preferably an integer greater than or equal to 1 and less than 3.

[0190] m represents an integer greater than or equal to 1, preferably an integer greater than or equal to 1 and less than 10, more preferably an integer greater than or equal to 1 and less than 5, and even more preferably an integer greater than or equal to 1 and less than 3.

[0191] E k E represents the pulsed electric field in V / cm. k The voltage is above 500V / cm and below 2000V / cm. By using E... k Setting it below 2000V / cm can prevent cell damage and the reduction in delivery efficiency caused by heating, boiling, or discharge. By using E... k Setting it to 500V / cm or higher ensures sufficient cell membrane potential and produces perforation.

[0192] T k The pulse duration is expressed in milliseconds (ms), preferably 0.01 ms to 100 ms, more preferably 0.05 ms to 20 ms, and even more preferably 0.1 ms to 10 ms. If the pulse duration is insufficient, adequate perforation cannot be formed on the cell membrane, resulting in a decrease in gene delivery efficiency. On the other hand, if the pulse duration is too long, excessive perforation of the cell membrane or boiling or discharge of the suspension due to heating may occur, leading to a decrease in gene delivery efficiency or cell viability.

[0193] E l E represents the pulsed electric field in V / cm. l It is above 50V / cm and less than 500V / cm. By E l Setting it to less than 500V / cm can prevent perforation (damage). By using E... l Setting it to 50V / cm or higher can prevent a decrease in electrophoresis speed, shorten the required time, and prevent a decrease in electrophoresis and importation efficiency.

[0194] T l The pulse duration is expressed in milliseconds (ms), preferably 0.01 ms to 1 s, more preferably 0.1 ms to 200 ms, and even more preferably 1 ms to 100 ms. Low-field kinetic pulses can be applied longer than perforation pulses because they inhibit cell damage and heating. However, applying excessively long pulses can lead to reduced efficiency in introducing genes into perforation sites on the cell membrane due to cell precipitation and rotation during pulse application, and reduced cell viability due to metal dissolution or free radical generation caused by electrode electrolysis.

[0195] In this invention, the CNET product is 1.0 × 10⁻⁶.4 Above and 1.0×10 7 The preferred value is 1.0 × 10⁻⁶. 5 Above and 1.0×10 6 Hereinafter, 2.0 × 10 is preferred. 5 Above and 1.0×10 6 The following is a further preferred value: 2.0 × 10 5 Above and 5.0×10 5 The following is particularly preferred: 2.0 × 10 5 Above and 4.0×10 5 the following.

[0196] If the required nucleic acid is not introduced into the cell, the virus cannot be generated. On the other hand, if excessive nucleic acid is introduced through excessive electroporation conditions, cell activity decreases due to perforation damage, cell survival rate decreases, and thus the titer decreases. Furthermore, cell activity decreases due to the toxicity of REP expression, cell survival rate decreases, and thus the titer decreases. Moreover, even within the range of conditions suitable for virus generation (high titer conditions), in order to further improve the Full rate (the ratio of the amount of capsid containing the complete gene to the total amount of capsid), it is preferable to avoid (inhibit) excessive capsid production (protein production) and to perform cellular and nuclear reaction control based on more precise control of the amount of plasmid introduced, so as to maintain the rate of gene replication (nucleic acid replication) and packaging reaction into the capsid for therapeutic or preventive purposes.

[0197] In this invention, the CNET product is preferably...

[0198] [Formula 11]

[0199]

[0200] express,

[0201] satisfy

[0202] [Formula 12]

[0203]

[0204] exist Figures 3-6 An example of a pulse waveform from electroporation is shown. Figures 3-6 In the diagram, the vertically elongated rectangle represents the perforation pulse, and the horizontally elongated rectangle represents the swimming pulse.

[0205] Figure 3 The upper part indicates that electroporation is performed in the order of perforation pulse, perforation pulse, and electrophoresis pulse. Figure 3 The middle section indicates that electroporation is performed in the order of perforation pulse, swimming pulse, and perforation pulse. Figure 3 The lower section indicates the integration of the perforation pulse and the swimming pulse.

