June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Efficient electrotransfer-mediated transfection of rd10 retinas using the non-viral Sleeping Beauty transposon system
Author Affiliations & Notes
  • Sabine Diarra
    Department of Experimental Ophthalmology, University Hospital RWTH Aachen, Aachen, Germany
  • Florian Waschkowski
    Institute for Materials in Electrical Engineering , RWTH Aachen University, Aachen, Germany
  • Aracelys Garcia Moreno
    Institute for Materials in Electrical Engineering , RWTH Aachen University, Aachen, Germany
  • Christine Haselier
    Department of Experimental Ophthalmology, University Hospital RWTH Aachen, Aachen, Germany
  • Stephan Hesse
    Department of Experimental Ophthalmology, University Hospital RWTH Aachen, Aachen, Germany
  • Zsuzsanna Izsvák
    Molecular Medicine in the Helmholtz Association, Max Delbrück Center, Berlin, Germany
  • Zoltán Ivics
    Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany
  • Gabriele Thumann
    Department of Ophthalmology, University Hospital of Geneva, Geneva, Switzerland
  • Frank Muller
    Institute of Complex Systems, Cellular Biophysics, ICS-4, Aachen, Germany
  • Wilfried Mokwa
    Institute for Materials in Electrical Engineering , RWTH Aachen University, Aachen, Germany
  • Sandra Johnen
    Department of Experimental Ophthalmology, University Hospital RWTH Aachen, Aachen, Germany
  • Peter Walter
    Department of Ophthalmology, RWTH Aachen University, Aachen, Germany
  • Footnotes
    Commercial Relationships   Sabine Diarra, None; Florian Waschkowski, None; Aracelys Garcia Moreno, None; Christine Haselier, None; Stephan Hesse, None; Zsuzsanna Izsvák, None; Zoltán Ivics, None; Gabriele Thumann, None; Frank Muller, None; Wilfried Mokwa, None; Sandra Johnen, None; Peter Walter, None
  • Footnotes
    Support  ERS Grant OPSF337
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 4537. doi:
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      Sabine Diarra, Florian Waschkowski, Aracelys Garcia Moreno, Christine Haselier, Stephan Hesse, Zsuzsanna Izsvák, Zoltán Ivics, Gabriele Thumann, Frank Muller, Wilfried Mokwa, Sandra Johnen, Peter Walter; Efficient electrotransfer-mediated transfection of rd10 retinas using the non-viral Sleeping Beauty transposon system. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4537.

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

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Abstract

Purpose : In several non-viral gene therapeutic approaches, the delivery of nucleic acids is mediated by the physically-based electroporation method. Here, cells are exposed to an electrical field in order to initiate a directed gene transfer. The distance between the electrode and the addressed target tissue is an essential electroporation parameter. To perform an efficient, well tolerable, temporally and spatially controlled transfection, specified electrode geometries, low voltage applications and suitable plasmid constructs are requested. Our approach aims at establishing an efficient delivery protocol of a transgene into retinal tissue using the non-viral Sleeping Beauty (SB100X) transposon system introduced by multielectrode arrays (MEA) specifically optimized for electroporation procedures. Here, we compare a protocol of a pipette-based electroporation system with commercially available and custom-made MEA structures using a Venus reporter gene in degenerated retinas of the rd10 mouse model.

Methods : Retinas were isolated from C57BL/6 and Pde6brd10 mice of different ages. Retinal explants were co-transfected with 30 ng of pT2-CMV-SB100X transposase plasmid and 470 ng of pT2-CAGGS-Venus transposon plasmid. Two days after transfection, Venus transgene expression was analysed via fluorescence microscopy. Immunohistochemical analysis was performed to identify the transfected cell types. For the MEA-based electroporation, different buffers were established and pulse parameters were tested with regard to survival of the transfected cells.

Results : Effective transfection was demonstrated in independent pipette-based experiments performed with explants of degenerated rd10 retinas. However, transfection of wild type retinas was less efficient. Using the MEA system, retinas well tolerated tested electroporation buffers and applied pulse parameters and showed high viability rates of at least 74%.

Conclusions : Electroporation-mediated transfection of retinas ex vivo with the plasmid DNA-based SB100X transposon system resulted in efficient and stable Venus transgene expression. Using multielectrode arrays as electroporation device to transfect retinal tissue is a very first step towards the development of an ocular prosthesis that, besides its electrical stimulation properties, can be used for a locally applied non-viral gene therapy to treat retinal degenerative diseases.

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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