Extracellular vesicles as drug delivery systems: Molecular mechanisms and therapeutic applications

Abstract

RNA therapeutics play a pivotal role in the treatment of various diseases by modulating disease-related gene expression. These therapies utilize RNA sequences to regulate the expression or activity of target proteins. While RNA therapeutics hold immense potential, particularly for addressing undruggable targets, their clinical application is hindered by the challenge of efficiently delivering RNA into the cell cytoplasm, which requires overcoming both extracellular and intracellular barriers. To address these obstacles, various engineered delivery vehicles have been explored to protect and deliver RNA. Among these, extracellular vesicles (EVs) have emerged as a promising drug delivery system. EVs are a heterogeneous population of membrane-enclosed vesicles secreted by cells into extracellular spaces. Their unique properties—biocompatibility, ability to traverse biological barriers, and reduced immunogenicity—make them attractive candidates for drug delivery. Moreover, EVs exhibit higher efficiency in RNA delivery compared to state-of-the-art lipid nanoparticles. To fully harness EVs for RNA therapeutics, it is crucial to understand the molecular pathways underlying EV-mediated functional RNA delivery. In this thesis, we focus on the role of integrins in EV-mediated RNA delivery and uptake, as they play a major role in cell signaling and are involved in various physiological processes. Specifically, we show that alpha4beta1 integrin mediates functional RNA delivery and EV uptake via binding to fibronectin on the EV surface. Additionally, we investigate the role of T-cell immunoglobulin and mucin domain 1 in EV-mediated functional RNA delivery. In later chapters, we explore various methods to enhance the loading efficiency of cargo inside EVs. We describe a novel modular platform for EV-mediated loading and delivery of various gene-editing tools, including Cas9-sgRNA ribonucleoprotein (RNP) complex, transcriptional activator dCas9-VRP, and Cas9 base editor ABE8e. Moreover, we engineer a microRNA backbone to actively load siRNA inside the EVs’ lumen.