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A. Subrizi, S. Van Vlierberghe, G. Subra, M. Amblard, J. Martinez, E. Schacht, P. Dubruel, M. Yliperttula, A. Urtti; Non-Viral Gene Therapy and Retinal Pigment Epithelium Tissue Engineering for the Treatment of Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4496.
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© ARVO (1962-2015); The Authors (2016-present)
Our approach to recovering visual function in AMD is the development of an ex vivo gene therapy in combination with retinal pigment epithelium (RPE) tissue engineering. We have studied the effect of substrate on RPE monolayer morphogenesis and electrophysiology. We have evaluated duration and direction of transgene secretion in RPE monolayers grown on selected scaffolds.
The scaffolds were prepared from aminopropylmethacrylamide-modified gelatine and decorated with several bioactive molecules: heparan sulphate, chondroitin sulphate, hyaluronic acid, fibronectin, laminin and the epitope RGD. Screening was carried out with Cell-IQ live cell imaging and analysis platform. Bioelectric measurements of RPE monolayers were performed with glass barrel micro reference Ag/AgCl electrodes and a voltage-current clamp. RPE cells grown on selected scaffolds were clamped inside vertical Ussing chambers and transfected with non-viral vectors by means of conventional and reverse transfection methods. A self-replicating plasmid and a plasmid with RPE tissue-specific promoter were used to prolong the expression of the transgene and to improve the transfection efficacy.
According to growth curves, morphology and viability of RPE cells, three scaffolds were selected for further studies: hyaluronic acid, heparan sulphate and laminin. The barrier properties of RPE monolayers and their viability were monitored by determining their bioelectric properties. Transgene expression in RPE monolayers lasted for several weeks. At the same time transepithelial electrical potential difference and transepithelial resistance were measured.
The reconstruction of a functional RPE and nanoparticulate-enhanced gene therapy providing efficient gene replacement may be a promising therapeutic option for the treatment of AMD.
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