Method for reducing host cell DNA residue in recombinant adeno-associated virus

Abstract

The invention belongs to the technical field of biology, and discloses a method for reducing host cell DNA (deoxyribonucleic acid) (Host Cell DNA, HCD) residues in recombinant adeno-associated viruses. An Emricasan reagent is added into a cell culture solution during rAAV production, so that the purpose of remarkably reducing HCD residues inside and outside rAAV particle capsids can be achieved. Compared with other rAAV production processes, the method has the advantages that the purity of rAAV products is improved, so that the safety and druggability of rAAV related gene therapy products are improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for reducing residual host cell DNA in recombinant adeno-associated viruses. 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 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, mainly including plasmid transfection production systems based on HEK293 / Hela cells, production systems based on insect cells and baculovirus, and packaging cells or inducible packaging cells. In the host cell, after the capsid protein and replication protein are expressed, the viral genome is replicated and packaged into the newly assembled capsid, thereby producing rAAV particles. In this process, host cell genomic DNA, the plasmid DNA used, etc. will also be randomly packaged into the capsid by mistake. Outside the capsid, these non-target genome contaminating DNA will also attach to the outside of the virus particle or the capsid. The generated rAAV undergoes further purification and concentration steps to remove various impurities generated during the rAAV packaging process and improve the concentration and purity of rAAV. During the purification process, the addition of Benzonase universal nuclease can effectively remove most of the contaminating DNA outside the capsid, but the contaminating DNA inside the capsid cannot be effectively removed. These residual contaminating DNA in rAAV products may stimulate unnecessary host immune responses, cause unpredictable biological effects of gene expression, and integrate into the host cell genome, which may have adverse effects on the efficacy and safety of gene therapy. Therefore, further research is urgently needed on how to reduce the residual contaminating DNA in rAAV production, so that the safety and effectiveness of rAAV can be improved, thereby accelerating the progress of rAAV-mediated gene therapy. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for reducing residual host cell DNA in recombinant adeno-associated virus, so as to effectively reduce the residual level of host cell DNA (Host Cell DNA, HCD) inside and outside the capsid in rAAV products.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a kit for reducing residual host cell DNA in a recombinant adeno-associated virus, comprising 1.0 μM to 5.0 μM Emricasan, or a salt thereof, or a stereochemical isomer thereof.

[0007] Emricasan is represented by formula (I):

[0008]

[0009] As a preferred embodiment of the kit of the present invention, 30U / mL-40U / mL of Benzonase is also included.

[0010] In a second aspect, the present invention provides a composition for reducing residual host cell DNA in a recombinant adeno-associated virus, comprising 1.0 μM-5.0 μM Emricasan and 30 U / mL-40 U / mL Benzonase.

[0011] In a third aspect, the present invention uses Emricasan in the preparation of a preparation for reducing residual host cell DNA in a recombinant adeno-associated virus.

[0012] As a preferred embodiment of the use of the present invention, the concentration of Emricasan is greater than 10 nM.

[0013] In a fourth aspect, the present invention provides a method for reducing residual host cell DNA in a recombinant adeno-associated virus, wherein Emricasan is added to the cell culture medium when culturing cells in rAAV.

[0014] As a preferred embodiment of the method described in the present invention, the concentration of Emricasan is greater than 10 nM.

[0015] As a further preferred embodiment of the method described in the present invention, the concentration of Emricasan is 1.0 μM-5.0 μM.

[0016] As a preferred embodiment of the method described in the present invention, the cell is at least one of HEK293, HEK-derived cells, CHO, CHO-derived cells, Hela cells, Vero cells, and SF-9 cells.

[0017] As a preferred embodiment of the method of the present invention, the cultured cells are cultured in suspension or adherently.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention can achieve the purpose of significantly reducing the HCD residue inside and outside the capsid of rAAV particles by adding Emricasan reagent to the cell culture medium during rAAV production. Compared with other rAAV production processes, this method can improve the purity of rAAV products, thereby improving the safety and drugability of rAAV-related gene therapy products. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Statistical graph of genomic titer and HCD residue in lysate samples with Emricasan added and without Benzonase treatment.

[0021] Figure 2 The figure shows the statistical graph of genomic titer and HCD residue of the primary purified sample with Emricasan added and without Benzonase treatment.

