Method for improving production efficiency of recombinant adeno-associated virus and application

Abstract

The invention belongs to the technical field of recombinant adeno-associated virus production, and discloses a method for improving the production efficiency of a recombinant adeno-associated virus and application. The method for improving the production efficiency of the recombinant adeno-associated virus comprises the following steps: adding a production promoting reagent into a cell culture solution; the production promoting reagent comprises at least one of a compound as shown in a formula (I) or a formula (II) and a derivative thereof. According to the method disclosed by the invention, the yield of rAAV produced by mammalian cells can be remarkably increased, so that the production cost is reduced, the large-scale production of rAAV is effectively promoted, and the method has important significance on rAAV-mediated gene therapy.

Description

Technical Field

[0001] The present invention relates to the technical field of recombinant adeno-associated virus production, and in particular to a method for improving the production efficiency of recombinant adeno-associated virus and its application. Background Art

[0002] Gene therapy is the process of introducing, modifying or removing specific genes into patient cells to repair or compensate for defective genes, thereby achieving the purpose of treating or preventing genetic diseases. In gene therapy, nucleic acid delivery mediated by viral vectors is currently the main way to introduce genes into cells. Recombinant adeno-associated virus (rAAV) vectors have many advantages, such as long-term stable expression of exogenous genes, no integration into the host genome, ability to infect dividing / non-dividing cells, a wide range of host cells, strong specificity, and low immunogenicity, making them a safe and effective gene therapy tool. Therefore, they are considered to be one of the most promising gene therapy vectors and have been applied to gene therapy and clinical research for a variety of diseases.

[0003] At present, multiple rAAV production platforms based on different host cell lines have been established, including: plasmid transfection production system based on HEK293 / Hela cells, production system based on insect cells and baculovirus, packaging cell or induced packaging cell method, etc. The plasmid transfection production system based on HEK293 cells is the mainstream technology for clinical / research-level rAAV production. The system uses a method of co-transfecting production cells with multiple plasmids containing all the necessary elements for rAAV packaging. The main process includes: preparation of plasmids, amplification of production cells, transfection of plasmids into production cells, cell lysis to harvest crude virus solution, virus purification and other steps.

[0004] However, the large-scale production of rAAV is limited by factors such as low productivity, making the production of rAAV far behind the dose required for clinical and commercial use, thus affecting the application of gene therapy drugs. Therefore, researching and developing production technologies to increase rAAV production in order to effectively promote the large-scale production of rAAV is of great significance for rAAV-mediated gene therapy. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method and application for improving the production efficiency of recombinant adeno-associated virus.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for improving the production efficiency of a recombinant adeno-associated virus, comprising: adding a production-promoting agent to a cell culture medium;

[0008] The production-promoting agent includes at least one of the compounds represented by formula (I) or formula (II) and their derivatives;

[0009]

[0010] The method of the present invention can significantly increase the yield of rAAV produced by mammalian cells, thereby reducing production costs and effectively promoting the large-scale production of rAAV, which is of great significance to rAAV-mediated gene therapy.

[0011] As a preferred embodiment of the method for improving the production efficiency of recombinant adeno-associated virus described in the present invention, the derivative includes at least one of a stereoisomer, a tautomer, a metabolite, a metabolic precursor, a prodrug, a solvate, a salt of a solvate, a crystal, and a pharmaceutically acceptable salt of a compound as shown in formula (I) or formula (II).

[0012] As a preferred embodiment of the method for improving the production efficiency of recombinant adeno-associated virus described in the present invention, Docetaxel and / or Abemaciclib are added to the cell culture medium.

[0013] Docetaxel is a taxane anti-tumor drug and immunomodulator, and is an M-phase cycle-specific drug. Abemaciclib is a cyclin-dependent kinase CDK4 / 6 inhibitor, commonly used in the treatment of HR+ / HER2- advanced or recurrent breast cancer. The method of the present invention adds it to the cell culture medium, which can increase the production of rAAV by mammalian cells by about 50%-70%.

[0014] As a further preferred embodiment of the method for improving the production efficiency of recombinant adeno-associated virus of the present invention, the final concentration of Docetaxel is 0.5 μM-5.0 μM.

[0015] Preferably, the final concentration of Docetaxel is any one or both of 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μM, 4.5 μM, and 5.0 μM.

[0016] As a further preferred embodiment of the method for improving the production efficiency of recombinant adeno-associated virus described in the present invention, the final concentration of Abemaciclib is 5.0 μM-50 μM.

[0017] Preferably, the final concentration of Docetaxel is any one or both of 5.0 μM, 6.0 μM, 7.0 μM, 8.0 μM, 9.0 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, and 50 μM.

[0018] As a preferred embodiment of the method for improving the production efficiency of recombinant adeno-associated virus described in the present invention, the production-promoting reagent is added to the cell culture fluid before the plasmid is transfected into the cells.

[0019] As a preferred embodiment of the method for improving the production efficiency of recombinant adeno-associated virus described in the present invention, the cells are mammalian cells.

