High-level production of replication-defective human immunodeficiency type 1 virus vector particles using helper-dependent adenovirus vectors.
High-level production of replication-defective human immunodeficiency type 1 virus vector particles using helper-dependent adenovirus vectors.
复制标题
使用辅助依赖性腺病毒载体高水平生产复制缺陷型人类免疫缺陷 1 型病毒载体颗粒。
DOI：
10.1038/mtm.2015.4
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Sutton,RichardE
中科院分区：
文献类型：
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作者：
Hu,Yani;O'Boyle,Kaitlin;Palmer,Donna;Ng,Philip;Sutton,RichardE
基于人类免疫缺陷病毒1型(HIV)的基因转移载体被广泛应用于实验室研究和临床研究，不仅可以引入新的基因，还可以减少宿主和病原体不需要的基因的表达。目前，绝大多数基于HIV的载体上清液是在293T细胞中通过最多5个DNA质粒共转染产生的，这一过程易受变异性的影响，难以规模化。在这里，我们报告了一种基于HIV的载体生产系统的开发，该系统利用助手依赖腺病毒(HDAd)。所有必要的HIV载体组分被插入到一个或多个HDAD中，然后被扩增到非常高的∼1013vp/ml滴度，然后被用于转导293细胞以产生基于HIV的载体上清，所得到的VSVG伪型慢病毒载体(LV)滴度和总IU比质粒转染法高10-30倍。基于HIV的载体生产的优化依赖于来自HDAd的所有HIV载体组分的最大化表达。培养上清中含有微量的HDAd，但不含复制能力强的慢病毒。该生产方法应适用于其他逆转录病毒载体系统。使用这种两步法生产基于艾滋病毒的载体的可规模生产应该会促进它们的临床进步。
Gene transfer vectors based upon human immunodeficiency virus type 1 (HIV) are widely used in bench research applications and increasingly in clinical investigations, both to introduce novel genes but also to reduce expression of unwanted genes of the host and pathogen. At present, the vast majority of HIV-based vector supernatants are produced in 293T cells by cotransfection of up to five DNA plasmids, which is subject to variability and difficult to scale. Here we report the development of a HIV-based vector production system that utilizes helper-dependent adenovirus (HDAd). All necessary HIV vector components were inserted into one or more HDAds, which were then amplified to very high titers of ∼1013vp/ml. These were then used to transduce 293-based cells to produce HIV-based vector supernatants, and resultant VSV G-pseudotyped lentiviral vector (LV) titers and total IU were 10- to 30-fold higher, compared to plasmid transfection. Optimization of HIV-based vector production depended upon maximizing expression of all HIV vector components from HDAd. Supernatants contained trace amounts of HDAd but were free of replication-competent lentivirus. This production method should be applicable to other retroviral vector systems. Scalable production of HIV-based vectors using this two-step procedure should facilitate their clinical advancement.