Development of a scalable process for high-yield lentiviral vector production by transient transfection of HEK293 suspension cultures.
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 10(8) 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 10(11) 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.
Ansorge
Sven
S
National Research Council Canada, Biotechnology Research Institute, Montréal, Québec H4P 2R2, Canada.
Lanthier
Stéphane
S
Transfiguracion
Julia
J
Durocher
Yves
Y
Henry
Olivier
O
Kamen
Amine
A
D019149:Bioreactors; D002087:Butyrates / Q000737:chemistry; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D003470:Culture Media / Q000737:chemistry; D005822:Genetic Vectors / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D014162:Transfection
D002087:Butyrates; D003470:Culture Media