Leveraging HTS Technologies To Develop Methods For In-Depth Characterization Of rAAV Products

By E Lecomte, S Maestro, S Stinus, DL Bowie, A Pérez San Vicente, A Navarro Nieto, O Aldasoro Zabala, L Agundez, F Bastida, N Pawlowski, A Lecanda Sanchez, R Palacios, S Cooper, R Fraser, S Brahma, J Smith, S Devi Velivela, C Trigueros Fernandez, A François

Gene therapy holds promise for patients with genetic diseases, especially as clinical trial success grows. Recombinant adeno-associated virus (rAAV) products undergo extensive characterization before release, with a focus on Critical Quality Attributes (CQAs) like identity and purity to ensure safety and efficacy. While quantitative PCR and Sanger sequencing remain gold standards, advancements in high-throughput sequencing (HTS) now enable a deeper analysis of rAAV DNA content.

Short-read sequencing offers high depth and low error rates, ideal for detecting low-level mutations and contaminating DNA. Long-read sequencing, however, provides single-molecule resolution, allowing for precise identification of rAAV genome truncations and contaminating DNA sizes. Our lab has developed complete protocols and bioinformatics pipelines, supporting CQA analysis that includes non-payload sequence purity (using Illumina and Oxford Nanopore), payload sequence integrity (assessing DNA length and truncation via Oxford Nanopore), and identity assessments of payload sequences (identifying SNPs and indels through Illumina).