[0022] Figure 3 The figure shows the statistical graph of the genomic titer and HCD residue of the initially purified samples treated with Emricasan and Benzonase.

[0023] Figure 4 This is a statistical graph of the genomic titer and HCD residue of the final purified and concentrated product after the addition of Emricasan and Benzonase treatment. DETAILED DESCRIPTION

[0024] 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.

[0025] 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.

[0026] Example 1: Genomic titer and HCD residue of lysate samples with Emricasan added and without Benzonase treatment

[0027] (1) Cell recovery and subculture

[0028] 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 125 mL culture flask containing 20 mL Dynamis (Gibco, Cat#A2661501) 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 2 to 3 days.

[0029] 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 to 3 days). With a final cell concentration of 3.0E+05 to 5.0E+5 cells / mL, take an appropriate volume of suspended cell culture and place it in a 250 mL culture flask, add Dynamis 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 2 to 3 days.

[0030] (2) Transfect cells with three rAAV plasmids

[0031] On the day of transfection, take 0.5 mL of suspended cells for cell counting and viability testing, and then dilute the cells to 2.6E+06 cells / mL using Dynamis medium. Dispense the cell dilution into 125 mL culture flasks at 20 mL / flask, and add Emricasan at final concentrations of 0 μM, 3.0 μM, and 5.0 μM, respectively. Then place the culture flasks at 37°C and 5% CO 2 The cells were cultured in an incubator with shaking at 100 rpm.

[0032] Mix pGOI-eGFP (Addgene: 68550), pRC-AAV9 (GenBank: KF032296), and pHelper (GenBank: AF369965) plasmids at a ratio of 28 μg: 33 μg: 7.0 μg per bottle, add 1.0 mL of DMEM medium, and mix by pipetting. Add 68 μL (Polyplus, Cat#101000044) transfection reagent, pipette to mix, and let stand at room temperature for 30 minutes. The transfection mixture was added to the flask containing the suspended cells and placed at 37°C with 5% CO 2 The cells were cultured in an incubator with shaking at 100 rpm.

[0033] (3) Genomic titer and HCD residual determination

[0034] On the third day after transfection, a final concentration of 10% cell lysis buffer was added to the culture flask and placed at 37°C and 5% CO 2 Oscillate and lyse at 100 rpm in an incubator for 2 h. Take 1.0 mL of lysate to a 1.5 mL centrifuge tube and centrifuge at 4000 rpm for 3 min. Take 100 μL of supernatant to determine the genomic titer and HCD residual level.

[0035] 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 4000 times as a qPCR template; ITR-qF (5'-GGAACCCCTAGTGATGGAGTT-3') and ITR-qR (5'-CGGCCTCAGTGAGCGA-3') were used as primers; a gradient dilution plasmid standard was set as a quantitative reference. A standard curve was prepared using the gradient dilution plasmid reference, and the genomic titer of the rAAV sample was calculated based on the standard curve. The results of the genomic titer test showed that there was no significant difference in the genomic titer of the groups adding 3.0μM and 5.0μM Emricasan compared with the Control group ( Figure 1 ).

[0036] The HEK293 residual DNA fragment analysis detection kit (Hubei Shenke, Cat#1103176) was used for qPCR to determine HCD residues: the rAAV lysis supernatant was diluted 200 times as a qPCR template; the reaction system was prepared according to the kit instructions, and a gradient dilution of HEK293 DNA quantitative reference was set. The standard curve was prepared with the gradient dilution of HEK293 DNA quantitative reference, and the HCD residue of the rAAV sample was calculated according to the standard curve, and the HCD residue level was calculated according to the corresponding genome titer.

[0037] The results of the HCD residue test showed that the HCD residue level in the rAAV lysate samples without Benzonase treatment was high; compared with the Control group, the HCD residue levels in the groups with 3.0μM and 5.0μM Emricasan were significantly reduced to 0.06 and 0.05 times ( Figure 1 ).

[0038] Example 2: Genomic titer and HCD residue of the primary purified sample with Emricasan added and without Benzonase treatment

[0039] (1) Cell recovery and subculture

[0040] 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 125 mL culture flask containing 20 mL Dynamis (Gibco, Cat#A2661501) 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 to 3 days.

