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

## TL;DR

Changing the timing of transfection steps improves AAV capsid filling and reveals differences in how AAV2 and AAV2/8 assemble.

## Contribution

A novel transfection schedule increases capsid filling efficiency by 7.5-fold and supports the temporal misalignment hypothesis.

## Key findings

- Chronologically distributed transfection increased capsid filling efficiency by 7.5-fold for AAV2 and AAV2/8.
- AAV2/8 production is more robust to transfection timing changes than AAV2.
- Non-standard chronologies reduced physical and biological titers.

## Abstract

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.

AAV gene therapy vectors suffer from low capsid filling, reducing efficacy. Nesbeth and colleagues tested chronologically distributed transfection timing for AAV2 and AAV2/8 serotypes. Physical and biological titers were always reduced, but delaying RepCap plasmid delivery increased capsid filling 7.5-fold for both serotypes, supporting temporal misalignment hypothesis and offering improved manufacturing methods.