(467h) Engineering Exosomes As Therapeutic Delivery Vehicles

Exosomes are a nanoscale class of extracellular vesicle (EV) that transfer proteins and nucleic acids between cells and have great potential as tunable therapeutic delivery vehicles; they are relatively easy to engineer, well-tolerated in vivo, and naturally capable of intracellular delivery of functional biomolecules. In order for this promise to be realized, robust and general methods for incorporating therapeutic cargo molecules into EVs and for targeting therapeutic EVs to specific destinations in vivo are required.  

Loading therapeutic RNA into EVs poses several challenges. Transfer of RNA into isolated EVs by electroporation can be inefficient and generates RNA precipitates.  Alternatively, mass-action-driven incorporation, in which the therapeutic RNA is overexpressed in EV producing cells, is attractive but has not been as widely explored. In particular, it is not known how native RNA sorting and processing mechanisms and biophysical limitations may impact the efficacy of this approach for different RNA cargo molecules. Thus, to provide a robust and orthogonal strategy for EV loading, we have developed a Targeted And Modular Exosome Loading (TAMEL) platform for directing the loading of a specific RNAs into EVs. The TAMEL system comprises a packaging protein and a cargo RNA. The packaging protein is an RNA binding protein targeted to EVs via fusion to an exosome-enriched protein. The cargo RNA is an RNA molecule displaying an RNA motif that is specifically bound by the packaging protein. Using this system, we have packaged small and large RNAs into EVs and characterized the relationship between RNA size and packaging into EVs by mass action alone or targeted means. This platform enables us to package mRNAs and RNAi-inducing RNA species into EVs and to quantitatively evaluate delivery and functional modulation of gene expression in recipient cells.  

To enhance delivery of therapeutic EVs to target recipient cells, we have also developed a technology for enhancing the display of targeting ligands on the exterior of EVs. Previous attempts to target EVs to specific recipient cells have been hindered by inconsistent targeting using different ligands or different EV-producing cell types. We determined that degradation of targeting peptides by endosomal proteases occurs during EV biogenesis. To overcome this challenge, we have also developed a glycosylation strategy that protects peptides from degradation while maintaining their ability to bind target proteins. We applied this peptide targeting strategy to engineer enhanced delivery of EVs to prostate cancer cells. The tools we have developed enable programming EVs for targeted delivery of RNA cargos in a robust and straightforward manner that can be applied to a wide variety of therapeutic targets.