학술논문
Development of a scalable process for high-yield lentiviral vector production by transient transfection of HEK293 suspension cultures.
Document Type
Academic Journal
Author
Ansorge S; National Research Council Canada, Biotechnology Research Institute, Montréal, Québec H4P 2R2, Canada.; Lanthier S; Transfiguracion J; Durocher Y; Henry O; Kamen A
Source
Publisher: John Wiley & Sons Country of Publication: England NLM ID: 9815764 Publication Model: Print Cited Medium: Internet ISSN: 1521-2254 (Electronic) Linking ISSN: 1099498X NLM ISO Abbreviation: J Gene Med Subsets: MEDLINE
Subject
Language
English
Abstract
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.
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.