Large quantities of extracellular vesicles produced via cellular nanoporation, and loaded with endogenously transcribed therapeutic mRNAs and targeting peptides, boost therapeutic outcomes in vivo.
Strategies for loading therapeutic mRNAs into EVs
a, Exogenous loading of mRNA into EVs. Pre-isolated EVs are exogenously loaded with a purified mRNA of interest. The resulting EVs retain their original cargo. b, Endogenous passive loading of mRNA into EVs. EV-producer cells are modified to overexpress the mRNA of interest, which is then loaded into vesicles during EV biogenesis, along with their original cargo and the protein translated from the overexpressed mRNA transcripts. c, Endogenous active loading of mRNA into EVs. EV-producer cells are modified to overexpress two constructs: the mRNA of interest with a specific binding domain (RBD) and an EV protein fused to an RNA-binding protein (RBP) capable of binding to the RBD of the co-expressed mRNA. The expression of the EV-protein–RBP fusion leads to the targeted binding of the engineered mRNA molecules before the fusion protein is incorporated into the vesicles, actively loading the bound mRNA along with it. EVs are also passively loaded with free mRNA molecules, proteins and natural EV cargo molecules. d, EV-mediated cargo delivery after mRNA loading. Bioengineered EVs are taken up by target cells. Endosomal degradation leads to the delivery of the cargo into the cytoplasm. Left, the delivery of mRNA molecules from exogenous loading leads to the translation of the protein of interest. Middle and right, EVs loaded endogenously with accompanying passive cargo deliver both mRNA and protein simultaneously to target cells. Protein resulting from the translation of delivered mRNA can only be distinguished from co-delivered, passively loaded protein when the RNA reaches translation and the accompanying protein cargo is degraded. Engineered molecules are depicted in red, green and blue. Native molecules are in grey and light blue. Figure created with BioRender.com.