HDX-MS reveals pH and temperature-responsive regions on AAV capsids and the structural basis for DNA release.

Recombinant adeno-associated viruses (AAVs) have become increasingly popular as gene therapy vectors in recent years. Like all viruses, AAVs undergo dynamic structural changes in response to varying temperature and pH conditions. However, the specific capsid regions involved in these processes remain unknown. In this study, we employed Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) to investigate the impact of pH and temperature on the structure and conformational dynamics of AAV capsids. Our analysis identified specific regions of the capsid that are sensitive to these environmental changes. Additionally, our data elucidated the structural basis for DNA uncoating or leakage triggered by low pH or high temperature. Detailed structural characterization of AAVs by HDX-MS in this study deepens our understanding of viral capsid conformational dynamics and stability in AAV transduction and manufacturing and storage conditions, paving the way for formulation development and next-generation capsid engineering.