Quality control of gene therapy vectors using nanopore direct RNA sequencing

Abstract

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 increasing 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 post-transcriptional 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 optimisation 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.

Biography

Kathleen is a third-year PhD student at the Walter and Eliza Hall Institute of Medical Research in Melbourne, Australia. She has a strong interest in long-read sequencing and bioinformatics, including their application to the development of new therapeutics.