Method for producing virus particles

Abstract

The present invention addresses the problem of providing a method for producing a virus to obtain high-purity virus particles, said method being more efficient than prior-art methods. Specifically, the present invention is a method for producing whole virus particles, the method including a step for refining whole virus particles from a virus particle mixed solution including hollow virus particles, intermediate virus particles, and whole virus particles, wherein the method includes: a step (a) for rotating a zonal rotor at low speed and arranging the virus particle mixed solution, a liquid (liquid L) having a lower density than the whole virus particles, and a liquid (liquid H1) having a higher density than the liquid L in the stated order from the rotor rotating-shaft side toward the outside; a step (b) for operating the zonal rotor in a centrifugation mode after step (a) and separating the hollow virus particles, the intermediate virus particles, and the whole virus particles; and a step (c) for retrieving the contents of the zonal rotor after step (b) while fractionating said contents, and recovering a fraction that contains the whole virus particles.

Description

Technical Field

[0001] The present invention relates to a method for purifying or producing virus particles. More specifically, it relates to a method for purifying or producing a large amount of virus particles.

Background Art

[0002] For the purpose of treating diseases, gene therapy that administers genes or cells into which genes have been introduced into the human body is one of the important treatment methods for treating intractable diseases. As a method for introducing genes into mammalian cells for gene therapy, currently, a biological method using a viral vector has become mainstream. A viral vector is a carrier for integrating a gene to be introduced for treatment into a viral strain that has lost or partially lost its replication ability and growth ability, and efficiently introducing and expressing the gene into cells. Viruses from which viral vectors are derived include enveloped viruses (viruses with envelopes) such as retroviruses, lentiviruses, herpesviruses, and Sendai viruses, and non-enveloped viruses (viruses without envelopes) such as adenoviruses and adeno-associated viruses (AAV). Among them, AAV is used for gene therapy for treating various diseases because it can infect many types of cells, has no pathogenicity to humans, and the virus particles are physically stable.

[0003] AAV vectors have been used as a means of gene delivery to target cells because they enable efficient gene transfer for stable intracellular expression of a target gene. Recently, clinical trials of AAV vector-based gene therapy have been conducted for various genetic diseases, such as Parkinson's disease, cystic fibrosis, rheumatoid arthritis, lipoprotein lipase deficiency, α1-antitrypsin deficiency, Duchenne muscular dystrophy (DMD), Leber's congenital amaurosis, hyperchloremia, hemophilia A, and hemophilia B. To safely and effectively perform gene therapy using AAV vectors, it is extremely important to overcome immune responses to the AAV vector (Non-Patent Document 1).

[0004] There have been various reports on gene therapy using AAV vectors. For example, in gene therapy for hemophilia A and hemophilia B using AAV2, AAV8, and AAV10 vectors, it has been reported that increased liver enzyme activity and AAV capsid-specific T cell activation were detected, and that the activity of Factor VIII and Factor IX was also reduced (Non-Patent Document 2, Non-Patent Document 3). On the other hand, it has been reported that when gene therapy for hemophilia was performed using a low-dose AAV5 vector, most patients did not experience an increase in liver enzyme activity, T cell activation was minimized, and blood coagulation factors were maintained (Non-Patent Document 1, Non-Patent Document 4). These findings suggest that the cellular immune response to AAV capsids induces the destruction of AAV vector-transduced hepatocytes. Therefore, for safe systemic administration of AAV vectors, it is important to use vectors with high transduction efficiency and reduce the dosage.

[0005] So far, as methods for purifying AAV vectors, chromatography using an ion exchange column or an affinity purification column, a method including a filtration step by tangential flow filtration (TFF), and methods combining these chromatography and TFF steps with sucrose density gradient centrifugation (Patent Document 1) or cesium chloride equilibrium density gradient centrifugation (Patent Document 2) have been reported. These methods have shown certain effects in removing impurities from the AAV vector fraction. However, in the method using ion exchange chromatography, it is impossible to completely remove hollow particles (AAV particles without a genome) and intermediate particles (AAV particles containing a fragment of the genome rather than the full-length genome). In addition, the method combining ion exchange chromatography and density gradient centrifugation is effective for removing impurities and separating intact particles (AAV particles containing the full-length genome) that do not contain hollow particles or intermediate particles, but the purification process of intact particles is complicated. Furthermore, the density gradient centrifugation reported so far has a long centrifugation process. Especially when using the cesium chloride equilibrium density gradient centrifugation method, if the AAV vector is contacted with cesium chloride for a long time, there has been a problem that the transduction efficiency of the AAV vector decreases (Non-Patent Documents 5 and 6). In addition, the conventional ultracentrifugation purification method has a limitation on the sample amount that can be handled at one time, and there are problems in large-scale purification. Recently, due to the progress of marketization and the shift of the starting material of the purified AAV vector from cell pellets to culture supernatants with the change of serotypes used, a purification method capable of handling a large amount of starting materials is required.

