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Producing high-quantity and high-quality recombinant adeno-associated virus by low-cis triple transfection.
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.
Afiliação
  • Liu H; Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
  • Zhang Y; Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
  • Yip M; Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
  • Ren L; Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
  • Liang J; Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
  • Chen X; Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
  • Liu N; Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
  • Du A; Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
  • Wang J; Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
  • Chang H; Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
  • Oh H; Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
  • Zhou C; Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
  • Xing R; Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
  • Xu M; Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
  • Guo P; Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
  • Gessler D; Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
  • Xie J; Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
  • Tai PWL; Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
  • Gao G; Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
  • Wang D; Department of Microbiology and Physiological Systems, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
Mol Ther Methods Clin Dev ; 32(2): 101230, 2024 Jun 13.
Article em En | MEDLINE | ID: mdl-38558570
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 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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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Idioma: En Revista: Mol Ther Methods Clin Dev Ano de publicação: 2024 Tipo de documento: Article País de afiliação: Estados Unidos
Texto completo
Adicionar na Minha BVS
Imprimir
XML
PubMed Links
Buscar no Google

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Idioma: En Revista: Mol Ther Methods Clin Dev Ano de publicação: 2024 Tipo de documento: Article País de afiliação: Estados Unidos