Therapeutic gene delivery into retinal cells by MEA-based electroporation

RWTH Publications (RWTH Aachen)

Abstract

Retinopathia pigmentosa (RP) is a group of hereditary retinal dystrophies that lead to blindness. Retinal remodeling that occurs during RP reduces the efficiency of retinal ganglion cell (RGC) stimulation. Currently, gene therapy using viral vectors is considered a promising approach for the treatment of genetic eye diseases. Despite its success, this technique also has disadvantages. In the present study, the non-viral Sleeping Beauty (SB) transposon system was used, which ensures stable integration of transgenes into the genome of the target cells. Since Müller glial cells are an appropriate and accessible target within the RP retina, the genetic modifiability of the glioblastoma cell line A 172 and the retinal progenitor cell line R28 were analyzed by electroporation. Electroporation of the cells was performed in suspension or in the adherent state. For this purpose, either an electrode was placed above the cells or the cells were cultivated on custom-made microelectrode arrays (MEAs), which are considered promising candidates for localized low voltage electroporation. In addition to the genes coding for the yellow fluorescent protein (YFP, Venus) and green fluorescent protein (GFP), the genes coding for the protective pigment epithelium-derived factor (PEDF) and brain-derived neurotrophic factor (BDNF) were used for transfection. The highest transfection efficiency for A 172 cells in suspension was 37.2 ± 22.5% and for R28 cells 16.1 ± 8.50%. Transfection with PEDF and BDNF yielded secretion rates of 1,090 ± 1,640 pg PEDF/h/104 cells and 71.0 ± 65.0 pg BDNF/h/104 cells, respectively. For the electroporation of adherent A 172 cells, a maximum transfection efficiency of 2.75 ± 0.87% was achieved; transfections with PEDF resulted in secretion rates of 68.9 ± 32.5 pg/h/104 cells. Unlike R28 cells, the MEA-based transfection of A 172 cells was not successful. The transfection of primary Müller cells in suspension, isolated from four week old C57BL/6J mice, provided first positive results. The results of this work show that transfection of glial-like cells on a MEA is possible, but far less efficient compared to the transfection of cells in suspension. The transfection of primary glial cells is also feasible. These results form an important basis for the development of retinal implants with integrated MEAs that enable both the stimulation of RGCs and the transfection of Müller cells.

Authors 1

1. Saishreyas Iyer corresponding Aachen

   RWTH Aachen University

   Affiliation as printed

   RWTH Aachen