Neurobiology Laboratory, U.S.A; Viral Vector Core, U.S.A.
Neurobiology Laboratory, U.S.A.
J Virol Methods. 2020 Jul;281:113863. doi: 10.1016/j.jviromet.2020.113863. Epub 2020 May 1.
Recombinant adeno-associated viruses (rAAVs) are robust and versatile tools for in vivo gene delivery. Natural and designer capsid variations in rAAVs allow for targeted gene delivery to specific cell types. Low immunogenicity and lack of pathogenesis also add to the popularity of this virus as an innocuous gene delivery vector for gene therapy. rAAVs are routinely used to express recombinases, sensors, detectors, CRISPR-Cas9 components, or to simply overexpress a gene of interest for functional studies. High production demand has given rise to multiple platforms for the production and purification of rAAVs. However, most platforms rely heavily on large amounts of starting material and multiple purification steps to produce highly purified viral particles. Often, researchers require several small-scale purified rAAVs. Here, we describe a simple and efficient technique for purification of recombinant rAAVs from small amounts of starting material in a two-step purification method. In this method, rAAVs are released into the packaging cell medium using high salt concentration, pelleted by ultracentrifugation to remove soluble impurities. Then, the resuspended pellet is purified using a protein spin-concentrator. In this protocol, we modify the conventional rAAV purification methods to eliminate the need for fraction collection and the labor-intensive steps for evaluating the titer and purity of individual fractions. The resulting rAAV preparations are comparable in titer and purity to commercially available samples. This simplified process can be used to generate highly purified rAAV particles on a small scale, thereby saving resources, generating less waste, and reducing a laboratory's environmental footprint.
重组腺相关病毒（rAAV）是体内基因传递的强大而多功能的工具。rAAV 的天然和设计衣壳变异允许针对特定细胞类型进行靶向基因传递。低免疫原性和缺乏发病机制也增加了这种病毒作为基因治疗中无毒性基因传递载体的受欢迎程度。rAAV 通常用于表达重组酶、传感器、探测器、CRISPR-Cas9 组件，或简单地上调感兴趣基因以进行功能研究。高生产需求促使出现了多种用于生产和纯化 rAAV 的平台。然而，大多数平台严重依赖大量起始材料和多个纯化步骤来生产高度纯化的病毒颗粒。通常，研究人员需要几个小规模纯化的 rAAV。在这里，我们描述了一种从少量起始材料中通过两步纯化方法纯化重组 rAAV 的简单高效技术。在该方法中，使用高盐浓度将 rAAV 释放到包装细胞培养基中，通过超速离心沉淀去除可溶性杂质。然后，将重新悬浮的沉淀使用蛋白质旋转浓缩器进行纯化。在本方案中，我们修改了常规的 rAAV 纯化方法，以消除对馏分收集和评估每个馏分的滴度和纯度的劳动密集型步骤的需求。所得 rAAV 制剂在滴度和纯度上与市售样品相当。该简化过程可用于小规模生成高度纯化的 rAAV 颗粒，从而节省资源、减少废物产生，并降低实验室的环境足迹。
