The Antibiotic-free pFAR4 Vector Paired with the Sleeping Beauty Transposon System Mediates Efficient Transgene Delivery in Human Cells

Molecular Therapy: Nucleic Acids

Marie Pastor¹  Mickäel Quiviger¹  Daniel Scherman¹  Corinne Marie¹  Julie Pailloux¹  Peter Walter²  Sandra Johnen²  Zoltán Ivics³  Nina Harmening⁴  Gabriele Thumann⁴  Martina Kropp⁴  Zsuzsanna Izsvák⁵ 

[1] CNRS, Unité de Technologies Chimiques et Biologiques pour la Santé (UTCBS), UMR 8258, 75006 Paris, France;Department of Ophthalmology, University Hospital RWTH Aachen, 52074 Aachen, Germany;Division of Medical Biotechnology, Paul Ehrlich Institute, 63225 Langen, Germany;Experimental Ophthalmology, University of Geneva, 1205 Geneva, Switzerland;Max Delbrück Center for Molecular Medicine in the Helmholtz Association, 13092 Berlin, Germany;

关键词: age-related macular degeneration;    electroporation;    RPE cells;    antibiotic-free pFAR4 vector;    Sleeping Beauty transposon;    ocular gene therapy;    transfection;    PEDF;    VEGF;   

DOI  :  10.1016/j.omtn.2017.12.017

来源: DOAJ

【 摘 要 】

The anti-angiogenic and neurogenic pigment epithelium-derived factor (PEDF) demonstrated a potency to control choroidal neovascularization in age-related macular degeneration (AMD) patients. The goal of the present study was the development of an efficient and safe technique to integrate, ex vivo, the PEDF gene into retinal pigment epithelial (RPE) cells for later transplantation to the subretinal space of AMD patients to allow continuous PEDF secretion in the vicinity of the affected macula. Because successful gene therapy approaches require efficient gene delivery and stable gene expression, we used the antibiotic-free pFAR4 mini-plasmid vector to deliver the hyperactive Sleeping Beauty transposon system, which mediates transgene integration into the genome of host cells. In an initial study, lipofection-mediated co-transfection of HeLa cells with the SB100X transposase gene and a reporter marker delivered by pFAR4 showed a 2-fold higher level of genetically modified cells than when using the pT2 vectors. Similarly, with the pFAR4 constructs, electroporation-mediated transfection of primary human RPE cells led to 2.4-fold higher secretion of recombinant PEDF protein, which was still maintained 8 months after transfection. Thus, our results show that the pFAR4 plasmid is a superior vector for the delivery and integration of transgenes into eukaryotic cells.

【 授权许可】

Unknown