Extracellular Vesicles for Therapeutic RNA Delivery

In recent years RNA therapeutics have emerged as a new class of highly promising therapeutics. These drugs function by treating disease at its genetic source by introducing, modifying or suppressing gene expression. In recent years, RNA therapeutics have achieved great success, most notably in the form of the recently introduced mRNA-based COVID vaccines.
Despite recent advances, the full potential of RNA therapeutics is yet to be fully realized largely due to difficulties involved in the delivery of therapeutic RNA to the cytosol of specific disease associated cells. Currently, RNA therapeutics are delivered via lipid nanoparticles (LNPs) which provide a protective envelope that protects the delicate RNA cargo from degradation and facilitates entry into target cells. However, LNPs possess several drawbacks. They rapidly accumulate in the liver post systemic administration and display dose-limiting toxicity and immunogenicity at high doses.
A potential solution to this problem can be found in the form of extracellular vesicles (EVs). These are lipid bound particles released by cells of all types which have been shown to be capable of functionally transferring RNA from one cell to another. Perhaps, this naturally occurring RNA transfer system could be exploited for the delivery of therapeutic RNA in a more efficient manner.
This thesis first provides an overview of the features of EVs which could be used to achieve this purpose and the ways in which EVs have been modified to function as therapeutic RNA delivery vehicles. EV-mediated RNA transfer is a relatively recently discovered phenomenon and the appropriate tools to study this process were lacking at the initiation of this PhD project. We therefore developed an EV-mediated transfer reporter system which was successfully utilized to study this process. This reporter system was also used to provide a direct comparison of EV and LNP-mediated RNA transfer which revealed that MDA-MB-231 and A431-derived EVs are capable of delivering RNA to reporter cells with an efficiency orders of magnitude greater than that of state-of-the-art LNPs. In later chapters, this thesis investigated possible explanations for this greater efficiency which revealed that this observation may not be due to differences in post-uptake intracellular trafficking, but could be related to fusogenic properties of the EV membrane in acidic compartments of the recipient cell.