Viral vector productivity enhancer and production method of viral vector

Abstract

The purpose of the present invention is to elevate viral vector productivity in producing a viral vector with the use of viral vector-producing cells. For this purpose, DNA replication is inhibited in the cells producing the viral vector.

Description

Viral vector productivity enhancer and method for producing viral vector

[0001] The present invention relates to a viral vector productivity enhancer that can be used in a viral vector production process using cultured cells and that can improve virus productivity, and a method for producing a viral vector.

[0002] Gene therapy using viral vectors is a therapeutic method in which a viral vector incorporating a gene encoding a therapeutically effective protein is administered to a patient, or cells transfected with the viral vector are administered to a patient. Viral vectors used in gene therapy include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, Sendai viruses, and herpes viruses. Although gene therapy is already in practical use, the establishment of technology that enables the efficient mass production of high-quality viral vectors is urgently needed for widespread use of gene therapy for various diseases.

[0003] Viral vector production typically involves first culturing and growing vector-producing cells, then infecting them with a small amount of virus. The cell culture is then continued to expand the viral infection, and the viral vector grows within the virus-infected cells. The viral vector that grows within the cells is then recovered and purified according to standard methods, and formulated as needed.

[0004] In such a viral vector production process, various ideas have been proposed to improve viral vector productivity. For example, Patent Document 1 discloses an enhancer that enhances the efficiency (transfection efficiency) of introducing a recombinant adeno-associated virus into cells in the above-mentioned viral vector production process. The enhancer disclosed in Patent Document 1 is valproic acid, a salt or derivative thereof; isobutyric acid, a salt or derivative thereof; or isovaleric acid, a salt or derivative thereof.

[0005] Furthermore, Patent Document 2 discloses an invention relating to a cell line used in the production process of adeno-associated virus vectors, in which the expression of at least one of YB1 (a gene encoding Y-box binding protein 1 (also known as a Y-box transcription factor and nuclease sensitive element binding protein 1)), NPM1 (a gene encoding nucleophosmin, nucleolar phosphorylation protein B23, and numatrin), and NCL (a gene encoding the nucleolar phosphorylation protein nucleolin) is reduced. Use of the cell line disclosed in Patent Document 2 improves the productivity of virus vectors.

[0006] Furthermore, Patent Document 3 discloses an invention relating to a cell line used in the production process of an adeno-associated virus vector, which is a cell line into which a specific miRNA has been introduced. By using the cell line disclosed in Patent Document 3, it is possible to produce an adeno-associated virus vector with a higher titer than conventional vectors without complicated procedures.

[0007] Special Publication No. 2020-524498 Publication Special Publication No. 2017-506885 Publication Patent No. 6093358

[0008] However, the above-mentioned known techniques for improving viral vector productivity have not been sufficient in terms of viral vector productivity, meaning that the technical challenge of improving viral vector productivity remains.

[0009] In view of the above-mentioned circumstances, an object of the present invention is to provide a novel viral vector productivity enhancer that can improve viral vector productivity, a method for producing a viral vector, and the like.

[0010] As a result of intensive research conducted by the present inventors to achieve the above-mentioned object, they discovered that viral vector productivity can be improved by inhibiting DNA replication in cells that produce viral vectors, and have thus completed the present invention.

[0011] (1) A viral vector productivity enhancer comprising a DNA replication inhibitor as a main component. (2) The viral vector productivity enhancer according to (1), characterized in that it is used for viral vector production by viral vector-producing cells. (3) The viral productivity enhancer according to (1), characterized in that the DNA replication inhibitor is a platinum preparation. (4) A method for producing a viral vector, comprising the steps of culturing viral vector-producing cells in which DNA replication has been inhibited, and recovering the viral vector from the cultured cells. (5) The method for producing a viral vector according to (4), characterized in that in the step of culturing the cells, the cells are cultured in a medium containing the viral vector productivity enhancer according to any one of (1) to (3). This specification incorporates the disclosures of Japanese Patent Application No. 2022-025036, from which the present application claims priority.

[0012] According to the present invention, a viral vector productivity enhancer that acts on viral vector-producing cells and significantly improves viral vector productivity by the cells can be provided. Furthermore, according to the present invention, a production method that significantly improves viral vector productivity in the production of viral vectors using viral vector-producing cells can be provided.

[0013] FIG. 1 is a characteristic diagram showing the results of measuring the effect of carboplatin on enhancing rAAV vector production. FIG. 2 is a characteristic diagram showing the results of measuring the effect of carboplatin on enhancing rAAV vector production over time. FIG. 3 is an electrophoresis photograph showing the results of SDS-PAGE analysis of rAAV vector constituent proteins when carboplatin is used. FIG. 4 is a photograph showing the results of an infectivity test of rAAV vectors produced using carboplatin. FIG. 5 is a characteristic diagram showing the results of measuring the effect of cisplatin on enhancing rAAV vector production. FIG. 6 is a characteristic diagram showing the results of measuring the effect of nedaplatin on enhancing rAAV vector production.

