Engineering Extracellular Vesicles for Protein-based Drug Delivery

This Ph.D. thesis delved into the intricate engineering of extracellular vesicles (EVs) for optimized protein-based drug delivery. The clinical utilization of EVs for drug delivery was contingent upon efficient drug loading methods, scalable EV production techniques, and improved methods to achieve targeted delivery for enhanced therapeutic efficacy. Thus, the research aimed to expedite the clinical translation of EVs for protein delivery by: (i) developing efficient loading approaches, (ii) upscaling EV production, and (iii) exploring the preclinical and therapeutic applications of EVs.

In the first section, novel strategies for enhancing EV-mediated protein delivery were developed. A versatile platform, TOP-EVs, was introduced, which leveraged the rapamycin-interacting FKBP/FRB complex and the vesicular stomatitis virus glycoprotein, thereby enhancing protein delivery both in vitro and in vivo. Further, a strategy using the FKBP12/FRB complex and the PTGFRN transmembrane protein was explored for efficient protein loading/unloading in target cells and concurrent EV-mediated targeted delivery of bioactive content. Furthermore, a new endogenous loading method, VINCI, was devised to improve protein delivery using engineered EVs.

The second section tackled the low yield issue prevalent in the current EV production process. Biofabrication of artificial cell-derived nanovesicles was shown to be a scalable and efficient solution, capable of producing EV mimics with functionality akin to natural EVs. However, more research was needed to enable effective protein loading inside these artificial nanovesicles.

The third section examined the preclinical and therapeutic applications of EVs. EVs' in vivo behavior and capacity for effective intracellular delivery of protein-based payloads were investigated using varied administration strategies. A thorough examination of the biodistribution of EVs and their ability to mediate intracellular protein delivery in the liver was performed. Moreover, evidence was provided that EVs could effectively deliver CRISPR/Cas9 to inactivate the Pcsk9 gene, a therapeutic target for cholesterol reduction, hence increasing LDL receptor expression.

In conclusion, this research underscored significant advancements in EV engineering for protein-based drug delivery. The strategies presented in this work could catalyze the clinical translation of EV-based nanomedicine, expanding the horizons of precision gene therapy and protein-based therapeutics. However, standardizing EV-isolation, -characterization, -purification, and -storage methods to decrease heterogeneity and ensure reproducibility remained fundamental challenges within the field. Future studies are warranted on identifying the most optimal loading and delivery strategies, scalable EV production platforms, and improved delivery to target organs. Advancements in these areas could propel the utilization of EVs as nanomedicine.