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

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

摘要/Abstract

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7.8×10¹¹ vg/mL，单细胞产毒量达到1.5×10⁵ 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。

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

李蕊蕊, 那道远, 王洪林, 赵亮, 谭文松, 叶倩. 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[J]. 生物技术通报, 2024, 40(12): 61-71.

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.

图/表 6

图1 质粒构建示意图 A：分体的rep基因和cap基因包装质粒构建示意图；B：辅助质粒构建示意图；C：新型三质粒系统构建示意图

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

图2 rep基因和cap基因的表达比例对rAAV产量的影响 A：转染12 h后病毒蛋白相对转录水平；B：转染效率和平均荧光强度；C：不同剂量rep基因对基因组滴度和CSVY的影响；D：不同剂量cap基因对基因组滴度和CSVY的影响；*、**、***分别表示在P<0.05、P<0.01、P<0.001水平上差异显著，下同

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

图3 辅助病毒基因关键序列对rAAV生产的影响 A：基因组滴度；B：单细胞产毒量；ns表示在P>0.05水平上无显著差异，下同

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

图4 合理优化质粒系统以提高rAAV产量 A：基因组滴度；B：单细胞产毒量

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

图5 新型三质粒系统的工艺参数优化 A：质粒与细胞的比例；B：质粒与转染试剂的比例；C：转染时细胞密度；D：三质粒的比例

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

图6 新型三质粒系统瞬时转染生产rAAV的基因组滴度（A）及CSVY（B）

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

参考文献 29

[1]	Araujo NM. A dual role for adeno-associated virus in human health[J]. Virol J, 2023, 20(1): 228.
[2]	Bretti A, Doong S. AAV production by transient transfection: strategies & challenges[J]. Cell Gene Therapy Insights, 2023, 9(5): 763-776.
[3]	Alomari O. Urgent call to action: making gene therapy for Duchenne muscular dystrophy(DMD)affordable and accessible[J]. Br J Clin Pharmacol, 2023, 89(11): 3463-3464. doi: 10.1111/bcp.15900 pmid: 37694346
[4]	Wang D, Tai PWL, Gao GP. Adeno-associated virus vector as a platform for gene therapy delivery[J]. Nat Rev Drug Discov, 2019, 18(5): 358-378. doi: 10.1038/s41573-019-0012-9 pmid: 30710128
[5]	Xiao X, Li J, Samulski RJ. Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus[J]. J Virol, 1998, 72(3): 2224-2232. doi: 10.1128/JVI.72.3.2224-2232.1998 pmid: 9499080
[6]	Ayuso E, Mingozzi F, Bosch F. Production, purification and characterization of adeno-associated vectors[J]. Curr Gene Ther, 2010, 10(6): 423-436. pmid: 21054248
[7]	Ohba K, Sehara Y, Enoki T, et al. Adeno-associated virus vector system controlling capsid expression improves viral quantity and quality[J]. iScience, 2023, 26(4): 106487.
[8]	Strobel B, Klauser B, Hartig JS, et al. Riboswitch-mediated attenuation of transgene cytotoxicity increases adeno-associated virus vector yields in HEK-293 cells[J]. Mol Ther, 2015, 23(10): 1582-1591. doi: 10.1038/mt.2015.123 pmid: 26137851
[9]	Mietzsch M, Eddington C, Jose A, et al. Improved genome packaging efficiency of adeno-associated virus vectors using rep hybrids[J]. J Virol, 2021, 95(19): e0077321.
[10]	Scarrott JM, Johari YB, Pohle TH, et al. Increased recombinant adeno-associated virus production by HEK293 cells using small molecule chemical additives[J]. Biotechnol J, 2023, 18(3): e2200450.
[11]	Zhao HR, Lee KJ, Daris M, et al. Creation of a high-yield AAV vector production platform in suspension cells using a design-of-experiment approach[J]. Mol Ther Methods Clin Dev, 2020, 18: 312-320.
[12]	Park S, Shin S, Lee H, et al. Enhancing the production of adeno-associated virus(AAV)₂ and AAV9 with high full capsid ratio in HEK293 cells through design-of-experiment optimization of triple plasmid ratio[J]. Biotechnol J, 2024, 19(3): e2300667.
