Engineering Adeno-Associated Virus Biology for Improved Vector Production

Abstract

Adeno-associated virus (AAV) has emerged as the leading gene delivery vector for use in clinical gene therapy. Since the first clinical study in 1995, AAV has been administered to over 3,000 patients living with genetic diseases. Eight therapies have received regulatory approval in the United States or European Union. The gene therapy community has witnessed the emergence of hundreds of engineered AAV serotypes promising to target specific cell types or limit transduction of others. While many of these remain actively investigated in clinical studies, most AAVs share a common clinical barrier: manufacturing inefficiencies.This dissertation investigates three fundamental bottlenecks in AAV vector manufacturing: genome replication, genome packaging, and viral egress. First, diversifying the AAV Rep protein through DNA shuffling and directed evolution revealed multiple functional Rep variants. Most variants retain their ability to produce vector, revealing previously unexplored sequence diversity of AAV Rep. Second, directed evolution of the AAV inverted terminal repeats (ITRs) uncovered a synthetic packaging element that enhanced the replicative ability and vector yield without compromising vector quality. Lastly, structure-guided evolution of the membrane-associated accessory protein (MAAP) revealed synthetic MAAP variants that markedly increased vector secretion, altered host trafficking dependencies, and holds promise to improve downstream vector production systems. Together, this work shows that previously underexplored areas of AAV biology can be engineered to overcome key manufacturing inefficiencies. These findings provide a foundation for improving the efficiency of manufacturing pipelines and safety of AAV.