Rational Design of Recombinant Adeno-associated Virus Transient Expressing System and Establishment of Its Efficient Production Process

doi:10.13560/j.cnki.biotech.bull.1985.2024-0547

Abstract

Abstract:

【Objective】This study focuses on understanding the impact of crucial viral and helper genes within the triple plasmid system on rAAV production to guide rational design of viral gene cassette and process optimization, thereby enhancing rAAV production efficiency.【Method】First, we clarified the composition and ratio of the rep and cap gene, as well as the critical sequences of helper viral genes that influenced the production of rAAV. Based on these insights, we rationally designed the viral gene cassettes on the plasmids and constructed a novel triple system. Finally, the critical process parameters of this new system were optimized to establish a high-efficiency rAAV production process.【Result】The proper ratio of rep and cap gene expression was crucial for rAAV production. Retaining only the essential helper protein sequences E4orf6（adenovirus early region 4 opening reading frame 6）and DBP（DNA binding protein）in the helper viral gene expression cassette achieved the same rAAV production efficiency. Herein, we developed a novel triple plasmid system, which led to a 2.6 fold increase in genome titer compared to the traditional triple plasmid system. Further the optimization of transfection parameters resulted, the genome titer reached 7.8×10¹¹ vg/mL, and the CSVY（cell specific virus yield）reached 1.5×10⁵ vg/cell, which were 3.7 fold and 2.4 fold compared to the traditional triple plasmid system, respectively.【Conclusion】By exploring the effects of the composition and ratio of rAAV viral genes and helper viral genes on the production of rAAV, and we re-designed the triple plasmid system. Finally, we established a new rAAV manufacturing process with a novel triple plasmid system, which were elevated 16-fold in genome titer and 10-fold in CSVY, respectively, compared with that of the traditional triple plasmid system. This study lays the foundation for efficient industrial production of rAAV.

Key words: recombinant adeno-associated virus, expressing system for transient transfection, plasmid design, HEK293 cells, process optimization

LI Rui-rui, NA Dao-yuan, WANG Hong-lin, ZHAO Liang, TAN Wen-Song, YE Qian. Rational Design of Recombinant Adeno-associated Virus Transient Expressing System and Establishment of Its Efficient Production Process[J]. Biotechnology Bulletin, 2024, 40(12): 61-71.

Figures/Tables 6

Fig. 1 Schematic diagram of plasmids A: Schematic diagram of rep/cap split-packing plasmids constructs. B: Schematic diagram of helper plasmids. C: Schematic diagram of novel triple plasmids system

Fig. 2 Effects of the expression ratio of rep gene and cap gene on the yield of rAAV A: Relative transcript levels of viral genes at 12 h. B: Transfection efficiency and mean fluorescent intensity. C: Genome titer and CSVY among various rep gene doses.D: Genome titer and CSVY among various cap gene doses. *, **, and *** indicate significant difference at P<0.05, P<0.01, and P<0.001 levels, respectively, the same below

Fig. 3 Effects of key sequences of adenoviral helper genes on rAAV production A: Genome titer. B: CSVY. ns indicate no significant difference at P>0.05 level, the same below

Fig. 4 Rational optimization of plasmid system for improving rAAV production A: Genome titer; B: CSVY

Fig. 5 Optimization of process parameters on novel triple plasmid system A: Ratio of plasmids and cell. B: Ratio of plasmids and PEI. C: Cell density when transfection. D: Molar ratio of plasmids

Fig. 6 Genome titer（A）and CSVY（B）of rAAV yielded in novel triple plasmid system

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