[0006] Based on the current situation regarding AAV vectors as described above, it is necessary to develop a new method for mass-producing vectors with high safety and high transduction efficiency.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

[0008] [Non-Patent Document 1] Muhuri et al., J Clin Invest. 131:e143780 doi:10.1172 / JCI143780. 2021. [Non-Patent Document 2] High et al., N Engl J Med. 381:455-464 2019. [Non-Patent Document 3] Batty et al., Hemasphere 5:e540 2021. [Non-Patent Document 4] Rangarajan et al., N Engl J Med. 377:2519-2530 2017. [Non-Patent Document 5] Hinderer et al., Hum Gene Ther. 29:285-298 2018. [Non-Patent Document 6] Auricchio et al., Hum Gene Ther. 12:71-76 2001. [Summary of the Invention] [Problems to be Solved by the Invention]

[0009] In view of the above circumstances, an object of the present invention is to provide a method for separating a high-purity and large amount of virus particles in a shorter time than conventional methods. [Means for Solving the Problems]

[0010] In order to purify intact AAV particles that maintain high purity and high transduction efficiency, the inventors investigated conditions such as the preparation conditions of the density gradient and the centrifugation time in cesium chloride equilibrium density gradient centrifugation. Specifically, when the inventors loaded the culture supernatant containing AAV particles onto a zonal rotor with two different concentrations of cesium chloride solutions and performed ultracentrifugation, they succeeded in separating / purifying intact particles that maintained high purity and high transduction efficiency from a larger volume of sample (starting sample containing AAV) in a shorter centrifugation time (about 4 to 5 hours).

[0011] That is, the present invention is as follows (1) to (11). (1) A method for producing intact virus particles, comprising a step of purifying intact virus particles from a virus particle mixture containing hollow virus particles, intermediate virus particles, and intact virus particles, a step (a) of rotating the zonal rotor at a low speed and arranging the virus particle mixture, a liquid (liquid L) having a density lower than that of the intact virus particles, and a liquid (liquid H1) having a density higher than that of the liquid L in this order from the rotation axis side of the rotor toward the outside; a step (b) of operating the zonal rotor in an ultracentrifugation mode after the step (a) to separate the hollow virus particles, the intermediate virus particles, and the intact virus particles; a step (c) of taking out the contents of the zonal rotor after the step (b) while fractionating and recovering the fraction containing the intact virus particles; The production method comprising the above. (2) The production method according to (1) above, wherein the step (a) is a step of rotating the zonal rotor at a low speed and arranging a liquid (liquid B) having a density lower than that of the liquid L and the liquid H1, the virus particle mixture, the liquid L, and the liquid H1 in this order from the rotation axis side of the rotor toward the outside. (3) In the step (c), the zonal rotor after the step (b) is rotated at a low speed, and a liquid (liquid H2) having a density higher than that of the liquid H1 is introduced from the radially outer side of the zonal rotor, so as to sequentially extrude the content of the zonal rotor and take it out while fractionating from the rotation axis side of the zonal rotor. The production method according to (1) or (2) above. (4) The production method according to (3) above, wherein the liquid L, the liquid H1, and the liquid H2 contain cesium chloride (CAS No.; 7647-17-8), iodixanol (CAS No.; 92339-11-2), iohexol (CAS No.; 66108-95-0), amidotrizoic acid (CAS No.; 737-31-5), or metrizamide (CAS No.; 31112-62-6). (5) The production method according to (1) above, wherein the virus particles are adeno-associated virus particles and the density of the liquid L is 1.21 to 1.38 g / mL. (6) The production method according to (1) above, wherein the virus particles are adeno-associated virus particles and the density of the liquid H1 is 1.39 g / mL or more. (7) A virus particle mixture containing hollow virus particles, intermediate virus particles, and complete virus particles, A liquid (liquid L) having a density lower than that of the complete virus particles, and A zonal rotor in which a liquid (liquid H1) having a density higher than that of the liquid L is arranged in this order from the rotation axis side toward the outside. (8) The zonal rotor according to (7) above, wherein a liquid (liquid B) having a density lower than that of the liquid L and the liquid H is arranged on the rotation axis side of the virus particle mixture. (9) The zonal rotor according to (7) or (8) above, wherein the liquid L, the liquid H1, and the liquid H2 contain cesium chloride (CAS No.; 7647-17-8), iodixanol (CAS No.; 92339-11-2), iohexol (CAS No.; 66108-95-0), amidotrizoic acid (CAS No.; 737-31-5), or metrizamide (CAS No.; 31112-62-6). (10) The zonal rotor according to (7) above, wherein the virus particles are adeno-associated virus particles and the density of the liquid L is 1.21 to 1.38 g / mL. (11) The zonal rotor according to (7) above, wherein the virus particles are adeno-associated virus particles and the density of the liquid H1 is 1.39 g / mL or more. In this specification, the symbol "~" indicates a numerical range including the values on both its left and right.

Advantages of the Invention

[0012] The present invention provides a method for producing complete virus particles with a high transduction efficiency. As a result, it becomes possible to improve the safety and effectiveness of gene therapy using virus vectors.

