Enhancing EV-AAV incorporation: insights into natural loading and active loading strategies for improved gene therapy vector delivery

Thesis

Gene therapy using adeno-associated viruses (AAVs) has emerged as a promising treatment for genetic disorders, with multiple FDA-approved therapies. However, challenges such as pre-existing immunity, the need for high doses, and manufacturing complexities limit their broader clinical application. This thesis investigates extracellular vesicle-associated AAVs (EV-AAVs) as a potential solution, focusing on their characterization and engineering for improved therapeutic delivery. Through systematic analysis using iodixanol density gradients and high-resolution size exclusion chromatography, we found that only 1.2-1.6% of EVs naturally incorporate AAVs, predominantly in larger vesicles enriched with phosphatidylserine. Multiple engineering strategies were explored to enhance EV-AAV yields, specifically surface loading, including using AAV receptor (AAVR), producer cell engineering through CD63 overexpression, and AAV9 capsid modification. CD63 overexpression resulted in a 10-fold increase in EV-AAV yields with 70% of associated AAVs protected within EVs. While surface-loaded AAVs showed promise in vitro, they demonstrated limited efficacy in vivo. Incorporation of the hepatitis A virus pX domain, and directed evolution of the AAV9 capsid, resulted in enhanced EV association. A comprehensive library screen revealed specific sequences, including LSGGLDILPKLC and LSDRKIERQGNVF, that significantly improved AAV incorporation into EVs. This work advances our understanding of EV-AAV biology and presents novel engineering approaches for enhancing their therapeutic potential, while highlighting critical challenges that must be addressed for clinical translation.

Authors: Vorobieva, I

• DOI: 10.5287/ora-xvozaqgwz
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: gene therapy; adeno-associated virus; extracellular vesicles
• Subjects: Gene therapy