Penaud-Budloo Magalie, Lecomte Emilie, Guy-Duché Aurélien, Saleun Sylvie, Roulet Alain, Lopez-Roques Céline, Tournaire Benoît, Cogné Benjamin, Léger Adrien, Blouin Véronique, Lindenbaum Pierre, Moullier Philippe, Ayuso Eduard
1 INSERM UMR1089, University of Nantes, Centre Hospitalier Universitaire, Nantes, France.
2 INRA, GeT-PlaGe, Genotoul, Castanet-Tolosan, France.
Hum Gene Ther Methods. 2017 Jun;28(3):148-162. doi: 10.1089/hgtb.2016.185. Epub 2017 Apr 21.
Recombinant adeno-associated viral (rAAV) vectors have proven excellent tools for the treatment of many genetic diseases and other complex diseases. However, the illegitimate encapsidation of DNA contaminants within viral particles constitutes a major safety concern for rAAV-based therapies. Moreover, the development of rAAV vectors for early-phase clinical trials has revealed the limited accuracy of the analytical tools used to characterize these new and complex drugs. Although most published data concerning residual DNA in rAAV preparations have been generated by quantitative PCR, we have developed a novel single-strand virus sequencing (SSV-Seq) method for quantification of DNA contaminants in AAV vectors produced in mammalian cells by next-generation sequencing (NGS). Here, we describe the adaptation of SSV-Seq for the accurate identification and quantification of DNA species in rAAV stocks produced in insect cells. We found that baculoviral DNA was the most abundant contaminant, representing less than 2.1% of NGS reads regardless of serotype (2, 8, or rh10). Sf9 producer cell DNA was detected at low frequency (≤0.03%) in rAAV lots. Advanced computational analyses revealed that (1) baculoviral sequences close to the inverted terminal repeats preferentially underwent illegitimate encapsidation, and (2) single-nucleotide variants were absent from the rAAV genome. The high-throughput sequencing protocol described here enables effective DNA quality control of rAAV vectors produced in insect cells, and is adapted to conform with regulatory agency safety requirements.
重组腺相关病毒(rAAV)载体已被证明是治疗多种遗传性疾病和其他复杂疾病的优秀工具。然而,病毒粒子内DNA污染物的非法包装是基于rAAV疗法的一个主要安全问题。此外,用于早期临床试验的rAAV载体的开发揭示了用于表征这些新型复杂药物的分析工具的准确性有限。尽管大多数关于rAAV制剂中残留DNA的已发表数据是通过定量PCR产生的,但我们已经开发了一种新颖的单链病毒测序(SSV-Seq)方法,用于通过下一代测序(NGS)对哺乳动物细胞中产生的AAV载体中的DNA污染物进行定量。在此,我们描述了SSV-Seq在准确鉴定和定量昆虫细胞中产生的rAAV储备中的DNA种类方面的应用。我们发现杆状病毒DNA是最丰富的污染物,无论血清型(2、8或rh10)如何,其占NGS读数的比例均小于2.1%。在rAAV批次中以低频(≤0.03%)检测到Sf9生产细胞DNA。先进的计算分析表明:(1)靠近反向末端重复序列的杆状病毒序列优先发生非法包装;(2)rAAV基因组中不存在单核苷酸变体。本文所述的高通量测序方案能够对昆虫细胞中产生的rAAV载体进行有效的DNA质量控制,并符合监管机构的安全要求。