Development of a laboratory scalable process for enhancing lentivirus production by transient transfection of hek293 adherent cultures

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. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS

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institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS A GUIDE IN LENTIVIRAL VECTOR PRODUCTION FOR HARD-TO-TRANSFECT CELLS, USING

CARDIAC-DERIVED C-KIT EXPRESSING CELLS AS A MODEL SYSTEM Article Open access 28 September 2021 ENGINEERED CHO CELLS AS A NOVEL AAV PRODUCTION PLATFORM FOR GENE THERAPY DELIVERY Article Open

access 06 November 2023 UNVEILING MOLECULAR SECRETS: ANALYSIS OF STABLE LENTIVIRAL PACKAGING CELL LINES ENABLES IDENTIFICATION OF NOVEL VIRAL GENE FUNCTIONS Article 15 April 2025 REFERENCES

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Biomacromolecules. 2006;7:2427–35. CAS  PubMed  Google Scholar  Download references 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). AUTHOR INFORMATION Author notes * These authors contributed equally: Y. K. Ho, H. P. Too AUTHORS AND AFFILIATIONS * Department of Biochemistry, National University

of Singapore, Singapore, 117596, Singapore Y. K. Ho & H. P. Too Authors * Y. K. Ho View author publications You can also search for this author inPubMed Google Scholar * H. P. Too View

author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Y. K. Ho. ETHICS DECLARATIONS CONFLICT OF INTEREST The authors declare

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affiliations. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 05 February 2020 *

Revised: 07 April 2020 * Accepted: 08 April 2020 * Published: 27 April 2020 * Issue Date: November 2020 * DOI: https://doi.org/10.1038/s41434-020-0152-x SHARE THIS ARTICLE Anyone you share

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