Assessment of long-read target enrichment sequencing for AAV integration site analysis
This study set out to evaluate how effectively long-read target enrichment sequencing (LR-TES) can characterize recombinant AAV (rAAV) integration events, which is an important aspect of understanding vector-related genotoxicity. Although rAAVs are generally considered safe, chromosomal integrations and associated structural alterations have been reported, making robust integration site analysis essential. LR-TES performance was assessed using 34 engineered MCF10A clones with predefined integration sites generated via CRISPR/Cas9-induced breaks. LR-TES uncovered several integrations at non-targeted sites and showed that only approx. 29% of integrated vector sequences remained intact, with most insertions exhibiting truncations, rearrangements, concatemers, or flanking genomic deletions. The method demonstrated a 1% limit of detection and provided detailed structural insights, highlighting its value as a useful tool to support rAAV safety assessment.