Molecular and Functional Characterization of BDNF-Overexpressing Human Retinal Pigment Epithelial Cells Established by Sleeping Beauty Transposon-Mediated Gene Transfer

Abstract

More and more patients suffer from multifactorial neurodegenerative diseases, such as age-related macular degeneration (AMD). However, their pathological mechanisms are still poorly understood, which complicates the development of effective therapies. To improve treatment of multifactorial diseases, cell-based gene therapy can be used to increase the expression of therapeutic factors. To date, there is no approved therapy for dry AMD, including late-stage geographic atrophy. We present a treatment option for dry AMD that transfers the brain-derived neurotrophic factor (BDNF) gene into retinal pigment epithelial (RPE) cells by electroporation using the plasmid-based Sleeping Beauty (SB) transposon system. ARPE-19 cells and primary human RPE cells were co-transfected with two plasmids encoding the SB100X transposase and the transposon carrying a BDNF transcription cassette. We demonstrated efficient expression and secretion of BDNF in both RPE cell types, which were further increased in ARPE-19 cell cultures exposed to hydrogen peroxide. BDNF-transfected cells exhibited lower apoptosis rates and stimulated neurite outgrowth in human SH-SY5Y cells. This study is an important step in the development of a cell-based BDNF gene therapy that could be applied as an advanced therapy medicinal product to treat dry AMD or other degenerative retinal diseases.

A flexible electrode Array for genetic transfection of different layers of the retina by electroporation

“…To overcome the cargo limitation of AAV or other similar viral vectors, many efforts have been paid to test the applicability of non-viral retinal gene delivery, including ion electroosmosis, 11 particle bombardment, 12 electroporation (EP), 13 magnetic transfection, 14 photoporation 15 and acoustic perforation. 16 In all non-viral delivery methods, EP has emerged as one of the most likely alternatives to AAV [17][18][19][20][21] because of its high transfection efficiency 22,23 and nontoxicity. 24 Electroporation, in which high-intensity electric fields are applied to destabilize cell membranes to facilitate transmembrane particle transport, 25 has been demonstrated to be capable of delivering DNA plasmids into the retina.…”

“…To overcome the cargo limitation of AAV or other similar viral vectors, many efforts have been paid to test the applicability of non-viral retinal gene delivery, including ion electroosmosis, 11 particle bombardment, 12 electroporation (EP), 13 magnetic transfection, 14 photoporation 15 and acoustic perforation. 16 In all non-viral delivery methods, EP has emerged as one of the most likely alternatives to AAV 17–21 because of its high transfection efficiency 22,23 and nontoxicity. 24…”