[0206] Figure 4 The polarity of a pulse can be either positive or negative.

[0207] Figure 5 This indicates that the pulse waveform can be a decaying waveform. That is, the pulse intensity can decay during one punch pulse and one swimming pulse. When using a decaying pulse, the pulse electric field (E)... k and E l It can be set to the time average value or the peak voltage value. The ET value is the time integral of the voltage waveform pulse.

[0208] Figure 6 Examples of pulse waveforms.

[0209] (5) Culture for generating viruses

[0210] In this invention, a virus is generated by culturing cells containing nucleic acids.

[0211] The cultivation method described above is not particularly limited and can be batch cultivation, fed batch cultivation, or any of the following: shaking cultivation, stirring cultivation, static cultivation, or bonding cultivation.

[0212] There is no particular limitation on the cell culture temperature; it is carried out at a temperature at which the cells can survive. The culture temperature is usually 25°C to 45°C, preferably 30°C to 42°C, more preferably 35°C to 40°C, and as an example, 37°C. The CO2 concentration is usually 3% to 10%, preferably 5% to 10%, and as an example, 8% CO2.

[0213] The pH of the culture medium is preferably 6.0–8.0, more preferably 6.5–7.5, and even more preferably 6.6–7.0. Furthermore, the pCO2 of the culture medium is preferably below 200 mmHg, more preferably below 160 mmHg.

[0214] As culture conditions, the conditions suitable for pre-culture can be directly applied. In addition, due to the influence of perforation damage based on electroporation, expression of introduced genes, immune response based on intracellular viral accumulation, toxicity, etc., culture conditions can be optimized to have less damage than those during pre-culture (proliferation culture).

[0215] The cell density is preferably the same as or lower than that of the pre-culture. Regarding cell density, in the case of batch culture, 1 × 10⁶ cells / year is preferred. 6 ~10×10 6 The optimal cell / mL ratio for fed-batch culture is 1×10⁻⁶. 6 ~20×10 6 cells / mL.

[0216] The cell concentration can be diluted during the formal culture process after electroporation.

[0217] The shear rate is preferably set to be the same as or lower than that of the pre-culture, and the oxygen concentration is preferably set to be the same as or higher than that of the pre-culture. The nutrient composition is the same as that of the pre-culture.

[0218] The duration of the virus generation process is preferably 24 hours to 90 days, more preferably 24 hours to 30 days, even more preferably 24 hours to 20 days, further preferably 24 hours to 10 days, even more preferably 24 hours to 7 days, and especially preferably 24 hours to 72 days. Electroporation differs from the PEI method in that it does not involve intracellular introduction, endosome extrusion, or PEI slow release; therefore, the rate of nuclear arrival and expression of the introduced gene is faster than in the PEI method. While the typical duration in the PEI method is 72 hours, in EP, approximately 48 hours is preferred.

[0219] Flasks or bioreactors can be used as culture vessels, and there are no particular limitations. The shape of the culture vessel is not particularly limited. The dimensions of the culture vessel are those suitable for the culture scale described later.

[0220] There is no particular limitation on the culture scale; cells can be cultured in any volume of culture medium, for example, cells can be cultured in a medium of 1 mL to 3000 L, preferably 1 L to 2500 L, more preferably 10 L to 1000 L, and especially preferably 50 L to 500 L.

[0221] In cell culture, an oxygen-containing gas can be introduced into the culture medium using an injector. It is preferable to adjust the dissolved oxygen concentration of the culture medium by introducing the oxygen-containing gas. The dissolved oxygen concentration in the culture medium can be appropriately set and is not particularly limited; typically, it is 10–150% when the saturated dissolved oxygen concentration in a 37°C solution at 1 atmosphere of air is set to 100%, preferably 15–120%, and more preferably 20–100%.

[0222] The orifice diameter of the injector is not particularly limited, but is preferably 5μm to 100μm, and more preferably 10μm to 50μm.

[0223] There is no particular limitation on the ventilation rate of oxygen-containing gas, which is usually 0.001 to 1.0 vvm, preferably 0.005 to 0.5 vvm. VVM stands for volume per volume per minute.

