转染
聚乙烯亚胺
HEK 293细胞
细胞培养
病毒载体
遗传增强
化学
效价
重组DNA
生物
质粒
分子生物学
细胞生物学
基因
病毒学
病毒
生物化学
遗传学
作者
Sven Ansorge,Stéphane Lanthier,Julia Transfiguracion,Yves Durocher,Olivier Henry,Amine Kamen
摘要
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. Copyright © 2009 John Wiley & Sons, Ltd.
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