Machine learning-assisted directed evolution yields dramatic improvement on novel AAV engineering task

Directed evolution enables the discovery of protein mutants with improved fitness through iterative rounds of selection and has been widely applied to adeno-associated virus (AAV) capsid engineering. Machine learning (ML) can augment this process by prioritising mutants for experimental validation, but whether its benefits outweigh the added cost of ML-designed library construction remains unclear. Here, we address this question by considering improvements in manufacturing efficiency of AAV capsids in the context of exosomal encapsulation, which is a technique for improving AAV immunogenicity. We generated a directed evolution dataset comprising 53,974 mutants across three rounds of selection and an independent assessment dataset of 472 ML-designed mutants with detailed profiling. Directed evolution alone yielded a 3-fold improvement, while ML-assisted directed evolution yielded a 41-fold improvement in manufacturing efficiency, demonstrating that ML-assisted directed evolution outperforms directed evolution alone. We show that data quality - specifically the number of selection rounds and the statistical power of mutant counts - has a greater impact on model performance than model architecture. Finally, we provide practical guidance on experimental design and implementation of ML tuning strategies that can augment model performance in protein engineering applications.