[0224] Cultivation can be carried out simultaneously with vibration stirring. The stirring speed during vibration stirring is typically 50 rpm to 200 rpm, preferably 80 to 180 rpm. Stirred cultivation can be based on rotational stirring of blades, propellers, or other similar components within the reactor. For example, a three-bladed propeller or a two-bladed propeller can be used. The size of the blades, propellers, or other components is determined according to the size of the cultivation tank.

[0225] The stirring speed during rotary stirring is typically 50 rpm to 400 rpm, preferably 80 to 350 rpm. Furthermore, stirring can be achieved through wave-like vibration or the up-and-down movement of the stirring blades, but there are no particular limitations.

[0226] During cultivation, a membrane separation process can be performed to separate the cell suspension extracted from the culture tank into a cell-containing liquid and an osmotic liquid by passing it through a separation membrane. In this operation, the cell suspension extracted from the culture tank is separated into a cell-containing liquid with a higher cell concentration than the original cell suspension and an osmotic liquid with a lower cell concentration than the original cell suspension.

[0227] The membrane separation process is preferably tangential filtration, more preferably alternating tangential flow (ATF) or tangential flow, and most preferably ATF. Filters capable of ATF include SuATF10-S02PES and F2RF02PES manufactured by Repligen Corporation.

[0228] Examples of membrane materials used in membrane separation processes include polyethersulfone, modified polyethersulfone, and mixed cellulose esters.

[0229] The pore size of the membrane used in the membrane separation process is preferably 0.1 μm to 0.4 μm, more preferably 0.15 μm to 0.3 μm.

[0230] In this invention, it is preferable that the cells are in a suspension during the nucleic acid introduction and culture processes, and the suspension is a culture medium.

[0231] In the culture of the present invention, including pre-culture, cell breeding can be performed by aspirating a portion of the culture medium. By performing cell breeding, the cell density can be maintained at a specified value.

[0232] As an example, cell propagation can be carried out using the following method: After determining the cell concentration, the pump is run at a rate of 0.3 vvd to 3 vvd when aspirating the culture medium. The cell solution to be propagated can be transferred to a different culture tank than the pre-cultured culture. Cell propagation is performed more than once a day, preferably within 5 hours after the first cell propagation of the day, and more preferably within 3 hours. After completing a day of cell propagation, the same amount of cell culture medium as the amount of cell suspension reduced due to cell propagation is added to restore the total amount of culture medium to the state before cell propagation, and the culture continues.

[0233] Cell propagation can also be performed through automatic control. In the case of automatic cell propagation, for example, the FUTURA sensor manufactured by ABER can be used to automatically and continuously propagate cells while monitoring the electrostatic capacitance of the culture medium. As an example, this can be implemented using the following method.

[0234] (1) Set the electrostatic capacitor as the target.

[0235] (2) Set the weight control value of the culture tank.

[0236] (3) The electrostatic capacitance of the culture medium was measured at a period of less than 0.1s.

[0237] (4) If the electrostatic capacitance of the culture medium exceeds the target, the pump for aspirating the culture medium shall be run at a speed of 0.3vvd to 3vvd.

[0238] (5) While measuring the weight of the culture tank, the culture medium is automatically supplied into the culture tank so that the volume of the culture medium is kept approximately constant.

[0239] (6) If the electrostatic capacitance of the culture medium is more than 0.01% below the target, stop pumping the culture medium.

[0240] The preferred cell proliferation rate is 10–50% / day, more preferably 20–50% / day. Cell proliferation rate is the proportion of culture medium aspirated daily relative to the total volume of culture medium.

[0241] (6) Virus recovery and purification

[0242] The method of the present invention preferably includes a step of recovering the generated virus.

[0243] Intracellular viral particles exist either in the culture medium or within the cell (nucleus). Extraction from the cell (disruption / dissolution of the cell membrane and nuclear membrane) can be performed via freeze-thaw cycles or by adding surfactants (such as Triton X) and stirring. Separation of the virus from cell debris can be achieved through centrifugation or deep filtration.

