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Florian Udry, Sarah Decembrini, Corinne Kostic, David M Gamm, Yvan Arsenijevic; Evaluation of iPS-derived retinal pigment epithelium (iRPE) as a model for in vitro lentiviral-based gene therapy. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4583.
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© ARVO (1962-2015); The Authors (2016-present)
Deficiencies in functions RPE give rise to several diseases, of which most of them result in visual impairments or blindness. The generation of hRPE for disease modelling is particularly worth to answer these major challenges. Here, we evaluate the suitability of iRPE as an in vitro model for lentiviral (LV) gene therapy (GT).
Human iPSCs were differentiated to iRPE using a protocol derived from Singh et al. (2013). All iRPE and foetal RPE (fRPE, Lonza) were P3 and matured 42 days on Transwell. hiPSCs, fRPE and iRPE mRNA expression was quantified by qPCR and analyzed by one-way ANOVA. Transepithelial resistance (TER) and growth factor secretion (by ELISA) were measured on iRPE cultures respectively and analyzed by Wilcoxon Signed Rank Test. iRPE and fRPE were transduced with different GFP constructs and FACSed 5 days later, DNA extracted, LTR sequence integration was quantified by qPCR and linear regressions were performed between the proportion of GFP+ cells and LTR-to-β-ACTIN ratio. All results are written as Mean±SD.
RPE mRNA markers were upregulated in iRPE compared to hiPSCs such as RPE65, MITF-H, PMEL-17, DCT, BEST1. Immunostaining assessed the presence of RPE65, CRALBP, OTX2 proteins and ZO-1 apical and BEST1 basolateral localization. Electron microscopy revealed apical microvilli, melanosomes and tight-junctions. TER values showed a consistent resistance of iRPE above 200Ω*cm2 from day 28 onwards (305.7±97.26Ω*cm2, Pv: <0.001). PEDF and VEGF secretion range was consistent with literature but not significantly polarized (apical vs basal ng/24h/cm2: 407.6±74.1 vs 453.6±120.3, Pv: 0.43, 8.1±2.8 vs 19.7±7.7, Pv: 0.06, respectively). Phagocytosis assay confirmed iRPE cells phagocytic ability. To test iRPE and fRPE cells reaction to LV GT, transduction of GFP constructs driven by different promoters was performed. Only EFS and R0.8 promoters showed a positive correlation between LV integration and GFP+ cells compared to GFAP, RHODOPSIN, SYNAPSIN and rNse which drive no reporter gene expression. In addition we performed a gene dosing test for RPE65 expression and observed a 3 fold RPE65 increase using LV.
In conclusion, iRPE revealed to be a suitable in vitro model to test GT constructs.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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