Abstract
Purpose :
Retinopathia pimentosa is a group of hereditary progressive diseases leading to blindness, starting with peripheral vision loss due to the death of rod photoreceptor cells. The efficacy of existing retinal prostheses is limited because of the remodeling processes that occur in the remaining retinal layers during disease progression, which interfere with electrical stimulation of the retinal ganglion cells. Gene therapy has emerged as promising treatment for several genetic diseases. The eye is an excellent target for gene therapy due to its enclosed structure. Currently, viral vectors represent the main gene transfer method, but among many advantages, they also carry disadvantages. Here, the non-viral Sleeping Beauty (SB100X) transposon system was used to introduce transgenes into cells by electroporation, resulting in a stable integration into the cells’ genome. Transfection was performed using the genes encoding yellow-fluorescent protein (YFP), pigment epithelium-derived factor, and brain-derived neurotrophic factor.
Methods :
The SB100X transposase plasmid and the corresponding transposon plasmid (1:16 ratio, 420 ng/µl) were introduced into the glioblastoma cell line A-172. Three different electroporation devices were used: a capillary-based system used for cells in suspension, a system for adherent cells, and a system using a custom-made multielectrode array (MEA). Transfection efficiency and cell viability were assessed by flow cytometry, ELISA, and a luciferase assay.
Results :
The best setting for A-172 cells in suspension was 1100 V, 30 ms, 2 pulses leading to a maximum efficiency of 62.4% YFP-positive cells. Increasing voltages resulted in an increased transfection efficiency but also in a decreased cell viability. For transfection of adherent cells, voltages ranged from 125 to 275 V. Two different pulses, a poring pulse and a transfer pulse with + polarity or +/- polarity were applied. A maximum transfection efficiency of 3.6% was observed when the poring pulse with + polarity and the transfer pulse with +/- polarity were used at 200 V for 5 ms. Electroporation using the custom-made MEA also showed transfected cells.
Conclusions :
Transfection of cells in suspension resulted in higher efficiencies than adherent cells. Transfection of adherent cells is of importance to further develop MEA-based intraocular chips with a dual function of electroporation and stimulation.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.