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Poster Presentations
Day 1, June 22（Sun.）
Room P (Maesato East, Foyer, Ocean Wing)
- 1P-PM-10
Structural analysis of recombinant adeno-associated virus capsids using hydrogen/deuterium exchange mass spectrometry
(Osaka Univ.)
oTomohiko Ikeda, Yuki Yamaguchi, Mitsuko Fukuhara, Yasuo Tsunaka, Aoba Matsushita, Tetsuo Torisu, Susumu Uchiyama
Recombinant Adeno-associated virus (rAAV) is widely used as a viral vector for gene therapy. While several rAAV-based drugs have been approved and commercialized, evaluating the rAAV capsid structure remains challenging due to the inherent flexibility of essential regions, including phospholipase A2 (PLA2), which plays a crucial role in rAAV functions during viral infection. In this study, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was applied for the structural analysis of rAAV8 capsids. Our HDX-MS results achieved 85% sequence coverage, including its structurally unresolved flexible regions. By comparing the deuterium uptake between DNA-packed full capsids and DNA-unpacked empty capsids, we found that the 5-fold symmetry axis and the PLA2 region of the capsid were significantly protected in full capsids. Furthermore, our HDX-MS revealed structural transitions during thermal stress, which caused genome leakage. By comparing HDX-MS results from non-heat-stressed and heat-stressed samples, we observed an increase in deuterium uptake after the incubation, indicating that the channel located in the 5-fold symmetry axis serves as a genome leakage pathway during thermal stress. This study represents the first application of HDX-MS to rAAV capsid structural analysis. Our findings demonstrate that HDX-MS is a powerful tool for monitoring rAAV capsid structural integrity and advancing viral vector development.