Biophysical and structural insights into AAV genome ejection.

Area of Science:

• Molecular biology
• Virology
• Biophysics

Background:

• Recombinant adeno-associated virus (rAAV) is a key gene therapy vector, but its efficiency depends on nuclear delivery.
• Understanding capsid biophysics during intracellular trafficking is crucial for optimizing rAAV efficacy.
• Low pH encountered during endo-lysosomal trafficking triggers viral protein externalization, aiding endosomal escape.

Purpose of the Study:

• To characterize the genome ejection (GE) process for AAV2 and AAV5 serotypes.
• To investigate the influence of temperature, pH, and capsid proteins on GE.
• To elucidate the structural dynamics of capsid-mediated genome release.

Main Methods:

• Temperature-dependent genome ejection assays.
• Quantitative PCR (qPCR) for transgene quantification.
• Transmission electron microscopy (TEM).
• Cryo-electron microscopy (cryo-EM) for structural analysis.

Main Results:

• The temperature for genome ejection (T_E) is significantly lower than the capsid disassembly temperature (T_M) for AAV2 and AAV5.
• Absence of VP1/VP2 proteins and decreased pH accelerate genome ejection.
• Cryo-EM revealed capsid structural changes consistent with concerted genome egress.

Conclusions:

• Genome ejection is a distinct process from capsid disassembly in rAAV.
• Environmental factors like pH and viral protein composition modulate genome release kinetics.
• Findings suggest a conserved genome ejection mechanism across rAAV serotypes, potentially improving gene therapy vector design.