Khedr, Moustafa Omar Ali Omar;
(2022)
Gene Editing and Human Induced Pluripotent Stem Cells for Advanced Modelling of Duchenne Muscular Dystrophy.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Duchenne muscular dystrophy (DMD) is a rare x-linked genetic disorder characterised by progressive muscle wasting and severe weakness. It is caused by absence or dysfunction of dystrophin, a protein encoded by the largest gene known in nature that protects muscle cell membrane from contraction-induced damage. To date DMD remains incurable. Several experimental therapies are being developed for DMD, including exon-skipping using anti-sense oligonucleotides (AONs), read-through technologies using small molecules, and gene therapy and genome editing strategies. However, the development and optimisation of these therapies and determining their relative efficiency is limited by the lack of an overarching (temporally, economically, and ethically viable) platform to model DMD and human dystrophin dynamics. Here CRISPR/Cas9 gene editing is utilised to generate human induced pluripotent stem (iPS) cells that can be used to detect dystrophin protein production qualitatively and quantitatively, by inserting a multifunctional reporter cassette capable of tracking dystrophin expression temporally and spatially, in real time and in fixed cells. Reporter functionality was assessed via transgenefree differentiation of iPS cells into skeletal muscle cells. Dystrophin-reporting iPS cells were also gene-edited to induce pathogenic DMD mutations in exon 52 which were targeted by mutation-specific AONs. In parallel, iPS cells were also differentiated in 3D artificial muscle constructs to model DMD in comparison to traditional monolayer cultures, with the aim of combining the potential of both platforms (multifunctional reporter and 3D tissue architecture) into a novel advanced DMD modelling platform to accelerate development of personalised and precision therapies for DMD.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Gene Editing and Human Induced Pluripotent Stem Cells for Advanced Modelling of Duchenne Muscular Dystrophy |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2022. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) Licence (https://creativecommons.org/licenses/by-nc-nd/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 > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology > Neurodegenerative Diseases UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10156298 |
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