Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1).
Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1).
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DOI：
10.1038/mtm.2015.58
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Okada T
中科院分区：
文献类型：
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作者：
Tomono T;Hirai Y;Okada H;Adachi K;Ishii A;Shimada T;Onodera M;Tamaoka A;Okada T
重组腺相关病毒（rAAV）是一种有吸引力的基因转移工具，在人类基因治疗中显示出潜在的应用前景。目前从转染的细胞裂解液中生产和纯化rAAV的方法主要是基于氯化铯和碘二醇密度超离心，尽管这些方法不具有可扩展性。同时，基于色谱的系统更具可扩展性。因此，在本研究中，我们开发了一种基于离子交换和凝胶过滤色谱的无血清培养上清生产和纯化rAAV血清型1 （rAAV1）的新方法，通过无超离心技术获得高纯度的产品，以达到良好生产规范（GMP）的要求。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，纯化的rAAV1显示出三条清晰的条带（VP1、VP2和VP3），阴性染色电镜分析显示，90%以上的rAAV1颗粒含有完全包装的病毒基因组。结果表明，从4 × 109 HEK293细胞中纯化得到的rAAV1基因组滴度为3.63 × 1013 vg /ml（总滴度为4.17 × 1013 vg /ml）。这种基于色谱的新方法将有助于扩大基因治疗临床应用的生产规模。
Recombinant adeno-associated virus (rAAV) is an attractive tool for gene transfer and shows potential for use in human gene therapies. The current methods for the production and purification of rAAV from the transfected cell lysate are mainly based on cesium chloride and iodixanol density ultracentrifugation, although those are not scalable. Meanwhile, chromatography-based systems are more scalable. Therefore, in this study, we developed a novel method for the production and purification of rAAV serotype 1 (rAAV1) from serum-free culture supernatant based on ion-exchange and gel-filtration chromatography to obtain highly purified products with an ultracentrifugation-free technique towards Good Manufacturing Practice (GMP) production. The purified rAAV1 displayed three clear and sharp bands (VP1, VP2, and VP3) following sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and more than 90% of rAAV1 particles contained fully packaged viral genomes according to negative-stain electron micrographic analysis. Consequently, the resultant genomic titer of the purified rAAV1 was 3.63 × 1013 v.g./ml (the total titer was 4.17 × 1013 v.g.) from the 4 × 109 HEK293 cells. This novel chromatography-based method will facilitate scale-up of manufacturing for clinical applications in gene therapy.