Protein Engineering and Directed Evolution for Nanocarrier Innovation<sup></sup>

The design of nonviral protein-based delivery systems has gained significant attention as an alternative to viral vectors and lipid nanoparticles (LNPs) for gene therapy. While viral vectors offer efficient gene delivery, they present challenges such as immunogenicity and size limitations. LNPs, though pivotal in recent advancements like messenger ribonucleic acid (mRNA)-based vaccines, cause inflammation and exhibit low endosomal escape efficiency. This article explores the development of engineered nonviral protein cages through rational design and directed evolution as an additional nanocarrier option for RNA packaging and delivery. We highlight key advances, including the design and evolution of capsidforming proteins capable of encapsulating and protecting their own encoding mRNA, a critical step in establishing a genotype-phenotype link for evolutionary optimization. Evolved protein cages, such as the I53-50-v4 and NC-4 variants, demonstrate enhanced stability and RNA protection, achieving structural transformations and packaging efficiencies akin to viral systems. The application of directed evolution has expanded the capacity of these nanocarriers enhancing their in vivo stability and biodistribution. This work underscores the potential of evolving nonviral capsids to become customizable platforms for therapeutic gene delivery, while also addressing current limitations in RNA cargo size and tissue targeting specificity. Future directions involve refining these systems to accommodate larger RNAs, and improving immunogenicity properties and dynamic control over cargo release.