Novel endogenous engineering platform for robust loading and delivery of functional mRNA by extracellular vesicles

ABSTRACT Messenger RNA (mRNA) has emerged as an attractive therapeutic molecule for a range of clinical applications. For in vivo functionality, mRNA therapeutics require encapsulation into effective, stable, and safe delivery systems to protect the cargo from degradation and reduce immunogenicity. Here, we developed a bioengineering platform for efficient mRNA loading and functional delivery using naturally-derived nanoparticles, Extracellular Vesicles (EVs). By expressing the highly specific PUFe RNA-binding domain fused to CD63 in EV producer cells stably expressing the target mRNA, our system exceeds mRNA loading efficiencies by up to 4.5-fold over previously reported EV-based approaches. By combining with an mRNA-stabilizing protein, PABPc, and a fusogenic endosomal escape moiety, VSVg, functional mRNA delivery in vivo is achieved at doses substantially lower than clinically used lipid nanoparticles. Our technology solves substantial drawbacks currently associated with EV-based nucleic acid delivery systems and could enable new applications for mRNA therapeutics.