GAPDH controls extracellular vesicle biogenesis and enhances the therapeutic potential of EV mediated siRNA delivery to the brain

Ghulam Hassan Dar,Clive Wilson,Wei-Li Kuan,Alfina A Speciale,Thomas C Roberts,Cláudia C Mendes,Roger A Barker,Wooi F Lim,Matthew J A Wood,Samir El Andaloussi,Imre Mäger,Deborah C I Goberdhan,André Görgens,Miguel J Lobo,Mariana Conceição,Inna Uliyakina

doi:10.1038/s41467-021-27056-3

Abstract

Extracellular vesicles (EVs) are biological nanoparticles with important roles in intercellular communication, and potential as drug delivery vehicles. Here we demonstrate a role for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in EV assembly and secretion. We observe high levels of GAPDH binding to the outer surface of EVs via a phosphatidylserine binding motif (G58), which promotes extensive EV clustering. Further studies in a Drosophila EV biogenesis model reveal that GAPDH is required for the normal generation of intraluminal vesicles in endosomal compartments, and promotes vesicle clustering. Fusion of the GAPDH-derived G58 peptide to dsRNA-binding motifs enables highly efficient loading of small interfering RNA (siRNA) onto the EV surface. Such vesicles efficiently deliver siRNA to multiple anatomical regions of the brain in a Huntington’s disease mouse model after systemic injection, resulting in silencing of the huntingtin gene in different regions of the brain.

Highlights

Extracellular vesicles (EVs) are biological nanoparticles with important roles in intercellular communication, and potential as drug delivery vehicles
In a set of experiments designed to express the N-terminal region of lactoferrin on the surface of EVs for the purposes of brain targeting[35], we observed that lactoferrin N, which was attached to the EV surface via fusion to DC-LAMP (Dendritic cell lysosomal associated membrane glycoprotein), was cleaved from this anchor
It was demonstrated that cells take-up extracellular iron by secreting EVs carrying surface glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which interacts with the iron-binding proteins lactoferrin and transferrin[33]

Summary

Introduction

Extracellular vesicles (EVs) are biological nanoparticles with important roles in intercellular communication, and potential as drug delivery vehicles. Fusion of the GAPDH-derived G58 peptide to dsRNA-binding motifs enables highly efficient loading of small interfering RNA (siRNA) onto the EV surface Such vesicles efficiently deliver siRNA to multiple anatomical regions of the brain in a Huntington’s disease mouse model after systemic injection, resulting in silencing of the huntingtin gene in different regions of the brain. EVs have attracted considerable interest as potential vehicles for drug delivery, as carriers of macromolecules like non-coding RNAs and proteins[13] Their immunological inertness and ability to cross biological barriers are two important features that can be exploited for therapeutic applications[14].

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Results

Conclusion