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M. R. Biswal, H. Prentice, G. Smith, C. Dorey, C. Robinson, T. House, K. Webster, J. Blanks; Hypoxia-Regulated Muller Cell-Specific Promoter for Use in Retinal Gene Therapy. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3101.
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Retinal ischemia and subsequent neovascularization leads to blindness and visual impairment in several eye diseases. Muller cells represent the functional link between blood vessels and neurons. Since Muller cells span the retina and secrete several trophic factors, it has been suggested that they would be excellent targets for gene therapy to the inner and outer retina. In this study, we constructed a hypoxia-regulated, Muller cell-specific vector and tested its response to hypoxia.
To restrict expression of a therapeutic gene to hypoxic Muller cells, we constructed a hybrid promoter using conserved domains of the human glial fibrillary acidic protein (GFAP) gene promoter and several hypoxia-responsive elements (HRE) and aerobically silenced elements (HRSE). Promoter domains were incorporated into plasmid vectors and transfected to primary mouse Muller cells and other cell lines. The promoter activation was quantified using dual luciferase reporter gene assay and GFP expression.
Our results demonstrated that the regulated promoter construct was silenced by 91% in aerobic conditions in primary mouse Muller cells and 20% in the M10M1 human Muller cell line compared to unregulated promoter. Exposure to hypoxia induced a 12-fold increase in expression in transfected primary mouse Muller cells and a 15-fold increase in the M10M1 Muller cell line. The regulated construct was not active in hypoxic or aerobic conditions in a control kidney HEK cell line, glioma C6 cell line or monkey choroidal endothelial RF6A cell line.
Our hypoxia-regulated, Muller cell-specific promoter provides a platform for exclusive delivery of AAV-based gene therapy to regions of retinal hypoxia. The in-vivo testing of the efficacy of these vectors for diabetic retinopathy is being continued in the murine model of oxygen-induced retinopathy.
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