Reinforcement-guided generative protein language models enable de novo design of highly diverse AAV capsids

Adeno-associated viral (AAV) vectors are widely used delivery platforms in gene therapy, and the design of improved capsids is key to expanding their therapeutic potential. A central challenge in AAV bioengineering, as in protein design more broadly, is the vast sequence design space relative to the scale of feasible experimental screening. Machine-guided generative approaches provide a powerful means of navigating this landscape and proposing novel protein sequences that satisfy functional constraints. Here, we develop a generative design framework based on protein language models and reinforcement learning to generate highly novel yet functionally plausible AAV capsids. A pretrained model was fine-tuned on experimentally validated capsid sequences to learn patterns associated with viability. Reinforcement learning was then used to guide sequence generation, with a reward function that jointly promoted predicted viability and sequence novelty, thereby enabling exploration beyond regions represented in the training data. Comparative analyses showed that fine-tuning alone produces sequences with high predicted viability but remains biased toward the training distribution, whereas reinforcement learining-guided generation reaches more distant regions of sequence space while maintaining high predicted viability. Finally, we propose a candidate selection strategy that integrates predicted viability, sequence novelty, and biophysical properties to prioritize variants for downstream evaluation. This work establishes a framework for the generative exploration of protein sequence space and advances the application of generative protein language models to AAV bioengineering.

Exploring the limits of pre-trained embeddings in machine-guided protein design: a case study on predicting AAV vector viability

Effective representations of protein sequences are widely recognized as a cornerstone of machine learning-based protein design. Yet, protein bioengineering poses unique challenges for sequence representation, as experimental datasets typically feature few mutations, which are either sparsely distributed across the entire sequence or densely concentrated within localized regions. This limits the ability of sequence-level representations to extract functionally meaningful signals. In addition, comprehensive comparative studies remain scarce, despite their crucial role in clarifying which representations best encode relevant information and ultimately support superior predictive performance. In this study, we systematically evaluate multiple ProtBERT and ESM2 embedding variants as sequence representations, using the adeno-associated virus capsid as a case study and prototypical example of bioengineering, where functional optimization is targeted through highly localized sequence variation within an otherwise large protein. Our results reveal that, prior to fine-tuning, amino acid-level embeddings outperform sequence-level representations in supervised predictive tasks, whereas the latter tend to be more effective in unsupervised settings. However, optimal performance is only achieved when embeddings are fine-tuned with task-specific labels, with sequence-level representations providing the best performance. Moreover, our findings indicate that the extent of sequence variation required to produce notable shifts in sequence representations exceeds what is typically explored in bioengineering studies, showing the need for fine-tuning in datasets characterized by sparse or highly localized mutations.