WO2016111343A1 - 非エンベロープウイルス粒子の製造方法 - Google Patents
非エンベロープウイルス粒子の製造方法 Download PDFInfo
- Publication number
- WO2016111343A1 WO2016111343A1 PCT/JP2016/050431 JP2016050431W WO2016111343A1 WO 2016111343 A1 WO2016111343 A1 WO 2016111343A1 JP 2016050431 W JP2016050431 W JP 2016050431W WO 2016111343 A1 WO2016111343 A1 WO 2016111343A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- enveloped virus
- substance
- virus particles
- raav
- virus
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
- C07K14/01—DNA viruses
- C07K14/075—Adenoviridae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
- C12N7/02—Recovery or purification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14141—Use of virus, viral particle or viral elements as a vector
- C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14151—Methods of production or purification of viral material
Definitions
- the present invention relates to a method for producing non-enveloped virus particles with high purity without complicated operations.
- virus vector a virus-derived gene transfer vector
- a viral vector is a vector in which a naturally occurring virus is modified so that a desired gene or the like can be transferred to a target.
- Technological development has progressed in recent years.
- a virus vector prepared using a gene recombination technique is called a gene recombinant virus vector.
- viruses having no envelope such as viruses having an envelope such as adenovirus and adeno-associated virus (hereinafter referred to as AAV) are well known.
- AAV can infect a wide variety of cells including humans, infects non-dividing cells such as blood cells, muscles, and nerve cells that have been differentiated, and is less susceptible to side effects because it is not pathogenic to humans.
- non-dividing cells such as blood cells, muscles, and nerve cells that have been differentiated
- side effects because it is not pathogenic to humans.
- the utility value as a vector for gene transfer used in gene therapy methods for the treatment of cancer and infectious diseases is It has attracted attention in recent years.
- an element essential for virus particle formation is usually introduced into a cell in the form of a nucleic acid construct, and a cell having the ability to produce a virus (hereinafter referred to as a virus producing cell) is prepared. This is done by culturing cells to express elements essential for virus particle formation.
- a virus producing cell a cell having the ability to produce a virus
- This is done by culturing cells to express elements essential for virus particle formation.
- those that require cis supply and those that can be supplied in trans are separated and introduced into cells for virus production, thereby producing wild-type virus and gene
- Patent Document 1 A method for preventing independent replication at the infection site of the recombinant virus is taken (Patent Document 1).
- rAAV genetically modified virus
- AAV vector derived from AAV will be specifically described as an example.
- a method using 3) adenovirus infection has been established because it requires the supply of auxiliary elements from any virus such as herpes virus or vaccinia virus.
- the virus-producing cells that have achieved virus production are then collected and crushed, and the resulting cell disruption solution containing rAAV particles is subjected to a process such as filter filtration, ultracentrifugation, chromatography, or ultrafiltration as appropriate. rAAV particles are purified to the final product.
- Patent Document 1 a method for obtaining rAAV particles with higher titer and purity is required, and various improved methods are disclosed.
- Patent Document 1 a method that promotes the production of rAAV particles and the release rate of the particles into the culture supernatant by culturing virus-producing cells under stress conditions in which the pH of the culture solution is increased.
- Patent Document 1 a method that promotes the production of rAAV particles and the release rate of the particles into the culture supernatant by culturing virus-producing cells under stress conditions in which the pH of the culture solution is increased.
- Patent Document 2 a method that promotes the production of rAAV particles and the release rate of the particles into the culture supernatant by culturing virus-producing cells under stress conditions in which the pH of the culture solution is increased.
- it is a device for the steps after purification of the produced rAAV particles.
- Non-Patent Document 1 and Non-Patent Documents methods for purifying rAAV particles of various serotypes (hereinafter also referred to as serotypes) without using ultracentrifugation or chromatography have been proposed (Non-Patent Document 1 and Non-Patent Documents). 2).
- this method is not only a complicated purification method including a two-phase separation method of a polyethylene glycol (PEG) phase and an aqueous phase, PEG precipitation, chloroform treatment, and dialysis, but also increases purity.
- PEG polyethylene glycol
- chloroform treatment chloroform treatment
- dialysis dialysis
- An object of the present invention is to provide a production method for obtaining high-purity non-enveloped virus particles without complicated operations.
- the present inventors have obtained a neutral or basic sample containing non-envelope virus particles.
- a substance that lowers the solubility of protein under acidic conditions and / or a substance that precipitates under acidic conditions By adding a substance that lowers the solubility of protein under acidic conditions and / or a substance that precipitates under acidic conditions, and removing the precipitate generated in the step of acidifying the sample after the addition of the substance, non-envelope
- the inventors have found that a fraction containing virus particles can be obtained, and completed the present invention.
- a method for producing non-enveloped virus particles comprising: (A) adding a substance that decreases protein solubility under acidic conditions and / or a substance that precipitates under acidic conditions to a neutral or basic sample containing non-enveloped virus particles; (B) a step of acidifying the sample to which the substance is added, and (c) removing a precipitate generated in the step (b) to obtain a fraction containing non-enveloped virus particles, Including methods, [2] The method according to [1], further comprising (d) a step of purifying non-enveloped virus particles after the step (c).
- a neutral or basic sample containing non-enveloped virus particles is a lysate or disrupted product of non-envelope virus-producing cells, a culture supernatant of non-enveloped virus-producing cells containing non-enveloped virus particles, or The method according to any one of [1] to [4], which is an extract obtained from a non-enveloped virus-producing cell, [6] The method according to [5], wherein the neutral or basic sample containing non-enveloped virus particles is a neutralized product of an extract obtained by treating non-enveloped virus-producing cells with an acidic solution.
- the present invention provides a production method for obtaining high-purity non-enveloped virus particles without complicated operations.
- FIG. 3 is a diagram showing the results of SDS-PAGE of the sample prepared in Example 1.
- 2 is a graph showing the recovery rate of the sample prepared in Example 1.
- FIG. 6 is a diagram showing the results of SDS-PAGE of the sample prepared in Example 2.
- FIG. 6 is a diagram showing the results of SDS-PAGE of the sample prepared in Example 3.
- FIG. 6 is a diagram showing the results of SDS-PAGE of the sample prepared in Example 6.
- a non-enveloped virus refers to a virus other than an enveloped virus.
- the envelope virus refers to a virus having a lipid layer or a lipid bilayer on the surface of the virus.
- Representative non-enveloped viruses include DNA genome viruses, such as adenoviruses, parvoviruses, papovaviruses, and human papillomaviruses, and RNA viruses such as rotaviruses, coxsackie viruses, enteroviruses, and sapoviruses.
- DNA genome viruses such as adenoviruses, parvoviruses, papovaviruses, and human papillomaviruses
- RNA viruses such as rotaviruses, coxsackie viruses, enteroviruses, and sapoviruses.
- non-enveloped virus there is no particular limitation on the non-enveloped virus to which the production method of the present invention is applied, and non-enveloped viruses that have already been known for production, non-enveloped viruses newly obtained from nature, or genetic recombination derived therefrom. Viral vectors may be used.
- the non-enveloped virus produced by the production method of the present invention is preferably exemplified by adenovirus or AAV belonging to Parvoviridae.
- the production method of the present invention can be applied to any known serotype of AAV, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13.
- the serotype of rAAV when the serotype of rAAV is described, the serotype from which capsid is derived is used as a reference. That is, the serotype of the rAAV is determined according to the origin of the cap gene used at the time of rAAV preparation, and does not depend on the origin of the serotype of the AAV genome encapsulated in the rAAV particles. For example, when the capsid is derived from AAV6 and the ITR in the AAV genome encapsulated in the rAAV particle is derived from AAV2, the rAAV is referred to as serotype 6 in the present specification. Furthermore, the production method of the present invention can also be applied to the production of AAV containing the AAV capsid mutant of each serotype.
- virus particle means a particle composed of a protein shell called capsid.
- the “virus vector” means a virus genome (nucleic acid form) contained in the virus particle.
- an rAAV particle is a particle composed of a protein shell called a capsid and contains an rAAV vector.
