Advancements In Nanopore Sequencing Allow In-Depth Characterization Of rAAV Vector Batches Comparable To SMRT™ Sequencing

By K. Breunig, F. Dunker-Seidler, F. Sonntag, M. Hoerer, RC. Feiner

This study highlights the application of long-read sequencing, particularly nanopore sequencing, for analyzing encapsidated DNA to optimize rAAV gene therapy platforms. Using a rAAV9 batch produced with the EpyQ™ plasmid system, three DNA conversion methods yielded consistent impurity distributions. Advances in nanopore technology, including V14 chemistry and dorado basecalling, demonstrated enhanced read quality and alignment accuracy compared to older methods.

The nanopore sequencing revealed high vector quality (>95% vector mapping) with minimal host cell DNA (HCD) impurities. Comparison with SMRT™ sequencing showed similar impurity profiles, though SMRT™ identified more plasmid backbone reads due to chimeric read differences. Truncation analysis pinpointed consistent vector hotspots, corroborated by independent SMRT™ data. These findings validate nanopore sequencing as a powerful tool for rAAV quality assessment and underscore its potential for improving gene therapy production.