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Development of Gene Therapy for Recessive Dystrophic Epidermolysis Bullosa

Georgiadis, C; (2016) Development of Gene Therapy for Recessive Dystrophic Epidermolysis Bullosa. Doctoral thesis , UCL (University College London). Green open access

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Abstract

Recessive dystrophic epidermolysis bullosa (RDEB) is a debilitating genetic cutaneous blistering condition caused by loss-of-function mutations in COL7A1, encoding type VII collagen (C7), central in anchoring fibril (AF) formation at the dermal-epidermal junction (DEJ). Presently no curative treatments exist for RDEB. Reconstitution of COL7A1 expression in autologous primary keratinocytes (KC) and fibroblasts (FB) by ex vivo gene therapy was hypothesised to restore C7 expression and AF construction at the DEJ and ameliorate the RDEB skin phenotype. Feasibility of this approach was demonstrated using a therapeutic grade, self-inactivating- lentiviral vector, encoding codon-optimised COL7A1 (LV-COL7) to transduce primary RDEB KCs and FBs. Expression and secretion of full-length de novo C7 was confirmed, with transduced cells exhibiting supranormal levels of protein expression and functional recovery in in vitro migration assays. A human:murine chimeric preclinical RDEB skin graft model was developed to assess functional correction mediated by the transduced cells in vivo. RDEB grafts lacking C7 expression exhibited severe blistering recapitulating the disease phenotype. Gene- modified grafts, showed C7 deposition at the DEJ, with re-establishment of basement membrane zone integrity. Functional correction was confirmed by an abundance of de novo synthesised AF structures throughout the DEJ securing dermal-epidermal attachment. Gene- corrected FBs were shown to mediate a superior therapeutic benefit. In addition, an alternative strategy was developed using zinc finger nucleases (ZFN) for the targeted editing of COL7A1 and site-specific restoration of endogenous C7 expression. Incorporation of ZFNs into non-integrating lentiviral vectors (NILV) resulted in a marked improvement of their cleavage activity. ZFN-mediated disruption of COL7A1 in KCs confirmed at both genomic and protein level initially enabled the in vitro modelling of RDEB, with observed regression of migration speeds. A dsDNA donor repair template was designed and codon optimised for co-delivery with the ZFNs. Sequencing across the ZFN binding site confirmed site-specific template insertion by targeted homologous recombination. In conclusion, gene correction of primary RDEB cells by LV-COL7 can mediate restoration of protein and structural defects in an RDEB model forming the proposal for therapeutic application in man. Furthermore, development of an alternative site-specific targeting strategy for correction of COL7A1 provides a promising insight into the realm of patient-tailored therapy.

Type: Thesis (Doctoral)
Title: Development of Gene Therapy for Recessive Dystrophic Epidermolysis Bullosa
Event: University College London (UCL)
Open access status: An open access version is available from UCL Discovery
Language: English
Keywords: COL7A1, Gene therapy, lentiviral vectors, Type VII collagen, Recessive Dystrophic Epidermolyis Bullosa, RDEB, gene editing, Zinc Finger Nuclease (ZFN)
UCL classification: UCL > Provost and Vice Provost Offices
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 Pop Health Sciences > UCL GOS Institute of Child Health
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Pop Health Sciences > UCL GOS Institute of Child Health > ICH Infect, Imm, Infla. and Physio Med
URI: http://discovery.ucl.ac.uk/id/eprint/1477579
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