Novel tools for production and purification of recombinant adenoassociated virus vectors
Novel tools for production and purification of recombinant adenoassociated virus vectors
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DOI：
10.1089/hum.1998.9.18-2745
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发表时间：
1998-12-10
影响因子：
4.2
通讯作者：
Kleinschmidt, JA
中科院分区：
文献类型：
--
作者：
Grimm, D;Kern, A;Kleinschmidt, JA
用于人类基因治疗的基于腺相关病毒2型（AAV-2）的载体的标准生成方案需要用重组AAV （rAAV）载体质粒和提供AAV Pep和cap基因的包装质粒共转染细胞。转染的细胞还必须被辅助病毒过度感染，例如腺病毒（Ad），它提供多种辅助功能，是产生rAAV所必需的。因此，使用这些方案生产的rAAV库存被辅助腺病毒污染。本文描述了一种新型包装/辅助质粒pDG的产生，它包含了AAV载体质粒扩增和包装所需的所有AAV和Ad功能。细胞与pDG和AAV载体质粒共转染足以产生感染性rAAV，从而产生无辅助病毒的rAAV库存。使用pDG作为包装质粒获得的rAAV滴度比使用常规rAAV生产方案获得的滴度高出100倍。在pDG中，MMTV-LTR启动子替代AAV-2 p5启动子导致Rep78/68的表达降低；然而，与标准包装质粒相比，VP蛋白的表达量显著增加。免疫荧光分析显示，在pdg转染的细胞中，VP蛋白的强烈积累导致AAV衣壳组装增强，这限制了高效生产rAAV。此外，利用AAV-2衣壳高特异性单克隆抗体（A20），建立了rAAV亲和纯化程序。这些工具的应用使重组AAV载体的生产和纯化有了显著的提高。
Standard protocols for the generation of adenoassociated virus type 2 (AAV-2)-based vectors for human gene therapy applications require cotransfection of cells with a recombinant AAV (rAAV) vector plasmid and a packaging plasmid that provides the AAV Pep and cap genes. The transfected cells must also be overinfected with a helper virus, e.g., adenovirus (Ad), which delivers multiple helper functions necessary for rAAV production. Therefore, rAAV stocks produced using these protocols are contaminated with helper adenovirus. The generation of a novel packaging/helper plasmid, pDG, containing all AAV and Ad functions required for amplification and packaging of AAV vector plasmids, is described here. Cotransfection of cells with pDG and an AAV vector plasmid was sufficient for production of infectious rAAV, resulting in helper virus-free rAAV stocks. The rAAV titers obtained using pDG as packaging plasmid were up to Ill-fold higher than those achieved using conventional protocols for rAAV production. Replacement of the AAV-2 p5 promoter by an MMTV-LTR promoter in pDG led to reduced expression of Rep78/68; however, expression of the VP proteins was significantly increased compared with VP levels from standard packaging plasmids. Immunofluorescence analyses showed that the strong accumulation of VP proteins in pDG-transfected cells resulted in enhanced AAV capsid assembly, which is limiting for efficient rAAV production. Furthermore, using a monoclonal antibody highly specific for AAV-2 capsids (A20), an rAAV affinity purification procedure protocol was established. The application of the tools described here led to a significant improvement in recombinant AAV vector production and purification.