Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 Plasmids

Gabriele Thumann; Nina Harmening; Cécile Prat-Souteyrand; Corinne Marie; Marie Pastor; Attila Sebe; Csaba Miskey; Laurence D Hurst; Sabine Diarra; Martina Kropp; Peter Walter; Daniel Scherman; Zoltán Ivics; Zsuzsanna Izsvák; Sandra Johnen

doi:10.1016/j.omtn.2017.02.002

Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 Plasmids

Mol Ther Nucleic Acids. 2017 Mar 17:6:302-314. doi: 10.1016/j.omtn.2017.02.002. Epub 2017 Feb 10.

Authors

Affiliations

¹ Department of Ophthalmology, University Hospitals of Geneva, 1205 Geneva, Switzerland; Laboratory of Ophthalmology, University of Geneva, 1205 Geneva, Switzerland. Electronic address: gabriele.thumann@hcuge.ch.
² Laboratory of Ophthalmology, University of Geneva, 1205 Geneva, Switzerland.
³ CNRS, Unité de Technologies Chimiques et Biologiques pour la Santé UMR 8258, 75006 Paris, France; Université Paris Descartes, Sorbonne-Paris-Cité, UTCBS, 75006 Paris, France; INSERM, UTCBS U 1022, 75006 Paris, France; Chimie ParisTech, PSL Research University, UTCBS, 75005 Paris, France.
⁴ Division of Medical Biotechnology, Paul-Ehrlich-Institute, 63225 Langen, Germany.
⁵ Department of Biology and Biochemistry, University of Bath, BA2 7AY Bath, UK.
⁶ Department of Ophthalmology, University Hospital RWTH Aachen, 52074 Aachen, Germany.
⁷ Max Delbrück Center for Molecular Medicine in the Helmholtz Association, 13092 Berlin, Germany.

Abstract

Neovascular age-related macular degeneration (nvAMD) is characterized by choroidal blood vessels growing into the subretinal space, leading to retinal pigment epithelial (RPE) cell degeneration and vision loss. Vessel growth results from an imbalance of pro-angiogenic (e.g., vascular endothelial growth factor [VEGF]) and anti-angiogenic factors (e.g., pigment epithelium-derived factor [PEDF]). Current treatment using intravitreal injections of anti-VEGF antibodies improves vision in about 30% of patients but may be accompanied by side effects and non-compliance. To avoid the difficulties posed by frequent intravitreal injections, we have proposed the transplantation of pigment epithelial cells modified to overexpress human PEDF. Stable transgene integration and expression is ensured by the hyperactive Sleeping Beauty transposon system delivered by pFAR4 miniplasmids, which have a backbone free of antibiotic resistance markers. We demonstrated efficient expression of the PEDF gene and an optimized PEDF cDNA sequence in as few as 5 × 10³ primary cells. At 3 weeks post-transfection, PEDF secretion was significantly elevated and long-term follow-up indicated a more stable secretion by cells transfected with the optimized PEDF transgene. Analysis of transgene insertion sites in human RPE cells showed an almost random genomic distribution. The results represent an important contribution toward a clinical trial aiming at a non-viral gene therapy of nvAMD.

Keywords: neovascular age-related macular degeneration; non-viral ex vivo gene therapy; pFAR4 miniplasmids; pigment epithelium-derived factor; primary pigment epithelial cells; sleeping beauty transposon system.

Abstract

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