Method for improving production of recombinant adeno-associated virus by baculovirus system

Abstract

The invention discloses a method for improving rAAV (recombinant adeno-associated virus) production of a baculovirus system, namely, sf9 cells are infected with a third-generation recombinant baculovirus solution containing an AAV (adeno-associated virus) genome without removing the cells by controlling the algebraic number, the infection mode and the sf9 cell density of the recombinant baculovirus, and the yield of rAAV can be further improved when the cell density is 2.5-4.0 * 10 cells / ml; better technical support is provided for cost reduction and benefit improvement in the fields of neuroscience research, disease model establishment, gene therapy and the like, and the application value and the market prospect are wide.

Description

Technical Field

[0001] The present invention belongs to the technical field of viral vectors, and in particular relates to a method for improving the production of recombinant adeno-associated viruses by a baculovirus system. Background Art

[0002] Recombinant adeno-associated virus (rAAV)-based gene delivery vectors are widely used in fields such as neural circuit structure and function analysis and gene therapy. rAAV vectors are one of the most promising and promising therapeutic tools. Currently, eight rAAV-based gene drugs have been approved for marketing, with an average selling price of over $1 million. Such high prices impose a significant financial burden on patients, primarily due to the high R&D and production costs.

[0003] Currently, the main method for producing rAAV virus is to co-transfect HEK-293T with three plasmids in adherent culture. However, in large-scale production, the cost of plasmid production is high and a large amount of relatively expensive transfection reagents is required. The use of baculovirus system to prepare rAAV is easier to scale up, has higher production capacity and lower cost. However, this method still has many variables, which is not conducive to the flexible operation of the next step of production. When used to produce rAAV virus, there is still a lot of room for improvement in yield. Therefore, it is necessary to develop new methods that can further improve the production of rAAV virus by baculovirus.

[0004] The present invention designs a new method for improving the production of rAAV virus by baculovirus, further improving the production capacity of rAAV virus, providing better technical support for neuroscience research, disease model establishment and gene therapy, etc., and has broad application value and market prospects. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a method for infecting sf9 cells with the third-generation recombinant baculovirus liquid containing the AAV genome without removing cells by controlling the generation number of recombinant baculovirus, the infection method and the sf9 cell density, thereby further improving the yield of rAAV.

[0006] The present invention adopts the following technical solutions:

[0007] A method for improving the production of recombinant adeno-associated virus by a baculovirus system comprises the following steps: transfecting Sf9 insect cells with recombinant bacmid DNA containing an adeno-associated virus genome, collecting the supernatant to obtain a P1-generation recombinant baculovirus; infecting Sf9 insect cells with the P1-generation recombinant baculovirus, collecting the supernatant to obtain a P2-generation recombinant baculovirus, further infecting Sf9 insect cells, and collecting a culture fluid containing the Sf9 insect cells to obtain a P3-generation recombinant baculovirus; and infecting Sf9 insect cells with the P3-generation recombinant baculovirus culture fluid to produce the recombinant adeno-associated virus.

[0008] Preferably, when the P3 generation recombinant baculovirus is infected, the density of the Sf9 insect cells is 2.5-4.0×10 6 cells / ml.

[0009] Compared with the existing technology, the present invention has the following advantages: the existing methods still have many variables, such as the number of infected baculovirus generations, infection methods, cell density at the time of infection, and other uncertain factors, which are not conducive to the flexible operation of the next step of production, and there is still a lot of room for improvement in rAAV virus yield. The present invention controls the number of infected baculovirus generations to P3, the infection method to baculovirus without removing cells, and the cell density at the time of infection to 2.5-4.0×10 6 cells / ml, the new method significantly improved the final rAAV yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is the vector map of the shuttle plasmid PFD-0012. DETAILED DESCRIPTION