- the rAAV vector contains viral genomic DNA present in rAAV particles.
- virus particle includes a virus-like particle not containing a virus genome (for example, AAV hollow particle: WO 2012/144446).
- rAAV vector-derived virus-like particles or AAV hollow particles are a category of rAAV particles.
- acidic means that the pH is less than 6.5
- neutral means that the pH is 6.5 to 7.5
- basic means that the pH is more than 7.5.
- a substance that lowers protein solubility under acidic conditions and / or a substance that precipitates under acidic conditions is first added to a neutral or basic sample containing non-enveloped virus particles.
- the addition of the substance can be carried out by adding a solid substance, but is preferably carried out by adding a solution prepared in advance so as to have a desired final concentration.
- the process may proceed to the next step (b).
- the substance is allowed to stand for a while after the addition of the substance.
- the standing temperature and the standing time may be appropriately determined and are not particularly limited.
- the standing temperature is, for example, 0 to 40 ° C., preferably 4 to 37 ° C.
- the standing time is, for example, 1 minute to 24 hours, preferably 5 minutes to 1 hour, for example, leave at 37 ° C. for 30 minutes.
- the “substance that reduces protein solubility under acidic conditions” used in the method of the present invention refers to a substance that has the property of reducing protein solubility under acidic conditions as compared to neutral or basic conditions. Say. Although the present invention is not particularly limited as the substance, those having the property of precipitating by lowering the solubility of the protein under acidic conditions by the interaction with the protein in the sample are suitable for the present invention. .
- the “substance that precipitates under acidic conditions” used in the method of the present invention refers to a substance that has the property of precipitating itself by changing from neutral or basic conditions to acidic conditions.
- the substance is not particularly limited to the present invention, but those having the property of precipitating proteins in the sample together when precipitated under acidic conditions are suitable for the present invention.
- the substance that lowers the protein solubility under acidic conditions and the substance that precipitates under acidic conditions used in the method of the present invention may be different substances or the same substance.
- the substance that lowers the solubility of the protein under the acidic condition and / or the substance that precipitates under the acidic condition is not particularly limited, but may be a surfactant.
- the said surfactant is divided roughly into an anionic surfactant, a nonionic surfactant, a cationic surfactant, and an amphoteric surfactant.
- An anionic surfactant is also referred to as an anionic surfactant and is ionized in water to become an organic anion.
- the anionic surfactant is classified into a carboxylic acid type, a sulfonic acid type, a sulfate ester type, and a phosphate ester type. Examples of those classified into the carboxylic acid type include deoxycholate, cholate or lauroyl sarcosine salt. Examples of the sulfonic acid type include dodecyl sulfate.
- the salt is preferably a sodium salt or a lithium salt.
- Non-ionic surfactant means a substance that does not turn into ions in water.
- the nonionic surfactant is classified into an ester type, an ether type, an ester ether type, an alkanolamide type, a sugar type, and a methylglucamine type.
- ester type include polyoxyethylene sorbitan monolaurate (Tween (registered trademark) 20), polyoxyethylene sorbitan monopalmitate (Tween (registered trademark) 40), polyoxyethylene sorbitan monostearate (Tween (registered trademark)).
- Tween (registered trademark) 40 polyoxyethylene sorbitan monostearate
- Examples of the ether type include polyoxyethylene nonylphenyl ether (Nonidet (registered trademark) P-40), polyoxyethylene alkyl ether (BriJ (registered trademark) L23, BriJ (registered trademark) L58).
- Examples of the ester ether type include polyoxyethylene sorbitan fatty acid ester, polyoxyethylene hexitan fatty acid ester or sorbitan fatty acid ester polyethylene glycol.
- Examples of the alkanolamide N, N-bis [3- (D-gluconamido) propyl] deoxycholamide is exemplified.
- Examples of the sugar type include alkyl maltoside and alkyl glucopyranoside.
- Examples of the methylglucamine type include alkyl-N-methylglucamine.
- the cationic surfactant is also called a cationic surfactant, which is ionized in water to become an organic cation.
- Cationic surfactants are classified into an alkylamine salt type and a quaternary ammonium salt type, and examples of the alkylamine salt type include monomethylamine hydrochloride, dimethylamine hydrochloride and trimethylamine hydrochloride. Further, examples of the quaternary ammonium salt type include hexadecyltrimethylammonium bromide and myristyltrimethylammonium bromide.
- amphoteric surfactant is a surfactant having both an anionic group and a cationic group in the molecule.
- the ionization state in an aqueous solution is similar to amino acids, and there are many amino acid derivatives in amphoteric surfactants. Therefore, it has an isoelectric point like an amino acid, and acts as an anionic surfactant when it is on the alkaline side from the isoelectric point and as a cationic surfactant when it is on the acidic side.
- amphoteric surfactant include sulfobetaine.
- lauryl sulfobetaine caprylyl sulfobetaine, octyl sulfobetaine, palmityl sulfobetaine, or myristyl sulfobetaine is mentioned.
- an anionic surfactant that is insolubilized at an acidic pH is exemplified. More preferably, sodium deoxycholate, sodium chenodeoxycholate, sodium cholate, sodium glycocholate, sodium taurocholate and sodium taurodeoxycholate, which are carboxylic acid type anionic surfactants, are preferably used. Particularly preferred are sodium deoxycholate, sodium chenodeoxycholate, and sodium cholate.
- a substance that lowers the solubility of the protein under acidic conditions and / or a substance that precipitates under acidic conditions may be added as a solid or prepared in advance.
- a solution of the substance may be added.
- the concentration of the solution of the substance is not particularly limited and can be appropriately determined by those skilled in the art.
- the solvent used for preparing the solution of the substance is not particularly limited, and water, a buffer solution, a cell culture medium, and the like can be appropriately selected.
- Substances that reduce protein solubility under acidic conditions and / or substances that precipitate under acidic conditions are added to neutral or basic samples containing non-enveloped virus particles to a desired final concentration.
- the desired final concentration of the substance is not particularly limited as long as it is a concentration that precipitates contaminating proteins in the sample under acidic conditions.
- the final concentration when sodium deoxycholate, chenodeoxycholate, or sodium cholate is used is 0.1-2%. (V / v), preferably 0.2 to 1% (v / v).
- the optimum concentration of a substance that reduces protein solubility under acidic conditions and / or a substance that precipitates under acidic conditions differs depending on individual non-enveloped viruses, it is natural to determine an appropriate concentration.
- the pH of the sample to which the substance is added is acidified.
- the method is not particularly limited as long as a neutral or basic sample can be acidified.
- a gaseous acid may be bubbled or a solid acid may be added.
- an acidic solution prepared in advance is added to a desired final concentration.
- the acid used in the present invention include citric acid, acetic acid, malic acid, phosphoric acid, hydrochloric acid, sulfuric acid, nitric acid, lactic acid, propionic acid, butyric acid, oxalic acid, malonic acid, succinic acid, fumaric acid, and maleic acid.
- the acidic solution for example, a solution containing the compound is used.
- the solvent used for the preparation of the acidic solution is not particularly limited, and water, buffer solution, cell culture medium and the like can be appropriately selected.
- an aqueous solution containing citric acid and salts thereof is used.
- the acid or acidic solution is added to the sample so as to have a desired final concentration.
- the desired final concentration is not particularly limited as long as the pH of the sample is acidic.
- the final pH after the acidification is preferably less than pH 6.5, and more preferably in the range of pH 4-6.
- the optimum pH differs depending on individual non-enveloped viruses, it is natural to determine an appropriate pH.
- the sample containing non-enveloped virus particles used in the method of the present invention may be a sample derived from non-enveloped virus-producing cells.
- the virus-producing cell is not particularly limited, and may be a virus-producing cell obtained from the environment, a clinical specimen of a patient with an infectious disease, or the like, or a virus-producing cell produced artificially.
- the cells for producing the non-enveloped virus-producing cells are not particularly limited, and are mammalian cells such as humans, monkeys, rodents, preferably 293 cells (ATCC CRL-1573) or 293T / high in transfection efficiency. 17 cells (ATCC CRL-11268), 293F cells, 293FT cells (all manufactured by Life Technologies), G3T-hi cells (International Publication No. 2006/035829 pamphlet), Sf9 cells (ATCC CRL-1711), commercially available viruses A production cell line, AAV293 cells (manufactured by Stratagene), is exemplified.
