Investigation of the use of extracellular vesicles for the treatment of congenital muscular dystrophy

Aberrant glycosylation of alpha-dystroglycan (α-DG) leads to a group of pathologies known as dystroglycanopathies. Mutations in the gene encoding fukutin-related protein (fkrp) are one of the most common causes of secondary dystroglycanopathy in the UK. Mutations in fkrp prevent complete glycosylation of α-DG, leading to a loss of extracellular matrix binding capacity. These patients present with a very broad spectrum of muscular dystrophies ranging from severe Walker Warburg syndrome (WWS), characterised by severe structural brain, muscle, and eye abnormalities, to mild adult-onset Limb-girdle Muscular Dystrophy 2I (LGMD2I). No effective treatment is currently available for these patients. Current research focuses on three main strategies, small molecule treatments, gene therapy and cell therapy. This thesis investigates the use of extracellular vesicles (EVs) as an alternative therapy to those currently under investigation. For this purpose, the size exclusion chromatography (SEC) technique was characterised and optimised. In this optimisation, it was shown that increasing the length of the SEC columns improves the resolution of the columns. Because the biodistribution mechanisms of EVs in vivo are currently not fully characterised, a model of EVs with a fluorescence tag was used to study the distribution in muscle after intravenous (IV) injection. The difficulty of tracking the EVs made in vivo characterisation impossible, but in vitro characterisation showed that both myoblasts and myotubes are able to uptake EVs. Since the benefits derived from exercise have been widely described, the possibility that these benefits could be derived from EVs circulating in the blood was investigated for possible use as a therapy for patients with dystroglyconopathies. To this end, healthy participants were exercised and EVs were extracted from their blood. The possible benefits of these EVs on C2C12 cells were analysed. We also investigated the generation of a cell line of muscle cells overexpressing fkrp for packaging into EVs. These cells were cultured in a co-culture system with non-transfected cells and it was found that these recipient cells presented higher levels of fkrp.

Grantor dc:publisher

Oxford Brookes University

Year dc:date

2022

Author dc:creator

• Pagalday-Vergara, Vicente

Contributors dc:contributor

• Pink, Ryan

Statement dc:rights

• All rights reserved

Language dc:language

en

DOI dc:identifier

https://doi.org/10.24384/2s0w-4v91

OAI identifier oai:identifier

tle:3143e907-21fd-48a6-9751-79b8bdb9d961:d6bd9758-527a-46cd-bfe2-c433766e8fca:1