[0041] 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 to 3 days). With a final cell concentration of 3.0E+05 to 5.0E+05 cells / mL, take an appropriate volume of suspended cell culture and place it in a 250 mL culture flask, add Dynamis 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 to 3 days.

[0042] (2) Transfect cells with three rAAV plasmids

[0043] On the day of transfection, take 0.5 mL of suspended cells for cell counting and viability testing, and then dilute the cells to 2.6E+06 cells / mL using Dynamis medium. Dispense the cell dilution into 125-mL culture flasks at 20 mL / bottle, and add Emricasan at final concentrations of 0 μM, 1.0 μM, and 2.0 μM, respectively. Then place the culture flasks at 37°C and 5% CO 2 The cells were cultured in an incubator with shaking at 100 rpm.

[0044] Mix pGOI-eGFP, pRC-AAV1 (Addgene: 112862), and pHelper plasmids at a ratio of 28 μg: 33 μg: 7.0 μg per bottle, add 1.0 mL of DMEM medium, and mix by pipetting. Add 68 μL (Polyplus, Cat#101000044) transfection reagent, pipette to mix, and let stand at room temperature for 30 minutes. The transfection mixture was added to the flask containing the suspended cells and placed at 37°C with 5% CO 2 The cells were cultured in an incubator with shaking at 100 rpm.

[0045] (3) Genomic titer and HCD residual determination

[0046] On the third day after transfection, add cell lysis buffer with a final concentration of 10% to the flask and place it at 37°C and 5% CO 2 The samples were shaken at 100 rpm in an incubator for 2 h. TM GoPure TM AAV9 pre-packed chromatography column (ThermoFisher, Cat#A36650) was used to wash impurities with 0.1M PBS buffer (pH=7.4), then eluted with 3mL glycine-HCl buffer (pH2.7), and collected in 1M Tris-HCl buffer (pH=8.0) pre-containing 8% for neutralization. 100 μL of this initial purification sample was taken for determination of genome titer and HCD residual level.

[0047] The genomic titer of rAAV samples was determined by qPCR using AceQ Universal SYBR qPCR master kit (Vazyme, Cat#Q511-03): first, the rAAV primary purification sample was digested with DNase I (TaKaRa, Cat#2270A) and diluted 40,000 times as a qPCR template; ITR-qF (5'-GGAACCCCTAGTGATGGAGTT-3') / ITR-qR (5'-CGGCCTCAGTGAGCGA-3') were used as primers; a gradient dilution plasmid standard was set as a quantitative reference. A standard curve was prepared using the gradient dilution plasmid reference, and the genomic titer of the rAAV sample was calculated based on the standard curve. The results of the genomic titer test showed that the genomic titer level in the rAAV primary purification sample that was not treated with Benzonase was low; compared with the Control group, the genomic titers of the groups adding 1.0μM and 2.0μM Emricasan were significantly different ( Figure 2 ).

[0048] The HEK293 residual DNA fragment analysis detection kit (Hubei Shenke, Cat#1103176) was used for qPCR to determine the HCD residue: the rAAV primary purification sample was diluted 100 and used as the qPCR template; the reaction system was prepared according to the kit instructions, and a gradient dilution of the HEK293 DNA quantitative reference was set. The standard curve was prepared with the gradient dilution of the HEK293 DNA quantitative reference, and the HCD residue of the rAAV sample was calculated according to the standard curve, and the HCD residue level was calculated according to the corresponding genome titer.

[0049] The results of the HCD residue test showed that the HCD residue level in the rAAV primary purification sample without Benzonase treatment was high; compared with the Control group, the HCD residue level in the group with 1.0μM and 2.0μM Emricasan was significantly reduced to 0.04 and 0.02 times ( Figure 2 ).

[0050] Example 3: Genomic titer and HCD residue of the primary purified sample treated with Emricasan and Benzonase

[0051] (1) Cell recovery and subculture

[0052] 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 125 mL culture flask containing 20 mL Dynamis (Gibco, Cat#A2661501) 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 to 3 days.

[0053] 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 to 3 days). With a final cell concentration of 3.0E+05 to 5.0E+5 cells / mL, take an appropriate volume of suspended cell culture and place it in a 250 mL culture flask, add Dynamis 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 to 3 days.