Brief Description of the Drawings

[0013]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Mode for Carrying Out the Invention

[0014] Hereinafter, the mode for carrying out the present invention will be described. The first embodiment is a method for producing intact virus particles, which includes forming a density gradient due to a solute in a solution in a zonal rotor using two solutions of different concentrations (densities), and separating and purifying the intact virus particles from hollow particles and intermediate virus particles by the density gradient. More specifically, the first embodiment is a method for producing intact virus particles, which includes a step of purifying intact virus particles from a virus particle mixture containing hollow virus particles, intermediate virus particles, and intact virus particles. Step (a) of rotating the zonal rotor at a low speed and arranging, in this order from the rotation axis side of the rotor toward the outside, the virus particle mixture, a liquid (liquid L) having a lower density than the intact virus particles, and a liquid (liquid H1) having a higher density than the liquid L. Step (b) of operating the zonal rotor in an ultracentrifugation mode after step (a) to separate the hollow virus particles, the intermediate virus particles, and the intact virus particles. Step (c) of fractionally removing the contents of the zonal rotor after step (b) and collecting the fraction containing the intact virus particles. The method for producing the intact virus particles includes the above steps. Optionally, step (a) may be a step of rotating the zonal rotor at a low speed and arranging, in this order from the rotation axis side of the rotor toward the outside, a liquid (liquid B) having a lower density than the liquid L and the liquid H1, the virus particle mixture, the liquid L, and the liquid H1, that is, in the order of liquid B, virus particle mixture, liquid L, liquid H1.

[0015] Here, a complete virus particle refers to a virus particle in which a full-length genome (in the case of a virus vector, a full-length vector genome containing a foreign gene) is packaged and which has the ability to infect target cells. An intermediate virus particle refers to a virus particle in which a part of the full-length genome is packaged but which has lost the ability to infect target cells or has a significantly reduced ability to infect target cells. A hollow virus particle refers to a virus particle that contains almost no genome and has almost lost the ability to infect target cells. In order to ensure safety and the like, there is a strong demand for preparing highly pure complete virus particles for virus particles used in gene therapy such as AAV vectors, and the need to separate complete virus particles from intermediate virus particles and hollow virus particles is extremely high.

[0016] In this embodiment, step (a) is a step of injecting a virus particle mixture into a zonal rotor while creating a density gradient using a solution (liquid L) having a lower density than the complete virus particle and a solution (liquid H1) having a higher density than liquid L in the zonal rotor. Step (a) can be carried out, for example, by rotating the zonal rotor at a low speed (for example, about 1,000 rpm to 4,000 rpm; about 100×g to 1,600×g for about 30 minutes to 1 hour) and loading the virus particle mixture, liquid L, and liquid H1 in this order, or a liquid (liquid B) having a lower density than the liquid L and the liquid H, the virus particle mixture, the liquid L, and the liquid H1 in this order (that is, in the order of liquid B, virus particle mixture, liquid L, liquid H1) into the zonal rotor. After the end of the low-speed rotation operation, a linear density gradient is created between the density of liquid L and the density of liquid H1 from the rotation axis side to the outside in the rotor. The virus particles stop on the rotation axis side in the rotor. When the virus particle is an AAV vector, the densities of the complete virus particle, the intermediate virus particle, and the hollow virus particle are approximately 1.39 g / cm 3 ~1.41 g / cm 3 respectively, with some error, approximately 1.35 g / cm 3 ~1.38 g / cm3 Degree, approximately 1.3 g / cm 3 3 to 1.35 g / cm 3 Degree. In order to separate complete virus particles, intermediate virus particles, and hollow virus particles, the density of solution L is, for example, 1.21 g / cm 3 ~1.38 g / cm 3 Degree, or 1.22 g / cm 3 ~1.29 g / cm 3 Degree may also be sufficient. Further, the density of solution H1 is desirably higher than the density of complete virus particles, for example, 1.39 g / cm 3 Degree or higher may be sufficient. Note that as the density of solution H1 increases, the resulting density gradient becomes steep, and although complete virus particles are concentrated and recovered, the possibility of contamination with intermediate virus particles and the like also increases. Therefore, the density of solution H1 is, for example, 1.39 g / cm 3 ~1.45 g / cm 3 Degree may be prepared.

[0017] In the present embodiment, the ratio of the volumes of liquid L and liquid H1 (the total volume of liquid L and liquid H1; liquid L + liquid H1) injected into the zonal rotor to the volume of the virus particle mixed solution (the volume of liquid L + liquid H1: the volume of the virus particle mixed solution) is not particularly limited, but may be, for example, about 1:1 to 4, about 1:1 to 3, or about 1:1 to 2. Further, the ratio of the volume of liquid H1 to the volume of liquid L (the volume of liquid H1: the volume of liquid L) injected into the zonal rotor is not particularly limited, but may be, for example, about 1:1 to 3, about 1:1 to 2, or about 1:1 to 1.5. Note that the total volume of the virus particle mixed solution, liquid L, and liquid H1 injected into the zonal rotor depends on the rotor volume of the zonal rotor used, and those skilled in the art can appropriately select an optimal injection amount.

[0018] In step (b) of the present embodiment, the zonal rotor after step (a) is operated in an ultracentrifugation mode (high-speed rotation) to separate the hollow virus particles, the intermediate virus particles, and the complete virus particles. The ultracentrifugation mode in step (b) is not particularly limited, but the rotation speed may be, for example, about 30,000 rpm to about 40,000 rpm (about 90,000×g to 160,000×g). The operation time in the ultracentrifugation mode may be, for example, about 3 hours to about 10 hours, or about 4 hours to about 6 hours.

