Fitness Landscape-Guided Engineering of Locally Supercharged Virus-like Particles with Enhanced Cell Uptake Properties

[Image: see text] Protein-based nanoparticles are useful models for the study of self-assembly and attractive candidates for drug delivery. Virus-like particles (VLPs) are especially promising platforms for expanding the repertoire of therapeutics that can be delivered effectively as they can deliver many copies of a molecule per particle for each delivery event. However, their use is often limited due to poor uptake of VLPs into mammalian cells. In this study, we use the fitness landscape of the bacteriophage MS2 VLP as a guide to engineer capsid variants with positively charged surface residues to enhance their uptake into mammalian cells. By combining mutations with positive fitness scores that were likely to produce assembled capsids, we identified two key double mutants with internalization efficiencies as much as 67-fold higher than that of wtMS2. Internalization of these variants with positively charged surface residues depends on interactions with cell surface sulfated proteoglycans, and yet, they are biophysically similar to wtMS2 with low cytotoxicity and an overall negative charge. Additionally, the best-performing engineered MS2 capsids can deliver a potent anticancer small-molecule therapeutic with efficacy levels similar to antibody-drug conjugates. Through this work, we were able to establish fitness landscape-based engineering as a successful method for designing VLPs with improved cell penetration. These findings suggest that VLPs with positive surface charge could be useful in improving the delivery of small-molecule- and nucleic acid-based therapeutics.