- the 293 cell or the like constantly expresses the adenovirus E1 protein so that one or several of these proteins necessary for rAAV production may be expressed transiently or constitutively. It may be a modified cell.
- a non-enveloped virus vector can be introduced into these various cells by introducing a non-enveloped virus vector using a known method or a commercially available kit.
- the cells can be cultured under known culture conditions. For example, culturing 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.
- the culture period is not particularly limited, and is, for example, 12 to 150 hours, preferably 48 to 120 hours.
- rAAV-producing cells will be described as non-enveloped virus-producing cells.
- A a nucleic acid encoding an AAV-derived Rep protein and a nucleic acid encoding a Cap protein as essential elements for rAAV particle formation
- B An rAAV-producing cell is produced by introducing a nucleic acid supplying an adenovirus-derived element such as E1a protein, E1b protein, E2 protein, E4 protein and VARNA, and
- C a nucleic acid encapsulated in rAAV particles into any cell. can do.
- a method of using an adenovirus or the like as a helper virus instead of the nucleic acid described in (B) is also known.
- nucleic acid encoding the AAV-derived Rep protein and the nucleic acid encoding the Cap protein can be mounted on a plasmid or viral vector and introduced into a cell.
- Introduction of these nucleic acids into cells can be performed by a known method using, for example, a commercially available or known plasmid or viral vector.
- the nucleic acid encapsulated in the rAAV particles is composed of an AAV-derived ITR sequence and a nucleic acid that is desired to be mounted on an rAAV vector.
- the nucleic acid desired to be loaded on the rAAV vector includes any foreign gene such as a polypeptide (enzyme, growth factor, cytokine, receptor, structural protein, etc.), antisense RNA, ribozyme, decoy, RNA that causes RNA interference, etc.
- the nucleic acid which supplies is illustrated.
- an appropriate promoter, enhancer, terminator or other transcriptional regulatory element may be inserted into the nucleic acid to control the expression of the foreign gene.
- the nucleic acid encapsulated in the rAAV particle may contain any foreign gene desired to be loaded into the rAAV vector between the two ITR sequences, or the rAAV vector between the two ITR sequences. It may contain any foreign gene desired to be loaded on and one or more elements for controlling the expression of the foreign gene.
- the nucleic acid encapsulated in the rAAV particles can be introduced into the cell as a nucleic acid construct in the form of a plasmid.
- the plasmid can be constructed using, for example, a commercially available rAAV vector plasmid, such as pAAV-CMV Vector (manufactured by Takara Bio Inc.).
- the neutral or basic sample containing non-enveloped virus particles used in the method of the present invention can be prepared, for example, by disrupting or lysing non-enveloped virus-producing cells cultured as described above.
- the culture supernatant containing the non-envelope virus particles may be used as a sample.
- the disruption or lysis of the non-enveloped virus-producing cells can be performed by a known method such as ultrasonic treatment, freeze-thaw treatment, enzyme treatment, osmotic pressure treatment or the like.
- neutralization is not particularly limited, but means adding a basic solution or pH buffer solution to an acidic solution, and the neutralized solution (neutralized product) is neutral. It may be basic or basic. Although it does not specifically limit as a basic solution, For example, sodium hydroxide aqueous solution, potassium hydroxide solution, etc. are mentioned. Further, the pH buffer solution is not particularly limited, and examples thereof include a Tris-HCl solution.
- the neutral or basic sample containing the non-enveloped virus particles used in the method of the present invention may be a neutralized product of an extract obtained by treating non-enveloped virus-producing cells with an acidic solution. . That is, a sample obtained by neutralizing a sample obtained by bringing a non-enveloped virus-producing cell into contact with an acidic solution can be suitably used.
- This step involves suspending the non-enveloped virus-producing cell pellet collected by removing the culture solution by centrifugation or filtration after culturing, or suspending the non-enveloped virus-producing cell culture in the culture solution. It is carried out by an operation of adding a component that can be made acidic.
- the acidic solution may be a solution that exhibits a pH lower than that during culture of non-enveloped virus-producing cells and that can obtain an extract containing non-enveloped virus particles from non-enveloped virus-producing cells that have achieved virus production.
- a solution similar to that exemplified as the acidic solution used in step (b) of the method of the present invention can be used.
- the component capable of acidifying the culture solution is a component that makes the pH of the culture solution of the non-enveloped virus-producing cells lower than the pH at the time of culturing the non-enveloped virus-producing cells, and the virus production has not been achieved.
- the temperature and time when contacting with the acidic solution or the component capable of being acidic are not particularly limited.
- the temperature is, for example, 0 to 40 ° C., preferably 4 to 37 ° C.
- the time is, for example, 1 minute to 48. Examples are time, preferably 5 minutes to 24 hours.
- it can be stored for a long time in an ultra-low temperature freezer such as ⁇ 80 ° C. in a state where it is in contact with an acidic solution or a component capable of being acidic or after neutralization. By this contact operation, the non-enveloped virus is released out of the virus-producing cells.
- the neutral or basic sample containing the non-enveloped virus particles used in the method of the present invention may be a sample that has been subjected to nuclease treatment in advance.
- nuclease treatment may be performed before adding a substance that lowers the solubility of a protein under acidic conditions and / or a substance that precipitates under acidic conditions.
- the nuclease used in the step is preferably one that acts on the DNA in the extract, and examples include benzonase (Benzonase registered trademark, Merck Millipore), Cryonase Cold-active nuclease (Takara Bio). Conditions such as the temperature and time of the nuclease treatment and the amount of nuclease may be appropriately determined according to the type of enzyme used, and are not particularly limited.
- the step of removing the precipitate generated in the method of the present invention and obtaining a fraction containing non-enveloped virus particles is the step of removing the precipitate generated in the acidifying step and removing the fraction containing non-enveloped virus particles.
- the removal of the precipitate may be performed by a known solid-liquid separation method such as filtration. It is preferably carried out by centrifugation.
- the fraction containing non-enveloped virus particles obtained through the step of removing the precipitate may be subjected to a further purification step.
- non-enveloped virus particles may be concentrated by the purification step. Examples of the purification method include ultracentrifugation, chromatography, ultrafiltration, and other known methods.
- the ultrafiltration is filtration using an ultrafiltration membrane.
- An ultrafiltration membrane is a separation membrane having many fine pores that are physically distinct.
- the fractionated molecular weight of the ultrafiltration membrane is preferably 50 to 300 kDa, more preferably 70 to 150 kDa, and even more preferably 100 kDa from the viewpoint of purification of non-enveloped virus and removal of contaminants. .
- it can be carried out using an ultrafiltration membrane Amicon (registered trademark) Ultra-15 (manufactured by Merck Millipore, Inc., hereinafter referred to as an ultrafiltration membrane of 100 kDa) having a fractional molecular weight of 100 kDa.
- the recovery rate of non-enveloped virus particles can be improved by adding polyethylene glycol (PEG) to the fraction containing non-envelope virus particles.
- PEG polyethylene glycol
- the PEG to be added is not particularly limited, and those having various average molecular weights can be used.
- PEG having an average molecular weight of 200 to 10,000, preferably PEG having an average molecular weight of 4,000 to 8,000 is used in the present invention. More preferably, PEG having an average molecular weight of 6,000 can be used.
- the purification of non-enveloped virus particles by the chromatography is performed by ion exchange columns (for example, Mustang Q (manufactured by Pall)), affinity columns (for example, AVB Sepharose (registered trademark) (manufactured by GE), heparin columns, etc.) It can be carried out with a hydroxyapatite column or the like.
- ion exchange columns for example, Mustang Q (manufactured by Pall)
- affinity columns for example, AVB Sepharose (registered trademark) (manufactured by GE), heparin columns, etc.
- the non-enveloped virus particles are preferably adeno-associated virus particles.
- the degree of acquisition of non-enveloped virus particles is indicated using the titer of non-envelope virus as an evaluation method.
