Chronologically distributed transfection improves AAV2 and AAV2/8 capsid filling and reveals assembly schedule divergence.

Adeno-associated virus (AAV) gene therapy vectors often suffer from low capsid filling, resulting in high proportions of empty capsids that reduce efficacy and complicate manufacturing processes. This study investigates whether chronologically distributed transfection could improve capsid filling for AAV2 and AAV2/8 serotypes. We used an empirical approach to test different transfection chronologies by varying the timing of Helper, RepCap, and Payload plasmid delivery across two time points, T1 and T2 (24 and 44 h post-seeding, respectively). Our results revealed distinct serotype-specific responses to altered transfection chronologies, with AAV2/8 production being robust to a broader range of chronologies than AAV2. All non-standard chronologies reduced physical and biological titers. Notably, T1 transfection with Helper and Payload plasmids, followed by RepCap plasmid at T2, increased capsid filling efficiency by approximately 7.5-fold for both AAV2 and AAV2/8. This finding provides empirical support for a temporal misalignment hypothesis, whereby suboptimal AAV capsid filling results from capsid assembly occurring before peak genome replication. Our study demonstrates a re-scheduled transfection procedure that can enhance AAV production outcomes and reveals fundamental differences in assembly dynamics between serotypes. These insights contribute to understanding AAV assembly mechanisms and offer a novel method for process development in gene therapy manufacturing.