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, a bioengineering platform for efficient mRNA loading and functional mRNA delivery was developed using extracellular vesicles (EVs) as naturally derived nanoparticles. Engineered EVs carrying the highly specific PUFe RNA-binding domain fused to CD63 were produced in cells stably expressing the target mRNA with compatible binding sites. Using this system, the target mRNA was actively loaded into the produced EVs during EV biogenesis with loading efficiencies exceeding those previously reported for other EV- based approaches. In combination with the expression of an mRNA-stabilizing protein, PABPc, and a fusogenic endosomal escape moiety, VSVg, functional extrahepatic mRNA delivery via EVs in vivo was achieved at mRNA doses substantially lower than currently used for lipid nanoparticles. Our technology overcomes major limitations currently associated with EV-based nucleic acid delivery systems and could enable new applications for mRNA therapeutics. TABLE OF CONTENTS This study developed a novel system for highly efficient mRNA loading using engineered extracellular vesicles (EVs). The synergic components of the mRNA loading platform also solve limitations in delivery by supporting mRNA stability and translation, as well as endosomal escape in recipient cells. Thus, functional mRNA delivery is achieved at mRNA doses substantially lower than currently used for lipid nanoparticles.

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