Despite recent advances made toward improving the efficacy of lentiviral gene therapies, a sizeable proportion of produced vector contains an incomplete and thus potentially nonfunctional 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 analyzed lentiviral vectors (LVs), which account for most of the detected RNA fragments. Finally, we show that these insights can be used for the optimization of LV design. In summary, nanopore direct RNA sequencing is a powerful tool for the quality control and optimization of LVs, which may help to improve lentivirus manufacturing and thus the development of higher quality lentiviral gene therapies.

中文翻译：

---

使用纳米孔直接 RNA 测序的基因治疗载体质量控制的优化方案


尽管最近在提高慢病毒基因疗法的疗效方面取得了进展，但相当大比例的生产载体包含不完整的 RNA 基因组，因此可能没有功能。这会破坏慢病毒的基因递送并增加制造成本，必须加以改进以促进慢病毒基因疗法的广泛临床实施。在这里，我们比较了三种长读长测序技术检测载体设计中问题的能力，并确定纳米孔直接 RNA 测序是最强大的。我们展示了这种方法如何识别和量化由隐蔽剪接和多聚腺苷酸化位点引起的不完整 RNA，包括广泛使用的土拨鼠肝炎病毒转录后调节元件 （WPRE） 中潜在的隐蔽多聚腺苷酸化位点。使用慢病毒 RNA 的人工多聚腺苷酸化，我们还在分析的慢病毒载体 （LVs） 中鉴定了多个发夹相关的截断，这些截断占检测到的 RNA 片段的大部分。最后，我们表明这些见解可用于优化 LV 设计。总之，纳米孔直接 RNA 测序是 LV 质量控制和优化的有力工具，这可能有助于改进慢病毒的制造，从而开发更高质量的慢病毒基因疗法。