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CRISPR/Cas9 genome editing of Recessive Dystrophic Epidermolysis Bullosa (RDEB) mutation hotspot

Naso, Gaetano; (2021) CRISPR/Cas9 genome editing of Recessive Dystrophic Epidermolysis Bullosa (RDEB) mutation hotspot. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe life-threatening skin adhesion disorder caused by loss-of-function mutations in the COL7A1-encoding type VII collagen (C7), a structural protein playing a crucial role in anchoring fibril (AF) formation at the dermal-epidermal junction (DEJ). Combinatorial cell and gene therapies based on the addition of a full-length copy of COL7A1 cDNA in RDEB keratinocytes, fibroblasts and skin equivalents have shown potential in preclinical and clinical settings although only modest and transient improvements have been reported. In parallel, induced pluripotent stem cells (iPSCs) are being investigated in preclinical studies for RDEB. iPSCs represent a valuable source of autologous patient material and can be differentiated into keratinocytes and fibroblasts for cellular therapy applications. Implementation of CRISPR/Cas9 and base editing-mediated gene correction in patient-derived iPSCs has allowed for the generation of autologous cellular models capable of overcoming barriers of conventional gene therapy. In this regards, the work described in this thesis aims to evaluate the feasibility of genome-editing approaches using CRISPR/Cas9 and Cytosine Base editing (BE) platforms to correct a mutation hotspot (c.425A>G, p.Lysl42Arg) within exon 3 of COL7A1 gene in patient-derived iPSCs. Gene repair by homology-directed recombination (HDR) following CRISPR/Cas9-induced double-strand breaks (DSBs) through viral and non-viral donor template deliveries resulted in a significant correction of the COL7A1 locus on genomic level. To avoid concerns surrounding the generation of DSBs, seamless BE-based G:C to A:T conversion resulted in a high restoration of the wild type COL7A1 sequence. Ultimately, capacity of gene- and base-corrected RDEB iPSCs to be differentiated in into keratinocytes (iKer) was evaluated in vitro and functional recovery of de novo C7 was assayed on protein level. Overall, this study explored the potential of CRISPR/Cas9 and BE site-specific correction of COL7A1 in RDEB-derived pluripotent stem cells. Furthermore, it demonstrated that gene-corrected iPSCs can be used as a source of epidermal progenitors thereby confirming their potential for future cell therapies for skin disorders.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: CRISPR/Cas9 genome editing of Recessive Dystrophic Epidermolysis Bullosa (RDEB) mutation hotspot
Event: UCL (University College London)
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2021. 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 > 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 > Infection, Immunity and Inflammation Dept
URI: https://discovery.ucl.ac.uk/id/eprint/10134745
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