October 6, 2025Molecular Therapy — Methods & Clinical Development1 citationsOpen Access
Adeno-associated virus serotype 9 structural heterogeneity and stability characterized by charge detection mass spectrometry
Key Result
Charge detection mass spectrometry revealed that AAV9 capsids undergo significant structural compaction and premature genome extrusion under acidic conditions in phosphate buffered saline.
Structured PICO
P
Population
Adeno-associated virus serotype 9 (AAV9) virus-like particles (VLPs) consisting of only viral protein (VP) 3 and wild-type AAV9 capsids
I
Intervention
Exposure to different buffer compositions (ammonium acetate vs. phosphate buffered saline), pH levels (7.4, 5.4, 4.0), and temperatures (4°C, 37°C)
O
Outcome
Structural heterogeneity and stability characterized by charge detection mass spectrometry (CDMS)surrogate
Charge detection mass spectrometry reveals that AAV9 capsid structure and stability are highly dependent on buffer composition, pH, and temperature, which has implications for viral vector purification and storage.
Limitations
- More detailed structural information requires modeling and/or additional experimental data from complimentary structural methods
Abstract
The variability in structure and stability of adeno-associated virus serotype 9 (AAV9) in response to changes in buffer composition, pH, and temperature was investigated using charge detection mass spectrometry (CDMS). AAV9 virus-like particles (VLPs) consisting of only viral protein (VP) 3 and wild-type AAV9 capsids (i.e., capsids containing varying stoichiometries of VP1, VP2, and VP3) showed differences in structure, indicating that these different VP stoichiometries and compositions may contribute substantially to conformational heterogeneity. Significant differences in AAV9 structure and stability were observed in ammonium acetate (AA) vs. phosphate buffered saline (PBS) solutions under some conditions. At 37°C under acidic conditions, AAV capsids fell apart in AA, whereas in PBS, capsids underwent structural compaction. Subsequent nuclease binding experiments indicated that partially extruded DNA was the likely origin of this structural compaction that occurred under different physical and chemical conditions. Results from one freeze-thaw cycle indicated that the capsids degraded by a similar mechanism to that in acidified solution. The structural complexity revealed by CDMS highlights the advantages of this biophysical characterization method in providing, for the first time, a holistic insight into the potential heterogeneous conformational transitions of AAV9 during purification, storage, and the natural infection process.