Purification of recombinant adeno-associated virus type 8 vectors by ion exchange chromatography generates clinical grade vector stock
Purification of recombinant adeno-associated virus type 8 vectors by ion exchange chromatography generates clinical grade vector stock
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DOI：
10.1016/j.jviromet.2004.07.001
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发表时间：
2004-11-01
影响因子：
3.1
通讯作者：
Nathwani, AC
中科院分区：
文献类型：
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作者：
Davidoff, AM;Ng, CYC;Nathwani, AC
基于最近分离的AAV血清型8（rAAV-8）的重组载体显示出基因治疗的巨大前景，特别是对于影响肝脏的疾病。然而，这种载体系统向临床的过渡受到缺乏有效的可规模化纯化方法的限制。在这份报告中，我们描述了一种简单的方法，用于纯化rAAV-8载体颗粒的基础上离子交换层析，产生载体股票大于90%的纯度。从五种不同载体制备物纯化的rAAV-8的平均产率为41。这些纯化的储备物的电子显微镜显示典型的二十面体病毒体，具有小于10%的空颗粒。编码人因子IX（hFIX）基因的离子交换纯化的rAAV-8载体的肝靶向递送导致免疫活性小鼠中血浆hFIX水平接近正常水平的30%，这比用相等数量的rAAV-5离子交换纯化的载体颗粒观察到的高20倍。该方法花费不到5小时从生产细胞中处理和纯化rAAV-8载体，代表了目前用于纯化rAAV-8载体系统的CsCl密度离心技术的显著进步，并可能促进rAAV-8载体系统向临床的过渡。(C)2004 Elsevier B. V.保留所有权利。
Recombinant vectors based on the recently isolated AAV serotype 8 (rAAV-8) shows great promise for gene therapy, particularly for disorders affecting the liver. Transition of this vector system to the clinic, however, is limited by the lack of an efficient scaleable purification method. In this report, we describe a simple method for purification of rAAV-8 vector particles based on ion exchange chromatography that generates vector stocks with greater than 90% purity. The average yield of purified rAAV-8 from five different vector preparation was 41 Electron microscopy of these purified stocks revealed typical icosohedral virions with less than 10% empty particles. Liver targeted delivery of ion-exchange purified rAAV-8 vector encoding the human factor IX (hFIX) gene, resulted in plasma hFIX levels approaching 30% of normal in immunocompetent mice, which is 20-fold higher than observed with an equivalent number of rAAV-5 ion exchange purified vector particles. The method takes less then 5 h to process and purify rAAV-8 vector from producer cells and represents a significant advance on the CsCl density centrifugation technique in current use for purification of rAAV-8 vector systems and will likely facilitate the transition of the rAAV-8 vector system to the clinic. (C) 2004 Elsevier B.V. All rights reserved.