[0011] Take 0.1 μg of recombinant shuttle plasmid PFD-0012 (plasmid map as shown Figure 1 The recombinant shuttle plasmid PFD-0012 was transformed into Stbl3 competent cells, cultured in LB liquid medium for 1 hour, and then plated onto solid culture medium in a culture dish. After incubation at 30°C overnight, a single colony was selected and placed in LB liquid medium, cultured overnight at 30°C, and the plasmid was extracted. Following the Bac-to-Bac system protocol, the recombinant shuttle plasmid PFD-0012 was transformed into DH10Bac-RC9 competent cells (obtained from Xu Fuqiang's laboratory at the Chinese Academy of Sciences, according to the method of patent CN109609552B) for Tn7 transposase-mediated homologous recombination. This generated recombinant E. coli DH10Bac-RC9-0012, which incorporates all rAAV packaging components (referring to the construction method of DH10Bac-Δ(Chia-Cath)-Rep2-Cap2 in CN109609552A, replacing Cap2 with Cap9). The recombinant bacmid DNA was extracted and transfected into adherent cultured Sf9 insect cells. The culture medium was replaced with complete medium 6 hours after transfection. The cell status was examined daily under a microscope. After 120 hours, the supernatant was collected to obtain the recombinant baculovirus BEV-P1. The recombinant baculovirus was further infected with adherent cultured Sf9 insect cells. After 72 hours, the supernatant was collected and centrifuged to remove cell debris. The recombinant baculovirus obtained was BEV-P2. 0.1 ml of BEV-P2 was used to infect 20 ml of a cell volume with a cell density of 2.0 × 10 6 Sf9 cells were cultured in suspension at 5 × 10 cells / ml. After 48 hours, the culture medium (including supernatant and cells) was collected. The recombinant baculovirus obtained was BEV-P3. 4 ml of BEV-P3 culture medium (titer 5 × 109 GC / mL), were evenly divided into 4 groups, each with 1 ml. After being treated with different methods, 100 ml of Sf9 cells cultured at different densities were infected to produce rAAV. The specific groups are as follows:

[0012] Experimental group a: 1 ml of BEV-P3 baculovirus solution was taken without removing cells. The cell density at the time of infection was 2.5×10 6 cells / ml;

[0013] Experimental group b: 1 ml of BEV-P3 baculovirus solution was taken and centrifuged to remove cells. The cell density at the time of infection was 2.5×10 6 cells / ml;

[0014] Experimental group c: 1 ml of BEV-P3 baculovirus solution was taken and centrifuged to remove cells. The cell density at the time of infection was 4.0×10 6 cells / ml;

[0015] Experimental group d: 1 ml of BEV-P3 baculovirus solution was taken without removing cells. The cell density at the time of infection was 4.0×10 6 cells / ml.

[0016] 72 hours after infection, the culture medium (including supernatant and cells) was collected and the virus was purified and concentrated by iodixanol gradient centrifugation and ultrafiltration. Finally, the titer of the recombinant adeno-associated virus was determined by SYBR Green qPCR. The average rAAV virus yields are shown in Table 1. These results demonstrate that the new baculovirus-based rAAV production method can further improve rAAV production capacity.

[0017] Table 1 Average yield of rAAV virus

[0018]

Claims

1. A method for improving the production of recombinant adeno-associated virus by a baculovirus system, characterized in that: The method comprises the following steps: transfecting Sf9 insect cells with recombinant bacmid DNA containing an adeno-associated virus genome, collecting the supernatant to obtain a P1-generation recombinant baculovirus; infecting Sf9 insect cells with the P1-generation recombinant baculovirus, collecting the supernatant to obtain a P2-generation recombinant baculovirus, and then infecting Sf9 insect cells, collecting the culture fluid containing the Sf9 insect cells, which is a P3-generation recombinant baculovirus; and infecting Sf9 insect cells with the P3-generation recombinant baculovirus to produce a recombinant adeno-associated virus.

2. The method according to claim 1, characterized in that When the P3 recombinant baculovirus is infected, the density of the Sf9 insect cells is 2.5-4.0×10 6 cells / ml.

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