Advanced characterization of DNA molecules in rAAV vector preparations by single-stranded virus next-generation sequencing

Recent successful clinical trials with recombinant adeno-associated viral vectors (rAAVs) have led to a renewed interest in gene therapy. However, despite extensive developments to improve vector-manufacturing processes, undesirable DNA contaminants in rAAV preparations remain a major safety concern. Indeed, the presence of DNA fragments containing antibiotic resistance genes, wild-type AAV, and packaging cell genomes has been found in previous studies using quantitative polymerase chain reaction (qPCR) analyses. However, because qPCR only provides a partial view of the DNA molecules in rAAV preparations, we developed a method based on next-generation sequencing (NGS) to extensively characterize single-stranded DNA virus preparations (SSV-Seq). In order to validate SSV-Seq, we analyzed three rAAV vector preparations produced by transient transfection of mammalian cells. Our data were consistent with qPCR results and showed a quasi-random distribution of contaminants originating from the...

近年来，重组腺相关病毒载体（recombinant adeno-associated viral vectors，rAAVs）相关临床试验的成功，使基因治疗领域再度掀起研究热潮。尽管载体生产工艺已取得长足进展，但重组腺相关病毒制剂中存在的有害DNA污染物仍是重大安全隐患。既往研究通过定量聚合酶链式反应（quantitative polymerase chain reaction，qPCR）分析已发现，此类制剂中存在携带抗生素抗性基因、野生型腺相关病毒（wild-type AAV）以及包装细胞基因组的DNA片段。然而，由于定量聚合酶链式反应仅能对重组腺相关病毒制剂中的DNA分子提供局部检测视角，我们开发了一种基于下一代测序（next-generation sequencing，NGS）的方法，用于全面表征单链DNA病毒制剂，即SSV-Seq。为验证该方法的可靠性，我们对三种通过哺乳动物细胞瞬时转染制备的重组腺相关病毒载体制剂进行了分析。所得数据与定量聚合酶链式反应结果一致，且显示出源自……的污染物呈准随机分布。