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

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 W.L. Tai; Guangping Gao; Dan Wang

Molecular Therapy: Methods & Clinical Development (Jun 2024)

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

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 W.L. Tai,
Guangping Gao,
Dan Wang

Affiliations

Hao Liu: Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
Yue Zhang: Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
Mitchell Yip: Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
Lingzhi Ren: Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
Jialing Liang: Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
Xiupeng Chen: Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
Nan Liu: Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
Ailing Du: Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
Jiaming Wang: Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
Hao Chang: Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
Hyejin Oh: Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
Chen Zhou: Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
Ruxiao Xing: Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
Mengyao Xu: Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
Peiyi Guo: Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
Dominic Gessler: Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
Jun Xie: Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
Phillip W.L. Tai: Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
Guangping Gao: 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; Corresponding author: Guangping Gao, PhD, Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
Dan Wang: Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA; RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA; Corresponding author: Dan Wang, PhD, Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.

Journal volume & issue: Vol. 32, no. 2
p. 101230

Abstract

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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.

Published in Molecular Therapy: Methods & Clinical Development

ISSN: 2329-0501 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Biology (General): Genetics; Science: Biology (General): Cytology
Website: http://www.cell.com/molecular-therapy-family/methods/latest-content

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