Rapid analysis of gene therapy vectors with the Oxford Nanopore MinION sequencer
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Adeno-associated virus (AAV) is a non-enveloped single-stranded DNA virus with a genome size of ~4.7 kb. The virus is primarily studied as a vector for gene therapy because of its non-pathogenic nature and ability to deliver engineered transgene cassettes as recombinant (r)AAV. Currently, two AAV-based therapies have been made commercially available with over 200 different rAAV-based gene therapy clinical trials ongoing. Despite their promise, quality control methods for rAAV vectors fall short in detecting contaminants and truncated genomes. These unwanted materials, which include viral and human DNA fragments related to the production process, can impact the efficacy and safety of these therapies. In order to develop a means to analyze full-length rAAV genomes, we evaluated the capacity of long-read sequencing by Oxford Nanopore Technology’s MinION platform to sequence two clinically relevant forms of rAAV genomes: the singlestranded (ss) and the self-complementary (sc)AAV vector genomes, as well as the parental plasmids used to produce these vectors. For this study, we used both MinION R9 and R10 flow cells to: 1) evaluate differences in sequencing ssAAV and scAAV genomes, 2) validate whether reads accurately reflect the vector genome population in preparations, and 3) assess the accuracy of nanopore sequencing across the inverted terminal repeats (ITRs), which are characterized by Tshaped hairpins that interfere with the processivity of standard polymerases used in sequencing. Our results show that both R9 and R10 can identify and quantify DNA contaminants and heterogeneous vector populations; however, R9 showed higher sequencing efficiency and coverage of the ITRs. Sequencing of the parental plasmids validated that abnormal vector genomes are not an artifact of the sequencing platform. Additionally, through MinION’s ability to sequence through the ITRs, we were able to reveal replication intermediates during vector production, which showed that genomes are predominantly amplified by conventional rolling-hairpin replication(RHR) and not rollingcircle replication (RCR).