In Vitro and in Vivo RNA Inhibition by CD9-HuR Functionalized Exosomes Encapsulated with miRNA or CRISPR/dCas9

Abstract

In vitro and in vivo delivery of RNAs of interest holds promise for gene therapy. Recently, exosomes are considered as the rational vehicle for RNA delivery, especially miRNA and/or siRNA, while the loading efficiency is limited. In this study, we engineered the exosomes for RNA loading by constructing a fusion protein, in which the exosomal membrane protein CD9 was fused with RNA binding protein, while the RNAs of interest either natively harbors or is engineered to have the elements for the binding. By proof-of-principle experiments, we here fused CD9 with HuR, an RNA binding protein interacts with miR-155 with a relatively high affinity. In the exosome packaging cells, the fused CD9-HuR successfully enriches miR-155 into exosomes when miR-155 was excessively expressed. Moreover, miR-155 encapsulated in the exosomes in turn could be efficiently delivered into the recipient cells and recognizes the endogenous targets. In addition, we also revealed that the CD9-HuR exosomes could enrich the functional miRNA inhibitor or CRISPR/dCas9 when the RNAs were engineered to have the AU rich elements. Taken together, we here have established a novel strategy for RNA cargo encapsulation into engineered exosome, which in turn functions in the recipient cells.