[0020] As a further preferred embodiment of the method for improving the production efficiency of recombinant adeno-associated virus described in the present invention, the cell is any one of HEK293, HEK-derived cells, CHO, CHO-derived cells, Hela cells, Vero cells, and SF-9 cells.

[0021] As a further preferred embodiment of the method for improving the production efficiency of recombinant adeno-associated virus of the present invention, the cells are cultured in suspension and / or adherent culture.

[0022] In a second aspect, the present invention applies the method for improving the production efficiency of recombinant adeno-associated virus in rAAV production.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The method of the present invention adds docetaxel or abemaciclib to the cell culture medium, which can increase the rAAV production of mammalian cells by about 50%-70%, thereby reducing production costs and effectively promoting the large-scale production of rAAV, which is of great significance to rAAV-mediated gene therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The genome titer of rAVV packaging was determined by adding Docetaxel as in Example 1.

[0026] Figure 2 The genome titer of rAVV packaging with the addition of Abemaciclib in Example 2. DETAILED DESCRIPTION

[0027] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0028] Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0029] Example 1: Adding Docetaxel for rAVV packaging

[0030] (1) Cell recovery and subculture

[0031] Take out the frozen Viral Production Cells 2.0 (Gibco, Cat#A49784) suspension cells from the liquid nitrogen tank, immediately place them in a 37°C water bath and shake gently to thaw the cells quickly. In a biosafety cabinet, transfer the thawed cells to a 125mL culture flask containing 20mL OPM293 CD05 (OPM, Cat#81075-001) culture medium, and then place the culture flask at 37°C and 5% CO. 2 The cells were cultured in an incubator with shaking at 100 rpm for about 2-3 days.

[0032] In a biosafety cabinet, take 0.5 mL of suspended cells for cell counting and viability testing. When the cell density is greater than 3.0E+06 cells / mL and the cell viability is greater than 90%, the cells are passaged (passaged every 2-3 days). With a final cell concentration of 3E5-6E5 cells / mL, take an appropriate volume of suspended cell culture and place it in a 250 mL culture flask, add OPM293CD05 culture medium to a total volume of 120 mL. Place the culture flask at 37°C and 5% CO 2 The cells were cultured in an incubator with shaking at 100 rpm for about 2-3 days.

[0033] (2) Transfect cells with three rAAV plasmids

[0034] On the day of transfection, take 0.5mL of suspended cells for cell counting and viability detection, and then dilute the cells to 2.6E6 cells / mL using OPM293 CD05 culture medium. Dispense the cell dilution into 6-well plates at 5mL / tube, add Docetaxel at final concentrations of 0.5μM, 1.0μM, and 5.0μM, respectively, and use an equal volume of DMSO as a control. Then place the 6-well plates at 37°C and 5% CO 2 The culture was shaken at 120 rpm in an incubator.

[0035] Mix pGOI-eGFP, pRC-AAV9, and pHelper plasmids at a ratio of 7.0 μg:8.3 μg:1.7 μg per well, add 250 μL DMEM medium, and mix by pipetting. Add 17 μL -AAV (Polyplus, Cat#101000044) transfection reagent, pipette to mix, and let stand at room temperature for 30 minutes. -AAV transfection mixture was added to a 6-well plate containing suspended cells and placed at 37°C with 5% CO 2 The culture was shaken at 120 rpm in an incubator.

[0036] (3) Genomic titer determination

[0037] On the third day after transfection, a final concentration of 10% cell lysis buffer was added to the 6-well plate and placed at 37°C and 5% CO 2 Incubate the cells in an incubator at 120 rpm for 2 h. Transfer the lysate to a 5.0 mL centrifuge tube and centrifuge at 4000 rpm for 3 min. Take 50 μL of the supernatant to measure the genomic titer.

[0038] The genomic titer of rAAV samples was determined by qPCR using AceQ Universal SYBR qPCR master kit (Vazyme, Cat#Q511-03): first, the rAAV lysate supernatant was digested with DNase I (TaKaRa, Cat#2270A) and diluted 1000 times as a qPCR template; the upstream primer ITR-qF (5'-GGAACCCCTAGTGATGGAGTT-3') and the downstream primer ITR-qR (5'-CGGCCTCAGTGAGCGA-3') were used as primers; the gradient dilution plasmid standard was set as a quantitative reference. The standard curve was prepared with the gradient dilution plasmid reference, and the genomic titer of the rAAV sample was calculated based on the standard curve.

[0039] The results of the genome titer test showed that compared with the DMSO control, the addition of 0.5 μM and 1.0 μM Docetaxel could increase the genome titer by about 50% (see Figure 1 ).

[0040] Example 2: Adding Abemaciclib for rAVV packaging

[0041] (1) Cell recovery and subculture

[0042] Take out the frozen Viral Production Cells 2.0 (Gibco, Cat#A49784) suspension cells from the liquid nitrogen tank, immediately place them in a 37°C water bath and shake gently to thaw the cells quickly. In a biosafety cabinet, transfer the thawed cells to a 125mL culture flask containing 20mL OPM293 CD05 (OPM, Cat#81075-001) culture medium, and then place the culture flask at 37°C and 5% CO. 2 The cells were cultured in an incubator with shaking at 100 rpm for about 2-3 days.