[0244] Virus purification can be performed using TFF (concentration / buffer exchange), affinity chromatography, or AEX (anion exchange chromatography) as needed. This removes cell debris, cellular nucleic acids, and cellular proteins. Alternatively, commercially available virus purification kits (such as AAV purification kits) can be used. For example, Takara Bio Inc.'s AAVpro (registered trademark) Purification Kit Maxi / Midi can be used.

[0245] (7) Analysis

[0246] The viral titer generated by the method of this invention can be determined using conventional methods known to those skilled in the art. For example, the culture medium after cell culture is recovered, and the cells are disrupted by freeze-thaw cycles, followed by centrifugation to recover the supernatant. The recovered supernatant is then reacted with MgCl2 and Benzonase to digest gDNA (genomic DNA) and residual plasmids. The sample containing the target virus obtained as described above is used as a ddPCR (Droplet Digital PCR) sample, and the viral titer (vg / mL) can be determined by measuring the viral genome copy number through ddPCR.

[0247] Furthermore, the capsid particle titer (Vp / mL) can be determined using an ELISA kit, and the Full rate can be calculated based on the ratio to the genomic titer (vg / mL) calculated by ddPCR. Alternatively, the Full / Empty ratio can be determined through analysis using virus extraction, HPLC analysis of purified sample solutions, or isoelectric point capillary electrophoresis, within the same assay system.

[0248] In this invention, the ratio of the amount of capsid containing the complete gene to the total amount of capsid (Full rate) in the virus generated during the culture process is preferably 5% or more, more preferably 10% or more, further preferably 20% or more, and especially preferably 30% or more.

[0249] Figure 9 This is a schematic diagram of a continuous production facility for AAVs, connecting a perfusion culture unit and a flow cytometry electroporation unit. The perfusion culture unit, equipped with an ATF, produces cells at high concentrations (e.g., 40 × 10⁻⁶ cells per 10⁻⁶). 6 HEK293 cells were pre-cultured (cells / ml). In the table below, M represents ×10⁻¹⁰ cells / ml. 6A specific proportion of cell-containing solution was aspirated from the perfusion culture device once daily, plasmids were mixed, and gene delivery was continuously performed using a sterile, connected flow cytometry electroporation device. Although a diagram of the concentration device is omitted, ATF can also be used to concentrate the cells to a concentration of 80 × 10⁻⁶ cells. 6 Cells / ml or higher. The same volume of culture medium as the aspirated volume is added to the perfusion culture device, and the temporarily decreased cell concentration is restored to the same level as the previous day after 24 hours of incubation. Cell-containing solutions are then aspirated again on subsequent days for gene delivery. By using a perfusion culture device and repeating this cycle, a large supply of high-concentration cells can be achieved. The oxygen concentration in the cell-containing solution after gene delivery is higher than that after pre-culture.

[0250] Cells infused with genes (plasmids) via flow cytometry are supplied with a solution to the subsequent formal culture unit for 48 hours of AAV production culture. The formal culture unit and control unit are switched according to the date. The unit can be prepared for several production days or a certain number of units can be reused. In the case of repeated use, the unit that has completed the 48-hour AAV production culture can be reused after cleaning and sterilization, or a new single-use reactor can be set up in the formal culture unit. Through this application, continuous AAV production can be achieved. In the PEI method, which requires control of the formation of the PEI-plasmid complex and the cell introduction reaction of the complex, there is a problem of reduced gene introduction efficiency under high cell concentration conditions. In the current batch AAV production using the PEI method, the cell concentration is 2–4 × 10⁻⁴. 6 The use of cells / ml is common. In contrast, electroporation has the advantage of enabling highly efficient gene delivery and expression at high cell concentrations.

[0251] Through perfusion culture, a concentration of 40 × 10⁻⁶ was continuously supplied to 40% of the volume of each device daily for 25 days. 6 With cells / ml, when all cells are cultured for gene introduction and AAV production based on flow cytometry electroporation, the cell concentration is 10-20 times, the cell suspension supply is 0.4 times / day × 25 days, and a total of 100-200 times the cells can be gene introduced. Even small-scale culture devices can produce large quantities of AAV.

