AAV capsid sites breakdown: large protein insertions impact on vector dynamics

Area of Science:

• Molecular Biology
• Biotechnology
• Gene Therapy

Background:

• Adeno-associated virus (AAV) vectors are widely used for gene delivery.
• Current capsid engineering primarily uses small peptide insertions, limiting large protein delivery.
• Integrating large proteins into AAV capsids presents challenges in protein folding and assembly.

Purpose of the Study:

• To identify engineerable hotspots in AAV capsids for large protein insertion.
• To expand AAV vector applications beyond traditional gene therapy by enabling large protein cargo delivery.
• To investigate the behavior and versatility of different capsid insertion sites.

Main Methods:

• A capsid mosaic approach was used, integrating mCherry protein (236 a.a.) into VP1 and VP2 capsid proteins.
• Systematic breakdown of different capsid insertion sites to analyze their behavior.
• Characterization of engineered AAV2/mCherry mosaic vectors for biophysical properties and particle functionality.

Main Results:

• Engineered AAV2/mCherry mosaic vectors showed no major impairments in functionality.
• Residues 138 and 453 were identified as permissive sites for functional protein integration.
• Residue 138 proved most flexible for inserting proteins up to 236 amino acids, followed by residue 453.

Conclusions:

• AAV capsids demonstrate robustness in hosting substantial domain insertions.
• The identified permissive sites (138 and 453) enable functional integration of large proteins.
• This research lays the foundation for novel AAV vector systems capable of delivering large protein cargo.