转基因
重组DNA
病毒学
质粒
转染
HEK 293细胞
遗传增强
分子生物学
腺相关病毒
生物
化学
DNA
细胞培养
遗传学
载体(分子生物学)
基因
作者
Fei Liu,Yue Zhang,M. T. Yip,Lingzhi Ren,Jialing Liang,X. Chen,Nan Li,Ailing Du,Jiaming Wang,Haiyan Chang,Hyejin Oh,Changyong Zhou,Ruiyun Xing,Mingxing Xu,Peiyi Guo,Dominic J. Gessler,Jun Xie,Phillip W.L. Tai,Guangping Gao,Dan Wang
标识
DOI:10.1016/j.omtm.2024.101230
摘要
Recombinant adeno-associated virus (rAAV)-based gene therapy is entering clinical and commercial stages at an unprecedented pace. Triple transfection of HEK293 cells is currently the most widely used platform for rAAV manufacturing. Here, we develop low-cis triple transfection that decreases transgene plasmid use by 10- to 100-fold and overcomes several major limitations associated with standard triple transfection. This new method improves packaging of yield-inhibiting transgenes by up to 10-fold, and generates rAAV batches with reduced plasmid backbone contamination that otherwise cannot be eliminated in downstream processing. When tested in mice and compared with rAAV produced by standard triple transfection, low-cis rAAV shows comparable or superior potency and results in diminished plasmid backbone DNA and RNA persistence in tissue. Mechanistically, low-cis triple transfection relies on the extensive replication of transgene cassette (i.e., inverted terminal repeat-flanked vector DNA) in HEK293 cells during production phase. This cost-effective method can be easily implemented and is widely applicable to producing rAAV of high quantity, purity, and potency.
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