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Enhanced Biosafety of the Sleeping Beauty Transposon System by Using mRNA as Source of Transposase to Efficiently and Stably Transfect Retinal Pigment Epithelial Cells

Neovascular age-related macular degeneration (nvAMD) is characterized by choroidal neovascularization (CNV), which leads to retinal pigment epithelial (RPE) cell and photoreceptor degeneration and blindness if untreated. Since blood vessel growth is mediated by endothelial cell growth factors, inclu...

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Autores principales: Harmening, Nina, Johnen, Sandra, Izsvák, Zsuzsanna, Ivics, Zoltan, Kropp, Martina, Bascuas, Thais, Walter, Peter, Kreis, Andreas, Pajic, Bojan, Thumann, Gabriele
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10135490/
https://www.ncbi.nlm.nih.gov/pubmed/37189405
http://dx.doi.org/10.3390/biom13040658
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author Harmening, Nina
Johnen, Sandra
Izsvák, Zsuzsanna
Ivics, Zoltan
Kropp, Martina
Bascuas, Thais
Walter, Peter
Kreis, Andreas
Pajic, Bojan
Thumann, Gabriele
author_facet Harmening, Nina
Johnen, Sandra
Izsvák, Zsuzsanna
Ivics, Zoltan
Kropp, Martina
Bascuas, Thais
Walter, Peter
Kreis, Andreas
Pajic, Bojan
Thumann, Gabriele
author_sort Harmening, Nina
collection PubMed
description Neovascular age-related macular degeneration (nvAMD) is characterized by choroidal neovascularization (CNV), which leads to retinal pigment epithelial (RPE) cell and photoreceptor degeneration and blindness if untreated. Since blood vessel growth is mediated by endothelial cell growth factors, including vascular endothelial growth factor (VEGF), treatment consists of repeated, often monthly, intravitreal injections of anti-angiogenic biopharmaceuticals. Frequent injections are costly and present logistic difficulties; therefore, our laboratories are developing a cell-based gene therapy based on autologous RPE cells transfected ex vivo with the pigment epithelium derived factor (PEDF), which is the most potent natural antagonist of VEGF. Gene delivery and long-term expression of the transgene are enabled by the use of the non-viral Sleeping Beauty (SB100X) transposon system that is introduced into the cells by electroporation. The transposase may have a cytotoxic effect and a low risk of remobilization of the transposon if supplied in the form of DNA. Here, we investigated the use of the SB100X transposase delivered as mRNA and showed that ARPE-19 cells as well as primary human RPE cells were successfully transfected with the Venus or the PEDF gene, followed by stable transgene expression. In human RPE cells, secretion of recombinant PEDF could be detected in cell culture up to one year. Non-viral ex vivo transfection using SB100X-mRNA in combination with electroporation increases the biosafety of our gene therapeutic approach to treat nvAMD while ensuring high transfection efficiency and long-term transgene expression in RPE cells.
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spelling pubmed-101354902023-04-28 Enhanced Biosafety of the Sleeping Beauty Transposon System by Using mRNA as Source of Transposase to Efficiently and Stably Transfect Retinal Pigment Epithelial Cells Harmening, Nina Johnen, Sandra Izsvák, Zsuzsanna Ivics, Zoltan Kropp, Martina Bascuas, Thais Walter, Peter Kreis, Andreas Pajic, Bojan Thumann, Gabriele Biomolecules Article Neovascular age-related macular degeneration (nvAMD) is characterized by choroidal neovascularization (CNV), which leads to retinal pigment epithelial (RPE) cell and photoreceptor degeneration and blindness if untreated. Since blood vessel growth is mediated by endothelial cell growth factors, including vascular endothelial growth factor (VEGF), treatment consists of repeated, often monthly, intravitreal injections of anti-angiogenic biopharmaceuticals. Frequent injections are costly and present logistic difficulties; therefore, our laboratories are developing a cell-based gene therapy based on autologous RPE cells transfected ex vivo with the pigment epithelium derived factor (PEDF), which is the most potent natural antagonist of VEGF. Gene delivery and long-term expression of the transgene are enabled by the use of the non-viral Sleeping Beauty (SB100X) transposon system that is introduced into the cells by electroporation. The transposase may have a cytotoxic effect and a low risk of remobilization of the transposon if supplied in the form of DNA. Here, we investigated the use of the SB100X transposase delivered as mRNA and showed that ARPE-19 cells as well as primary human RPE cells were successfully transfected with the Venus or the PEDF gene, followed by stable transgene expression. In human RPE cells, secretion of recombinant PEDF could be detected in cell culture up to one year. Non-viral ex vivo transfection using SB100X-mRNA in combination with electroporation increases the biosafety of our gene therapeutic approach to treat nvAMD while ensuring high transfection efficiency and long-term transgene expression in RPE cells. MDPI 2023-04-07 /pmc/articles/PMC10135490/ /pubmed/37189405 http://dx.doi.org/10.3390/biom13040658 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Harmening, Nina
Johnen, Sandra
Izsvák, Zsuzsanna
Ivics, Zoltan
Kropp, Martina
Bascuas, Thais
Walter, Peter
Kreis, Andreas
Pajic, Bojan
Thumann, Gabriele
Enhanced Biosafety of the Sleeping Beauty Transposon System by Using mRNA as Source of Transposase to Efficiently and Stably Transfect Retinal Pigment Epithelial Cells
title Enhanced Biosafety of the Sleeping Beauty Transposon System by Using mRNA as Source of Transposase to Efficiently and Stably Transfect Retinal Pigment Epithelial Cells
title_full Enhanced Biosafety of the Sleeping Beauty Transposon System by Using mRNA as Source of Transposase to Efficiently and Stably Transfect Retinal Pigment Epithelial Cells
title_fullStr Enhanced Biosafety of the Sleeping Beauty Transposon System by Using mRNA as Source of Transposase to Efficiently and Stably Transfect Retinal Pigment Epithelial Cells
title_full_unstemmed Enhanced Biosafety of the Sleeping Beauty Transposon System by Using mRNA as Source of Transposase to Efficiently and Stably Transfect Retinal Pigment Epithelial Cells
title_short Enhanced Biosafety of the Sleeping Beauty Transposon System by Using mRNA as Source of Transposase to Efficiently and Stably Transfect Retinal Pigment Epithelial Cells
title_sort enhanced biosafety of the sleeping beauty transposon system by using mrna as source of transposase to efficiently and stably transfect retinal pigment epithelial cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10135490/
https://www.ncbi.nlm.nih.gov/pubmed/37189405
http://dx.doi.org/10.3390/biom13040658
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