Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct

• Main Authors: Yu Hua Chen, Celeste Pallant, Christopher J. Sampson, Alessia Boiti, Sabine Johnson, Pijus Brazauskas, Philip Hardwicke, Michela Marongiu, Vanesa M. Marinova, Marlene Carmo, Nathan P. Sweeney, Ashkenaz Richard, Anthony Shillings, Peter Archibald, Eva Puschmann, Bernadette Mouzon, David Grose, Miriam Mendez-Tavio, Mao Xiang Chen, Stephen R.C. Warr, Tarik Senussi, Paul S. Carter, Sean Baker, Cindy Jung, Martijn H. Brugman, Steven J. Howe, Conrad A. Vink
• Format: Article
• Language: English
• Published: Elsevier 2020-12-01
• Series: Molecular Therapy: Methods & Clinical Development
• Subjects: lentiviral vector; cell line development; producer cell line; manufacturing
• Online Access: http://www.sciencedirect.com/science/article/pii/S2329050120301777

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.