Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 Plasmids
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 gro...
| Main Authors: | , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2017-03-01
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| Series: | Molecular Therapy: Nucleic Acids |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2162253117301282 |
| _version_ | 1818842899112001536 |
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| author | 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 |
| author_facet | 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 |
| author_sort | Gabriele Thumann |
| collection | DOAJ |
| description | 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, sleeping beauty transposon system, pFAR4 miniplasmids, primary pigment epithelial cells, pigment epithelium-derived factor |
| first_indexed | 2024-12-19T04:49:18Z |
| format | Article |
| id | doaj.art-3ef7533fe13543258264496282a1f989 |
| institution | Directory Open Access Journal |
| issn | 2162-2531 |
| language | English |
| last_indexed | 2024-12-19T04:49:18Z |
| publishDate | 2017-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj.art-3ef7533fe13543258264496282a1f9892022-12-21T20:35:24ZengElsevierMolecular Therapy: Nucleic Acids2162-25312017-03-016302314Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 PlasmidsGabriele Thumann0Nina Harmening1Cécile Prat-Souteyrand2Corinne Marie3Marie Pastor4Attila Sebe5Csaba Miskey6Laurence D. Hurst7Sabine Diarra8Martina Kropp9Peter Walter10Daniel Scherman11Zoltán Ivics12Zsuzsanna Izsvák13Sandra Johnen14Department of Ophthalmology, University Hospitals of Geneva, 1205 Geneva, Switzerland; Laboratory of Ophthalmology, University of Geneva, 1205 Geneva, Switzerland; Corresponding author: Gabriele Thumann, Department of Ophthalmology, University Hospitals of Geneva, 22 Rue Alcide-Jentzer, 1205 Geneva, Switzerland.Laboratory of Ophthalmology, University of Geneva, 1205 Geneva, SwitzerlandLaboratory of Ophthalmology, University of Geneva, 1205 Geneva, SwitzerlandCNRS, 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, FranceCNRS, 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, FranceDivision of Medical Biotechnology, Paul-Ehrlich-Institute, 63225 Langen, GermanyDivision of Medical Biotechnology, Paul-Ehrlich-Institute, 63225 Langen, GermanyDepartment of Biology and Biochemistry, University of Bath, BA2 7AY Bath, UKDepartment of Ophthalmology, University Hospital RWTH Aachen, 52074 Aachen, GermanyLaboratory of Ophthalmology, University of Geneva, 1205 Geneva, SwitzerlandDepartment of Ophthalmology, University Hospital RWTH Aachen, 52074 Aachen, GermanyCNRS, 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, FranceDivision of Medical Biotechnology, Paul-Ehrlich-Institute, 63225 Langen, GermanyMax Delbrück Center for Molecular Medicine in the Helmholtz Association, 13092 Berlin, GermanyDepartment of Ophthalmology, University Hospital RWTH Aachen, 52074 Aachen, GermanyNeovascular 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, sleeping beauty transposon system, pFAR4 miniplasmids, primary pigment epithelial cells, pigment epithelium-derived factorhttp://www.sciencedirect.com/science/article/pii/S2162253117301282 |
| spellingShingle | 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 Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 Plasmids Molecular Therapy: Nucleic Acids |
| title | Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 Plasmids |
| title_full | Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 Plasmids |
| title_fullStr | Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 Plasmids |
| title_full_unstemmed | Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 Plasmids |
| title_short | Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 Plasmids |
| title_sort | engineering of pedf expressing primary pigment epithelial cells by the sb transposon system delivered by pfar4 plasmids |
| url | http://www.sciencedirect.com/science/article/pii/S2162253117301282 |
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