基于色谱法的腺相关病毒纯化

Chromatography-based purification of adeno-associated virus.

作者信息

Smith Richard H, Yang Linda, Kotin Robert M

机构信息

Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD, USA.

出版信息

Methods Mol Biol. 2008;434:37-54. doi: 10.1007/978-1-60327-248-3_4.

DOI:10.1007/978-1-60327-248-3_4

PMID:18470638

Abstract

Virus-mediated gene transfer shows great potential as a therapeutic strategy for the management of various inherited and acquired human diseases. Among the current viral vectors, adeno-associated virus (AAV) has become the vector of choice for numerous gene therapy applications. As AAV-based vectors approach the clinic, the need for scalable methods of production and purification is steadily increasing. In this chapter, we present a column chromatography-based protocol for the purification of recombinant AAV type 1 (AAV-1) to near homogeneity. The protocol, which can be completed within one working day, employs three major purification steps: (1) polyethylene glycol-mediated vector precipitation, (2) anion-exchange chromatography, and (3) gel filtration chromatography. This method provides a basic strategy, or "platform," that can be adapted to the purification of other recombinant AAV vector serotypes.

摘要

病毒介导的基因转移作为治疗各种遗传性和获得性人类疾病的一种治疗策略显示出巨大潜力。在当前的病毒载体中，腺相关病毒（AAV）已成为众多基因治疗应用的首选载体。随着基于AAV的载体进入临床，对可扩展的生产和纯化方法的需求正在稳步增加。在本章中，我们介绍一种基于柱色谱的方法，用于将重组1型腺相关病毒（AAV-1）纯化至接近均一性。该方法可在一个工作日内完成，采用三个主要纯化步骤：（1）聚乙二醇介导的载体沉淀，（2）阴离子交换色谱，以及（3）凝胶过滤色谱。此方法提供了一种基本策略或“平台”，可适用于其他重组AAV载体血清型的纯化。