[0054] (2) Transfect cells with three rAAV plasmids

[0055] On the day of transfection, take 0.5 mL of suspended cells for cell counting and viability testing, and then dilute the cells to 2.6E+06 cells / mL using Dynamis medium. Dispense the cell dilution into 125-mL culture flasks at 20 mL / bottle, and add Emricasan at final concentrations of 0 μM, 1.0 μM, and 2.0 μM, respectively. Then place the culture flasks at 37°C and 5% CO 2 The cells were cultured in an incubator with shaking at 100 rpm.

[0056] Mix pGOI-eGFP, pRC-AAV1, and pHelper plasmids at a ratio of 28 μg:33 μg:7.0 μg per bottle, add 1.0 mL of DMEM medium, and mix by pipetting. Add 68 μL (Polyplus, Cat#101000044) transfection reagent, pipette to mix, and let stand at room temperature for 30 minutes. The transfection mixture was added to the flask containing the suspended cells and placed at 37°C with 5% CO 2 The cells were cultured in an incubator with shaking at 100 rpm.

[0057] (3) Genomic titer and HCD residual determination

[0058] On the third day after transfection, add 10% cell lysis buffer and 40U / mL Benzonase to the flask and place it at 37°C and 5% CO. 2 The samples were shaken at 100 rpm in an incubator for 2 h. TM GoPure TM AAV9 pre-packed chromatography column (ThermoFisher, Cat#A36650) was used to wash impurities with 0.1M PBS buffer (pH=7.4), then eluted with 3mL glycine-HCl buffer (pH2.7), and collected in 1M Tris-HCl buffer (pH=8.0) pre-containing 8% for neutralization. 100 μL of this initial purification sample was taken for determination of genome titer and HCD residual level.

[0059] The genomic titer of rAAV samples was determined by qPCR using AceQ Universal SYBR qPCR master kit (Vazyme, Cat#Q511-03): first, the rAAV primary purification sample was digested with DNase I (TaKaRa, Cat#2270A) and diluted 40,000 times as a qPCR template; ITR-qF (5'-GGAACCCCTAGTGATGGAGTT-3') / ITR-qR (5'-CGGCCTCAGTGAGCGA-3') were used as primers; a gradient dilution plasmid standard was set as a quantitative reference. A standard curve was prepared using the gradient dilution plasmid reference, and the genomic titer of the rAAV sample was calculated based on the standard curve. The results of the genomic titer test showed that compared with the Control group, the genomic titer of the group adding 1.0μM and 2.0μM Emricasan was slightly increased ( Figure 3 ).

[0060] The HEK293 residual DNA fragment analysis detection kit (Hubei Shenke, Cat#1103176) was used for qPCR to determine the HCD residue: the rAAV primary purification sample was diluted 100 and used as the qPCR template; the reaction system was prepared according to the kit instructions, and a gradient dilution of the HEK293 DNA quantitative reference was set. The standard curve was prepared with the gradient dilution of the HEK293 DNA quantitative reference, and the HCD residue of the rAAV sample was calculated according to the standard curve, and the HCD residue level was calculated according to the corresponding genome titer.

[0061] The results of the HCD residue test showed that the HCD residue level in the rAAV primary purification sample treated with Benzonase was very low; compared with the Control group, the HCD residue level in the group adding 1.0μM and 2.0μM Emricasan was significantly reduced to 0.08 and 0.07 times ( Figure 3 ).

[0062] Example 4: Genomic titer and HCD residue of the final purified concentrated product after adding Emricasan and Benzonase treatment

[0063] (1) Cell recovery and subculture

[0064] 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 125 mL culture flask containing 20 mL Dynamis (Gibco, Cat#A2661501) 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 to 3 days.

[0065] 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 to 3 days). With a final cell concentration of 3.0E+05 to 5.0E+05 cells / mL, take an appropriate volume of suspended cell culture and place it in a 250 mL culture flask, add Dynamis 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 to 3 days.

[0066] (2) Transfect cells with three rAAV plasmids

[0067] On the day of transfection, take 0.5 mL of suspended cells for cell counting and viability testing, and then dilute the cells to 2.6E+06 cells / mL using Dynamis medium. Dispense the cell dilution into 125-mL culture flasks at 20 mL / bottle, and add Emricasan at final concentrations of 0 μM, 3.0 μM, and 5.0 μM, respectively. Then place the culture flasks at 37°C and 5% CO 2 The cells were cultured in an incubator with shaking at 100 rpm.

