Auswirkungen der Elektroporation auf das Genexpressionsmuster boviner retinaler Pigmentepithelzellen zur gentherapeutischen Behandlung der altersbedingten Makuladegeneration

Age-related macular degeneration (AMD) is the third common cause of blindness worldwide. Blindness is caused in particular by wet AMD, characterized by the development of choroidal neovascularisations (CNVs). Beside the standard anti-VEGF treatment, alternative therapy models are established. One approach is the genetic modification of autologous iris pigment epithelial (IPE) cells and their transplantation into the subretinal space. The genetic modification consists of the overexpression of the pigment epithelium-derived factor (PEDF) gene. PEDF is known to possess anti-angiogenic, neuroprotective und anti-tumorigenic properties. This thesis examined the question, if the electroporation is a useful method to insert additional genetic information into target primary pigment epithelial cells. Therefore, the impact of the electroporation on the gene expression pattern of bovine retinal pigment epithelial (RPE) cells was examined. Furthermore, the differences in gene expression of retinal and iris pigment epithelial cells as well as the impact of cultivation of these cells on the expression of selected genes was analysed. The comparison of different genes in RPE und IPE in vivo and in vitro showed that freshly isolated primary IPE cells are the most favourable cells for subretinal transplantation. Cultivation has a clear impact on the gene expression pattern of both cell types. The analysis of cultivated RPE cells, individually treated with the single steps of the transfection process, showed that the incubation with the electroporation buffer as well as the exposure to the chosen electric field parameters did not lead to changes in gene expression. Contrary to the expectations and the previous results of the research group, the cell cultures transfected with the PEDF encoding plasmid tended to showed a more pro-angiogenic gene expression pattern than non-transfected control cells. The reduced expression of the immunesuppressive-acting CD86 gene in cells transfected with linearized pMS-N-PEDF-H-IV plasmid could be disadvantageous. The analysis of the other examined genes showed no expression changes. The successful transfection was confirmed by detection of the expression of the recombinant PEDF gene in transfected cell cultures. The secretion of the recombinant PEDF protein was proven by Western blot analysis. PEDF-transfected cells are able to provide anti-angiogenic properties, which was demonstrated by the research group of Professor Thumann in various in vitro and in vivo experiments. The experiments of this thesis show that primary pigment epithelial cells can be efficiently transfected using electroporation and provide another reference that PEDF-transfected RPE and IPE cells are suitable for subretinal transplantation to treat wet AMD