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. To improve this approach, we evaluate a new reverse transcriptase (Induro) and show that it generates significantly longer direct RNA reads that often span the entire lentiviral genome.
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 Post-transcriptional Regulatory Element (WPRE). Using artificial polyadenylation of the lentiviral RNA, we also identify multiple hairpin-associated truncations in the analysed lentiviral vectors (LVs), which account for most of the detected RNA fragments and may be the result of cleavage by AGO2. Finally, we show that these insights can be used for the optimisation of LV design in an iterative, data-driven development approach. In summary, nanopore direct RNA sequencing is a powerful tool for the quality control and optimisation of LVs, which may help to improve lentivirus manufacturing and thus the development of higher quality lentiviral gene therapies. This work can also be extended to lentiviruses used for other purposes (e.g. in research) and even to the quality control of mRNA vaccines.