- the titer of the non-enveloped virus is not particularly limited, but (a) the number of non-enveloped virus genomes (genome titer) in a fixed amount of sample, (b) the non-enveloped virus measured experimentally Examples thereof include a method for measuring the ability to infect cells (infection titer), or (c) the purity of a protein constituting a non-enveloped virus.
- a method of measuring the copy number of the viral genome in the sample by the PCR method is exemplified.
- AAVpro registered trademark
- Titration Kit for Real Time PCR
- Ver. 2 manufactured by Takara Bio Inc.
- a method for detecting a change in cell shape (cytopathy) by infecting an appropriate target cell with a serial dilution of a sample for example, a method for detecting expression of a transgene, or introduction into a cell
- cytopathy a change in cell shape
- examples of the method (c) include a method of analyzing the protein by SDS-PAGE or a method of quantifying by an immunological method.
- kit for the production method of the present invention is a kit for carrying out the method for producing the non-viral envelope virus particles described in (1) above, and (1) in an acidic condition It contains a substance that lowers protein solubility and / or a substance that precipitates under acidic conditions, and (2) an acidic solution.
- the substance that precipitates under the acidic condition is preferably the substance that reduces the solubility of the protein under the acidic condition and / or the substance that precipitates under the acidic condition described in (1) the method for producing non-enveloped virus particles of the present invention. Can be used.
- the acidic solution was added with the substance that decreases the solubility of the protein under acidic conditions and / or the substance that precipitates under acidic conditions, as described in the above (1) method for producing non-enveloped virus particles of the present invention.
- the kit of the present invention may further contain a neutralizing solution.
- the neutralizing solution is preferably a basic solution or a pH buffer solution. Although it does not specifically limit as a basic solution, For example, sodium hydroxide aqueous solution, potassium hydroxide solution, etc. are mentioned.
- the pH buffer solution is not particularly limited, and examples thereof include a Tris-HCl solution.
- a plasmid containing a reagent used for preparing an extract from a non-enveloped virus-producing cell, a nucleic acid supplying an element essential for the formation of a non-enveloped virus particle, and a plasmid containing a nucleic acid encapsulated in a non-enveloped virus particle It is good also as a kit containing etc.
- kits used in the production method and non-enveloped virus particles produced by the production method are also provided.
- the non-enveloped virus particles obtained using the production method of the present invention can be used as an active ingredient of a pharmaceutical composition.
- the pharmaceutical composition can be used ex vivo on patient-derived cells or administered directly to the patient.
- Example 1 Purification of rAAV5 by addition of sodium deoxycholate and addition of citric acid (1) Inoculation of cells for production of rAAV DMEM (Sigma) containing 10% FBS (Gibco) in a tissue culture treated T225 flask (Corning) 293T / 17 cells suspended in the company). Thereafter, the cells were cultured for 2 days in a CO 2 incubator at 37 ° C. and a CO 2 concentration of 5% (v / v).
- Example 3- (2) Obtaining the extract by citrate buffer treatment
- 0.5 mL of 0.5 M EDTA manufactured by Wako Pure Chemical Industries, Ltd.
- 0.5 M EDTA manufactured by Wako Pure Chemical Industries, Ltd.
- the cell pellet is resuspended in 2 mL citrate buffer (38.1 mM citrate, 74.8 mM sodium citrate, 75 mM sodium chloride, 100 mM magnesium chloride, pH 5), mixed with a vortex mixer for 15 seconds, and 5 minutes at room temperature.
- the mixture was allowed to stand and mixed again with a vortex mixer for 15 seconds. Thereafter, centrifugation was performed at 4,000 ⁇ g, 4 ° C. for 10 minutes, and the supernatant was collected. A solution obtained by adding 1/10 amount of 2M Tris-HCl (pH 9.5) to the obtained supernatant was used as an extract.
- Example 1- (3) and the five pre-concentration rAAV5 obtained in Example 1- (4) were each equidistant in 5 ⁇ sample buffer. (Takara Bio Inc.) was added and mixed, followed by treatment at 95 ° C. for 10 minutes. 4 ⁇ L of each treatment solution was applied to a 4-12% polyacrylamide gel and subjected to electrophoresis. After completion of the electrophoresis, the gel was immersed in an appropriate amount of Oriole fluorescent gel stain solution (manufactured by Bio-Rad) and shaken for 90 minutes while being shielded from light. The gel after shaking was photographed with Luminoshot 400 (manufactured by Takara Bio Inc.). The result is shown in FIG. In FIG. 1, lane C is an extract, and lanes 1 to 5 are rAAV5 (samples 1 to 5) before concentration.
- FIG. 1 All bands in FIG. 1 are contaminating protein bands. Since the concentration of rAAV5 is low at the stage before concentration, the rAAV5 band cannot be confirmed. As shown in FIG. 1, Lane 1 and Lane 2 had fewer contaminating protein bands than Lane C. This indicates that the contaminating protein can be precipitated and removed by adding citric acid or sodium deoxycholate to the mixture of rAAV5 and the contaminating protein. Further, as shown in FIG. 1, lanes 3 to 5 had fewer contaminating protein bands than lanes 1 and 2. This indicates that contaminating proteins can be precipitated and removed more efficiently by combining sodium deoxycholate addition and citric acid addition.
- Total genome titer (VG) genome titer (VG / mL) ⁇ total liquid volume (mL)
- the relative value (recovery rate) when the total genome titer of the extract is 100 is shown in FIG.
- the vertical axis represents the recovery rate (%)
- the horizontal axis lane C represents the extract
- lanes 1 to 5 represent rAAV5 (samples 1 to 5) before concentration.
- the recoveries of samples 1 to 5 were 99% to 113%, which was close to 100%. This indicates that the recovery rate of rAAV5 is very high in the purification of rAAV5 by adding sodium deoxycholate and citric acid.
- FIG. 2 revealed that the purification of rAAV5 by adding sodium deoxycholate and citric acid was a very efficient purification method. That is, contaminating proteins can be precipitated and removed by adding sodium deoxycholate and citric acid.
- rAAV5 can be purified at a high recovery rate by adding citric acid so that the final concentration of sodium deoxycholate is 0.5% (v / v) and the pH is 4-6.
- Example 2 Purification and Concentration of rAAV5 (1) Examination of Addition Amounts of Sodium Deoxycholate and Citric Acid Extracts were obtained in the same manner as in Examples 1- (1) to (3). 1/100 amount of 20 U / ⁇ L Cryonase (registered trademark) Cold-active Nuclease was added to the resulting extract (final concentration 0.2 U / ⁇ L) and reacted at 37 ° C. for 1 hour. Divide the sample into 4 equal parts, add 1/10 or 1/20 amount of 5% (v / v) sodium deoxycholate, let stand at 37 ° C. for 30 minutes, and then add 1/20 or 1/30 An amount of 1M citric acid was added. The results of measuring the pH for each sample are shown in Table 2.
- the samples were centrifuged at 5,000 ⁇ g, 4 ° C. for 5 minutes, and the supernatant was collected to remove the precipitates.
- the recovered supernatant was filtered through a filter having a pore size of 0.45 ⁇ m and designated rAAV5 before concentration.
- Example 2- (2) Purification and concentration of rAAV5 by ultrafiltration membrane
- the pre-concentration rAAV5 obtained in Example 2- (2) was placed in a 100 kDa ultrafiltration membrane (manufactured by Merck Millipore), 2,000 ⁇ g, 4
- the solution was centrifuged at 10 ° C. for 10 minutes, and the filtrate was discarded. Further, “the operation of adding 10 mL of D-PBS to the ultrafiltration membrane, suspending it, centrifuging under the above conditions and discarding the filtrate (washing operation)” was repeated 5 times, and the final concentrated solution was purified rAAV5 ( Samples a to d) were used. By these series of operations, low molecular weight impurities were removed and rAAV5 was concentrated 10 times.
- Example 2- (2) Purity confirmation of rAAV5
- lane M is Protein Molecular Weight Marker (Broad) (Takara Bio Inc., 200, 116, 97, 66, 44, 29, 20 kDa from the top), and lanes a to d are purified rAAV5 (samples a to d). It is.
