July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Efficient choroidal neovascularization reduction by Sleeping Beauty Transposon-mediated PEDF gene delivery in rat iris and retinal pigment epithelial cells by using a clinical electroporator
Author Affiliations & Notes
  • Patricia Fernandez
    Experimental Ophthalmology Laboratory, Clinica Universidad de Navarra, Pamplona, Spain
    IdiSNA, Pamplona, Spain
  • Sergio Recalde
    Experimental Ophthalmology Laboratory, Clinica Universidad de Navarra, Pamplona, Spain
    IdiSNA, Pamplona, Spain
  • Maria Hernandez
    Experimental Ophthalmology Laboratory, Clinica Universidad de Navarra, Pamplona, Spain
    IdiSNA, Pamplona, Spain
  • Laura Garcia-Garcia
    Experimental Ophthalmology Laboratory, Clinica Universidad de Navarra, Pamplona, Spain
  • Jaione Bezunartea
    Experimental Ophthalmology Laboratory, Clinica Universidad de Navarra, Pamplona, Spain
  • Juan Roberto Rodriguez
    Cell Therapy Area. Division of Cancer., Center for Applied Medical Research (CIMA), Pamplona, Spain
  • Corinne Marie
    Unité de Technologies Chimiques et Biologiques pour la Santé, INSERM U1022 – CNRS UMR8258, Paris, France
  • Daniel Scherman
    Unité de Technologies Chimiques et Biologiques pour la Santé, INSERM U1022 – CNRS UMR8258, Paris, France
  • Zsuzsanna Izsvak
    Max Delbrück Center for Molecular Medicine in the Helmholtz Society, Berlin, Germany
  • Martina Kropp
    Experimental Ophthalmology, University of Geneva, Geneva, Switzerland
    Department of Ophthalmology, University Hospitals of Geneva, Geneva, Switzerland
  • Sandra Johnen
    RWTH Aachen University, Aachen, Germany
  • Gabriele Thumann
    Experimental Ophthalmology, University of Geneva, Geneva, Switzerland
    Department of Ophthalmology, University Hospitals of Geneva, Geneva, Switzerland
  • Alfredo García-Layana
    Experimental Ophthalmology Laboratory, Clinica Universidad de Navarra, Pamplona, Spain
    IdiSNA, Pamplona, Spain
  • Footnotes
    Commercial Relationships   Patricia Fernandez, None; Sergio Recalde, None; Maria Hernandez, None; Laura Garcia-Garcia, None; Jaione Bezunartea, None; Juan Rodriguez, None; Corinne Marie, None; Daniel Scherman, None; Zsuzsanna Izsvak, None; Martina Kropp, None; Sandra Johnen, None; Gabriele Thumann, None; Alfredo García-Layana, None
  • Footnotes
    Support  FP7 HEALTH 2012-305134. LGG received ADA predoctoral grant from University of Navarra
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 223. doi:
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      Patricia Fernandez, Sergio Recalde, Maria Hernandez, Laura Garcia-Garcia, Jaione Bezunartea, Juan Roberto Rodriguez, Corinne Marie, Daniel Scherman, Zsuzsanna Izsvak, Martina Kropp, Sandra Johnen, Gabriele Thumann, Alfredo García-Layana; Efficient choroidal neovascularization reduction by Sleeping Beauty Transposon-mediated PEDF gene delivery in rat iris and retinal pigment epithelial cells by using a clinical electroporator. Invest. Ophthalmol. Vis. Sci. 2018;59(9):223.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : We aimed to test the efficacy of a clinical electroporator device (Igea, Italy) to transfect rat primary cells to release pigment epithelium derived factor (PEDF) by the non-viral Sleeping Beauty (SB100X) transposon system and miniplasmids free of antibiotic resistance markers (pFAR4). Moreover, we assessed in vivo the efficiency of injecting electroporated rat iris (IPE) and retinal pigment epithelial (RPE) primary cells in a laser-induced choroidal neovascularization (CNV) rat model

Methods : Electroporation parameters were optimized for RPE and IPE cells. The pFAR4-ITRs CAGGS Venus and pFAR4-CMV SB100X SV40 plasmids and were analyzed at day 3, 6, 10 and 14. Brown Norway rats were lasered and after 48 h, 10,000 rat IPE or RPE primary cells co-transfected with GMP-grade pFAR4-ITRs CMV PEDF BGH and pFAR4-CMV SB100X SV40 plasmids were subretinally injected. At day 5 the human PEDF expression was confirmed (PCR) and VEGF and PEDF protein expression (Western blot, n=3). Fluorescein angiography (FA) was performed at day 7 and animals were subjected to endothelial (lectin) staining to measure CNV areas in flatmounts, and apoptosis (caspase-3), microglia (Iba1) and macrophage (CD68) markers (immunofluorescence confocal microscopy, n=3) to quantify the signal using Fiji software. ANOVA and post hoc or Kruskal Wallis and U Mann-Whitney were applied (SPSS v.20)

Results : Both IPE and RPE cells showed successful transfection efficacy, being IPE more efficient. CNV area was reduced in the rats injected with RPE (p<0.05) and IPE (p<0.05) vs. saline. FA leakage did not show statistical difference in any of the groups injected vs. saline. IPE and RPE injected groups showed human PEDF gene. The antiangiogenic status (PEDF/VEGF) was similar in both IPE and RPE groups. Iba1 was reduced in CNV areas of IPE (p<0.01) and RPE (p<0.01) groups and CD68 staining was decreased in the RPE group (p<0.05). Caspase-3 decreased in RPE (p<0.01) and IPE (p<0.01) groups

Conclusions : Combining the SB100X transposon and the pFAR4 to release PEDF using an electroporator for clinical practice was effective reducing CNV and beneficial decreasing microglia activation and macrophages and increasing cell survival in the CNV area. This cell and gene PEDF-release therapeutic strategy could be considered for retinal diseases involving angiogenesis

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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