gms | German Medical Science

Artificial Vision 2017

The International Symposium on Visual Prosthetics

01.12. - 02.12.2017, Aachen

The use of microelectrode arrays for non-viral, transposon-mediated, electroporation-based gene transfer in rd10 mouse retinae – An interdisciplinary approach

Meeting Abstract

  • Sabine Diarra - Department of Ophthalmology, University Hospital RWTH Aachen, Aachen, Germany
  • F. Waschkowski - Institute for Materials in Electrical Engineering I, RWTH Aachen University, Aachen, Germany
  • A. Garcia Moreno - Institute for Materials in Electrical Engineering I, RWTH Aachen University, Aachen, Germany
  • Z. Izsvák - Max Delbrück Center for Molecular Medicine in the Helmholtz Society, Berlin, Germany
  • Z. Ivics - Paul-Ehrlich-Institute, Langen, Germany
  • G. Thumann - University of Geneva, University Hospitals of Geneva, Department of Ophthalmology, Geneva, CH
  • U. Schnakenberg - Institute for Materials in Electrical Engineering I, RWTH Aachen University, Aachen, Germany
  • F. Müller - Institute of Complex Systems, Cellular Biophysics, ICS-4, Forschungszentrum Jülich GmbH, Jülich, Germany
  • W. Mokwa - Institute for Materials in Electrical Engineering I, RWTH Aachen University, Aachen, Germany
  • P. Walter - Department of Ophthalmology, University Hospital RWTH Aachen, Aachen, Germany
  • S. Johnen - Department of Ophthalmology, University Hospital RWTH Aachen, Aachen, Germany

Artificial Vision 2017. Aachen, 01.-02.12.2017. Düsseldorf: German Medical Science GMS Publishing House; 2017. Doc17artvis39

doi: 10.3205/17artvis40, urn:nbn:de:0183-17artvis400

Published: November 30, 2017

© 2017 Diarra et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 License. See license information at http://creativecommons.org/licenses/by/4.0/.


Outline

Text

Objective: The use of multielectrode arrays (MEAs) allows the physiological characterisation of the retinal neural network. Additionally, MEAs have been described for the electroporation-based transfer of exogenously added molecules. Cells are exposed to an electrical field which results in a reversible pore formation, allowing the molecules (e.g., transgene-encoding plasmid DANN) to enter the cell and the nucleus. We evaluate the electroporation process carried out by specialized MEA structures in order to expand the function of a retinal prosthesis to a temporally and spatially controlled transfection device. MEA experiments were performed with the non-viral Sleeping Beauty (SB100X) transposon system that can mediate stable insertion of the transgene into the host cell genome. We aim to transfect cells of the rd10 retina, a mouse model of Retinitis pigmentosa, with the DANN constructs ex vivo. New MEAs were designed, in which the geometry of the electrodes was optimized in order to achieve sufficient electric field strengths at the lowest possible voltages to gain good transfection efficiency and cell viability.

Materials and Methods: Retinae were isolated from C57BL/6 and Pde6brd10 mice of different ages. Explants were transfected with 30 ng of pCMV-SB100X transposase and 470 ng of pT2-CAGGS-Venus transposon plasmid. One day after transfection, expression of the Venus reporter transgene was analysed by fluorescence microscopy and by immunohistochemistry. Different parameters of electrical pulses were tested to optimize cell survival and transfection efficiency.

Results: In first experiments Venus fluorescent cells were detected in the rd10 retina after transfection with the MEA system. Retinae tolerated voltage applications of up to 1.6 V with high viability rates.

Discussion: The first results point towards the feasibility to establish an interdisciplinary approach to combine ocular prosthetics with non-viral gene transfer in order to treat retinal degenerative diseases.

Acknowledgements: This project was funded by the Exploratory Research Space at the RWTH Aachen University (grant OPSF337).