Programmable Extracellular Vesicles for Macromolecule Delivery and Genome Modifications.

Abstract

Getting large macromolecules through the plasma membrane and endosomal barriers remains a major challenge. Here, we report a generalizable method of delivering proteins and ribonucleoproteins (RNPs) to cells invitro and mouse liver tissue invivo with engineered ectosomes. These ectosomes, referred to as "Gectosomes," are designed to co-encapsulate vesicular stomatitis virus G protein (VSV-G) with bioactive macromolecules via split GFP complementation. We found that this method enables active cargo loading, improves the specific activity of cargo delivery, and facilitates Gectosome purification. Experimental and mathematical modeling analyses suggest that active cargo loading reduces non-specific encapsulation of cellular proteins, particularly nucleic-acid-binding proteins. Using Gectosomes that encapsulate Cre, Ago2, and SaCas9, we demonstrate their ability to execute designed modifications of endogenous genes in cell lines invitro and mouse liver tissue invivo, paving the way toward applications of this technology for the treatment of a wide range of human diseases.