Recombinant adeno-associated virus (rAAV) has become a leading vector for in vivo gene delivery. The quality of the AAV product is crucial for the success of gene therapy, and thorough understanding of the viral genome sequence is becoming essential for effective quality control. During AAV production, non-targeted sequences may be inadvertently packaged alongside the gene of interest (GOI). To ensure the safety of the final product, it is also critical to evaluate and characterize these non-GOI sequences.
In this study, we evaluate and develop optimized nanopore long-read sequencing workflow to analyze our AAV viral genome produced from our triple transfection platform. This method confirms the correct gene of interest (GOI) sequence for identity, its integrity and provides insights into the impurity profile of the AAV product. Specifically, we assess the presence of mis-packaged sequences from the host cell genome or vector backbone used in a triple transfection platform. Nanopore sequencing offers a comprehensive view of the AAV genome and delivers robust, informative analysis of both in-process and purified products.