June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Rat Primary Cells transfected with PEDF using the Sleeping Beauty Transposon system are effective in reducing the proangiogenic status in choroidal neovascularization
Author Affiliations & Notes
  • Sergio Recalde
    Experimental Ophthalmology laboratory, Universidad de Navarra, Pamplona, Navarra, Spain
    IDISNA, Navarra Institute for Health Research , Pamplona, Navarra, Spain
  • Maria Hernandez
    Experimental Ophthalmology laboratory, Universidad de Navarra, Pamplona, Navarra, Spain
    IDISNA, Navarra Institute for Health Research , Pamplona, Navarra, Spain
  • Patricia Fernandez
    Experimental Ophthalmology laboratory, Universidad de Navarra, Pamplona, Navarra, Spain
    IDISNA, Navarra Institute for Health Research , Pamplona, Navarra, Spain
  • Laura García-Garcia
    Experimental Ophthalmology laboratory, Universidad de Navarra, Pamplona, Navarra, Spain
    IDISNA, Navarra Institute for Health Research , Pamplona, Navarra, Spain
  • Juan Roberto Rodriguez
    Cell Therapy Area. Division of cancer., Center of Applied Medical Research (CIMA), Pamplona, Navarra, Spain
    IDISNA, Navarra Institute for Health Research , Pamplona, Navarra, Spain
  • Jaione Bezunartea
    Experimental Ophthalmology laboratory, Universidad de Navarra, Pamplona, Navarra, Spain
    IDISNA, Navarra Institute for Health Research , Pamplona, Navarra, Spain
  • Maite Moreno
    Experimental Ophthalmology laboratory, Universidad de Navarra, Pamplona, Navarra, Spain
    IDISNA, Navarra Institute for Health Research , Pamplona, Navarra, Spain
  • Idoia Belza
    Experimental Ophthalmology laboratory, Universidad de Navarra, Pamplona, Navarra, 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 Izsvák
    Molecular Medicine in the Helmholtz Society, Max Delbrück Center, Berlin, Germany
  • Sandra Johnen
    Deparment of Ophthalmology, University Hospital RWTH Aachen, Aachen, Germany
  • Gabriele Thumann
    déparment des neurosciences clinique, service dOphtalmologie, Hôpitaux Universitaires de Genève, Genève, Switzerland
  • Alfredo Garcia-Layana
    Experimental Ophthalmology laboratory, Universidad de Navarra, Pamplona, Navarra, Spain
    IDISNA, Navarra Institute for Health Research , Pamplona, Navarra, Spain
  • Footnotes
    Commercial Relationships   Sergio Recalde, None; Maria Hernandez, None; Patricia Fernandez, None; Laura García-Garcia, None; Juan Rodriguez, None; Jaione Bezunartea, None; Maite Moreno, None; Idoia Belza, None; Corinne Marie, None; Daniel Scherman, None; Zsuzsanna Izsvák, None; Sandra Johnen, None; Gabriele Thumann, None; Alfredo Garcia-Layana, Alcon (C), Allergan (C), Bayer (C), Novartis (C), Thea (C)
  • Footnotes
    Support  FP7 HEALTH 2012-305134. LGG received ADA predoctoral grant from University of Navarra
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 1964. doi:
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      Sergio Recalde, Maria Hernandez, Patricia Fernandez, Laura García-Garcia, Juan Roberto Rodriguez, Jaione Bezunartea, Maite Moreno, Idoia Belza, Corinne Marie, Daniel Scherman, Zsuzsanna Izsvák, Sandra Johnen, Gabriele Thumann, Alfredo Garcia-Layana; Rat Primary Cells transfected with PEDF using the Sleeping Beauty Transposon system are effective in reducing the proangiogenic status in choroidal neovascularization. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1964.

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

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Abstract

Purpose : There is a need to explore alternative molecular targets as well as delivery systems for the development of new therapeutic strategies in wet AMD. Our approach comprises the effect of pigment epithelium derived factor (PEDF) release using the non-viral Sleeping Beauty (SB100X) transposon system delivered by miniplasmids free of antibiotic resistance markers (pFAR4) in rat primary cells subretinally injected in a model of laser-induced choroidal neovascularization (CNV).

Methods : Retinal pigment epithelial cells (RPEs) and iris pigment epithelial cells (IPEs) derived from Brown Norway rats were co-transfected with pT2-pFAR4-PEDF and pFAR4-SB100X plasmids. Laser-induced CNV was performed and 48 h later, the transfected cells were injected in the subretinal space of Brown Norway rats. The animals were sacrificed at day 5 for the evaluation of VEGF and PEDF protein expression by Western blot (n=3) and zymographies for MMP-2/9 activity were performed (n=3). At day 7 post-injection, animals were sacrificed to undergo immunofluorescence with microglial/macrophage markers (isolectin, Iba1, CD68) in the CNV area and visualized using a confocal microscope (n=3).

Results : While no differences in VEGF expression were observed, there was a significant increase (p<0.05) in PEDF secretion in the treated groups that resulted in an increased in PEDF/VEGF ratio. These data indicate that the treatment improved a favorable antiangiogenic environment, that was observed, especially the group of 10,000 RPE cells transfected with PEDF. MMP-2 activity was significantly reduced (p<0.05) in the groups that had the highest PEDF levels, while there were no statistical differences for MMP-9 levels. Microglial and macrophage cell activation was localized in the angiogenesis area after subretinal injection; no alterations were observed compared to the saline group

Conclusions : The combined use of the SB100X transposon system and the pFAR4 miniplasmid was effective in optimizing the anti-angiogenic status in the retina by increasing PEDF secretion and reducing metalloproteinase activity. Moreover, the transfected transplanted cells did not activate microglia and macrophages in the CNV surrounding area. This is an important step for the establishment of a safe, specific, and effective gene therapeutic treatment for angiogenic retinal diseases, such as wet AMD.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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