[13]	Lu M, Lin YC, Kuo HJ, et al. Tuning capsid formation dynamics in recombinant adeno-associated virus producing synthetic cell lines to enhance full particle productivity[J]. Biotechnol J, 2024, 19(3): e2400051.
[14]	Li CW, Samulski RJ. Serotype-specific replicating AAV helper constructs increase recombinant AAV type 2 vector production[J]. Virology, 2005, 335(1): 10-21. pmid: 15823602
[15]	Emmerling VV, Pegel A, Milian EG, et al. Rational plasmid design and bioprocess optimization to enhance recombinant adeno-associated virus(AAV)productivity in mammalian cells[J]. Biotechnol J, 2016, 11(2): 290-297. doi: 10.1002/biot.201500176 pmid: 26284700
[16]	Meier AF, Fraefel C, Seyffert M. The interplay between adeno-associated virus and its helper viruses[J]. Viruses, 2020, 12(6): 662.
[17]	Ward P, Dean FB, O'Donnell ME, et al. Role of the adenovirus DNA-binding protein in in vitro adeno-associated virus DNA replication[J]. J Virol, 1998, 72(1): 420-427. pmid: 9420241
[18]	Allen JM, Halbert CL, Miller AD. Improved adeno-associated virus vector production with transfection of a single helper adenovirus gene, E4orf6[J]. Mol Ther, 2000, 1(1): 88-95. pmid: 10933916
[19]	Liu H, Zhang Y, Yip M, et al. Producing high-quantity and high-quality recombinant adeno-associated virus by low-cis triple transfection[J]. Mol Ther Methods Clin Dev, 2024, 32(2): 101230.
[20]	Wang YD, Fu Q, Park SY, et al. Decoding cellular mechanism of recombinant adeno-associated virus(rAAV)and engineering host-cell factories toward intensified viral vector manufacturing[J]. Biotechnol Adv, 2024, 71: 108322.
[21]	Lu M, Lee Z, Lin YC, et al. Enhancing the production of recombinant adeno-associated virus in synthetic cell lines through systematic characterization[J]. Biotechnol Bioeng, 2024, 121(1): 341-354.
[22]	Moreno F, Lip F, Rojas H, et al. Development of an insect cell-based adeno-associated virus packaging cell line employing advanced Rep gene expression control system[J]. Mol Ther Methods Clin Dev, 2022, 27: 391-403.
[23]	付强, 李艳, 郑昭芬, 等. 聚乙烯亚胺介导三质粒共转染HEK293细胞的条件优化[J]. 生物医学工程学杂志, 2015, 32(1): 137-141.
	Fu Q, Li Y, Zheng ZF, et al. Optimization of triple plasmids transfection into HEK293 cells mediated by polyethylenimine[J]. J Biomed Eng, 2015, 32(1): 137-141.
[24]	Moghimi SM, Symonds P, Murray JC, et al. A two-stage poly(ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy[J]. Mol Ther, 2005, 11(6): 990-995. doi: 10.1016/j.ymthe.2005.02.010 pmid: 15922971
[25]	Moço PD, Xu XG, Silva CAT, et al. Production of adeno-associated viral vector serotype 6 by triple transfection of suspension HEK293 cells at higher cell densities[J]. Biotechnol J, 2023, 18(9): e2300051.
[26]	Fu Q, Polanco A, Lee YS, et al. Critical challenges and advances in recombinant adeno-associated virus(rAAV)biomanufacturing[J]. Biotechnol Bioeng, 2023, 120(9): 2601-2621.
[27]	Marwidi Y, Nguyen HO B, Santos D, et al. A robust and flexible baculovirus-insect cell system for AAV vector production with improved yield, capsid ratios and potency[J]. Mol Ther Methods Clin Dev, 2024, 32(2): 101228.
[28]	张峒, 蒋卓含, 吴奕凡, 等. 利用多基因缺失型杆状病毒在昆虫细胞中生产腺相关病毒[J]. 生物工程学报, 2024, 40(2): 473-484.
	Zhang T, Jiang ZH, Wu YF, et al. Production of adeno-associated virus in insect cells using multiple gene deleted baculovirus[J]. Chinese Journal of Biotechnology, 2024, 40(2): 473-484. doi: 10.13345/j.cjb.230325 pmid: 38369834
[29]	Dobrowsky T, Gianni D, Pieracci J, et al. AAV manufacturing for clinical use: insights on current challenges from the upstream process perspective[J]. Curr Opin Biomed Eng, 2021, 20: 100353.