[0252] The present invention will be described in more detail through the following embodiments, but the present invention is not limited to the embodiments.

[0253] Example

[0254] (1) Pre-culture

[0255] Thermo Fisher Scientific's HEK293 suspension cell line, Viral Production Cells 2.0 (VPCs 2.0), was used as the cell line. The culture medium used was FISI's Balan-CD-HEK293 medium. The culture apparatus used was a Biott BCP1L animal cell culture reactor. The culture was carried out at 40 × 10⁻⁶. 6 Cells were cultured at a density of 40% cells / mL, and perfused at 37℃ ± 1.5℃. A schematic diagram of the perfusion culture is shown below. Figure 7 .

[0256] (2) Concentration and Culture Medium Replacement

[0257] In batch electroporation (BEP) experiments, 40 × 10⁻⁶ cells extracted from the perfusion culture reactor were centrifuged at 200 x g for 5 minutes. 6 After removing the supernatant from the cell suspension, the cells were resuspended in fresh Balan-CD-HEK293 medium to adjust the cell concentration to 120 M cells / mL.

[0258] In the flow electroporation (FEP) experiment, 40 × 10⁻⁶ samples extracted from the perfusion culture reactor were used directly. 6 Cell suspension with cells / mL.

[0259] (3) Plasmids

[0260] AAV generation plasmid was used.

[0261] pAAV-GFP (serial number 1)

[0262] pAAV-RC5 (Serial Number 2)

[0263] pHelper (Serial Number 3)

[0264] A schematic diagram of pAAV-GFP, pAAV-RC5, and pHelper is shown below. Figure 8 .

[0265] (Serial Number 1)

[0266]

[0267]

[0268]

[0269]

[0270] (Serial Number 2)

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278] (Serial Number 3)

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286] The above-mentioned plasmid stock solutions (TE buffer) proliferated in E. coli were mixed in equimolar ratios at a concentration of 2 mg / mL. The mass ratio (g) of the mixture is the ratio of plasmid sizes (base length: bp).

[0287] In the case of intermittent electroporation (BEP), adjust to 120×10 6 The plasmid mixture was added to cell suspensions at cell / mL to prepare cell / plasmid mixtures with plasmid concentrations of 60, 150, and 375 μg / mL.

[0288] In the case of flow electroporation (FEP), the same applies as in the case of BEP, or continuous mixed perfusion. For 40 × 10⁻⁶ samples extracted from the culture reactor... 6 Cell suspension at a concentration of cells / mL is continuously mixed with plasmid stock solution at a flow rate ratio of 12.3:1, and a cell / plasmid mixture with a plasmid concentration of 150 μg / mL is continuously supplied (liquid delivery) to the downstream FEP unit.

[0289] (4) Electroporation and (5) Formal culture

[0290] <Intermittent electroporation>

[0291] Electroporation was performed under the conditions described in the table below.

[0292] Two colorimetric cells were prepared, each filled with 0.2 mL of cell / plasmid mixture in an aluminum electrode cell with a 2 mm gap.

[0293] The desired pulse was applied using a NEPAGENE NEPA21 electroporator. The EP-treated mixture was recovered with a dropper and transferred to a 125 mL flask.

[0294] After standing for 30 minutes in an incubator at 8% CO2 and 37°C, 12 mL of Balan-CD-HEK293 medium was added. The mixture was then returned to the incubator and cultured for 48 hours at 8% CO2, 37°C, and 150 rpm.

[0295] <Flow electroporation case>

[0296] Fill a 50 mL syringe with the cell plasmid mixture.

[0297] exist Figure 1 and Figure 2 In the FEP device shown, with an electrode gap of 3 mm, a flow path width of 20 mm, and an electrode length of 20 mm, 20 mL of liquid was delivered at a rate of 86 mL / min. For continuous plasmid mixing, the rates were set at 79.55 mL / min for cell solution and 6.45 mL / min for plasmid stock solution.

[0298] A pulse voltage of 412.5 V (electric field 1375 V / cm), a pulse width of 3.5 ms, and a pulse period of 450 ms was applied to the FEP electrode. EP was continuously applied to cells flowing within the electrode pair, with an average of one pulse applied to each cell.

