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E. Ostad-Saffari, M. Moosajee, S. P. Wong, D. C. Tracey-White, T. Tolmachova, R. P. Harbottle, M. C. Seabra; Persistent Expression of Non-Viral S/MAR Vectors in the RPE for Choroideremia Gene Therapy. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3122.
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Novel non-viral plasmids harbouring scaffold/matrix attachment regions (S/MARs) provide sustained expression and functional persistence within cells. S/MAR gene therapy vectors are an attractive alternative to conventional viral delivery systems due to their low toxicity and reduced immunogenicity. Choroideremia (CHM) is an X-linked disorder caused by the loss of function of Rab Escort Protein 1 (REP1/CHM gene). The aim of our current study is to investigate whether S/MAR vectors can persistently express within the retina and provide a potential therapeutic system for CHM.
We generated S/MAR vectors containing either REP1/CHM cDNA or reporter EGFP and Luciferase genes, driven by the human elongation factor 1 short (EFS), Ubiquitin-C or CMV promoter. CHM fibroblasts were transfected with DNA constructs, expressing EGFP and REP1/CHM and analysed by western blot analysis, FACS and in vitro prenylation. Vector DNA was formulated with linear polyethylenimine (PEI). DNA:PEI complex (2ul) was subretinally injected into one eye of MF1 mice (1-2 months old), the contralateral eye served as a control. The temporal expression of luciferase was followed utilising in vivo bioluminescent imaging. Histological and immunofluorescence analysis was used to evaluate integration of the vector within the retina.
pEOS-Rep1 plasmids were able to successfully rescue the phenotype of CHM derived cells and provide expression of hREP1 after transfection of human and mouse CHM fibroblasts. DNA:PEI complexes were successfully delivered to the eye via subretinal injections. We were able to observe steady and persistent transgene expression in vivo for up to 4 months after one single vector administration. Immunohistochemistry demonstrated RPE localisation of transgene.
We have developed a novel S/MAR vector that provides effective and sustained long term gene delivery to the RPE. Our S/MAR vectors could also be used as a therapeutic delivery of the REP1/CHM gene for the treatment of CHM, with applications to other genetic RPE diseases.
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