Uncovering the role of heparan sulphate proteoglycans in small extracellular vesicle production: potential tools for optimised regenerative medicine therapies

Abstract

Cells secrete small membrane-bound extracellular vesicles (sEVs) called exosomes. These vesicles contain a mixture of protein, lipids, nucleic acids and saccharides (known collectively as cargo) that mediate therapeutic effects similar to their producer cell. Exosomes may be optimised to generate potent therapies through manipulating exosome biogenesis mechanisms. This project aims to prove this concept by altering the global heparan sulphate (HS) present in MCF-7 cells, in order to modify syndecan, a key component in the syndecan-syntenin-ALIX mechanism for exosome production. We predict that HS may be involved in exosome cargo selection, due to its ability to form interactions with a wide range of factors. In addition, the structure of HS influences the activity of heparanase, a regulator in the rate of exosome production. Therefore, structural alterations to HS could allow the cargo (thus therapeutic activity) to be modulated, whilst simultaneously increasing exosome yields.
To initiate this project, techniques to characterise the HS structures on MCF-7 cells and their EVs were established. Methods included compositional disaccharide analysis and flow cytometry to detect HS-protein binding epitopes. A bead-free flow cytometry-based method was developed to characterise individual sEVs based on sEV enriched proteins and the 10E4 epitope. Knockouts (KO) in three key HS biosynthetic enzymes were generated using CRISPR-Cas9 technology. Subsequent alterations to HS structures were confirmed. Wildtype and KO MCF-7 cells were cultured in CELLine Integra bioreactors to obtain high sEV yields. While more work is required to demonstrate the potential of manipulating HS biosynthesis to optimise sEVs, the techniques developed throughout this project make such investigations possible.