Citation
- Authors: Shiina S. et al.
- Year: 2022
- Journal: J Biosci Bioeng S1389-1723 00194-3
- Applications: in vitro / DNA / FectoVIR-AAV
- Cell type: Expi293F
Description: Human embryonic kidney Fibroblast
Known as: Expi 293-F, Expi, HEK-293 Expi
Method
To produce rAAV2 in Expi293F cells, three plasmid DNAs were used in each experiment: (i) a packaging plasmid containing the adeno-associated virus (AAV) serotype rep 2 and cap 2 gene (pRC2-mi342, Takara Bio Inc., Shiga, Japan); (ii) a helper plasmid containing the helper gene E2A, VA, and E4 genes originating from adenovirus (pHelper, Takara Bio Inc.); and (iii) an AAV genome plasmid DNA containing the GFP gene as a gene of interest (GOI) flanked by the inverted terminal repeats (ITRs) (pAAV-GFP, Cell Biolabs Inc., San Diego, CA, USA). The transfection complex was prepared in a volume of liquid containing 10% of the culture medium after transfection. The three plasmids were added to DMEM (Sigma-Aldrich) at the following concentrations. pRC2-mi342: 0.5 μg per 106 cells, pHelper: 0.25 μg per 106 cells, pAAV-GFP: 0.25 μg per 106 cells. To this plasmid DNA suspension, FectoVIR-AAV (101000022; Polyplus, Illkirch, France) was added at a concentration of 2 μL per 106 cells, mixed by inverting 20 times, and allowed to stand for 30 min at room temperature.
Abstract
Investigation of enhancers to improve recombinant adeno-associated virus 2 (rAAV2) productivity by human embryonic kidney 293 cells (HEK293) suspension culture showed that the addition of ethanol improved the productivity and packaged genome integrity of rAAV2. Further optimization showed that adding ethanol in the range of 0.09%-1.11% (v/v) during rAAV2 production effectively improved rAAV2 productivity and quality. In addition, ethanol addition improved cell viability. Furthermore, proteome and pathway analysis of the cells during rAAV2 production showed that the addition of ethanol resulted in the upregulation of pathways related to intercellular signaling, gene expression, cell morphology, intercellular maintenance, and others. In contrast, pathways related to cell death, immunity, and reactions to infection were downregulated. These changes in pathway regulation were responsible for the improvement in rAAV2 productivity, packaged genome integrity, and cell viability during rAAV2 production. The results of this study can be applied to the production of viral vectors for in vivo gene therapy in an inexpensive and safe manner.