Engineered extracellular vesicles for biomedical applications

Abstract

Nature's very own nanoparticle, Extracellular vesicles (EVs), are lipid membrane-enclosed vesicles encapsulated with diverse biomolecules and are actively secreted by all cell types for intercellular communication. The unique properties of EVs, such as stability in circulation, biocompatibility, immune tolerance, and the ability to cross biological barriers, render EVs a next-generation drug delivery tool. Therapeutic EV research has seen tremendous development in the past decade, from in vitro studies towards pre-clinical models to various clinical trials. Even so, the road towards successful clinical translation has faced various hurdles primarily due to the lack of technology to address the knowledge gap in EV biology. Hence, this thesis is focused on addressing some of these critical challenges and exploring novel biomedical applications for EVs.

EVs are considered as essential mediators in physiology and disease pathology. However, to elucidate their important role in pathophysiology or as therapeutics, sensitive tools for visualising them are much needed. Here, in paper I, we have developed a sensitive bioluminescent labelling system for tracking EVs in vitro and in vivo. By genetically modifying the producer cells with EV-associated tetraspanins-fusions, we could efficiently load luciferase enzymes (Nanoluciferase and Thermoluciferase) into EVs. Utilising the Nanoluciferase labelling system, we could detect as low as 5000 EVs in a solution, and the naked eye could visualise the luminescence generated from these EVs. With this level of sensitivity, we explored various in vivo applications and observed that exogenous EVs are rapidly distributed throughout the body, primarily to the liver, lung, and spleen. In addition, we identified that EV subpopulations differ in their in vivo biodistribution profile. In summary, this system allows for highly sensitive detection of EVs in vivo and reflects the true fate of EVs.

Despite tremendous advancement in understanding EV biology or engineering, techniques to surface engineer EVs with large protein biotherapeutics without altering their innate properties are largely lacking. Here in paper II, we developed a novel surface display technology for EVs, which allows for efficient display of several membrane proteins on the EV surface simultaneously. Using this platform, we decorated EVs' surface with cytokine receptors that can decoy pro-inflammatory cytokines such as TNF-α or IL-6/sIL-6R complexes. These cytokine decoy EVs were more active than a clinically approved biologic against TNF-α in vitro. Importantly, these cytokine decoy EVs ameliorated the disease phenotype in three different mice inflammation models, including neuroinflammation. In paper III, we have applied interleukin 6 signal transducer (IL-6ST) decoy EVs to tackle inflammation in muscle pathologies to enhance the muscle regeneration process. Using decoy EVs as a therapeutic intervention in mdx mice mimicking Duchene Muscular Dystrophy (DMD), we could achieve significant downregulation of phosphorylation of the proinflammatory transcription factor STAT3 in muscles.

In conclusion, the tools developed in this thesis, from highly sensitive detection of EV subtype to efficient display of biotherapeutics cargo on EV surfaces, holds great future potential and applicability in numerous biomedical applications of EVs.