[0019] In step (c) of the present embodiment, the contents of the zonal rotor after step (b) are taken out while fractionating, and a fraction containing the complete virus particles is recovered. Here, those skilled in the art can easily select a method for taking out the contents of the zonal rotor to the outside of the rotor. For example, a liquid (liquid H2) having a higher density than liquid H1 is introduced from the radially outer side of the zonal rotor, and the contents of the zonal rotor are sequentially extruded to take out the contents to the outside of the rotor. The contents extruded to the outside of the rotor can be fractionated, for example, by a fraction collector or the like at a constant volume from the side of the rotation axis of the rotor, so that the complete virus particles can be separated from the intermediate virus particles and the hollow virus particles. The contents of the zonal rotor can be extruded to the outside of the rotor by rotating the rotor at a low speed (for example, about 1,000 rpm to 4,000 rpm; about 100×g to 1,600×g) while injecting liquid H2 into the zonal rotor. The contents extruded to the outside of the rotor may be recovered while fractionating with a fraction collector or the like from the side of the rotation axis of the rotor. Here, the density of liquid H2 may be higher than that of H1.

[0020] In this embodiment, the virus particle mixture, liquid L, liquid H1, and liquid H2 may contain cesium chloride (CAS number: 7647-17-8), iodixanol (CAS number: 92339-11-2), iohexol (CAS number: 66108-95-0), amidotrizoic acid (CAS number: 737-31-5), or metrizamide (CAS number: 31112-62-6), etc. The density gradient formed in the zonal rotor may be a density gradient formed by cesium chloride, iodixanol, iohexol, amidotrizoic acid, metrizamide, etc., and preferably, it is a density gradient formed by cesium chloride. Further, in addition to water, the liquid B, virus particle mixture, liquid L, liquid H1, and liquid H2 may contain a buffer solution (for example, phosphate buffer, HEPES buffer, Tris buffer), salts, etc.

[0021] The purity (proportion in all virus particles) of the complete virus particles obtained after step (c) of this embodiment is, for example, 80% or more, preferably 90% or more, and more preferably 95% or more.

[0022] The virus (particles) in this embodiment includes, but is not limited to, wild-type viruses and viruses (virus vectors) that carry foreign genes used as vectors. Also, the type of virus is not particularly limited, and both enveloped viruses and non-enveloped viruses are included.

[0023] An enveloped virus is a virus in which the viral genome and a protein shell called a capsid are covered by a membranous structure (envelope), while a non-enveloped virus is a virus without an envelope. Examples of enveloped viruses include DNA viruses such as herpesvirus, poxvirus, and hepadnavirus, and RNA viruses such as flavivirus, togavirus, coronavirus, orthomyxovirus, paramyxovirus, rhabdovirus, bunyavirus, and retrovirus. Examples of non-enveloped viruses include DNA viruses such as adenovirus, adeno-associated virus (AAV), and papillomavirus, and RNA viruses such as picornavirus, calicivirus, norovirus, and rotavirus. A preferred virus in this embodiment is adeno-associated virus (including an adeno-associated virus vector).

[0024] This embodiment will be further described by taking as an example the case of producing complete virus particles of an adeno-associated virus vector (AAV vector). As the density gradient medium, for example, cesium chloride can be used. As the liquid L, for example, a cesium chloride solution of about 25 wt% to 28 wt% (1.22 g / cm 3 ~1.29 g / cm 3 about), as the liquid H1, for example, a cesium chloride solution of about 40 wt% to 42 wt% (1.42 g / cm 3 ~1.45 g / cm 3Prepare a cesium chloride solution of a certain concentration (e.g., P32ZT or P35ZT; both rotors have a maximum diameter of 24 cm for the rotor lid, a maximum inner diameter of 17.78 cm, and a maximum rotation radius (Rmax) of 8.89 cm), and it may be injected into a zonal rotor such as Eppendorf Himac Technologies. Into the rotor, the virus particle mixture, liquid L, and liquid H1 may be injected in this order while operating at a low speed rotation (e.g., about 3,000 rpm when using P32ZT or P35ZT). By operating the rotor at a low speed rotation, a density gradient of cesium chloride solution from the density of the virus particle mixture and liquid L to the density of liquid H1 is formed from the side of the rotor rotation axis towards the outside (the above is step (a) of this embodiment). Once the density gradient of cesium chloride is formed, it may be operated at a high speed rotation (e.g., about 30,000 rpm to 35,000 rpm when using P32ZT or P35ZT) for about 4 to 5 hours. By operating in the ultracentrifugation mode of high speed rotation, the hollow virus particles, intermediate virus particles, and complete virus particles are banded in order from the side of the rotor rotation axis towards the outside (the edge side of the rotor) (the above is step (b) of this embodiment). The contents in the rotor are extruded out of the rotor in order from the solution closer to the rotor axis side while injecting a liquid H2 (e.g., about 42 wt% to 45 wt% (1.45 g / cm 3 ~1.49 g / cm 3 degree) cesium chloride solution) and operating at a low speed rotation (e.g., about 3,000 rpm when using P32ZT or P35ZT). The solution extruded out of the rotor can be collected while fractionating with a fraction collector or the like to obtain a fraction containing complete virus particles (the above is step (c) of this embodiment).