[0014] The viral vector productivity enhancer and the method for producing a viral vector according to the present invention improve viral vector productivity in cells that produce viral vectors.

[0015] In an embodiment of the present invention, viral vector productivity is improved by inhibiting DNA replication in cells that produce viral vectors. Viral vector productivity means that the amount of viral vector is higher when the cells are cultured under specified conditions compared to a control, or that the viral titer is higher when the cells are cultured under specified conditions compared to a control. Measurement of the viral vector amount and viral titer can be performed using conventionally known methods as appropriate. The amount of viral vector and viral titer can be measured by, but are not limited to, plaque assay, TCID50 assay (tissue culture infectious dose), quantitative RT-PCR assay, etc.

[0016] In the present invention, a viral vector refers to a virus or a genetically modified virus that is artificially produced and amplified for research or medical purposes. The type of virus is not particularly limited, and examples include non-enveloped viruses such as adeno-associated virus (AAV), adenovirus, enterovirus, parvovirus, papovavirus, human papillomavirus, rotavirus, coxsackievirus, sapovirus, norovirus, poliovirus, echovirus, hepatitis A virus, hepatitis E virus, rhinovirus, astrovirus, circovirus, and simian virus; herpes viruses such as retrovirus, lentivirus, Sendai virus, and herpes simplex virus; and enveloped viruses such as vaccinia virus, measles virus, baculovirus, influenza virus, leukemia virus, and Sindbis virus and poxvirus. Among these, viral vectors used in gene therapy, such as adeno-associated viruses, are preferred.

[0017] Adeno-associated viruses are viruses belonging to the Parvoviridae family that contain linear, single-stranded DNA in the capsid. Examples of adeno-associated viruses include AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), and AAV type 10 (AAV10). Artificially engineered AAV viral vectors, such as AAV-DJ and AAV-PHP.B, are also included. Furthermore, technologies for packaging and amplifying artificial nucleic acids within adeno-associated virus capsids are also considered.

[0018] Furthermore, the term "cells capable of producing these viral vectors" is synonymous with "packaging cells capable of producing these viral vectors," and examples thereof include human embryonic kidney 293 (HEK293) cells transfected with viral vectors (packaging cells). Cells transfected with viral vectors are not limited to HEK293 cells, but include HEK293T cells, HEK293S cells, HEK293F cells, HEK293FT cells, HEK293FTM cells, HEK293SG cells, HEK293SGGD cells, HEK293H cells, HEK293E cells, and HEK293MSR cells derived from HEK293 cells. Commercially available cells developed for adenovirus (e.g., Adeno-X 293 Cell Line) and adeno-associated virus (e.g., AAVpro 293T Cell Line) can also be used. Other mammalian-derived packaging cells include human cervical cancer-derived HeLa cells, human lung cancer-derived A549 cells, human fibrosarcoma HT-1080 cells, human retinal pigment epithelium-derived cells such as PER. C6 cells, hamster-derived BHK cells and CHO cells, and African green monkey-derived Vero cells and COS cells.

[0019] Methods for producing viral vectors are not limited to transfecting a plasmid into packaging cells, but also include infecting packaging cells with a helper virus such as adenovirus or herpesvirus to amplify the virus, and establishing and amplifying producer cells by incorporating components for packaging the viral vector into the cellular genome.

[0020] Furthermore, viral vector-producing cells are not limited to mammalian cells, and can also be amplified using insect-derived cells, such as Lepidopteran cells, such as Sf9 and Sf21 cells derived from the ovaries of the armyworm moth, Tni cells derived from the nettle looper moth, and High Five cells, by transfecting the viral vector or infecting the cells with a baculovirus carrying components for packaging the viral vector.

[0021] The medium for culturing the viral vector-producing cells described above is not particularly limited, and an appropriate medium can be selected depending on the cells used. For example, Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (DMEM containing 10% FBS, DMEM containing 10% FCS) can be used, or serum-free Eagle's minimum essential medium (E-MEM) can be used. Furthermore, the viral vector-producing cells can be cultured by either adherent culture or suspension culture.

[0022] [Inhibition of DNA replication] In the present invention, "inhibiting DNA replication" means, but is not limited to, allowing a DNA replication inhibitor such as a platinum agent to act on the viral vector-producing cells. Platinum agents that can be used in the present invention include, but are not limited to, carboplatin, oxaliplatin, cisplatin, nedaplatin, and analogs of these compounds. Among these, it is preferable to use carboplatin or its analogs having the following structural formula as the DNA replication inhibitor.

