The Antibiotic-free pFAR4 Vector Paired with the Sleeping Beauty Transposon System Mediates Efficient Transgene Delivery in Human Cells
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, t...
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doaj-c0102cc49ae944f381497e5360d1481b2020-11-25T02:29:36ZengElsevierMolecular Therapy: Nucleic Acids2162-25312018-06-0111C576710.1016/j.omtn.2017.12.017The Antibiotic-free pFAR4 Vector Paired with the Sleeping Beauty Transposon System Mediates Efficient Transgene Delivery in Human CellsMarie Pastor0Sandra Johnen1Nina Harmening2Mickäel Quiviger3Julie Pailloux4Martina Kropp5Peter Walter6Zoltán Ivics7Zsuzsanna Izsvák8Gabriele Thumann9Daniel Scherman10Corinne Marie11CNRS, Unité de Technologies Chimiques et Biologiques pour la Santé (UTCBS), UMR 8258, 75006 Paris, FranceDepartment of Ophthalmology, University Hospital RWTH Aachen, 52074 Aachen, GermanyExperimental Ophthalmology, University of Geneva, 1205 Geneva, SwitzerlandCNRS, Unité de Technologies Chimiques et Biologiques pour la Santé (UTCBS), UMR 8258, 75006 Paris, FranceCNRS, Unité de Technologies Chimiques et Biologiques pour la Santé (UTCBS), UMR 8258, 75006 Paris, FranceExperimental Ophthalmology, University of Geneva, 1205 Geneva, SwitzerlandDepartment of Ophthalmology, University Hospital RWTH Aachen, 52074 Aachen, GermanyDivision of Medical Biotechnology, Paul Ehrlich Institute, 63225 Langen, GermanyMax Delbrück Center for Molecular Medicine in the Helmholtz Association, 13092 Berlin, GermanyExperimental Ophthalmology, University of Geneva, 1205 Geneva, SwitzerlandCNRS, Unité de Technologies Chimiques et Biologiques pour la Santé (UTCBS), UMR 8258, 75006 Paris, FranceCNRS, Unité de Technologies Chimiques et Biologiques pour la Santé (UTCBS), UMR 8258, 75006 Paris, FranceThe 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.http://www.sciencedirect.com/science/article/pii/S2162253117303189age-related macular degenerationelectroporationRPE cellsantibiotic-free pFAR4 vectorSleeping Beauty transposonocular gene therapytransfectionPEDFVEGF |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Marie Pastor Sandra Johnen Nina Harmening Mickäel Quiviger Julie Pailloux Martina Kropp Peter Walter Zoltán Ivics Zsuzsanna Izsvák Gabriele Thumann Daniel Scherman Corinne Marie |
spellingShingle |
Marie Pastor Sandra Johnen Nina Harmening Mickäel Quiviger Julie Pailloux Martina Kropp Peter Walter Zoltán Ivics Zsuzsanna Izsvák Gabriele Thumann Daniel Scherman Corinne Marie The Antibiotic-free pFAR4 Vector Paired with the Sleeping Beauty Transposon System Mediates Efficient Transgene Delivery in Human Cells Molecular Therapy: Nucleic Acids age-related macular degeneration electroporation RPE cells antibiotic-free pFAR4 vector Sleeping Beauty transposon ocular gene therapy transfection PEDF VEGF |
author_facet |
Marie Pastor Sandra Johnen Nina Harmening Mickäel Quiviger Julie Pailloux Martina Kropp Peter Walter Zoltán Ivics Zsuzsanna Izsvák Gabriele Thumann Daniel Scherman Corinne Marie |
author_sort |
Marie Pastor |
title |
The Antibiotic-free pFAR4 Vector Paired with the Sleeping Beauty Transposon System Mediates Efficient Transgene Delivery in Human Cells |
title_short |
The Antibiotic-free pFAR4 Vector Paired with the Sleeping Beauty Transposon System Mediates Efficient Transgene Delivery in Human Cells |
title_full |
The Antibiotic-free pFAR4 Vector Paired with the Sleeping Beauty Transposon System Mediates Efficient Transgene Delivery in Human Cells |
title_fullStr |
The Antibiotic-free pFAR4 Vector Paired with the Sleeping Beauty Transposon System Mediates Efficient Transgene Delivery in Human Cells |
title_full_unstemmed |
The Antibiotic-free pFAR4 Vector Paired with the Sleeping Beauty Transposon System Mediates Efficient Transgene Delivery in Human Cells |
title_sort |
antibiotic-free pfar4 vector paired with the sleeping beauty transposon system mediates efficient transgene delivery in human cells |
publisher |
Elsevier |
series |
Molecular Therapy: Nucleic Acids |
issn |
2162-2531 |
publishDate |
2018-06-01 |
description |
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. |
topic |
age-related macular degeneration electroporation RPE cells antibiotic-free pFAR4 vector Sleeping Beauty transposon ocular gene therapy transfection PEDF VEGF |
url |
http://www.sciencedirect.com/science/article/pii/S2162253117303189 |
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