- rAAV5 structural protein VP1, VP2, VP3 bands
- Lane a had few contaminating protein bands. This is because rAAV5 was further treated by ultrafiltration membrane treatment of 100 kDa after removing precipitates produced by addition of sodium deoxycholate (final concentration 0.5% (v / v)) and citric acid (pH 5). Indicates that can be purified and concentrated.
- Example 3 Purification of rAAV of various serotypes (rAAV1, rAAV2, rAAV3, rAAV6) (1) Seeding of cells for producing rAAV Cells for producing rAAV were seeded in the same manner as in Example 1- (1).
- Plasmid transfection for rAAV production Plasmid for rAAV production was transfected into the cells obtained in Example 3- (1) in the same manner as in Example 1- (2). However, according to the serotype of rAAV to be prepared, instead of pRC5 Vector, pRep-Cap1 AAV Helper Plasmid (Applied Viromics), pRC2-mi342 Vector (manufactured by Takara Bio Inc.), pRep-Cap3 AAV HelperPlasPs PRC6 Vector (manufactured by Takara Bio Inc.).
- the samples were centrifuged at 5,000 ⁇ g, 4 ° C. for 5 minutes, and the supernatant was collected to remove the precipitates.
- the recovered supernatant was filtered through a filter with a pore size of 0.45 ⁇ m and used as rAAV before concentration.
- the bands of Lane C and Lane Pre are all contaminating protein bands. Since rAAV has a low concentration at this stage, the rAAV band cannot be confirmed.
- the three thick bands in Lane F are bands of rAAV structural proteins (VP1, VP2, VP3). As shown in FIG. 4, in each serotype of rAAV, Lane Pre had fewer contaminating protein bands than Lane C. This indicates that contaminant proteins can be precipitated and removed by adding sodium deoxycholate and citric acid regardless of the serotype of rAAV. In each serotype of rAAV, Lane F had few contaminating protein bands other than rAAV structural proteins (VP1, VP2, VP3). This indicates that, regardless of the serotype of rAAV, rAAV can be concentrated and low molecular weight contaminating proteins can be removed by 100 kDa ultrafiltration membrane treatment.
- the recovery rate of rAAV (Pre) before concentration was 89% to 120%, which was close to 100%. This indicates that in the purification of rAAV by adding sodium deoxycholate and citric acid, the recovery rate of rAAV is very high regardless of the serotype of rAAV.
- Example 5 Examination of various types of acidic solutions (1) Acquisition of rAAV6 extract An rAAV6 extract was obtained in the same manner as in Examples 3- (1) to (3).
- the samples were centrifuged at 5,000 ⁇ g, 4 ° C. for 5 minutes, and the supernatant was collected to remove the precipitates.
- the recovered supernatant was filtered through a filter with a pore size of 0.45 ⁇ m and designated rAAV6 before concentration.
- Example 6 Examination of various types of surfactants (1) Acquisition of rAAV2 extract A rAAV2 extract was obtained in the same manner as in Examples 3- (1) to (3).
- the samples were centrifuged at 5,000 ⁇ g, 4 ° C. for 5 minutes, and the supernatant was collected to remove the precipitates.
- the recovered supernatant was filtered through a filter having a pore size of 0.45 ⁇ m and designated rAAV2 before concentration.
- the bands of Lane C and Lane Pre are all contaminating protein bands. Since rAAV2 has a low concentration at this stage, the rAAV2 band cannot be confirmed.
- the three thick bands in lane F are bands of rAAV2 structural proteins (VP1, VP2, VP3). As shown in FIG. 5, in all the surfactants, Lane Pre had fewer contaminating protein bands than Lane C. This indicates that contaminant proteins can be precipitated and removed by addition of surfactant and citric acid, regardless of the type of surfactant.
- the highly non-enveloped virus particles can be obtained without complicated operations by the non-enveloped virus production method of the present invention.
- the non-enveloped virus particles prepared by the production method of the present invention and compositions containing such particles as active ingredients are very useful as gene transfer methods in basic research of gene therapy or clinical applications.
Abstract
Description
[1] 非エンベロープウイルス粒子の製造方法であって、
(a)非エンベロープウイルス粒子を含有する中性又は塩基性の試料に、酸性条件でのタンパク質の溶解度を低下させる物質及び/又は酸性条件で沈殿する物質を添加する工程、
(b)前記物質を添加した試料を酸性にする工程、及び
(c)前記(b)工程で生じた沈殿物を除去し、非エンベロープウイルス粒子を含む画分を取得する工程、
を含む方法、
[2](c)工程の後にさらに、(d)非エンベロープウイルス粒子を精製する工程、を含むことを特徴とする[1]に記載の方法、
[3] 酸性条件でのタンパク質の溶解度を低下させる物質及び/又は酸性条件で沈殿する物質が、界面活性剤である[1]に記載の方法、
[4] 酸性にする工程が、酸性の溶液を添加する工程である[1]~[3]のいずれかに記載の方法、
[5] 非エンベロープウイルス粒子を含有する中性又は塩基性の試料が、非エンベロープウイルス産生細胞の溶解物もしくは破砕物、非エンベロープウイルス粒子を含有する非エンベロープウイルス産生細胞の培養液上清、または非エンベロープウイルス産生細胞から得られる抽出物である[1]~[4]のいずれかに記載の方法、
[6] 非エンベロープウイルス粒子を含有する中性又は塩基性の試料が、非エンベロープウイルス産生細胞を酸性の溶液で処理して得られる抽出物の中和物である[5]に記載の方法、
[7] 非エンベロープウイルス粒子を含有する中性又は塩基性の試料が、さらにヌクレアーゼ処理を施された試料である[1]~[6]のいずれかに記載の方法、
[8] 非エンベロープウイルス粒子がアデノ随伴ウイルス粒子である[1]~[7]のいずれかに記載の方法、