[0299] The liquid from the EP process was collected and returned to a recovery bottle. 1.5 mL of the recovered EP solution was pipetted into a 125 mL flask. After incubation for 30 minutes in an incubator at 8% CO2 and 37°C, 12 mL of Balan-CD-HEK293 medium was added. The flask was then returned to the incubator and incubated for 48 hours at 8% CO2, 37°C, and 150 rpm.

[0300] (6) AAV recovery and (7) analysis

[0301] <Titer Determination>

[0302] After 48 hours of culture, aliquot 2 mL of the cell suspension into a 50 mL centrifuge tube. Add 20 μL of Triton X (final concentration 0.1%) and 1.25 x 10⁻⁶ mg / L of the solution to the cell suspension. -5 Benzoinase from U / cells was added with 20 μL of 200 mmol / L MgCl2 (final concentration 2 mmol / L), and the mixture was stirred at 37 °C / 180 rpm to decompose the nucleic acids (genome and plasmids derived from cells) in the suspension. The supernatant was collected by centrifugation at 10625 g for 10 min using a 1.5 mL tubing. The genome within the AAV was extracted by thermally decomposing the capsid of the AAV. The ITR sequence number was determined by ddPCR, and the AAV titer (vg / mL) was calculated.

[0303] <Determination of Full Rate>

[0304] AAV was extracted and purified using the AAVpro (registered trademark) Purification Kit from Takara Bio Inc. The purified conditioning solution was analyzed by high-performance liquid chromatography (HPLC) to determine the full fraction. An anion-exchange column was used for separation, and a fluorescence detector (excitation: 280 nm, detection: 348 nm) was used for detection. The full fraction was calculated based on the area values ​​of the empty and full peaks of AAV.

[0305] The evaluation is based on the following criteria.

[0306] <CNET's review>

[0307] A: 2.0E+05 or higher and 4.0E+05 or lower

[0308] B: Greater than 4.0E+05 and less than 5.0E+05

[0309] C: 1.0E+5 or higher and less than 2.0E+05, or greater than 5.0E+05 and less than 1.0E+6

[0310] D: 1.0E+4 or higher but less than 1.0E+5, or 1.0E+6 or higher but less than 1.0E+7.

[0311] E: Less than 1.0E+4 or greater than 1.0E+7

[0312] <Titer Evaluation>

[0313] A: Above 1.0E+11vg / mL

[0314] B: Above 5.0E+10 vg / mL and less than 1.0E+11 vg / mL

[0315] C: Above 1.0E+10vg / mL and less than 5.0E+10vg / mL

[0316] D: Above 1.0E+9 vg / mL and less than 1.0E+10 vg / mL

[0317] E: Less than 1.0E+9 vg / mL

[0318] <Evaluation of Full Rate>

[0319] A: More than 30%

[0320] B: 20% or more but less than 30%

[0321] C: 10% or more but less than 20%

[0322] D: 5% or more but less than 10%

[0323] E: Less than 5%

[0324] <Overall Evaluation>

[0325] A: Case where the titer is rated A to C, and the full rate is rated A.

[0326] B: Case where the titer is rated A to C, and the full rate is rated B.

[0327] C: Cases where the titer is rated A to C, and the full rate is rated C or D.

[0328] D: Case where the titer is rated A to C and the full rate is rated E.

[0329] E: Case where the titer is evaluated as D.

[0330] F: Case where the titer is evaluated as E.

[0331]

[0332] [Table 2]

[0333]

[0334] ※1μg / ml·V / cm·ms

[0335] Symbol Explanation

[0336] 10 Upper electrode holding plate

[0337] 20 Lower electrode holding plate

[0338] 30 Manifold board

[0339] 32 Opening (flow path)

[0340] 41 Electrode pairs

[0341] 41a Upper electrode

[0342] 41b Lower electrode

[0343] 50 Flow Inlets

[0344] 52 Outlet

[0345] 100 Electroporation Devices

[0346] a1 Surface portion of the upper electrode

[0347] a2 Back side of the upper electrode

[0348] b1 Surface portion of the lower electrode

[0349] b2 The back part of the lower electrode

[0350] L electrode length

[0351] W - Electrode width (flow path width)