[0025] In this embodiment, the virus particle mixture (a mixture containing at least complete virus particles) can be prepared from virus-producing cells or cell culture supernatants by culturing the virus-producing cells, and those skilled in the art can easily prepare it by appropriate means. Further, a virus particle mixture obtained by concentrating or roughly purifying the culture supernatant of virus-producing cells or the lysate of the cells by TFF (Tangential Flow Filtration) or column chromatography or the like may be used as the starting sample in this embodiment. In this embodiment, the "virus-producing cell" is a cell that produces elements necessary for forming virus particles and has the ability to produce a virus. The virus-producing cell may be a cell artificially created so as to be able to produce a virus, or a cell that has become able to produce a virus after being infected with a virus in a natural environment. As the virus-producing cell in this embodiment, preferably, it is an artificially created virus-producing cell, and particularly preferably, the virus is a non-enveloped virus.

[0026] The method of artificially creating virus-producing cells varies depending on the virus, and details have already been described in many reviews and the like, so please refer to those reviews. Here, only an outline of the preparation of cells that produce virus vectors will be described. When producing virus particles that function as vectors, a plasmid lacking a region encoding a non-structural protein of the virus (a protein involved in virus replication, etc.) and a region encoding a structural protein of the virus (a protein such as a capsid), and instead inserting a gene of interest, a plasmid encoding a non-structural protein and a structural protein of the virus, a plasmid encoding other necessary genes depending on the type of virus vector, etc. are introduced into an arbitrary cell, whereby virus-producing cells can be prepared. For example, in the case of an AAV vector, an AAV vector-producing cell can be prepared by introducing a plasmid containing a target gene, a plasmid containing a gene encoding a Rep protein (a protein necessary for virus replication) and a Cap protein (a protein constituting the capsid), and a plasmid containing a gene encoding an E1a protein, an E1b protein, an E2 protein, and an E4 protein derived from adenovirus into HEK293 cells, HEK293T cells, or the like.

[0027] The culture conditions for virus-producing cells are already known, and those skilled in the art can appropriately select them according to the type of virus. Although not particularly limited, for example, culture may be performed for about several days to about 20 days at about 30 to 38 °C and a CO2 concentration of about 5 to 10% in a medium such as DMEM or IMDM containing necessary supplements (growth factors, amino acids, etc.) and serum.

[0028] The sample containing the virus may be an extract obtained by extracting the virus from virus-producing cells or a product obtained by roughly purifying the extract. For example, in the case of a virus released into the medium, the culture supernatant after culturing virus-producing cells may be collected as a sample. In the case of a virus accumulated in cells, the collected virus-producing cells may be disrupted by a freeze-thaw method, an ultrasonic disruption method, or the like, and the debris and the like may be removed and used as a sample. Since many reagents and kits for preparing a sample containing a virus from virus-producing cells are commercially available, these reagents and kits may be used to prepare a sample.

[0029] The second embodiment is a zonal rotor in which a virus particle mixture containing hollow virus particles, intermediate virus particles, and complete virus particles, a liquid L, and a liquid H1, or a liquid B, the virus particle mixture, the liquid L, and the liquid H1 are arranged in this order from the rotation axis side toward the outside. The zonal rotor according to the second embodiment is the rotor in step (a) of the first embodiment, for example, a rotor in a state of rotating at a low speed. A liquid having a density equal to or higher than the density of the liquid L and equal to or lower than the density of the liquid H1 may be stored in the zonal rotor according to the second embodiment in a state where a density gradient is formed. For the liquid B, the liquid L, and the liquid H1, refer to the description regarding the first embodiment.

[0030] When this specification is translated into English and contains the words "a", "an", and "the" in the singular form, unless the context clearly indicates otherwise, it shall be construed to include not only the singular but also the plural. Examples are shown below to further explain the present invention. However, these examples are merely illustrative of the embodiments of the present invention and do not limit the scope of the present invention.

Examples

[0031] 1. Method 1-1. Preparation of AAV Vector The AAV vector was prepared and recovered on a large scale from the culture supernatant (conditioned medium) according to a previously reported method (Verdera et al., Mol Ther 28: 723-746 2020). Adenovirus E1a, adenovirus E1b, and Bcl- XLThe 293EB cell line that expresses (Tomono et al., Hum Gene Ther Methods 30:137-143 2019) was cultured for 4 days in Dulbecco's Modified Eagle Medium (DMEM, FUJIFILM Wako) supplemented with 10% fetal bovine serum (Thermo Fisher) using 2 × 500 mL flasks (HYPERFlask, MilliporeSigma) or a 1 L bioreactor (iCELLis Nano Bioreactor). Next, in DMEM (serum-free) containing 2 mM L-glutamine, 12.1% (w / v) NaHCO3, and 12.9% D-glucose, the AAV vector plasmid (encoding ZsGreen1 or ZsGreen1-DR) and the helper plasmid were transfected into 293EB cells using polyethyleneimine hydrochloride (PEI MAX, Polysciences). Five days after transfection, the AAV vector was recovered from the culture supernatant (810 mL), and in the presence of 5 mM MgCl2, the AAV vector was treated with 18.5 U / mL endonuclease (Kaneka) at 37°C for 30 minutes.

