DNA ejection and AAV capsid degradation under thermal stress: Insights from CDMS and native digestion strategies

Adeno-associated viruses (AAVs) are the predominant vectors for in vivo gene therapy, enabling tissue-specific gene delivery and contributing to a rapidly expanding landscape of approved therapies. As AAV-based products advance toward widespread clinical use, ensuring capsid stability throughout manufacturing, storage, and administration has become a critical quality and regulatory consideration. Despite this importance, the molecular mechanisms governing AAV capsid degradation under moderate, prolonged thermal stress remain insufficiently characterized. Here, we integrate charge detection mass spectrometry (CDMS) and native digestion strategies to elucidate AAV degradation pathways under extended thermal stress. Using AAV1 and AAV8 as model systems, we demonstrate that genome release proceeds through intermediate DNA-extrusion states that retain the mass of full capsids while exhibiting elevated charge states. DNA ejection ultimately drives the conversion of full capsids into empty forms, generating distinct, structurally compromised intermediates influenced by serotype, genome size, and the degree of thermal stress. Furthermore, DNA extrusion/ejection occurs cooperatively with VP1u externalization, facilitating DNA-viral protein-mediated aggregation that can be largely reversed by native enzymatic digestion of either the DNA or protein components. Together, these findings offer new insights into AAV degradation pathways under thermal stress and establish an effective analytical framework for AAV stability assessment.