Efficient choroidal neovascularization reduction by Sleeping Beauty Transposon-mediated PEDF gene delivery in rat iris and retinal pigment epithelial cells by using a clinical electroporator

Abstract

Purpose : We aimed to test the efficacy of a clinical electroporator device (Igea, Italy) to transfect rat primary cells to release pigment epithelium derived factor (PEDF) by the non-viral Sleeping Beauty (SB100X) transposon system and miniplasmids free of antibiotic resistance markers (pFAR4). Moreover, we assessed in vivo the efficiency of injecting electroporated rat iris (IPE) and retinal pigment epithelial (RPE) primary cells in a laser-induced choroidal neovascularization (CNV) rat model

Methods : Electroporation parameters were optimized for RPE and IPE cells. The pFAR4-ITRs CAGGS Venus and pFAR4-CMV SB100X SV40 plasmids and were analyzed at day 3, 6, 10 and 14. Brown Norway rats were lasered and after 48 h, 10,000 rat IPE or RPE primary cells co-transfected with GMP-grade pFAR4-ITRs CMV PEDF BGH and pFAR4-CMV SB100X SV40 plasmids were subretinally injected. At day 5 the human PEDF expression was confirmed (PCR) and VEGF and PEDF protein expression (Western blot, n=3). Fluorescein angiography (FA) was performed at day 7 and animals were subjected to endothelial (lectin) staining to measure CNV areas in flatmounts, and apoptosis (caspase-3), microglia (Iba1) and macrophage (CD68) markers (immunofluorescence confocal microscopy, n=3) to quantify the signal using Fiji software. ANOVA and post hoc or Kruskal Wallis and U Mann-Whitney were applied (SPSS v.20)

Results : Both IPE and RPE cells showed successful transfection efficacy, being IPE more efficient. CNV area was reduced in the rats injected with RPE (p<0.05) and IPE (p<0.05) vs. saline. FA leakage did not show statistical difference in any of the groups injected vs. saline. IPE and RPE injected groups showed human PEDF gene. The antiangiogenic status (PEDF/VEGF) was similar in both IPE and RPE groups. Iba1 was reduced in CNV areas of IPE (p<0.01) and RPE (p<0.01) groups and CD68 staining was decreased in the RPE group (p<0.05). Caspase-3 decreased in RPE (p<0.01) and IPE (p<0.01) groups

Conclusions : Combining the SB100X transposon and the pFAR4 to release PEDF using an electroporator for clinical practice was effective reducing CNV and beneficial decreasing microglia activation and macrophages and increasing cell survival in the CNV area. This cell and gene PEDF-release therapeutic strategy could be considered for retinal diseases involving angiogenesis

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.