In vitro and in vivo evaluation of genetically corrected RPE cells for autologous cell therapy of hereditary retinal dystrophies

Abstract

Purpose: The generation of genetically corrected RPE cells from patient‐derived human induced pluripotent stem cells (hiPSCs) may provide a source for autologous therapeutically‐relevant therapy for this rare retinal dystrophy as well to create the human cellular disease models. Recently we generated a faithful RPE model from patient bearing mutation in MERTK gene and genetically corrected these cells using CRISPR/Cas9 system. For further application in humans is necessary in vivo validation for survival.Methods: The RPE cells from the genetically corrected patient‐derived hiPSC were characterized using immunocytological methods for the differentiation and polarization markers, electronic microscopy, RT‐PCR, TEER measurement, ELISA test, Western Blot and phagocytic assay. The implantations of RPE‐scaffolds were carried out into the minipig eyes. Due to the xenogeneic transplantation of human cells into minipig eyes, tacrolimus immunosuppression therapy was used. At the 2‐week follow‐up from transplantation, non‐invasive OCT and fundus camera examinations of the implanted RPE‐scaffolds were performed. Consequently, euthanasia, histological and immunohistochemical investigations of the implanted retina were performed.Results: All generated RPE cells exhibit typical features of RPE cells. We demonstrated the reestablishment of the expression of full‐length MERTK protein as well as the reversion of lost phagocyte function of hiPSC‐RPE in vitro, which represents the first example of its kind in this field. Histological and immunohistochemical investigation of the RPE implants and adjacent retina showed structurally healthy RPE‐scaffold implant and neuroretinal cells above the implant.Conclusions: The generated hiPSC‐derived RPE in clinical grade conditions display the typical characteristics of mature RPE cells re‐establishing the function of phagocytosis in genetically corrected patient‐derived RPE cells in vitro. Functional in vivo studies in minipigs confirmed the RPE survival as well as survival of adjacent photoreceptors and thus can be employed as a potential treatment for patients.