[0023]

[0024] Here, a carboplatin analog means a compound having a structure in which a specific atom or atomic group in the above structural formula is replaced with another atom or atomic group, and which inhibits DNA synthesis by covalently binding to DNA in the same way as carboplatin.

[0025] When carboplatin is used as a viral vector productivity enhancer, the concentration of carboplatin in the medium for culturing viral vector-producing cells is not particularly limited, but can be, for example, 5 to 150 μg / ml, preferably 10 to 100 μg / ml, more preferably 10 to 50 μg / ml, and even more preferably 20 to 40 μg / ml.

[0026] Furthermore, as the DNA replication inhibitor, it is preferable to use cisplatin or its analogues having the following structural formula:

[0027]

[0028] Here, a cisplatin analog means a compound having a structure in which a specific atom or atomic group in the above structural formula is replaced with another atom or atomic group, and which inhibits DNA synthesis by covalently binding to DNA in the same way as cisplatin.

[0029] When cisplatin is used as a viral vector productivity enhancer, the concentration of cisplatin in the medium for culturing viral vector-producing cells is not particularly limited, but can be, for example, 1 to 10 μg / ml, preferably 1 to 5 μg / ml, and more preferably 1 to 3 μg / ml.

[0030] Furthermore, as the DNA replication inhibitor, it is preferable to use nedaplatin having the following structural formula or an analog thereof.

[0031]

[0032] Here, an analog of nedaplatin refers to a compound having a structure in which a specific atom or atomic group in the above structural formula is replaced with another atom or atomic group, and which inhibits DNA synthesis by covalently binding to DNA in the same way as nedaplatin.

[0033] When nedaplatin is used as a viral vector productivity enhancer, the concentration of nedaplatin in the medium for culturing viral vector-producing cells is not particularly limited, but can be, for example, 1 to 20 μg / ml, preferably 2 to 10 μg / ml, more preferably 2 to 7 μg / ml, and even more preferably 2 to 5 μg / ml.

[0034] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0035] [Experimental Method] 1) Cell Culture HEK293 cells, an immortalized human fetal kidney cell line purchased from ATCC, were cultured in E-MEM containing 10% fetal bovine serum, penicillin, and streptomycin at 37°C in a 5% CO2 environment.

[0036] 2) Construction of Type 1 AAV Vector (rAAV1) Recombinant adeno-associated virus 1 (rAAV1) was constructed using three plasmids: pAAV-ZsGreen1 (TaKaRa Bio), pAAV2 / 1, and pHelper. These three rAAV1 constructing plasmids were mixed in OPTI-MEM with Polyethylenimine Max (PEI; Polysciences) and DNA at a DNA:PEI ratio of 1:2 and incubated at room temperature for 15 minutes. HEK293 cells (>90% confluent) were transfected in E-MEM containing 10% FBS for 6 hours and then cultured in serum-free E-MEM to construct rAAV1.

[0037] 3) Measurement of viral titer by quantitative PCR (qPCR) rAAV1 genomic DNA was extracted to measure viral titer. Specifically, the culture supernatant was collected and treated with benzonase at 37°C for 1 hour, after which viral genomic DNA was extracted and purified using the DNeasy Blood & Tissue kit (QIAGEN). Viral titer was measured in triplicate by real-time PCR. qPCR was performed using primers targeting Zs-Green1.

[0038] 4) Evaluation of AAV vector production by chemical treatment. HEK293 cells were placed in a 24-well plate at 4 × 10 per well. 5 The day after seeding, the AAV vector-producing plasmid was transfected. Six hours later, the transfection solution was removed and the medium was replaced with serum-free E-MEM supplemented with various concentrations of the DNA replication inhibitors carboplatin, cisplatin, or nedaplatin, as shown in Table 1, and the cells were cultured for three days. Viral titers were compared using a control group supplemented with serum-free E-MEM without any added chemicals. Changes in AAV production over time with carboplatin were also monitored over a 4-7 day period.

[0039]

[0040] 5) Quality evaluation of AAV vectors treated with chemicals The quality of AAV vectors produced by HEK293 cells treated with chemicals was evaluated by measuring purity by SDS-PAGE and infectivity.

