Characterization of AAV vector particle stability at the single-capsid level

Area of Science:

• Molecular Biology
• Biophysics
• Gene Therapy Vectorology

Background:

• Adeno-associated viruses (AAV) are leading vectors for in vivo gene transfer and gene therapy.
• Understanding AAV vector genome uncoating is critical for optimizing gene delivery efficiency.
• Viral genome release strategies vary across virus families and impact vector performance.

Purpose of the Study:

• To investigate the physical properties and stability of AAV vector particles.
• To characterize the pathways and kinetics of single-stranded DNA (ssDNA) uncoating from AAV capsids.
• To compare the stability of AAV serotypes 8 and 9 (AAV8 and AAV9) during genome release.

Main Methods:

• Atomic force microscopy (AFM) for single-particle analysis.
• Biochemical experiments to assess capsid integrity and DNA release.
• Physical modeling and kinetic analysis to describe uncoating pathways.
• Controlled temperature incubations to trigger and study ssDNA uncoating.

Main Results:

• AAV genome release occurs via two distinct pathways: intact capsid ejection or capsid rupture with entangled ssDNA.
• ssDNA ejection exhibits a two-step behavior, analyzed by genome length distributions.
• A kinetic model was developed to quantitatively describe uncoating pathways over time and temperature.
• The model enabled quantification of the relative stability between AAV8 and AAV9 particles.

Conclusions:

• Genome uncoating from AAV vectors is a complex process involving multiple pathways.
• The developed kinetic model provides quantitative insights into AAV vector stability.
• Understanding these uncoating mechanisms is essential for the rational design of improved gene therapy vectors.