Cell-Based Gene Therapy Using the Sleeping Beauty Transposon System Reduces Neovascularisation in Rats

Background: Age-related macular degeneration (AMD) is a progressive retinal disorder, characterised by imbalanced pro- and anti-angiogenic signals, leading vision loss. The aim of this study was to evaluate the effect of ex vivo cell-based gene therapy on primary cells with stable expression of human pigment epithelium derived factor (PEDF) release using the non-viral Sleeping Beauty (SB100X) transposon system delivered by miniplasmids free of antibiotic resistance markers (pFAR4). Methods: Rat retinal pigment epithelial cells (RPEs) and iris pigment epithelial cells (IPEs) were co-transfected with pFAR4-ITRs CMV PEDF BGH and pFAR4-CMV-SB100X-SV40 plasmids. Laser-induced choroidal neovascularization (CNV) was performed in rats and transfected primary cells (tRPE and tIPE) were injected into the subretinal space. The leakage and CNV areas, vascular endothelial growth factor (VEGF), PEDF protein expression, metalloproteinase 2 and 9 (MMP-2/9) and microglial/macrophage markers were measured and evaluated. Findings: The animals injected with tRPE/IPE cells significantly showed a reduction in the leakage area at 14 days while at 7 days only tIPE cells showed a significant reduction. The CNV area at 7 days was reduced with tRPE/IPE cells. There was a significant increase in PEDF and PEDF/VEGF ratio with tRPE cells along with a reduction in the MMP-2 activity. Activated microglial fluorescence intensity increased in all groups analyzed. Interpretation: Our data demonstrate that a single administration of SB100X-engineered RPE/IPE cells release enough hPEDF to favour an anti-angiogenic environment that ultimately reduces CNV suggest that ex vivo non-viral gene therapy could be an effective and safe therapeutic option for angiogenic retinal diseases. Funding: This work was supported by the European Union’s Seventh Framework Programme for research, technological development and demonstration, grant agreement no. 305134 and Fundacion Jesus Gangoiti Barrera. LGG received a predoctoral grant from the Asociacion de Amigos de la Universidad de Navarra. Declaration of Interest: A. Garcia-Layana is consultant in Thea, Allergan, Bayer, Novartis and Roche Laboratories. Z. Izsvak and Z. Ivics are co-inventors on several patents on Sleeping Beauty transposon technology. Ethical Approval: The study was performed according to the Association for Research in Vision and Ophthalmology (ARVO) Resolution on the Use of Animals in Ophthalmic and Vision Research and approved by the Ethics Committee for Animal Research of the University of Navarra (protocol approval number 023-13).