Abstract
The neuronal ceroid lipofuscinoses (NCLs/Batten disease) are a group of fatal autosomal recessive lysosomal storage disorders (LSDs). There are no effective treatments available for any form of NCL. This thesis focuses on modelling late infantile CLN2 disease (LINCL), which is caused by mutations in the CLN2 gene, resulting in deficiency of the lysosomal enzyme tripeptidyl peptidase I (TPP1). Potential treatments of this disorder are based upon delivering this missing enzyme to the brain but in order to judge the efficacy of future therapies, pre-clinical studies must be carried out in disease models. A mouse model of CLN2 disease exists, which is deficient in TPP1 and in this thesis I characterised the disease progression of this Tpp1-/- mouse. This analysis revealed a pattern of disease progression that is unique in the NCLs to these Tpp1-/- mice, where neuronal cell loss was initiated in the thalamus and preceded microglial and astrocytic activation. Subsequently, neuronal cell loss spread to the cortical target regions and was accompanied by glial activation. In addition, the effects of the adaptive immune system on CLN2 disease progression were explored by examining the same Tpp1-/- mice on an immunodeficient background. These immunocompromised mice had a more pronounced disease phenotype in terms of atrophy of regional brain structures and neuronal cell loss in the somatosensory cortex and specific thalamic nuclei than the equivalent TPP1-deficient mice with an intact immune system. The TPP1-deficient mice which lacked the adaptive immune responses lived longer, however this extended lifespan allowed a more severe neurodegerative phenotype to develop.This thesis also presents the process of developing a human neural progenitor cell (hNPC) model of late infantile CLN2 disease by introducing a CLN2 disease-causing mutation into a hNPC line using genome editing. Transcription activator-like effector nucleases (TALENs) are genome-editing tools that fuse naturally occurring DNAbinding proteins that can be designed and arranged according to a simple code to target a specific DNA sequence, to a DNA cleavage domain. These TALENs can be used to edit genomes by inducing double strand breaks. In this thesis I present my attempts to produce TALENs to target the CLN2 gene and to produce a clonal hNPC cell line that harbours a CLN2 disease mutation. I show that a culture of hNPCs was isolated that contained largely wild-type hNPCs and a smaller population of hNPCs containing the desired CLN2 disease-causing mutation. I also describe an optimised method for transfection of hNPCs that is now being used by other researchers wishing to carry out genome editing in this hNPC line.
| Date of Award | 2015 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Jonathan Cooper (Supervisor) & Jack Price (Supervisor) |