文献类型：期刊文献

中文题名：重组腺相关病毒瞬时表达系统的理性设计及其高效生产工艺的建立

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

作者：李蕊蕊[1];那道远[1];王洪林[1];赵亮[1];谭文松[1];叶倩[1]

机构：[1]华东理工大学生物工程学院,上海200237

年份：2024

卷号：40

期号：12

起止页码：61

中文期刊名：生物技术通报

外文期刊名：Biotechnology Bulletin

收录：;北大核心:【北大核心2023】；CSCD:【CSCD2023_2024】；

语种：中文

中文关键词：重组腺相关病毒;瞬时转染表达系统;质粒设计;HEK293细胞;工艺优化

外文关键词：recombinant adeno-associated virus;expressing system for transient transfection;plasmid design;HEK293 cells;process optimization

摘要：【目的】探究关键病毒基因和辅助基因对rAAV生产的影响以指导病毒基因表达框的理性设计和工艺优化,实现rAAV生产效率的提升。【方法】首先,明确用于生产rAAV的关键基因元件rep和cap基因的组成和比例,以及辅助病毒基因的关键序列对rAAV生产的影响。然后,据此理性设计质粒上rAAV病毒基因表达框,构建新型三质粒系统。最后,通过对新型三质粒系统关键工艺参数进行优化,建立基于新型三质粒系统的rAAV高效生产工艺。【结果】合理的rep基因和cap基因表达比例对rAAV生产至关重要,辅助病毒基因表达框中仅保留关键蛋白序列E4orf6(adenovirus early region 4 open reading frame 6,E4orf6)和DBP(DNA binding protein,DBP)即可实现同样的rAAV生产效率。据此结果重新设计的新型三质粒系统瞬时转染人胚胎肾细胞(human embryonic kidney 293 cells,HEK293)时,rAAV基因组滴度较传统三质粒系统提高2.6倍。在进一步优化该系统的转染参数后,基因组滴度达到7.8×10^(11)vg/mL,单细胞产毒量达到1.5×10^(5)vg/cell,相较于优化前分别提高3.7倍和2.4倍。【结论】通过探究rAAV病毒基因和辅助病毒基因的组成与比例对病毒载体包装的影响,重新设计了rAAV的瞬时表达系统,优化并建立了新型三质粒系统的rAAV生产工艺,显著提高了rAAV的产量,其中基因组滴度提高16倍,单细胞产量提高10倍,为rAAV的高效工业化生产奠定了基础。
【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 7.8×10^(11)vg/mL,and the CSVY(cell specific virus yield)reached 1.5×10^(5)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.