Development of a scalable process for high-yield lentiviral vector production by transient transfection of HEK293 suspension cultures.

Autor: Ansorge, Sven, Lanthier, Stéphane, Transfiguracion, Julia, Durocher, Yves, Henry, Olivier, Kamen, Amine
Zdroj: Journal of Gene Medicine; Oct2009, Vol. 11 Issue 10, p868-876, 9p
Abstrakt: Background Lentiviral vectors (LV) offer several advantages over other gene delivery vectors. Their potential for the integration and long-term expression of therapeutic genes renders them an interesting tool for gene and cell therapy interventions. However, large-scale LV production remains an important challenge for the translation of LV-based therapeutic strategies to the clinic. The development of robust processes for mass production of LV is needed. Methods A suspension-grown HEK293 cell line was exploited for the production of green fluorescent protein-expressing LV by transient polyethylenimine (PEI)-based transfection with LV-encoding plasmid constructs. Using third-generation packaging plasmids ( Gag/Pol, Rev), a vesicular stomatitis virus G envelope and a self-inactivating transfer vector, we employed strategies to increase volumetric and specific productivity. Functional LV titers were determined using a flow cytometry-based gene transfer assay. Results A combination of the most promising conditions (increase in cell density, medium selection, reduction of PEI-DNA complexes per cell, addition of sodium butyrate) resulted in significantly increased LV titers of more than 150-fold compared to non-optimized small-scale conditions, reaching infectious titers of approximately 108 transducing units/ml. These conditions are readily scalable and were validated in 3-liter scale perfusion cultures. Conclusions Our process produces LV in suspension cultures and is consequently easily scalable, industrially viable and generated more than 1011 total functional LV particles in a single bioreactor run. This process will allow the production of LV by transient transfection in sufficiently large quantities for phase I clinical trials at the 10-20-liter bioreactor scale. Copyright © 2009 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]
Databáze: Complementary Index
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