Modelling and investigating treatments of childhood inherited mitochondrial diseases in zebrafish.
Mitochondrial DNA depletion syndromes (MDDS) are a group of severe, individually rare, clinically heterogeneous disorders that primarily affect children. Affected tissues tend to be those with a high energy demand such as the central nervous system and skeletal muscle. MDDS stem from disruption to the replication and maintenance of mitochondrial DNA (mtDNA), which encodes key components of the mitochondrial respiratory chain needed for oxidative phosphorylation. There are currently no cures for MDDS, only supportive therapies that try to alleviate symptoms. Due to their clinical heterogeneity and diverse pathomechanism, there is currently a lack of suitable *in vivo* models that allow for disease characterisation and studying treatment. In this thesis I aimed to develop new zebrafish disease models to try and recapitulate features of MDDS to then trial a therapy, nucleoside supplementation, which has been shown to have positive effects in other MDDS disease models and in recent patient trials. Here, using CRISPR/Cas9 mutagenesis I developed three new zebrafish models of MDDS. The first, focused on RRM2B, a protein involved in maintaining mitochondrial nucleotide pools, where I show nucleoside supplementation rescues several hallmarks of MDDS including mtDNA depletion, movement defects and elevated lactate. The second two models focus on POLG, the key catalytic protein for mtDNA replication, where I was also able to recapitulate several features of POLG MDDS and show that nucleoside supplementation increase mtDNA copy number in a model with a mutation in the linker region, an area associated with some of the most common forms of POLG MDDS. These results help to ratify nucleoside supplementation as a leading therapy prospect for treating MDDS. Lastly, utilising a CRISPR/Cas9 F0 knockout method, I modelled a novel mitochondrial disease in zebrafish, recapitulating patient disease features, including a neuromuscular transmission defect, helping to strengthen the disease genotype-phenotype correlation and highlight the neuromuscular junction as feature of mitochondrial disease.
Mitochondria, Mitochondrial Disease, Mitochondrial DNA depletion syndromes, Zebrafish
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