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Title: The use of the CRISPR-Cas9 system and iPSC-derived neurons with a SNCA mutation to model neurodegeneration
Author: Mosaku, Olukunbi Eniola
ISNI:       0000 0004 7661 2986
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2018
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Parkinson's disease (PD) is characterised by the selective loss of dopaminergic neurons of the substantia nigra pars compacta. Patients suffer from a progressive motor disorder, defined by the presence of rigidity, resting tremor and bradykinesia. Current treatment options, relieve symptoms for a limited period, but are not curative, as the underlying molecular causes of neurodegeneration are unknown. Several causative PD mutations have been identified and could provide insight into the defective molecular pathways in PD. Multiplication or missense mutation of the SNCA gene leads to autosomal dominant PD. Alpha-synuclein, encoded by the SNCA gene, is a defining component of proteinaceous deposits found in surviving neurons in PD and a central protein in PD aetiology. Induced pluripotent stem cells (iPSCs) self-renew indefinitely and generate all germ layer lineages. Human iPSCs derived from an individual with a genetic variant known to cause disease, provide a platform to investigate the molecular basis of disease. However, genetic variation between iPSC lines can lead to functional disparities, masking or accentuating disease-specific phenotypes. Genome engineering facilitates the generation of iPSCs which differ exclusively at the locus of interest, providing a genetically stable cellular model. iPSCs from an individual with a SNCA missense mutation, G51D, and an unaffected relative were characterised, demonstrating ex vivo pluripotency was established and dopaminergic neurons could be derived. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system was exploited to introduce the G51D mutation into control iPSCs. Changes in dopamine turnover and protein metabolism were detected after differentiation of CRISPR-Cas9 generated iPSCs, now harbouring the heterozygote G51D mutation. A CRISPR-Cas9 G51D homozygote iPSC clone was generated and a reduction in the number of dopaminergic neurons produced observed. This study demonstrates human iPSCs can be used to detect phenotypic differences in specialised cells, despite the latency of PD, and before overt neurodegeneration.
Supervisor: Gissen, P. ; Hardy, J. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available