[9] [1]~[8]のいずれかに記載の方法を実施するためのキットであって、
(1)酸性条件でのタンパク質の溶解度を低下させる物質及び/又は酸性条件で沈殿する物質、及び
(2)酸性の溶液、
を含有することを特徴とするキット、
[10] さらに、中和液を含有することを特徴とする[9]に記載のキット、
に関する。
本発明の非エンベロープウイルス粒子の製造方法は、
(a)非エンベロープウイルス粒子を含有する中性又は塩基性の試料に、酸性条件でのタンパク質の溶解度を低下させる物質及び/又は酸性条件で沈殿する物質を添加する工程、
(b)前記物質を添加した試料を酸性にする工程、及び
(c)前記(b)工程で生じた沈殿物を除去し、非エンベロープウイルス粒子を含む画分を取得する工程、
を含むことを特徴とする。
本発明のキットは、上記(1)記載の非ウイルスエンベロープウイルス粒子を製造する方法を実施するためのキットであって、(1)酸性条件でのタンパク質の溶解度を低下させる物質及び/又は酸性条件で沈殿する物質、及び(2)酸性の溶液、を含有することを特徴とする。当該酸性条件で沈殿する物質は、上記(1)本発明の非エンベロープウイルス粒子の製造方法で説明した、酸性条件でのタンパク質の溶解度を低下させる物質及び/又は酸性条件で沈殿する物質が好適に使用できる。また、当該酸性の溶液は、上記(1)本発明の非エンベロープウイルス粒子の製造方法で説明した、酸性条件でのタンパク質の溶解度を低下させる物質及び/又は酸性条件で沈殿する物質が添加された試料のpHを酸性にする工程で使用できるものであれば特に限定はされない。本発明のキットについては、さらに中和液を含んでいてもよい。当該中和液は、好適には塩基性の溶液やpH緩衝液である。塩基性の溶液としては、特に限定はされないが、例えば水酸化ナトリウム水溶液、水酸化カリウム溶液などが挙げられる。また、pH緩衝液としては、特に限定はされないが、例えばTris-HCl溶液などが挙げられる。さらに、非エンベロープウイルス産生細胞からの抽出物の調製に使用される試薬や、非エンベロープウイルス粒子の形成に必須な要素を供給する核酸を含むプラスミド、非エンベロープウイルス粒子に封入される核酸を含むプラスミド等を含むキットとしてもよい。
(1)rAAV製造用細胞の播種
組織培養処理T225フラスコ(コーニング社製)に、10%FBS(ギブコ社製)を含むDMEM(シグマ社製)に懸濁した293T/17細胞を播種した。その後、37℃、CO2濃度5%(v/v)のCO2インキュベーターで2日間培養した。
実施例1-(1)で得られた細胞に、リン酸カルシウム法を用いて、AAV2のRepタンパク質及びAAV5のCapタンパク質をコードする配列を含むpRC5 Vector(タカラバイオ社製)、アデノウイルスのE2A、VA、V4をコードする配列を含むpHelper Vector(タカラバイオ社製)、AAV2ゲノムの2つのITRの間に蛍光タンパク質ZsGreen1の発現カセットを含むpAAV-ZsGreen1 Vector(タカラバイオ社製)をそれぞれ25μgずつトランスフェクションした。トランスフェクション6時間後、培地を完全に除去し、2%胎仔ウシ血清(FBS)を含むDMEMをフラスコ1枚当たり40mL添加し、37℃、CO2濃度5%(v/v)のCO2インキュベーターで2日間培養した。
実施例1-(2)で得られた各フラスコに、0.5M EDTA(和光純薬社製)0.5mLを添加し、室温で数分間静置することで細胞を剥離させた。その後、溶液ごと細胞を回収し、1,750×g、4℃、10分間遠心後、上清を除去した。細胞ペレットを2mLのクエン酸バッファー(38.1mM クエン酸、74.8mM クエン酸ナトリウム、75mM 塩化ナトリウム、100mM 塩化マグネシウム、pH5)で再懸濁し、15秒間ボルテックスミキサーで混和を行い、室温で5分間静置、再び15秒間ボルテックスミキサーで混和を行った。その後、4,000×g、4℃、10分間遠心を行い、上清を回収した。得られた上清に1/10量の2M Tris-HCl(pH9.5)を添加した溶液を抽出液とした。
実施例1-(3)で得られた抽出液に、1/100量の20U/μL Cryonase(登録商標) Cold-active Nuclease(タカラバイオ社製)を添加後(終濃度0.2U/μL)、37℃で1時間反応させた。サンプルを5等分し、サンプル1には1/10量の1M クエン酸のみを添加した。サンプル2には、1/10量の5%(v/v) デオキシコール酸ナトリウムのみを添加した。サンプル3には1/10量の5%(v/v) デオキシコール酸ナトリウムを添加し、37℃で30分間静置後、さらに1/40量の1M クエン酸を添加した。サンプル4には1/10量の5%(v/v) デオキシコール酸ナトリウムを添加し、37℃で30分間静置後、さらに1/20量の1M クエン酸を添加した。サンプル5には1/10量の5%(v/v) デオキシコール酸ナトリウムを添加し、37℃で30分間静置後、さらに1/10量の1M クエン酸を添加した。それぞれのサンプルについて、pHを測定した結果を表1に示す。
実施例1-(3)で得られた抽出液及び実施例1-(4)で得られた5種の濃縮前rAAV5に、それぞれ等量の5×サンプルバッファー(タカラバイオ社製)を加えて混合し、95℃、10分間処理を行った。各処理液4μLを4-12%ポリアクリルアミドゲルにアプライし電気泳動を行った。泳動終了後、ゲルを適当量のOriole蛍光ゲルステイン溶液(バイオラッド社製)に浸し、遮光して90分間振とうした。振とう後のゲルをLuminoshot400(タカラバイオ社製)にて撮影した。その結果を図1に示す。図1中、レーンCは抽出液、レーン1~5は濃縮前rAAV5(サンプル1~5)である。
実施例1-(3)で得られた抽出液及び実施例1-(4)で得られた5種の濃縮前rAAV5のゲノム力価を測定した。ゲノム力価測定には、AAVpro(登録商標) Titration Kit(for Real Time PCR) Ver.2(タカラバイオ社製)を使用し、反応液の調製等の操作はキット添付の説明書に従った。得られたゲノム力価のデータを用いて、下記式に従って総ゲノム力価を算出した。
(1)デオキシコール酸ナトリウム及びクエン酸の添加量の検討
実施例1-(1)~(3)と同様の方法で抽出液を取得した。得られた抽出液に、1/100量の20U/μL Cryonase(登録商標) Cold-active Nucleaseを添加後(終濃度0.2U/μL)、37℃で1時間反応させた。サンプルを4等分し、1/10量又は1/20量の5%(v/v) デオキシコール酸ナトリウムを添加し、37℃で30分間静置後、さらに1/20量又は1/30量の1M クエン酸を添加した。それぞれのサンプルについて、pHを測定した結果を表2に示す。
実施例2-(2)で得られた濃縮前rAAV5を100kDaKの限外ろ過膜(メルクミリポア社製)に入れ、2,000×g、4℃、10分間遠心し、そのろ過液を廃棄した。さらに「限外ろ過膜に10mLのD-PBSを添加後、懸濁し、上記の条件で遠心し、そのろ過液を廃棄する操作(洗浄操作)」を5回繰り返し、最終濃縮液を精製rAAV5(サンプルa~d)とした。これら一連の操作により、低分子量の夾雑物を除去するとともにrAAV5を10倍に濃縮した。
実施例1-(5)と同様の方法で実施例2-(2)で得られた精製rAAV5の純度を確認した。その結果を図3に示す。図3中、レーンMはProtein Molecular Weight Marker (Broad)(タカラバイオ社製、上から200、116、97、66、44、29、20kDa)、レーンa~dは精製rAAV5(サンプルa~d)である。
(1)rAAV製造用細胞の播種
実施例1-(1)と同様の方法でrAAV製造用細胞を播種した。
実施例1-(2)と同様の方法でrAAV製造用プラスミドを実施例3-(1)で得られた細胞にトランスフェクションした。ただし、調製するrAAVのセロタイプに合わせて、pRC5 Vectorの代わりに、pRep-Cap1 AAV Helper Plasmid(Applied Viromics社製)、pRC2-mi342 Vector(タカラバイオ社製)、pRep-Cap3 AAV Helper Plasmid(Applied Viromics社製)、pRC6 Vector(タカラバイオ社製)を使用した。
実施例1-(3)と同様の方法で各セロタイプのrAAVの抽出液を取得した。
実施例3-(3)で得られた各抽出液に、1/100量の20U/μL Cryonase(登録商標) Cold-active Nucleaseを添加後(終濃度0.2U/μL)、37℃で1時間反応させた。その後、各サンプルに1/10量の5%(v/v) デオキシコール酸ナトリウムを添加し、37℃で30分間以上静置後、さらに1/20量の1M クエン酸を各サンプルに添加した。それぞれのサンプルについて、pHを測定したところ、pH4~6であった。
実施例3-(4)で得られた濃縮前rAAVを100kDaKの限外ろ過膜に入れ、2,000×g、4℃、5分間以上遠心し、そのろ過液を廃棄した。さらに「限外ろ過膜に14mLのD-PBSを添加後、懸濁し、上記の条件で遠心し、そのろ過液を廃棄する操作(洗浄操作)」を5回繰り返し、最終濃縮液を精製rAAVとした。これら一連の操作により、低分子量の夾雑物を除去するとともにrAAVを3~5倍に濃縮した。
実施例1-(5)と同様の方法で各セロタイプのrAAVの抽出液(レーンC)、濃縮前rAAV(レーンPre)及び精製rAAV(レーンF)の純度を確認した。その結果を図4に示す。
実施例1-(6)と同様の方法で各セロタイプのrAAVのゲノム力価を測定し、各工程のrAAVの総ゲノム力価を算出した。抽出液の総ゲノム力価を100とした時の相対値(回収率)を表3に示す。
(1)濃縮前rAAV6の取得
実施例3-(1)~(4)と同様の方法で濃縮前rAAV6を取得した。
濃縮前rAAV6を3等分し、サンプル1はポリエチレングリコール添加無しサンプルとした。サンプル2には終濃度0.4%(w/v)になるように、ポリエチレングリコール(PEG)-6000(SIGMA社製)溶液を添加した。サンプル3に終濃度0.8%(w/v)になるように、PEG-6000溶液を添加した。それぞれのサンプルを100kDaKの限外ろ過膜に入れ、2,380×g、室温、10分間以上遠心し、ろ過液を廃棄した。さらに「限外ろ過膜にそれぞれの終濃度のポリエチレングリコールを含有した10~14mLのPBSを添加後、懸濁し、上記の条件で遠心し、そのろ過液を廃棄する操作(洗浄操作)」を3回繰り返した。サンプル2とサンプル3だけPBSによる洗浄操作を追加で行い、最終濃縮液を精製rAAV6とした。これら一連の操作により、低分子量の夾雑物を除去するとともにrAAV6を6~12倍に濃縮した。
実施例1-(6)と同様の方法で各rAAV6のゲノム力価を測定し、各工程のrAAV6の総ゲノム力価を算出した。抽出液の総ゲノム力価を100とした時の相対値(回収率)を表4に示す。
(1)rAAV6抽出液の取得
実施例3-(1)~(3)と同様の方法でrAAV6抽出液を取得した。
