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Development of a non-viral gene therapy strategy for choroideremia

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Title: Development of a non-viral gene therapy strategy for choroideremia
Authors: Ostad-Saffari, Elham
Item Type: Thesis or dissertation
Abstract: Non-viral plasmids harbouring scaffold matrix attachment regions (S/MARs) provide stable and functional episomal maintenance, resistance to epigenetic silencing and have been shown to be capable of sustained expression in murine tissues. S/MARs are an attractive alternative to conventional viral gene delivery vectors due to their low toxicity and reduced immunogenicity. This thesis documents the development of this novel non-viral episomal plasmid DNA (pDNA) expression system for persistently expressing vectors for Choroideremia (CHM) gene therapy. To this end, our goal was to analyse the ability of S/MAR plasmids to generate efficient and stable transgene expression in the mouse retina following subretinal injections. We generated and analysed a series of S/MAR constructs expressing either REP1 cDNA or the reporter enhanced green fluorescent protein (EGFP) or Luciferase (Luc) genes driven by the elongation factor-1 short (EFS) or cytomegalovirus (CMV) early enhancer/chicken beta actin (CAG) promoter. We demonstrate using the AtT20 cell line, the principle that S/MAR containing plasmids can provide long-term episomal EGFP and REP1 transgene expression in vitro. Furthermore, long-term REP1 expression was also analysed in CHM fibroblasts, where we showed in preliminary experiments that DNA constructs expressing REP1 were able to rescue CHM derived cells by rescue of their Rab27a prenylation defect and provide long-term expression of hREP1 after transfection of human and mouse CHM fibroblasts. We also developed an optimised method for delivering S/MAR containing plasmids in the eye via subretinal injections of complexed pDNAs, and provide evidence for the utility and versatility of S/MAR plasmids in ocular tissue. We show for the first time the longitudinal transgene expression of Luc in the retina as measured using bioluminescent imaging. Long- term maintenance and expression of EGFP and REP1 transgenes were also observed by PCR and Western blot analysis. Further analysis provided evidence for the long term episomal maintenance of these vectors in the eye as shown by Southern analysis. We provide evidence for the superiority of an S/MAR containing plasmid in providing long-term expression in the eye. All control pDNA constructs were shown to be incapable of sustaining significant transgene expression beyond one-month following pDNA delivery. We further demonstrate the lack of toxicity within the eye and show that fundus examinations as well as detailed histological examinations of retinal sections do not elicit an inflammatory response to our plasmids once subretinally injected in the eye. Finally, we explored the therapeutic potential of the REP1-S/MAR episome, by subretinally injecting mouse models of CHM. Further repeat experiments in CHM mouse models would add confidence to the overall experiments; however initial findings were encouraging where a partial correction of the REP1 protein and functional rescue in the eye was shown.
Content Version: Open Access
Issue Date: Oct-2011
Date Awarded: Mar-2014
URI: http://hdl.handle.net/10044/1/24912
DOI: https://doi.org/10.25560/24912
Supervisor: Seabra, Miguel
Tolmachova, Tanya
Harbottle, Richard
Sponsor/Funder: Fight for Sight (Organization)
Choroideremia Research Foundation Canada
Department: National Heart and Lung Institute
Publisher: Imperial College London
Qualification Level: Doctoral
Qualification Name: Doctor of Philosophy (PhD)
Appears in Collections:National Heart and Lung Institute PhD theses

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