[0041] 1. Purity measurement by SDS-PAGE. HEK293 cells were cultured in two square dishes until they reached 90% or more confluence. These HEK293 cells were transfected with an AAV vector production plasmid. After 6 hours, the medium was replaced with serum-free E-MEM (20 μg / ml, 72 ml) supplemented with carboplatin, and the cells were cultured for 5 days. HEK293 cells not treated with chemicals served as a control. On the fifth day of culture, cells were harvested using a scraper. The cells were centrifuged at 2000 g for 10 minutes, and the AAV vector was extracted, purified, and concentrated using the AAVpro Purification Kit (TaKaRa Bio). Subsequently, viral titers were measured by qPCR, and a 5-20% polyacrylamide gradient gel was used to measure the viral titer of 6.1 × 10 cells. 9 SDS-PAGE was performed at 1000µg / lane. After electrophoresis, the gel was stained with Oriole Fluorescent Gel Stain (Bio-RAD). The major bands of AAV capsid proteins (VP1, VP2, and VP3) were photographed using a chemiluminescence imager, and the ratio (1:1:10) and purity of each band were analyzed.

[0042] 2. Infectivity assessment: HEK293 cells were placed in a 24-well plate at 4 x 10 cells per well. 5 The cells were seeded at 6.25 x 10 cells per well of a 96-well plate, transfected with an AAV vector-producing plasmid, and then treated with chemicals for 5 days. The culture supernatant was then collected. HEK293 cells not treated with chemicals served as a control. The virus titer was measured by qPCR, and 6.25 x 10 cells were plated in each well of a 96-well plate. 3 HEK293 cells were seeded at 1 × 10 5 Three days after transduction, green fluorescence was observed and photographed using an inverted research microscope.

[0043] [Experimental Results] The results of measuring the effect of carboplatin on enhancing rAAV vector production are shown in Figure 1. In Figure 1, the vertical axis represents the amount of AAV vector produced (vg), and the horizontal axis represents the concentration of the chemical substance. As can be seen from Figure 1, carboplatin has the effect of enhancing AAV vector production, and a particularly high AAV vector production enhancing effect was observed in the concentration range of 20 to 40 μg / ml.

[0044] The results of measuring the effect of carboplatin on enhancing rAAV vector production over time are shown in Figure 2. In Figure 2, the vertical axis represents the amount of AAV vector produced (vg), and the horizontal axis represents the time (days) elapsed since the start of vector production. As can be seen from Figure 2, the amount of AAV vector production increased over time from the start of vector production, and it was confirmed that the amount of AAV vector production continued to increase even 7 days after treatment.

[0045] The results of SDS-PAGE analysis of the rAAV vector component proteins when carboplatin was used are shown in Figure 3. As can be seen from Figure 3, the rAAV vector produced by carboplatin treatment was confirmed to contain the same amount of rAAV component proteins (VP1, VP2, and VP3) as the untreated vector.

[0046] The results of an infectivity test of rAAV vectors produced using carboplatin are shown in Figure 4. As shown in Figure 4, it was confirmed that the rAAV vectors produced by treatment with carboplatin retained the same infectivity as AAV vectors produced without carboplatin treatment.

[0047] The results of measuring the rAAV vector production enhancement effect of cisplatin, which inhibits DNA replication like carboplatin, are shown in Figure 5. In Figure 5, the vertical axis represents AAV vector production (vg), and the horizontal axis represents chemical concentration. As can be seen from Figure 5, cisplatin, like carboplatin, has the effect of enhancing AAV vector production, with a particularly high enhancement effect observed at concentrations of 1 to 5 μg / ml.

[0048] Furthermore, the effect of nedaplatin, which inhibits DNA replication, on enhancing rAAV vector production was measured and the results are shown in Figure 6. In Figure 6, the vertical axis represents the amount of AAV vector produced (vg), and the horizontal axis represents the concentration of the chemical substance. As can be seen from Figure 6, nedaplatin, like carboplatin, has the effect of enhancing AAV vector production, and a particularly high enhancement effect on AAV vector production was observed at concentrations of 2 to 10 μg / ml.

[0049] These results demonstrated that the use of DNA replication inhibitors such as carboplatin, cisplatin, and nedaplatin can enhance rAAV vector production.

Claims

1. A viral vector productivity enhancer whose main ingredient is a DNA replication inhibitor.

2. 2. The viral vector productivity enhancer according to claim 1, which is used for producing a viral vector using viral vector-producing cells.

3. 2. The viral vector productivity enhancer according to claim 1, wherein the DNA replication inhibitor is a platinum agent.

4. A method for producing a viral vector, comprising the steps of culturing cells that produce viral vectors and in which DNA replication is inhibited, and recovering the viral vector from the cultured cells.

5. The method for producing a viral vector according to claim 4, characterized in that in the step of culturing the cells, the cells are cultured in a medium containing the viral vector productivity enhancer according to any one of claims 1 to 3.