A Scalable and Accurate Method for Quantifying Vector Genomes of Recombinant Adeno-Associated Viruses in Crude Lysate
A Scalable and Accurate Method for Quantifying Vector Genomes of Recombinant Adeno-Associated Viruses in Crude Lysate
复制标题
DOI：
10.1089/hgtb.2016.173
复制
发表时间：
2017-06-01
影响因子：
--
通讯作者：
Gao, Guangping
中科院分区：
文献类型：
--
作者：
Ai, Jianzhong;Ibraheim, Raed;Gao, Guangping
重组腺相关病毒（rAAV）在基础和临床研究中的应用日益增加，促使人们努力提高rAAV的生产质量和产量。标准载体生产工作流程要求在生产终点对纯化载体进行基因组滴定以评估产率。不幸的是，缺乏在中间生产步骤期间的制备物的质量控制措施和评估多批rAAV制备物的保真度的经济手段。在这里，我们描述了一种可扩展的和准确的方法，直接定量聚合酶链反应（qPCR）滴定的rAAV基因组在粗裂解液。裂解物样品用DNA酶I预处理以去除载体和包装质粒DNA，然后用蛋白酶K预处理以释放耐内切核酸酶的包装病毒基因组并蛋白水解粗裂解物固有的可影响定量PCR效率的因子。我们表明，这种方法是精确的，可扩展的，适用于载体基因组滴定的单链和自身互补的AAV基因组，无论血清型差异的主要限制标准裂解物转导方法，间接筛选载体包装效率。因此，我们所描述的方法在减轻时间和成本负担、过程中质量控制评估、大规模制备中的批次/批次监测和良好制造规范方面代表了对rAAV载体生产的显著改进。
Increasing interest and application of recombinant adeno-associated viruses (rAAVs) in basic and clinical research have urged efforts to improve rAAV production quality and yield. Standard vector production workflows call for genome titration of purified vectors at the endpoint of production to assess yield. Unfortunately, quality control measures for preparations during mid-production steps and economical means to assess the fidelity of multiple batches of rAAV preparations are lacking. Here we describe a scalable and accurate method for the direct quantitative polymerase chain reaction (qPCR) titration of rAAV genomes in crude lysate. Lysate samples are pretreated with DNase I to remove vector and packaging plasmid DNAs, followed by proteinase K to release endonuclease-resistant packaged viral genomes and to proteolyze factors inherent to crude lysates that can impinge upon quantitative PCR efficiencies. We show that this method is precise, scalable, and applicable for vector genome titrations of both single-stranded and self-complementary AAV genomes irrespective of serotype differences-a major limitation for standard lysate transduction methods that indirectly screen for vector packaging efficiency. Our described method therefore represents a significant improvement to rAAV vector production in terms of alleviating time and cost burdens, in-process quality control assessment, batch/lot monitoring in large-scale preparations, and good manufacturing practices.