Assessing Codon Language Models for Context-Aware Codon Optimization in Nucleic Acid-Based Medicines

<jats:p>Codon optimization uses synonymous sequence changes to improve the expression and therapeutic performance of nucleic acid-based medicines. Masked language models (MLMs) have recently been proposed as alternatives to traditional, frequency-based codon optimization approaches, yet whether they offer a meaningful advantage over such simpler methods remains unclear. Here we benchmark three prominent MLMs --- CaLM, EnCodon and CodonTransformer --- across backtranslation fidelity, sequence generation and nine molecular phenotype prediction tasks, and experimentally evaluate model-designed sequences using a secreted embryonic alkaline phosphatase (SEAP) reporter. The models differed markedly in amino-acid fidelity and generated distinct synonymous sequence variants. However, no single model performed best across all benchmark tasks and simple sequence features remained competitive in several settings. Our interpretability analysis revealed that the models integrate a large window of codon context for making predictions, as opposed to frequency-based approaches. Our in vitro data showed that MLM-designed variants outperformed conventional and commercial vendor-derived sequences in both transient and stably integrated expression, supporting the models' ability to capture translational context beyond codon frequency. Together, our results establish MLMs as effective and complementary tools for codon optimization and suggest that sampling across multiple models may improve the likelihood of identifying high-performing therapeutic sequences.</jats:p>