腺相关病毒载体（AAV）的可扩展生产与纯化

Scalable Production and Purification of Adeno-Associated Viral Vectors (AAV).

作者信息

Blessing Daniel, Déglon Nicole, Schneider Bernard L

机构信息

Laboratory of Neurotherapies and Neuromodulation, Department of Clinical Neurosciences, Lausanne University Hospital, Lausanne, Switzerland.

Neurosciences Research Center (CRN), LNTM, Lausanne University Hospital, Lausanne, Switzerland.

出版信息

Methods Mol Biol. 2018;1850:259-274. doi: 10.1007/978-1-4939-8730-6_17.

DOI:10.1007/978-1-4939-8730-6_17

PMID:30242692

Abstract

Here we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium in orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI) mediated transfection of a 2-plasmid system and is specified for production in milliliter to liter scales. After PEI and plasmid DNA (pDNA) complex formation the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 3-day batch process, cell cultures are further processed using different methods for lysis and recovery. Methods for the purification of viral particles are described, including iodixanol gradient purification, immunoaffinity chromatography, and ultrafiltration, as well as quantitative PCR to quantify vector titer.

摘要

在此，我们描述了通过在轨道振荡生物反应器中无血清培养基中培养的HEK293细胞进行瞬时转染来生产腺相关病毒（AAV）载体以及随后纯化载体颗粒的方法。AAV组件表达方案基于聚乙烯亚胺（PEI）介导的双质粒系统转染，并指定用于毫升至升规模的生产。在形成PEI与质粒DNA（pDNA）复合物后，稀释的细胞培养物无需预先浓缩步骤或更换培养基即可进行转染。经过3天的分批培养过程后，使用不同的裂解和回收方法对细胞培养物进行进一步处理。描述了病毒颗粒的纯化方法，包括碘克沙醇梯度纯化、免疫亲和色谱和超滤，以及用于定量载体滴度的定量PCR。