[0043] In a biosafety cabinet, take 0.5 mL of suspended cells for cell counting and viability testing. When the cell density is greater than 3.0E+06 cells / mL and the cell viability is greater than 90%, the cells are passaged (passaged every 2-3 days). With a final cell concentration of 3E5-6E5 cells / mL, take an appropriate volume of suspended cell culture and place it in a 250 mL culture flask, add OPM293CD05 culture medium to a total volume of 120 mL. Place the culture flask at 37°C and 5% CO 2 The cells were cultured in an incubator with shaking at 100 rpm for about 2-3 days.

[0044] (2) Transfect cells with three rAAV plasmids

[0045] On the day of transfection, 0.5 mL of suspended cells was taken for cell counting and viability testing, and then the cells were diluted to 2.6E6 cells / mL using OPM293 CD05 medium. The cell dilution was dispensed into 6-well plates at 5 mL / tube, and Abemaciclib was added at final concentrations of 5.0 μM, 10 μM, and 50 μM, respectively, with an equal volume of DMSO as a control. The 6-well plates were then placed at 37°C and 5% CO 2 The culture was shaken at 120 rpm in an incubator.

[0046] Mix pGOI-eGFP, pRC-AAV9, and pHelper plasmids at a ratio of 7.0 μg:8.3 μg:1.7 μg per well, add 250 μL DMEM medium, and mix by pipetting. Add 17 μL -AAV (Polyplus, Cat#101000044) transfection reagent, pipette to mix, and let stand at room temperature for 30 minutes. -AAV transfection mixture was added to a 6-well plate containing suspended cells and placed at 37°C with 5% CO 2 The culture was shaken at 120 rpm in an incubator.

[0047] (3) Genomic titer determination

[0048] On the third day after transfection, a final concentration of 10% cell lysis buffer was added to the 6-well plate and placed at 37°C and 5% CO 2 Incubate the cells in an incubator at 120 rpm for 2 h. Transfer the lysate to a 5.0 mL centrifuge tube and centrifuge at 4000 rpm for 3 min. Take 50 μL of the supernatant to measure the genomic titer.

[0049] The genomic titer of rAAV samples was determined by qPCR using AceQ Universal SYBR qPCR master kit (Vazyme, Cat#Q511-03): first, the rAAV lysate supernatant was digested with DNase I (TaKaRa, Cat#2270A) and diluted 1000 times as a qPCR template; the upstream primer ITR-qF (5'-GGAACCCCTAGTGATGGAGTT-3') and the downstream primer ITR-qR (5'-CGGCCTCAGTGAGCGA-3') were used as primers; the gradient dilution plasmid standard was set as a quantitative reference. The standard curve was prepared with the gradient dilution plasmid reference, and the genomic titer of the rAAV sample was calculated based on the standard curve.

[0050] The results of the genome titer test showed that the addition of 10 μM Abemaciclib could increase the genome titer by about 70% compared with the DMSO control (see Figure 2 ).

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for improving the production efficiency of recombinant adeno-associated virus, characterized in that: adding a production-promoting agent to the cell culture medium; The production-promoting agent includes at least one of the compounds represented by formula (I) or formula (II) and their derivatives; 2. The method for improving the production efficiency of recombinant adeno-associated virus according to claim 1, characterized in that: The derivatives include at least one of stereoisomers, tautomers, metabolites, metabolic precursors, prodrugs, solvates, salts of solvates, crystals, and pharmaceutically acceptable salts of the compounds represented by formula (I) or formula (II).

3. The method for improving the production efficiency of recombinant adeno-associated virus according to claim 1, characterized in that: Docetaxel and / or Abemaciclib were added to the cell culture medium.

4. The method for improving the production efficiency of recombinant adeno-associated virus according to claim 3, characterized in that: The final concentration of Docetaxel is 0.5 μM-5.0 μM.

5. The method for improving the production efficiency of recombinant adeno-associated virus according to claim 3, characterized in that: The final concentration of Abemaciclib is 5.0 μM-50 μM.

6. The method for improving the production efficiency of recombinant adeno-associated virus according to claim 1, characterized in that: The production-promoting reagent is added to the cell culture medium before the plasmid is transfected into the cells.

7. The method for improving the production efficiency of recombinant adeno-associated virus according to any one of claims 1 to 6, characterized in that: The cells are mammalian cells.

8. The method for improving the production efficiency of recombinant adeno-associated virus according to claim 7, characterized in that: The cell is any one of HEK293, HEK-derived cells, CHO, CHO-derived cells, Hela cells, Vero cells, and SF-9 cells.

9. The method for improving the production efficiency of recombinant adeno-associated virus according to claim 8, characterized in that: The cells are cultured in suspension and / or adherent culture.

10. Use of the method for improving the production efficiency of recombinant adeno-associated virus according to any one of claims 1 to 9 in rAAV production.

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