Producing high-quantity and high-quality recombinant adeno-associated virus by low-cis triple transfection

IF 4.6 2区医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-03-12 DOI:10.1016/j.omtm.2024.101230

Hao Liu, Yue Zhang, Mitchell Yip, Lingzhi Ren, Jialing Liang, Xiupeng Chen, Nan Liu, Ailing Du, Jiaming Wang, Hao Chang, Hyejin Oh, Chen Zhou, Ruxiao Xing, Mengyao Xu, Peiyi Guo, Dominic Gessler, Jun Xie, Phillip WL. Tai, Guangping Gao, Dan Wang

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Abstract

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 reduces the transgene plasmid usage 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., ITR-flanked vector DNA) in HEK293 cells during production phase. This cost-effective method can be easily implemented and widely applicable to producing rAAV of high quantity, purity, and potency.

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通过低顺式三重转染技术生产高数量和高质量的重组腺相关病毒

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最广泛使用的 rAAV 生产平台。在这里，我们开发了低顺式三重转染技术，可将转基因质粒用量减少 10 到 100 倍，并克服了与标准三重转染相关的几个主要限制。这种新方法可将产量抑制型转基因的包装提高 10 倍，并能生产出减少质粒骨架污染的 rAAV 批次，而这种污染在下游处理过程中是无法消除的。在小鼠体内进行测试并与标准三重转染产生的 rAAV 进行比较时，低顺式 rAAV 显示出相当或更高的效力，并减少了质粒骨架 DNA 和 RNA 在组织中的持久性。从机理上讲，低顺式三重转染依赖于转基因盒（即 ITR 侧翼载体 DNA）在 HEK293 细胞中生产阶段的广泛复制。这种经济有效的方法易于实施，可广泛用于生产高数量、高纯度和高效力的 rAAV。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Molecular Therapy-Methods & Clinical Development Biochemistry, Genetics and Molecular Biology-Molecular Biology

CiteScore

9.90

自引率

4.30%

发文量

163

审稿时长

12 weeks

期刊介绍： The aim of Molecular Therapy—Methods & Clinical Development is to build upon the success of Molecular Therapy in publishing important peer-reviewed methods and procedures, as well as translational advances in the broad array of fields under the molecular therapy umbrella. Topics of particular interest within the journal''s scope include: Gene vector engineering and production, Methods for targeted genome editing and engineering, Methods and technology development for cell reprogramming and directed differentiation of pluripotent cells, Methods for gene and cell vector delivery, Development of biomaterials and nanoparticles for applications in gene and cell therapy and regenerative medicine, Analysis of gene and cell vector biodistribution and tracking, Pharmacology/toxicology studies of new and next-generation vectors, Methods for cell isolation, engineering, culture, expansion, and transplantation, Cell processing, storage, and banking for therapeutic application, Preclinical and QC/QA assay development, Translational and clinical scale-up and Good Manufacturing procedures and process development, Clinical protocol development, Computational and bioinformatic methods for analysis, modeling, or visualization of biological data, Negotiating the regulatory approval process and obtaining such approval for clinical trials.