Purification of Adeno-Associated Viruses Using an Electrochemically Controllable Polyelectrolyte Brush - Separation of Genome-Filled Adeno Associated Viruses from Non-Genome-Corporated Virus Capsids

Abstract

Adeno-associated Viruses (AAVs) are a promising gene therapy vector due to their low toxicity, long-term gene expression, versatility in transducing various cell types, and site-specific integration into the chromosome. However, several challenges remain in AAV production, such as low yield and difficulties in gene incorporation, which necessitate a thorough purification process that is both labor-intensive and expensive. Conventional techniques, that rely on chromatography column resins, face severe limitations, especially with diverse AAV serotypes. This thesis explores a novel purification approach using an electrochemical controllable polyelectrolyte brush (PE) brush. The PE brush serves as an adhesive surface for biomolecules, with high protein-binding capacity, and electrochemical signals induce controlled release. The electrochemical method avoids the drawbacks of conventional pH changes and hence, offers the potential to resolve impurity challenges. The objective was to validate the electrochemical PE brush system for AAV purification. Three milestones were established: confirming immobilization and elution, evaluating product purity and quantity, and optimizing the system. Results demonstrate successful immobilization and electrochemical elution using both PAA and PDEA brushes. The system shows the potential to up-concentrate capsids and enhance the percentage of filled capsids relative to the total amount of capsids. Recirculation further improves binding efficiency. Even though this study shows great potential to improve purification, the need for further purity enhancement needs to be acknowledged to unlock the advancement of the method in the aspect of AAV purification. Further refinements can put this innovative approach in a competitive position with conventional purification methods for AAVs.