[0032] 2. Purification of intact AAV vectors by cesium chloride equilibrium density gradient ultracentrifugation using a zonal rotor CsCl (FUJIFILM Wako), HNE buffer (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.4, 0.15 M NaCl, 25 mM ethylenediaminetetraacetic acid (EDTA)) or HN buffer (50 mM HEPES pH 7.4, 0.15 M NaCl) was added to the prepared AAV vector (5% CsCl) (Table 1). For the cesium chloride density gradients of experimental IDs, Z2 to Z7, the following solutions were injected in the order of (1), (2), (3), (4) from the outside (edge side) of the zonal rotor (P32ZT or P35ZT, Eppendorf Himac Technologies) and centrifuged at 3,000 rpm to create; (1) 200 mL of 5% (w / v) CsCl (in HNE or HN buffer) (2) 5% (w / v) CsCl in HNE or HN buffer containing AAV vector, 900 mL - 1 L (3) 25 - 27% CsCl in HNE or HN buffer, 300 mL (4) 40% CsCl in HNE or HN buffer, 200 - 300 mL

Table 1

[0033] After creating the density gradient, the zonal rotor was centrifuged at 30,000 rpm - 35,000 rpm for 4 - 10 hours (himac CP 80NX, Eppendorf Himac Technologies) to separate the intact AAV vector from the intermediate and empty vectors. After ultracentrifugation, while centrifuging the zonal rotor at 3,000 rpm, 42 - 45% CsCl (HNE or HN buffer) was slowly injected into the rotor from the outside of the rotor into the rotor, and the solution in the rotor (the density gradient solution of cesium chloride containing the AAV vector) was pushed out of the rotor, and the pushed-out solution was collected while fractionating with a fraction collector (Figure 1A). The refractive index of each fraction was measured using an Abbe refractometer (NAR - 1T LIQUID, Atago) or a digital refractometer (RX 5000i, Atago) (Figure 1B, Table 2 and Table 3). Each fraction collected was dialyzed against 0.5 mM MgCl2 (aqueous solution) at 4°C for about 2 hours using a 20 kDa molecular weight cut-off dialysis cassette (#66003, Thermo Fisher), and then dialyzed against 0.5 mM MgCl2 (PBS solution) at 4°C overnight.

[0034]

Table 2

Table 3

[0035] 1-3. Evaluation of genomic copy number, capsid protein, and transduction efficiency of AAV vectors by quantitative PCR (qPCR), Western blotting, and flow cytometry After ultracentrifugation using a zonal rotor, the AAV genomic copy number of each fraction collected was calculated using the AAVpro Titration Kit (for Real Time PCR) Ver. 2 (TaKaRa Bio) and the QuantStudio 3 Real-Time PCR System (Applied Biosystems). The presence or absence of AAV capsid protein in each fraction was confirmed by Western blotting. Samples were treated with NuPAGE LDS sample buffer (Thermo Fisher) and NuPAGE Reducing Agent (Thermo Fisher), loaded onto a 4-25% gradient polyacrylamide gel (Criterion TG Precast Gels, Bio-Rad), and then electrophoresed with SDS running buffer (Nacalai Tesque). After electrophoresis, the electrophoresed proteins were transferred to a PVDF membrane (Trans-Blot Turbo Midi PVDF Transfer Packs, Bio-Rad), and proteins were detected using anti-AAV VP1 / VP2 / VP3 mouse antibody (clone B1, Progen) and Amersham ECL Mouse IgG, HRP-linked whole Ab (Cytiva). Transduction efficiency was evaluated by the percentage of ZsGreen1-positive cells (%ZsGreen1) in the transduced 293EB cells. 293EB cells (1×10 5Cells were cultured overnight in a 24-well plate. After culturing, the cells were transduced with each fraction sample (300 μL / well) in serum-free DMEM (300 μL / well) containing 2 mM L-glutamine, 12.1% NaHCO3, and 12.9% D-glucose. On the day after transduction, 600 μL of medium (the same as above) was added to each well. %ZsGreen1 was evaluated by flow cytometry (FACSMelody, Becton Dickinson) on the third day after transduction. Analysis of the results was performed using FlowJo Version 7.1 (Becton Dickinson).

[0036] 1-4. Evaluation of full particles (including full-length genome) and empty particles by analytical ultracentrifugation (AUC) The purity of the AAV vector was analyzed using a Proteome Lab XL-I ultracentrifuge (Beckman Coulter). 400 μL of the AAV vector sample was added to the centerpiece of the cell housing. Three cell housings with the sample added and one counterbalance were set in the AUC rotor. After setting the temperature of the rotor to 20°C, ultracentrifugation was performed at 12,000 rpm at 20°C, and light absorption (260 nm) and interference were measured at 92 time points for 4 - 5 hours. Full particles, intermediates, and empty particles of AAV were analyzed using Sedfit (National Institutes of Health) and visualized using GUSSI (UT Southwestern Medical Center).

