An extracellular vesicle biogenesis-inspired engineering platform for efficient protein delivery and therapeutic base editing

Abstract

Efficient and controllable delivery of genome-editing proteins remains a central challenge for therapeutic translation of gene-editing technologies. Extracellular vesicles (EVs) offer an attractive non-viral delivery modality due to their biocompatibility and large capacity for cytosolic cargo delivery. Yet, rational strategies to achieve controlled and programmable protein loading are still lacking. Here, we present NEO-TOP-EVs, an EV biogenesis-guided engineering platform that systematically integrates key features of three design principles inspired by vesicle formation: 1) PI(4,5)P2-mediated plasma membrane targeting, 2) ESCRT-dependent membrane scission, and 3) self-assembly-driven cargo clustering for enabling efficient encapsulation of genome-editing ribonucleoproteins. Together, the NEO design increased cargo incorporation and enhanced functional delivery of gene editing modalities under particle-normalized conditions. Using NEO-TOP-EVs, we achieve efficient delivery of Cas9 and adenine base editor ribonucleoproteins without nucleic acid templates. In an in vitro proof-of-concept, delivery of an adenine base editor targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) induces efficient splice-site disruption, resulting in reduced PCSK9 expression and enhanced LDL receptor activity. Proof-of-concept in vivo experiments provide preliminary evidence of functional Cre protein delivery to the liver. Together, these findings establish NEO-TOP-EVs as a modular platform for protein-based genome editing, demonstrating how biogenesis-informed EV engineering yields functional protein delivery at levels relevant to therapeutic development.