Rat Primary Cells transfected with PEDF using the Sleeping Beauty Transposon system are effective in reducing the proangiogenic status in choroidal neovascularization

Abstract

Purpose : There is a need to explore alternative molecular targets as well as delivery systems for the development of new therapeutic strategies in wet AMD. Our approach comprises the effect of pigment epithelium derived factor (PEDF) release using the non-viral Sleeping Beauty (SB100X) transposon system delivered by miniplasmids free of antibiotic resistance markers (pFAR4) in rat primary cells subretinally injected in a model of laser-induced choroidal neovascularization (CNV).

Methods : Retinal pigment epithelial cells (RPEs) and iris pigment epithelial cells (IPEs) derived from Brown Norway rats were co-transfected with pT2-pFAR4-PEDF and pFAR4-SB100X plasmids. Laser-induced CNV was performed and 48 h later, the transfected cells were injected in the subretinal space of Brown Norway rats. The animals were sacrificed at day 5 for the evaluation of VEGF and PEDF protein expression by Western blot (n=3) and zymographies for MMP-2/9 activity were performed (n=3). At day 7 post-injection, animals were sacrificed to undergo immunofluorescence with microglial/macrophage markers (isolectin, Iba1, CD68) in the CNV area and visualized using a confocal microscope (n=3).

Results : While no differences in VEGF expression were observed, there was a significant increase (p<0.05) in PEDF secretion in the treated groups that resulted in an increased in PEDF/VEGF ratio. These data indicate that the treatment improved a favorable antiangiogenic environment, that was observed, especially the group of 10,000 RPE cells transfected with PEDF. MMP-2 activity was significantly reduced (p<0.05) in the groups that had the highest PEDF levels, while there were no statistical differences for MMP-9 levels. Microglial and macrophage cell activation was localized in the angiogenesis area after subretinal injection; no alterations were observed compared to the saline group

Conclusions : The combined use of the SB100X transposon system and the pFAR4 miniplasmid was effective in optimizing the anti-angiogenic status in the retina by increasing PEDF secretion and reducing metalloproteinase activity. Moreover, the transfected transplanted cells did not activate microglia and macrophages in the CNV surrounding area. This is an important step for the establishment of a safe, specific, and effective gene therapeutic treatment for angiogenic retinal diseases, such as wet AMD.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.