Liu Hao, Zhang Yue, Yip Mitchell, Ren Lingzhi, Liang Jialing, Chen Xiupeng, Liu Nan, Du Ailing, Wang Jiaming, Chang Hao, Oh Hyejin, Zhou Chen, Xing Ruxiao, Xu Mengyao, Guo Peiyi, Gessler Dominic, Xie Jun, Tai Phillip W L, Gao Guangping, Wang Dan
Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
Department of Microbiology and Physiological Systems, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
Mol Ther Methods Clin Dev. 2024 Mar 12;32(2):101230. doi: 10.1016/j.omtm.2024.101230. eCollection 2024 Jun 13.
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.
基于重组腺相关病毒（rAAV）的基因疗法正以前所未有的速度进入临床和商业阶段。HEK293细胞的三重转染是目前rAAV生产中使用最广泛的平台。在此，我们开发了低顺式三重转染方法，该方法可将转基因质粒的使用量减少10至100倍，并克服了与标准三重转染相关的几个主要限制。这种新方法将抑制产量的转基因包装效率提高了多达10倍，并产生了质粒骨架污染减少的rAAV批次，否则在下游加工过程中无法消除这些污染。在小鼠中进行测试并与标准三重转染产生的rAAV相比时，低顺式rAAV显示出相当或更高的效力，并减少了组织中质粒骨架DNA和RNA的残留。从机制上讲，低顺式三重转染依赖于生产阶段HEK293细胞中转基因盒（即反向末端重复序列侧翼的载体DNA）的大量复制。这种经济高效的方法易于实施，广泛适用于生产高质量、高纯度和高效力的rAAV。