実施例5-(1)で得られたrAAV6抽出液に、1/100量の20U/μL Cryonase(登録商標) Cold-active Nucleaseを添加後(終濃度0.2U/μL)、37℃で1時間反応させた。サンプルの一部を3等分した後、各サンプルに1/10量の5%(v/v) デオキシコール酸ナトリウムを添加し、37℃で30分間以上静置後、さらに1/20量の1M クエン酸、1/20量の1M 塩酸、又は1/20量の1M 酢酸をそれぞれ添加した。それぞれのサンプルのpHを測定し、酸性であることを確認した。それぞれのサンプルの条件を表5に示す。
実施例1-(5)と同様の方法で、濃縮前rAAV6の純度を確認したところ、酸性の溶液の種類に関わらず同等の純度を示した。また、実施例1-(6)と同様の方法で濃縮前rAAV6のゲノム力価を測定し、回収率を算出したところ、酸性の溶液の種類に関わらず同等の回収率を示した。これらのことは、rAAVと夾雑タンパク質の混合物にデオキシコール酸ナトリウム及び酸性の溶液を添加する際に、どのような種類の酸性の溶液を使用しても、夾雑タンパク質を沈殿させ、除去できることを示す。
(1)rAAV2抽出液の取得
実施例3-(1)~(3)と同様の方法でrAAV2抽出液を取得した。
実施例6-(1)で得られたrAAV2抽出液に、1/100量の20U/μL Cryonase(登録商標) Cold-active Nucleaseを添加後(終濃度0.2U/μL)、37℃で1時間反応させた。サンプルの一部を3等分した後、各サンプルに1/10量の5%(v/v) デオキシコール酸ナトリウム、5% (v/v)ケノデオキシコール酸ナトリウム、又は5%(v/v) コール酸ナトリウムを添加し、37℃で30分間以上静置後、さらに1/20量の1M クエン酸をそれぞれ添加した。それぞれのサンプルのpHを測定し、酸性であることを確認した。それぞれのサンプルの条件を表6に示す。
実施例6-(2)で得られた濃縮前rAAV2を100kDaの限外ろ過膜に入れ、2,000×g、4℃、5分間以上遠心し、そのろ過液を廃棄した。さらに「限外ろ過膜に14mLのD-PBSを添加後、懸濁し、上記の条件で遠心し、そのろ過液を廃棄する操作(洗浄操作)」を5回繰り返し、最終濃縮液を精製rAAV2とした。これら一連の操作により、低分子量の夾雑物を除去するとともにrAAV2を濃縮した。
実施例1-(5)と同様の方法でrAAV2抽出液(レーンC)、濃縮前rAAV2(レーンPre)及び精製rAAV2(レーンF)の純度を確認した。その結果を図5に示す。
Claims (10)
- 非エンベロープウイルス粒子の製造方法であって、
(a)非エンベロープウイルス粒子を含有する中性又は塩基性の試料に、酸性条件でのタンパク質の溶解度を低下させる物質及び/又は酸性条件で沈殿する物質を添加する工程、
(b)前記物質を添加した試料を酸性にする工程、及び
(c)前記(b)工程で生じた沈殿物を除去し、非エンベロープウイルス粒子を含む画分を取得する工程、
を含む方法。 - (c)工程の後にさらに、(d)非エンベロープウイルス粒子を精製する工程、を含むことを特徴とする請求項1に記載の方法。
- 酸性条件でのタンパク質の溶解度を低下させる物質及び/又は酸性条件で沈殿する物質が、界面活性剤である請求項1に記載の方法。
- 酸性にする工程が、酸性の溶液を添加する工程である請求項1~3のいずれか1項に記載の方法。
- 非エンベロープウイルス粒子を含有する中性又は塩基性の試料が、非エンベロープウイルス産生細胞の溶解物もしくは破砕物、非エンベロープウイルス粒子を含有する非エンベロープウイルス産生細胞の培養液上清、または非エンベロープウイルス産生細胞から得られる抽出物である請求項1~4のいずれか1項に記載の方法。
- 非エンベロープウイルス粒子を含有する中性又は塩基性の試料が、非エンベロープウイルス産生細胞を酸性の溶液で処理して得られる抽出物の中和物である請求項5に記載の方法。
- 非エンベロープウイルス粒子を含有する中性又は塩基性の試料が、ヌクレアーゼ処理を施された試料である請求項1~6のいずれか1項に記載の方法。
- 非エンベロープウイルス粒子がアデノ随伴ウイルス粒子である請求項1~7のいずれか1項に記載の方法。
- 請求項1~8のいずれか1項に記載の方法を実施するためのキットであって、
(1)酸性条件でのタンパク質の溶解度を低下させる物質及び/又は酸性条件で沈殿する物質、及び
(2)酸性の溶液、
を含有することを特徴とするキット。 - さらに、中和液を含有することを特徴とする請求項9に記載のキット。
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP16735082.6A EP3243904A4 (en) | 2015-01-09 | 2016-01-08 | Method for producing non-enveloped viral particles |
US15/540,606 US10415020B2 (en) | 2015-01-09 | 2016-01-08 | Method for producing non-enveloped viral particles |
CN201680005210.7A CN107109374B (zh) | 2015-01-09 | 2016-01-08 | 用于生产无包膜病毒颗粒的方法 |
JP2016568750A JP6616329B2 (ja) | 2015-01-09 | 2016-01-08 | 非エンベロープウイルス粒子の製造方法 |
KR1020177021403A KR102493350B1 (ko) | 2015-01-09 | 2016-01-08 | 비엔벨로프 바이러스 입자의 제조방법 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2015-002883 | 2015-01-09 | ||
JP2015002883 | 2015-01-09 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2016111343A1 true WO2016111343A1 (ja) | 2016-07-14 |
Family
ID=56356028
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2016/050431 WO2016111343A1 (ja) | 2015-01-09 | 2016-01-08 | 非エンベロープウイルス粒子の製造方法 |
Country Status (6)
Country | Link |
---|---|
US (1) | US10415020B2 (ja) |
EP (1) | EP3243904A4 (ja) |
JP (1) | JP6616329B2 (ja) |
KR (1) | KR102493350B1 (ja) |
CN (1) | CN107109374B (ja) |
WO (1) | WO2016111343A1 (ja) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023182428A1 (ja) * | 2022-03-24 | 2023-09-28 | 国立大学法人 東京大学 | ウイルスの精製方法 |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2023532544A (ja) * | 2020-07-01 | 2023-07-28 | メディミューン,エルエルシー | 生物学的製剤を精製するための洗浄剤及び方法 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040110266A1 (en) * | 2002-05-17 | 2004-06-10 | Chiorini John A. | Scalable purification of AAV2, AAV4 or AAV5 using ion-exchange chromatography |
WO2013192604A1 (en) * | 2012-06-22 | 2013-12-27 | Takeda Vaccines (Montana), Inc. | Purification of virus like particles |
WO2015005430A1 (ja) * | 2013-07-11 | 2015-01-15 | タカラバイオ株式会社 | 非エンベロープウイルスの製造方法 |
Family Cites Families (20)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0245949B1 (en) | 1986-04-09 | 1997-10-29 | Eli Lilly And Company | A method of using eukaryotic expression vectors comprising the bk virus enhancer |
CA1318619C (en) | 1986-04-09 | 1993-06-01 | Brian William Grinnell | Method of using eukaryotic expression vectors comprising the bk virus enhancer |
DE3622089A1 (de) | 1986-07-02 | 1988-01-07 | Krueger Gmbh & Co Kg | Viruzides mittel mit breitbandwirkung |
ES2210391T3 (es) | 1995-08-03 | 2004-07-01 | Avigen, Inc. | Sistema auxiliar de alta eficacia para la produccion de vectores aav. |
CA2625279A1 (en) * | 1995-08-30 | 1997-03-06 | Genzyme Corporation | Chromatographic purification of adenovirus and aav |
US6004797A (en) | 1995-11-09 | 1999-12-21 | Avigen, Inc. | Adenovirus helper-free recombinant AAV Virion production |
CA2267207C (en) * | 1997-08-04 | 2008-10-28 | Tonen Corporation | Methods for detecting or assaying virus |
US7244828B2 (en) * | 1997-11-07 | 2007-07-17 | Aftab Alam | Agent for protein precipitation, a method of protein precipitation, a method of protein assay using protein precipitation agent, and a kit for protein assay |
