Probing recombinant AAV capsid integrity and genome release under thermal stress by single-molecule interferometric scattering microscopy

Adeno-associated viruses (AAVs) are gaining traction as delivery vehicles for gene therapy although the molecular understanding of AAV-transgene release is still limited. Typically, the process of viral uncoating is investigated ( in vitro ) through thermal stress, revealing capsid disintegration at elevated temperatures. Here, we used single-molecule interferometric scattering microscopy to assess the (in)stability of different empty and filled AAV preparations. By introducing a heat-stable DNA plasmid as an internal standard, we quantitatively probed the impact of heat on AAVs. Generally, empty AAVs exhibited greater heat resistance than genome-filled particles. Our data also indicate that upon DNA release, the capsids do not transform into empty AAVs, but seem to aggregate or disintegrate. Strikingly, some AAVs exhibited an intermediate state with disrupted capsids but preserved bound genome, a feature that experimentally only emerged following incubation with a nuclease. Our data demonstrate that the thermal uncoating process is highly AAV specific ( i . e ., can be influenced by serotype, genome, host system). We argue that nuclease treatment in combination with mass photometry can be used as an additional analytical tool for assessing structural integrity of recombinant and/or clinical AAV vectors.