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The Use of the CRISPR-Cas9 System and iPSC-derived Neurons with a SNCA Mutation to Model Neurodegeneration

Mosaku, Olukunbi Eniola; (2018) The Use of the CRISPR-Cas9 System and iPSC-derived Neurons with a SNCA Mutation to Model Neurodegeneration. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

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.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: The Use of the CRISPR-Cas9 System and iPSC-derived Neurons with a SNCA Mutation to Model Neurodegeneration
Event: UCL (University College London)
Language: English
Additional information: Copyright © The Author 2018. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health > Genetics and Genomic Medicine Dept
URI: https://discovery.ucl.ac.uk/id/eprint/10062570
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