Intracellular Delivery of Functional RNA by Designed Protein Assemblies

Abstract

Synthetic nucleocapsids have great potential to be developed into next generation medicines, however like all macromolecules they become trapped in the endosome-lysosome pathway and are degraded by lysosomal proteases. For this reason, endosomal escape has been a major longstanding bottleneck for drug delivery. In order to deliver therapeutics intracellularly, this limiting step must be overcome. Viruses have evolved mechanisms over long time periods to escape the endosome, therefore it is not surprising that many efforts at intracellular delivery and gene editing have taken a ‘top-down’ approach of re-engineering naturally occurring viruses. However, this approach comes with its own challenges and safety risks. Here we build on recent advances in protein design to create synthetic targeted nucleocapsids capable of delivering functional RNA cargo intracellularly. To this end we computationally designed proteins capable of disrupting membranes in a pH-driven manner and displayed these components on nucleocapsids encapsulating an RNA payload. To assess and quantify the ability of nucleocapsids to deliver RNA, we describe a novel prime-editing CRISPR/Cas9-based reporter system for high-throughput screening and sensitive detection of intracellular RNA delivery. This assay can detect functional RNA delivery as low as 5 fmols, and is capable of screening large RNA-barcoded libraries. We generated several nucleocapsid assemblies composed of purely computationally designed components, with both targeting and membrane-disrupting domains, and screened them for cytosolic RNA delivery. We report that EGFR and transferrin targeted assemblies containing membrane-disrupting domains delivered 31-fold and 12-fold more RNA, respectively, than their untargeted counterparts. These delivery vehicles offer a modular ‘bottom-up’ approach to creating tailor-made therapeutics and make significant headway towards developing next generation medicines.