[0037] 1-5. Analysis of the entire genomic region packaged in AAV by droplet digital PCR (ddPCR) The entire genomic region of the AAV vector in each fraction obtained after performing ultracentrifugation with a zonal rotor was analyzed by the ddPCR method. A 100-fold diluted sample (1.1 μL) was mixed with 0.25 mM probe, 0.5 mM forward primer, and 0.5 mM reverse primer (ddPCR Copy Number Assy, BioRad) (see Table 4), droplets were generated with an Automated Droplet Generator (BioRad), and a PCR reaction was performed in a C1000 Touch Thermal Cycler (BioRad). The fluorescence signals from each droplet were detected with a QX200 droplet reader (BioRad) using the QuantaSoft software package (BioRad).

Table 4

[0038] 1-6. Morphological analysis of AAV vectors using transmission electron microscopy (TEM) The AAV vectors were hydrophilized with an ion bombarder (Nisshin EM Co., type PIB-10), 3 μL of the hydrophilized sample was placed on a collodion membrane (Nisshin EM), and left standing for 1 minute. After washing 3 times with 3 μL of water, the sample was stained with PTA (Phosphotungstic acid) for 10 seconds. The stained sample on the collodion membrane was observed with a TEM (HT7800, Hitachi High-Tech).

[0039] 2. Results 2-1. Separation of intact (including full-length genome), intermediate (including genomic fragments), and empty particles (without genome) of AAV vectors by cesium chloride density gradient ultracentrifugation using a zonal rotor To establish a method for separating intact AAV vector particles from intermediate and empty particles in a large quantity and in a short time, a zonal rotor was used to form a density gradient with two concentrations of cesium chloride solutions and attempt to separate the intact particles by equilibrium density gradient (Figure 1A). Since the infection efficiency of the AAV vector into cells decreases as the contact time with cesium chloride increases, it was necessary to shorten the centrifugation time as much as possible. First, a density gradient was created with four concentrations (15 wt%, 25 wt%, 33 wt%, and 40 wt%) of cesium chloride using 300 mL of the culture supernatant containing the AAV vector, and then centrifuged at 35,000 rpm for 10 hours in ultracentrifugation mode (Table 1). As a result, a nearly linear density gradient of cesium chloride was formed (Figure 1B, Table 2). The AAV capsid protein was detected in fractions 17 and 25. In contrast, the transduction activity of the AAV genome and ZsGreen1 was detected from fraction 25 but not from fraction 17. This result indicates that intact particles with transduction ability (fraction 25) and empty particles without transduction ability (fraction 17) were separated. Intermediate particles are considered to be contained in the fractions between fraction 17 (empty particles) and fraction 25 (intact particles).

[0040] Separation among intact particles, intermediate particles, and empty particles was also possible by cesium chloride density gradient ultracentrifugation using four concentrations of cesium chloride solutions. However, it is considered that the biological activity (transduction ability) of the AAV vector decreased due to the long exposure of the AAV vector to cesium chloride for 10 hours. Therefore, it was thought that by forming a density gradient with two concentrations of cesium chloride solutions, it would be possible to form a steep density gradient that could concentrate intact AAV particles in a narrow density gradient range and also shorten the centrifugation time. Furthermore, by reducing the amount of cesium chloride solution injected into the zonal rotor, it is also possible to increase the amount of the sample containing the AAV vector (Table 1). Therefore, when cesium chloride at two concentrations (25-27 wt% and 40 wt%, experimental ID1; Z2-Z5) was injected into the rotor to form a density gradient and ultracentrifugation was performed, it was possible to process a larger volume of AAV vector (900-1,000 mL) in a shorter centrifugation time (4-5 hours) compared to the case of using a cesium chloride solution at four concentrations. The refractive index (density gradient) increased more sharply within a narrow range in the zonal rotor (Figure 1 and Table 3). Peaks in the AAV genome copy number (Figure 2A) and transduction efficiency (Figure 2B) were detected in fractions 16-17 (RI values; 1.369 and 1.370), and peaks in the capsid protein (Figure 2C) were also detected in these fractions. This result indicates that intact particles were recovered in fractions 16-17. On the other hand, fractions 11-12, in which intermediate particles or hollow particles were considered to be included although capsid proteins and a small number of genome copies were detected but no ZsGreen1 transformation activity was detected (Figure 2A, B, and C, RI values; 1.365 and 1.366). The above results indicate that the density gradient formed by the cesium chloride solution at two concentrations enables the separation of intact particles, intermediate particles, and hollow particles in a short time (4-5 hours) and also enables the processing of a large amount of virus particle mixed samples.

