无血清生产及柱纯化5型腺相关病毒

Serum-free production and column purification of adeno-associated virus type 5.

作者信息

Smith Richard H, Ding Chuantian, Kotin Robert M

机构信息

Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, Building 10, Room 7N-264, 9000 Rockville Pike, Bethesda, MD 20892, USA.

出版信息

J Virol Methods. 2003 Dec;114(2):115-24. doi: 10.1016/j.jviromet.2003.09.002.

DOI:10.1016/j.jviromet.2003.09.002

PMID:14625046

Abstract

Viral vectors derived from adeno-associated virus (AAV) are rapidly becoming the vehicles of choice for gene therapy applications. AAV-2 is the adeno-associated virus serotype most commonly employed in AAV-mediated gene therapy studies; however, recently developed vectors derived from alternative serotypes of AAV, such as AAV-5, are receiving special attention due to their disparate tissue tropisms and potential for serial administration. In this report, we describe a rapid and efficient method for the serum-free production and column purification of recombinant AAV-5 particles. This method utilizes a combination of anion-exchange chromatography and gel filtration chromatography to purify recombinant AAV particles to near homogeneity. Importantly, viral particles are captured directly from cellular extracts with high efficiency, and vector purification is achieved in less than one working day with a minimal amount of sample manipulation. The method described in this report does not require partial purification by density centrifugation, detergent treatment, or solvent extraction to achieve efficient levels of column binding and vector purification.

摘要

源自腺相关病毒（AAV）的病毒载体正迅速成为基因治疗应用的首选载体。AAV-2是AAV介导的基因治疗研究中最常用的腺相关病毒血清型；然而，最近开发的源自AAV其他血清型（如AAV-5）的载体因其不同的组织嗜性和连续给药的潜力而受到特别关注。在本报告中，我们描述了一种用于无血清生产和柱纯化重组AAV-5颗粒的快速有效方法。该方法利用阴离子交换色谱和凝胶过滤色谱相结合的方式将重组AAV颗粒纯化至接近均一性。重要的是，病毒颗粒可直接从细胞提取物中高效捕获，并且在不到一个工作日的时间内，通过最少的样品处理即可实现载体纯化。本报告中描述的方法不需要通过密度离心、去污剂处理或溶剂萃取进行部分纯化，就能实现高效的柱结合和载体纯化。