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

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

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Title		Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 Plasmids
Authors		Thumann, Gabriele Harmening, Nina Prat-Souteyrand, Cécile Marie, Corinne Pastor, Marie Sebe, Attila Miskey, Csaba Hurst, Laurence D Diarra, Sabine Kropp, Martina Walter, Peter Scherman, Daniel Ivics, Zoltán Izsvák, Zsuzsanna Johnen, Sandra show all authors [1 - 15]
Published in		Molecular Therapy - Nucleic Acids. 2017, vol. 6, p. 302-314
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 × 10(3) 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.
Identifiers		DOI: 10.1016/j.omtn.2017.02.002 PMID: 28325297
Full text		Article (Published version) (1.3 MB) - Limited access to UNIGE
Structures		Faculté de médecine / Section de médecine clinique / Département des neurosciences cliniques
Research group		Ophtalmologie expérimentale (925)
Citation (ISO format)		THUMANN, Gabriele et al. Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 Plasmids. In: Molecular Therapy - Nucleic Acids, 2017, vol. 6, p. 302-314. https://archive-ouverte.unige.ch/unige:94436