[0352] D. Electrode spacing (gap)

Claims

1. A method for producing a virus, comprising: The nucleic acid introduction process involves introducing nucleic acids into cells via electroporation to obtain cells that have been infused with nucleic acids. and The culturing process involves culturing the cells in which nucleic acids have been introduced. In the method for generating the virus, The CNET product in the electroporation, expressed by the following formula, is 1 × 10⁻⁶. 4 Above and 1×10 7 the following, [Formula 1] or [Formula 2] In the formula, C represents the nucleic acid concentration in μg / mL. N k N represents the number of pulses. k Represents integers greater than or equal to 1. E k E represents the pulsed electric field in V / cm. k The voltage is above 500V / cm and below 2000V / cm. T k Indicates pulse duration in milliseconds (ms). N l N represents the number of pulses. l Represents integers greater than or equal to 1. E l E represents the pulsed electric field in V / cm. l It is above 50V / cm and less than 500V / cm. T l Indicates pulse duration in milliseconds (ms). k represents an integer from 1 to n. l represents an integer from 1 to m. n represents an integer greater than or equal to 1. m represents an integer greater than or equal to 1.

2. The virus generation method according to claim 1, wherein, Nucleic acid is introduced solely through electroporation.

3. The virus generation method according to claim 1 or 2, wherein, The CNET product is 1×10 5 Above and 1×10 6 the following.

4. The virus generation method according to any one of claims 1 to 3, wherein, The CNET product is represented by the following formula. [Formula 3] And satisfy the following formula, [Formula 4] 5. The virus generation method according to any one of claims 1 to 4, wherein, The nucleic acid concentration C is 10–500 μg / mL.

6. The method for generating a virus according to any one of claims 1 to 5, wherein, In the viruses produced during the cultivation process, the ratio of capsid containing the complete genome to the total capsid is more than 30%.

7. The virus generation method according to any one of claims 1 to 6, wherein, Nucleic acids are introduced into cells in the absence of an introduction reagent.

8. The method for generating a virus according to any one of claims 1 to 7, wherein, In the nucleic acid introduction process, the cells are in a suspension with a conductivity of 5–20 mS / cm.

9. The method for generating a virus according to any one of claims 1 to 8, wherein, In the nucleic acid introduction and culture processes, the cells are in a suspension, which is a culture medium.

10. The method for generating a virus according to any one of claims 1 to 9, wherein, No additional nucleic acid introduction is performed after the nucleic acid introduction process.

11. The method for generating a virus according to any one of claims 1 to 10, wherein, The virus is an adeno-associated virus.

12. The method for generating a virus according to any one of claims 1 to 11, wherein, The nucleic acid contains one or more of the adeno-associated virus gene and viral helper genes.

13. The method for generating a virus according to any one of claims 1 to 12, wherein, Nucleic acid contains at least one viral helper gene.

14. The method for generating a virus according to any one of claims 1 to 13, wherein, Nucleic acid contains four or more genes from the group consisting of therapeutic or preventative genes, Rep genes, Cap genes, E2 genes, E4 genes, and VA-RNA1 genes.

15. The method for generating a virus according to any one of claims 1 to 14, wherein, Nucleic acids are introduced into cells using multiple plasmids.

16. The method for generating a virus according to any one of claims 1 to 15, wherein, The electroporation is a flow electroporation.

17. The method for generating a virus according to any one of claims 1 to 16, wherein, The cell concentration during nucleic acid introduction was 10 × 10⁻⁶. 6 cells / mL ~200×10 6 cells / mL.

18. The method for generating a virus according to any one of claims 1 to 17, wherein, Liquid is transported aseptically during the nucleic acid introduction and culture processes.

19. The method for generating a virus according to any one of claims 1 to 18, wherein, The cells are animal cells.

20. The method for generating a virus according to any one of claims 1 to 19, wherein, The cells were HEK cells.

21. The method for generating a virus according to any one of claims 1 to 20, comprising the step of recovering the generated virus.

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