Long read sequencing of lentiviral vectors. null

Despite recent advances made towards improving the efficacy of lentiviral gene therapies, a sizeable proportion of produced vector contains an incomplete and thus potentially non-functional RNA genome. This can undermine gene delivery by the lentivirus as well as increase manufacturing costs and must be improved to facilitate the widespread clinical implementation of lentiviral gene therapies. Here, we compare three long-read sequencing technologies for their ability to detect issues in vector design and determine Nanopore direct RNA sequencing to be the most powerful. We show how this approach identifies and quantifies incomplete RNA caused by cryptic splicing and polyadenylation sites, including a potential cryptic polyadenylation site in the widely used Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). Using artificial polyadenylation of the lentiviral RNA, we also identify multiple hairpin-associated truncations in the analysed lentiviral vectors, which account for most of the detected RNA fragments. Finally, we show that these insights can be used for optimization of lentiviral vector design. In summary, Nanopore direct RNA sequencing is a powerful tool for the quality control and optimisation of lentiviral vectors, which may help to improve lentivirus manufacturing and thus the development of higher quality lentiviral gene therapies.

尽管慢病毒基因治疗的疗效优化已取得诸多进展，但量产的慢病毒载体中仍有相当比例携带不完整的RNA基因组，因此可能丧失功能。这不仅会削弱慢病毒的基因递送能力，还会推高制造成本，亟需通过优化加以改进，以推动慢病毒基因治疗的大规模临床应用。在此研究中，我们对比了三种长读长测序技术检测载体设计缺陷的能力，最终确定纳米孔直接RNA测序（Nanopore direct RNA sequencing）为其中性能最优的方案。本研究阐明了该方法如何识别并定量由隐蔽剪接位点与隐蔽多聚腺苷酸化位点引发的不完整RNA，其中包括广泛使用的土拨鼠肝炎病毒转录后调控元件（Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element, WPRE）中潜在的隐蔽多聚腺苷酸化位点。通过对慢病毒RNA进行人工多聚腺苷酸化处理，我们还在分析的慢病毒载体中发现了多个发夹结构相关的截短产物，这类产物占检测到的RNA片段的绝大多数。最后，我们证实上述研究结论可用于优化慢病毒载体的设计方案。综上，纳米孔直接RNA测序是一种用于慢病毒载体质量控制与优化的高效工具，有望助力改善慢病毒的量产工艺，进而推动更高质量慢病毒基因治疗的研发。