[0041] 2-2. Detection of highly pure intact particles by analytical ultracentrifugation (AUC) Analytical ultracentrifugation was performed to evaluate the purity of the intact AAV particle fraction and the hollow particle fraction separated by the density gradient formed by the cesium chloride solution at two concentrations using a zonal rotor. Interestingly, intact particles (80S) (Figure 3B) and hollow particles (60S) (Figure 3A) with different sedimentation coefficients were detected as single peaks, respectively. Moreover, a higher purity intact particle fraction could be separated from the medium containing the AAV vector than from the cell lysate containing the AAV vector (compare Figures 3B and C). In addition, the intact AAV particles and the hollow AAV particles separated by cesium chloride density gradient ultracentrifugation were stained with phosphotungstic acid, and their morphological characteristics were observed under a transmission electron microscope. The intact AAV particles were observed as hexagonal particles with a white interior (Figure 4B), and the hollow AAV particles were observed as hexagonal particles with a black dot in the center (Figure 4A). From the observation results by electron microscopy, it is suggested that the hollow AAV particles were partially compressed and more strongly stained by phosphotungstic acid. The above results indicate that a large amount of intact AAV particles were separated with high purity by density gradient using a zonal rotor with two concentrations of cesium chloride.

[0042] 2-3. Analysis of DNA packaged in AAV particles by droplet digital PCR (ddPCR) To analyze the DNA packaged in intact AAV particles, intermediate AAV particles, and hollow AAV particles, 22 probes / primers for detecting the entire region of the AAV genome and the backbone region of the plasmid were designed and evaluated by ddPCR. It was confirmed that the full-length genomic (intact) AAV particles contained the entire region of the AAV genome and a partial ITR region (Figure 5). On the other hand, short-sized ITRs were detected in the intermediate particles and the hollow particles. These results suggest that during the production process of AAV vectors, short DNA fragments containing only ITRs were generated and packaged into the capsids.

Industrial Applicability

[0043] The present invention provides a method for efficiently preparing a virus (particle) such as a viral vector with high purity. Therefore, it is expected to be used in medical fields such as gene therapy.

Claims

1. A method for producing intact virus particles, comprising a step of purifying intact virus particles from a virus particle mixture containing hollow virus particles, intermediate virus particles, and intact virus particles, a step of rotating a zonal rotor at a low speed and arranging the virus particle mixture, a liquid having a density lower than that of the intact virus particles (liquid L), and a liquid having a density higher than that of the liquid L (liquid H1) in this order from the rotation axis side of the rotor toward the outside, wherein only the liquid L and the liquid H1 are arranged outside the virus particle mixture (step (a)), a step of operating the zonal rotor in an ultracentrifugation mode after step (a) to separate the hollow virus particles, the intermediate virus particles, and the intact virus particles (step (b)), a step of fractionating and taking out the contents of the zonal rotor after step (b) and recovering a fraction containing the intact virus particles (step (c)), wherein the virus is a DNA virus without an envelope, the density of the liquid L is 1.22 g / cm3 to 1.29 g / cm3, and the density of the liquid H1 is 1.39 g / cm3 to 1.45 g / cm3.

2. The production method according to claim 1, wherein step (a) is a step of rotating a zonal rotor at a low speed and arranging a liquid having a density lower than that of the liquid L and the liquid H1 (liquid B), the virus particle mixture, the liquid L, and the liquid H1 in this order from the rotation axis side of the rotor toward the outside.

3. The production method according to claim 1 or 2, wherein in step (c), the zonal rotor after step (b) is rotated at a low speed, and a liquid having a density higher than that of the liquid H1 (liquid H2) is introduced from the radially outer side of the zonal rotor, whereby the contents of the zonal rotor are sequentially pushed out and taken out while fractionating from the rotation axis side of the zonal rotor.

4. The production method according to claim 3, wherein the liquid L, the liquid H1, and the liquid H2 contain cesium chloride (CAS number: 7647-17-8), iodixanol (CAS number: 92339-11-2), iohexol (CAS number: 66108-95-0), amidotrizoic acid (CAS number: 737-31-5), or metrizamide (CAS number: 31112-62-6).

5. The manufacturing method according to claim 1, wherein the liquid L and the liquid H1 contain cesium chloride.

6. The manufacturing method according to claim 1 or claim 5, wherein the virus is an adeno-associated virus.

7. A zonal rotor in which a virus particle mixture containing hollow virus particles, intermediate virus particles, and complete virus particles, a liquid (liquid L) having a density lower than that of the complete virus particles, and a liquid (liquid H1) having a density higher than that of the liquid L are arranged in this order from the rotation axis side toward the outside, and only the liquid L and the liquid H1 are arranged outside the virus particle mixture, the virus particles are DNA viruses without an envelope, the density of the liquid L is 1.22 g / cm3 to 1.29 g / cm3, and the density of the liquid H1 is 1.39 g / cm3 to 1.45 g / cm3.

8. The zonal rotor according to claim 7, wherein a liquid (liquid B) having a density lower than that of the liquid L and the liquid H1 is arranged on the rotation axis side of the virus particle mixture.

9. The zonal rotor according to claim 8, wherein the liquid L and the liquid H1 contain cesium chloride (CAS number: 7647-17-8), iodixanol (CAS number: 92339-11-2), iohexol (CAS number: 66108-95-0), amidotrizoic acid (CAS number: 737-31-5), or metrizamide (CAS number: 31112-62-6).

10. The zonal rotor according to claim 7, wherein the liquid L and the liquid H1 contain cesium chloride.

11. The zonal rotor according to claim 7 or claim 10, wherein the virus is an adeno-associated virus.

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