DK1109892T3 (da) | 1998-09-04 | 2009-02-09 | Targeted Genetics Corp | Fremgangsmåder til at frembringe höjtitrede, hjælpevirusfrie præparater af frie rekombinante AAV-vektorer |
US6593123B1 (en) | 2000-08-07 | 2003-07-15 | Avigen, Inc. | Large-scale recombinant adeno-associated virus (rAAV) production and purification |
DK1506287T3 (da) * | 2002-05-14 | 2007-08-20 | Merck & Co Inc | Fremgangsmåder til oprensning af adenovirus |
JP5268890B2 (ja) | 2006-04-28 | 2013-08-21 | ザ・トラステイーズ・オブ・ザ・ユニバーシテイ・オブ・ペンシルベニア | Aavの規模適応性の製造方法 |
WO2008036899A2 (en) | 2006-09-22 | 2008-03-27 | Amgen Inc. | Methods for removing viral contaminants during protein purification |
WO2009123183A1 (ja) | 2008-03-31 | 2009-10-08 | 国立大学法人広島大学 | 抗ウイルス剤および抗ウイルス用組成物 |
ES2693194T3 (es) | 2009-06-16 | 2018-12-10 | Genzyme Corporation | Métodos mejorados para la purificación de vectores de AAV recombinantes |
WO2011074564A1 (ja) | 2009-12-15 | 2011-06-23 | タカラバイオ株式会社 | アデノウイルスベクターの製造方法 |
CN102205132B (zh) | 2011-05-12 | 2014-04-02 | 华侨大学 | 一种以重组腺相关病毒为载体的基因治疗药物的制剂处方 |
CN102260651A (zh) | 2011-06-24 | 2011-11-30 | 华中科技大学同济医学院附属协和医院 | 一种纯化病毒样颗粒蛋白的方法及其应用 |
JP6093358B2 (ja) | 2012-07-06 | 2017-03-08 | タカラバイオ株式会社 | アデノ随伴ウイルスベクターの産生細胞 |
KR102245861B1 (ko) | 2013-11-29 | 2021-04-28 | 다카라 바이오 가부시키가이샤 | 아데노 수반 바이러스의 정량 방법 |
-
2016
- 2016-01-08 WO PCT/JP2016/050431 patent/WO2016111343A1/ja active Application Filing
- 2016-01-08 KR KR1020177021403A patent/KR102493350B1/ko active IP Right Grant
- 2016-01-08 US US15/540,606 patent/US10415020B2/en active Active
- 2016-01-08 CN CN201680005210.7A patent/CN107109374B/zh active Active
- 2016-01-08 JP JP2016568750A patent/JP6616329B2/ja active Active
- 2016-01-08 EP EP16735082.6A patent/EP3243904A4/en active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040110266A1 (en) * | 2002-05-17 | 2004-06-10 | Chiorini John A. | Scalable purification of AAV2, AAV4 or AAV5 using ion-exchange chromatography |
WO2013192604A1 (en) * | 2012-06-22 | 2013-12-27 | Takeda Vaccines (Montana), Inc. | Purification of virus like particles |
WO2015005430A1 (ja) * | 2013-07-11 | 2015-01-15 | タカラバイオ株式会社 | 非エンベロープウイルスの製造方法 |
Non-Patent Citations (3)
Title |
---|
See also references of EP3243904A4 * |
WANG DAOWEN ET AL.: "Procedures for the efficient purification of pea seed-borne mosaic virus and its genomic RNA", J. VIROL. METHOD., vol. 36, no. 3, 1992, pages 223 - 230, XP023659017, ISSN: 0166-0934 * |
ZHIRNOV O. P.: "Isolation of Matrix Protein Ml from Influenza Viruses by Acid-Dependent Extraction with Nonionic Detergent", VIROLOGY, vol. 186, no. 1, 1992, pages 324 - 330, ISSN: 0042-6822 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023182428A1 (ja) * | 2022-03-24 | 2023-09-28 | 国立大学法人 東京大学 | ウイルスの精製方法 |
Also Published As
Publication number | Publication date |
---|---|
CN107109374A (zh) | 2017-08-29 |
JP6616329B2 (ja) | 2019-12-04 |
EP3243904A4 (en) | 2018-06-27 |
KR102493350B1 (ko) | 2023-01-27 |
US10415020B2 (en) | 2019-09-17 |
CN107109374B (zh) | 2021-04-06 |
JPWO2016111343A1 (ja) | 2017-10-19 |
EP3243904A1 (en) | 2017-11-15 |
KR20170100659A (ko) | 2017-09-04 |
US20180273907A1 (en) | 2018-09-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6521965B2 (ja) | 非エンベロープウイルス粒子の製造方法 | |
JP6322605B2 (ja) | 組換えaavベクターの高力価ヘルパーなし調製物を生成するための方法 | |
Robert et al. | Manufacturing of recombinant adeno‐associated viruses using mammalian expression platforms | |
BR112019025792A2 (pt) | agentes de aperfeiçoamento para transfecção de células melhoradas e/ou produção de vetor raav | |
JP2018518164A (ja) | 多段階陰イオン交換クロマトグラフィーによる組換えアデノ随伴ウイルス粒子の精製 | |
US10294452B2 (en) | Lysis, extraction and purification of adeno-associated virus and adenovirus from host cells | |
US20220162642A1 (en) | Plasmid system | |
JP5861001B2 (ja) | 非エンベロープウイルスの製造方法 | |
CN108085301B (zh) | 从宿主细胞提取和纯化腺相关病毒和腺病毒的方法及其组分和试剂盒 | |
JP6616329B2 (ja) | 非エンベロープウイルス粒子の製造方法 | |
JP2023525119A (ja) | アデノ随伴ウイルス粒子またはアデノウイルスを精製するための方法および組成物 | |
WO2023074877A1 (ja) | 細胞添加用組成物 | |
Nagy et al. | Engineered CHO cells as a novel AAV production platform for gene therapy delivery | |
Chen et al. | A simple, two-step, small-scale purification of recombinant adeno-associated viruses | |
WO2023085382A1 (ja) | ウイルスの精製方法 | |
JP2023513892A (ja) | Dna増幅方法 | |
WO2022045055A1 (ja) | pHの違いによる非エンベロープウイルスベクター粒子の調製方法 | |
JP2024016295A (ja) | 二価の陽イオンを利用した非エンベロープウイルスの製造方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 16735082 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2016568750 Country of ref document: JP Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 15540606 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
REEP | Request for entry into the european phase |
Ref document number: 2016735082 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref document number: 20177021403 Country of ref document: KR Kind code of ref document: A |