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Development of a laboratory scalable process for enhancing lentivirus production by transient transfection of HEK293 adherent cultures

Gene Therapy volume 27, pages 482–494 (2020)Cite this article

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Abstract

Transfection of surface adherent cells remain as a standard methodology for lentiviral production for early phase clinical studies and research purposes. Production today is based on transient co-transfection of three or four plasmids, where the viral elements are encoded separately for safety reasons. Assembly of functional lentiviral particles requires all plasmids to be efficiently transfected into each cell, a notable challenge with many currently available methods for transient transfection. We have previously demonstrated the significant improvement of cationic polymer-based transfection in various cell types using a combination of fusogenic lipids and histone deacetylase 6 inhibitor (Enhancers). In this report, we focused on the transfection step and the feasibility of improving lentiviral production using the Enhancers. After optimization of DNA amount and N/P ratio, transfection using seven commercial gene carriers showed comparable maximal efficiency of production with high cell viability. In the presence of Enhancers, the production of functional lentivirus using LPEI was increased by as much as tenfold and outperformed lentiviral production using Lipofectamine 3000. We demonstrate a scalable and optimized workflow where the use of the Enhancers significantly improved the lentiviral particle production in various HEK293 cell lines.

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Fig. 1: Determination of the transfection efficiencies of various commercial carriers in HEK293FT.
Fig. 2: Enhancers enhanced total protein production in HEK293FT transfected with PEIMAX (1 µg of DNA to 3 µL of PEIMAX).
Fig. 3: High amount of viral packaging mix and expression plasmid is detrimental to lentiviral production with PEIMAX-mediated transfection.
Fig. 4: Enhancers increased functional viral titre.
Fig. 5: Enhances significantly improved the quality of the lentiviral titres.
Fig. 6: Prolonged production of LV.
Fig. 7: Enhancers improved lentiviral production with different commercial packaging mix.
Fig. 8: Enhancers improved lentiviral production in HEK293 variants.

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Acknowledgements

This study is funded and supported by SMART Innovation Grant, Technology Adoption Programme and the Department of Biochemistry (National University of Singapore), respectively. The authors would like to thank Justin Tan Bing Quan for his contribution in assisting this study by producing the materials and conducting experiments.

Funding

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This project is supported by Yong Loo Lin School of Medicine, Adhoc funding from the National University Health System (NUHSRO/2019/085) and SMART Innovation Centre (Grant number: ING-000144 BIO).

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  1. These authors contributed equally: Y. K. Ho, H. P. Too

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  1. Department of Biochemistry, National University of Singapore, Singapore, 117596, Singapore

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Ho, Y.K., Too, H.P. Development of a laboratory scalable process for enhancing lentivirus production by transient transfection of HEK293 adherent cultures. Gene Ther 27, 482–494 (2020). https://doi.org/10.1038/s41434-020-0152-x

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