Comparison of Wildtype and Recombinant Adeno‐Associated Virus (AAV) Production Reveals Distinct Productivity and Content Ratio Profiles

ABSTRACT Recombinant adeno‐associated virus is a leading viral vector for gene therapy. However, recombinant adeno‐associated virus production systems have low titer and content ratio (ratio of genome‐containing to total adeno‐associated virus capsids), making it difficult to generate the large number of vector genomes required for clinical or commercial use. In contrast, wildtype adeno‐associated virus achieves over ten‐fold higher titer compared to recombinant adeno‐associated virus and a content ratio of over 95% full. Despite the higher productivity of wildtype adeno‐associated virus, the mechanisms behind its productivity remain poorly understood. In this study, we developed and characterized a novel wildtype adeno‐associated virus production platform in suspension human embryonic kidney cells across varying multiplicities of infection of adeno‐associated virus to adenovirus. The impact of cellular viral genome delivery mechanisms and the interplay between adeno‐associated virus and its adenoviral helper were investigated by comparing infection‐based systems with transfection‐based and infection–transfection hybrid systems. Wildtype and hybrid infection systems had remarkably different host cell growth profiles and viral gene expression patterns, and achieved over a log‐fold higher titers and 45% higher content ratios, reaching 8.1 × 10 12 vector genomes/mL culture and 72.2% full, compared to recombinant transfection systems (1.63 × 10 11 vector genomes/mL and 26.9% full).