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

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DOIResolve DOI: http://doi.org/10.1002/jgm.1370
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TypeArticle
Journal titleThe journal of gene medicine
Volume11
Issue10
Pages868876; # of pages: 9
Subjectbio; Biotechnology; Cell Line; Genes; methods; Plasmids; Sodium; therapy; Transfection; gene therapy; HEK293; lentiviral vectors; scalable production; suspension culture
AbstractBackground: 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.
Publication date
LanguageEnglish
AffiliationNational Research Council Canada; NRC Biotechnology Research Institute
Peer reviewedYes
NRC number52728
NPARC number12405291
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Record identifierafc77b24-dd8e-4868-aaff-15462330cbb1
Record created2009-10-26
Record modified2016-05-09
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