Otx2-genetically modified retinal pigment epithelial cells rescue photoreceptors after transplantation

Abstract

Purpose: Dysfunction or death of retinal pigment epithelial (RPE) cells leads to photoreceptor loss and blindness in several retinal diseases, including retinitis pigmentosa. Transplantation of healthy RPE cells is consequently a promising approach to treat these diseases. The benefice for visual function is generally limited because of dedifferentiation of RPE when cultured. This transformation is undesirable as it may be a precursor to proliferative vitreoretinopathy (PVR).

Methods: We studied gene expression change in primary pig RPE cultured using RT-PCR and western blotting. We over-expressed OTX2 in these cells as well as in human induced pluripotent stem-derived RPE using an AAV2.1 recombinant vector and measured the expression of candidate target genes by RT-PCR. Binding of OTX2 to promoter of some of the regulated genes was studied by chromatin immunoprecipitation. Furthermore we study the benefice of OTX2 in cultured RPE cells by evaluating the photoreceptor rescue in the royal college of surgeons (RCS) rats after transplantation. We measured the effect on photoreceptor function by optokinetic response and electroretinogram. Photoreceptor survival was also assesed by optical coherence tomography and histological analysis on plastic sections. The protective effect on cone was also studied using cone-enriched cultures from chicken embryos.

Results: We found that OTX2 is down-regulated during RPE epithelial to mesenchymal transition and retinal detachment. Infecting RPE cells with an AAV encoding OTX2 in-vitro, can prevent gene expression change associated to dedifferentiation of RPE cells. We identified some of these genes as direct targets of the transcription factor OTX2. Transplantation of Otx2-genetically modified RPE cells in the RCS eye results in an increase rescue of photoreceptors cells and their function as compared to control, GFP-genetically modified RPE cells. The conditioned media of Otx2-genetically modified RPE cells contains secreted factors that protect cone photoreceptors in vitro.

Conclusions: Our results demonstrate that OTX2 increases the benefice of grafted RPE cells in photorecepotr rescue in a model of rod-cone degeneration. This effect is madiated through downregulation of RPE dedifferentiation, RPE gene rescue and promoting the expression of trophic factors. This study opens a novel therapeutic approach for treating inherited retinal diseases.