[0068] Mix pGOI-eGFP, pRC-AAV9, and pHelper plasmids at a ratio of 28 μg:33 μg:7.0 μg per bottle, add 1.0 mL of DMEM medium, and mix by pipetting. Add 68 μL (Polyplus, Cat#101000044) transfection reagent, pipette to mix, and let stand at room temperature for 30 minutes. The transfection mixture was added to the flask containing the suspended cells and placed at 37°C with 5% CO 2 The cells were cultured in an incubator with shaking at 100 rpm.

[0069] (3) Genomic titer and HCD residual determination

[0070] On the third day after transfection, add a final concentration of 10% cell lysis buffer to the culture flask and shake for about 30 minutes. After the lysate is initially filtered through sterile filter paper, it is dissolved in 20 mL of virus diluent, and Benzonase is added at a final concentration of 40 U / mL for digestion, and large particles are removed by centrifugation. Then, iodixanol is used as a medium for density gradient centrifugation, and the rAAV virus particles are further purified and concentrated by ultrafiltration, and the volume is fixed to 0.2 mL. Take 50 μL of this final purified and concentrated sample for determination of genome titer and HCD residual level.

[0071] The genomic titer of rAAV samples was determined by qPCR using AceQ Universal SYBR qPCR master kit (Vazyme, Cat#Q511-03): first, the rAAV final purified and concentrated sample was digested with DNase I (TaKaRa, Cat#2270A), and diluted 80,000 times as a qPCR template; ITR-qF (5'-GGAACCCCTAGTGATGGAGTT-3') / ITR-qR (5'-CGGCCTCAGTGAGCGA-3') were used as primers; a gradient dilution plasmid standard was set as a quantitative reference. A standard curve was prepared using the gradient dilution plasmid reference, and the genomic titer of the rAAV sample was calculated based on the standard curve. The results of the genomic titer test showed that there was no significant difference in the genomic titer of the groups adding 3.0μM and 5.0μM Emricasan compared with the Control group ( Figure 4 ).

[0072] The HEK293 residual DNA fragment analysis detection kit (Hubei Shenke, Cat#1103176) was used for qPCR to determine the HCD residue: the rAAV final purified concentrated sample was diluted 200 times as the qPCR template; the reaction system was prepared according to the kit instructions, and a gradient dilution of the HEK293 DNA quantitative reference was set. The standard curve was prepared with the gradient dilution of the HEK293 DNA quantitative reference, and the HCD residue of the rAAV sample was calculated according to the standard curve, and the HCD residue level was calculated according to the corresponding genome titer.

[0073] The results of the HCD residue test showed that the HCD residue level in the final purified and concentrated rAAV samples treated with Benzonase was very low; compared with the Control group, the HCD residue levels in the groups adding 3.0μM and 5.0μM Emricasan were significantly reduced to 0.02 and 0.03 times ( Figure 4 ).

[0074] 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 kit for reducing residual host cell DNA in a recombinant adeno-associated virus, characterized in that: The present invention comprises 1.0 μM to 5.0 μM Emricasan, or a salt thereof, or a stereochemical isomer thereof.

2. The kit according to claim 1, characterized in that Also included is 30U / mL-40U / mL of Benzonase.

3. A composition for reducing residual host cell DNA in a recombinant adeno-associated virus, characterized in that: Includes 1.0μM-5.0μM Emricasan and 30U / mL-40U / mL Benzonase.

4. Use of Emricasan in the preparation of a preparation for reducing residual host cell DNA in recombinant adeno-associated viruses.

5. The use according to claim 4, characterized in that: The concentration of Emricasan is greater than 10 nM.

6. A method for reducing residual host cell DNA in a recombinant adeno-associated virus, characterized in that: In rAAV, Emricasan is added to the cell culture medium when the cells are cultured.

7. The method according to claim 6, characterized in that The concentration of Emricasan is greater than 10 nM.

8. The method according to claim 7, characterized in that The concentration of Emricasan is 1.0 μM-5.0 μM.

9. The method according to claim 6, characterized in that The cell is at least one of HEK293, HEK-derived cells, CHO, CHO-derived cells, Hela cells, Vero cells, and SF-9 cells.

10. The method according to claim 6, characterized in that The cultured cells are cultured in suspension or adherently.

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