利用流电穿孔技术高效大规模生产慢病毒载体。
Efficient large volume lentiviral vector production using flow electroporation.
机构信息
Department of Medical and Molecular Genetics, Indiana University School of Medicine , Indianapolis, IN 46202, USA.
出版信息
Hum Gene Ther. 2012 Feb;23(2):243-9. doi: 10.1089/hum.2011.088. Epub 2011 Oct 24.
Abstract
Lentiviral vectors are beginning to emerge as a viable choice for human gene therapy. Here, we describe a method that combines the convenience of a suspension cell line with a scalable, nonchemically based, and GMP-compliant transfection technique known as flow electroporation (EP). Flow EP parameters for serum-free adapted HEK293FT cells were optimized to limit toxicity and maximize titers. Using a third generation, HIV-based, lentiviral vector system pseudotyped with the vesicular stomatitis glycoprotein envelope, both small- and large-volume transfections produced titers over 1×10(8) infectious units/mL. Therefore, an excellent option for implementing large-scale, clinical lentiviral productions is flow EP of suspension cell lines.
摘要
慢病毒载体开始成为人类基因治疗的可行选择。在这里，我们描述了一种结合悬浮细胞系的便利性与可扩展、非化学基础且符合 GMP 的转染技术（即流电泳（EP））的方法。针对无血清适应的 HEK293FT 细胞优化了流 EP 的参数，以限制毒性并最大限度地提高滴度。使用第三代、基于 HIV 的、假型为水疱性口炎病毒糖蛋白包膜的慢病毒载体系统，小体积和大体积转染均产生超过 1×10(8)感染性单位/mL 的滴度。因此，流电泳转染悬浮细胞系是实施大规模临床慢病毒生产的绝佳选择。
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