Muwaffak, Maha; (2021) Extracellular vesicles as potential therapies for paediatric neurovisceral diseases. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

There are a large number of lethal diseases that affect the brain and visceral organs of children. Many of these neurovisceral conditions are caused by mutations in single genes leading to a lack of a particular protein or enzyme. These are intractable conditions for which there is no effective treatment available, and hence there is an overwhelming need to develop new therapies. Extracellular vesicles (EVs) offer one possible solution to this. EVs are anuclear cell-derived bodies that can deliver a desired therapeutic cargo to both the visceral organs and brain. Here we describe how a gene delivery strategy can be used to develop cellular factories that produce EVs loaded with proteins and enzymes of interest as prospective treatments for neurovisceral diseases. Various EV producer cell types were first evaluated for transduction efficiency with lentiviral vectors carrying marker genes for enhanced green fluorescent protein (eGFP) and luciferase enzyme. HEK293T cells had the highest transduction efficiency and were hence used to produce EVs loaded with eGFP and luciferase. These loaded-EVs were isolated using an optimised differential ultracentrifugation method, and characterised for size, concentration, morphology, eGFP and enzyme content. In vitro experiments demonstrated that loaded-EVs can deliver active luciferase enzyme to HEK293T cells and eGFP to primary neurons. Additionally, eGFP was shown to be co-localised with lysosomes in primary neurons. Injections of loaded-EVs into neonate mice through different routes of administration revealed that EVs delivered eGFP to both the brain and visceral organs, although to different extents. Significant eGFP content in the brain was only achieved following intracerebroventricular (ICV) injections. EVs were shown to cross the blood brain barrier following systemic administration but to a lower extent in comparison to the ICV route. Similar injections in adult mice resulted in eGFP only being detected in visceral organs at the same time points. The possibility of targeting delivery to the brain was evaluated through the use of the neuronal cell line SH-SY5Y to produce loaded-EVs. However, EVs derived from HEK293T cells delivered a higher eGFP cargo to both the brain and visceral organs than those derived from SH-SY5Y cells. Subsequently, Gaucher disease, a lysosomal storage disease caused by mutations in the GBA1 gene leading to a defective glucocerebrosidase (GCase) enzyme, was chosen as a paediatric neurovisceral disease model. HEK293T cells were used to generate EVs loaded with the GCase enzyme, which were characterised and tested in vitro. These experiments revealed that the EVs can upregulate lysosomal GCase enzyme activity in cells, indicating that they can be used to deliver lysosomal enzymes to the lysosomal compartment. Finally, the stability of HEK293T-derived EVs was evaluated, and it was determined that they are stable for two weeks at –80 °C but degrade over this time at 20 °C. The feasibility of freeze-drying using the lyoprotectant trehalose to improve stability of these EVs at higher storage temperatures was investigated, and freeze-drying with 2 %w/v trehalose enabled storage at 4 °C for two weeks and at 20 °C for one week without significant deleterious effects on the biological cargo. Overall, the work detailed in this thesis suggests that loaded-EVs could comprise potent therapeutic interventions for neurological disorders.

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