用于残留DNA和腺相关病毒载体基因组特征分析的单链DNA病毒测序（SSV-Seq）

Single-Stranded DNA Virus Sequencing (SSV-Seq) for Characterization of Residual DNA and AAV Vector Genomes.

作者信息

Lecomte Emilie, Leger Adrien, Penaud-Budloo Magalie, Ayuso Eduard

机构信息

INSERM UMR1089, University of Nantes, Centre Hospitalier Universitaire de Nantes, Nantes, France.

European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI), Cambridge, UK.

出版信息

Methods Mol Biol. 2019;1950:85-106. doi: 10.1007/978-1-4939-9139-6_5.

DOI:10.1007/978-1-4939-9139-6_5

PMID:30783969

Abstract

With the success of clinical trials using recombinant adeno-associated viral vectors (rAAV), regulatory agencies ask for a more comprehensive characterization of process- and product- related impurities found in rAAV stocks in order to assess the potential risks for patients. During production, rAAV capsids are known to internalize illegitimate DNA fragments in addition to their recombinant genome. These contaminants can come from plasmid or helper virus DNA as well as from the producer host cell. Here, we describe a method based on high-throughput sequencing to identify and quantify residual DNA in rAAV vector lots. Contrary to qPCR, SSV-Seq (Single-Stranded DNA Virus Sequencing) offers a nonselective approach to determine the percentage of each DNA contaminant and analyze rAAV vector genome identity.

摘要

随着使用重组腺相关病毒载体（rAAV）的临床试验取得成功，监管机构要求对rAAV原液中发现的与工艺和产品相关的杂质进行更全面的表征，以评估对患者的潜在风险。在生产过程中，已知rAAV衣壳除了其重组基因组外，还会内化非法DNA片段。这些污染物可能来自质粒或辅助病毒DNA，也可能来自生产宿主细胞。在此，我们描述了一种基于高通量测序的方法，用于鉴定和定量rAAV载体批次中的残留DNA。与qPCR不同，SSV-Seq（单链DNA病毒测序）提供了一种非选择性方法，可确定每种DNA污染物的百分比并分析rAAV载体基因组同一性。