Preclinical Evaluation of a Cell-Based Gene Therapy Using the Sleeping Beauty Transposon System in Choroidal Neovascularization

Maria Hernandez; Sergio Recalde; Laura Garcia-Garcia; Jaione Bezunartea; Csaba Miskey; Sandra Johnen; Sabine Diarra; Attila Sebe; Juan Roberto Rodriguez-Madoz; Severine Pouillot; Corinne Marie; Zsuzsanna Izsvák; Daniel Scherman; Martina Kropp; Felipe Prosper; Gabriele Thumann; Zoltán Ivics; Alfredo Garcia-Layana; Patricia Fernandez-Robredo

doi:10.1016/j.omtm.2019.10.013

Preclinical Evaluation of a Cell-Based Gene Therapy Using the Sleeping Beauty Transposon System in Choroidal Neovascularization

Mol Ther Methods Clin Dev. 2019 Nov 9:15:403-417. doi: 10.1016/j.omtm.2019.10.013. eCollection 2019 Dec 13.

Authors

Maria Hernandez^{1

2}, Sergio Recalde^{1

2}, Laura Garcia-Garcia^{1

2}, Jaione Bezunartea¹, Csaba Miskey³, Sandra Johnen⁴, Sabine Diarra⁴, Attila Sebe³, Juan Roberto Rodriguez-Madoz⁵, Severine Pouillot⁶, Corinne Marie^{7

8}, Zsuzsanna Izsvák^{9

10}, Daniel Scherman⁷, Martina Kropp^{11

12}, Felipe Prosper^{5

13}, Gabriele Thumann^{11

12}, Zoltán Ivics³, Alfredo Garcia-Layana^{1

2}, Patricia Fernandez-Robredo^{1

2}

Affiliations

¹ Experimental Ophthalmology Laboratory, Ophthalmology, Clínica Universidad de Navarra, IdiSNA, Pamplona, Spain.
² Red Temática de Investigación Cooperativa Sanitaria en Enfermedades Oculares, Oftared, ISCIII, Madrid, Spain.
³ Division of Medical Biotechnology, Paul Ehrlich-Institut, 63225 Langen, Germany.
⁴ Department of Ophthalmology, University Hospital RWTH Aachen, 52074 Aachen, Germany.
⁵ Regenerative Medicine Program, Center for Applied Medical Research (CIMA), University of Navarra, IdiSNA, Pamplona 31008, Spain.
⁶ GenoSafe, Evry 91000, France.
⁷ Université de Paris, UTCBS, CNRS, INSERM, F-75006 Paris, France.
⁸ Chimie ParisTech, PSL Research University, F-75005 Paris, France.
⁹ Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany.
¹⁰ Berlin Institute of Health, Berlin, Germany.
¹¹ Experimental Ophthalmology, University of Geneva, 1205 Geneva, Switzerland.
¹² Department of Ophthalmology, University Hospitals of Geneva, 1205 Geneva, Switzerland.
¹³ Area of Cell Therapy, Clínica Universidad de Navarra, University of Navarra, IdiSNA, Pamplona 31008, Spain.

Abstract

Age-related macular degeneration (AMD) is a progressive retinal disorder characterized by imbalanced pro- and antiangiogenic signals. The aim of this study was to evaluate the effect of ex vivo cell-based gene therapy 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). Retinal pigment epithelial (RPE) cells and iris pigment epithelial (IPE) cells were co-transfected with pFAR4-inverted terminal repeats (ITRs) CMV-PEDF-BGH and pFAR4-CMV-SB100X-SV40 plasmids. Laser-induced choroidal neovascularization (CNV) was performed in rats, and transfected primary cells (transfected RPE [tRPE] and transfected IPE [tIPE] cells) were injected into the subretinal space. The leakage and CNV areas, vascular endothelial growth factor (VEGF), PEDF protein expression, metalloproteinases 2 and 9 (MMP-2/9), and microglial/macrophage markers were measured. Injection with tRPE/IPE cells significantly reduced the leakage area at 7 and 14 days and the CNV area at 7 days. There was a significant increase in PEDF and the PEDF/VEGF ratio with tRPE cells and a reduction in the MMP-2 activity. Our data demonstrated that ex vivo non-viral gene therapy reduces CNV and could be an effective and safe therapeutic option for angiogenic retinal diseases.

Keywords: SB100X transposase; angiogenesis; iris pigment epithelial cells (IPE); pigment epithelium derived factor (PEDF); retinal pigment epithelium (RPE); vascular endothelial growth factor (VEGF).