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24th Annual Meeting of the German Retina Society

German Retina Society

17.06. - 18.06.2011, Aachen

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Sleeping beauty-mediated transfection of pigment epithelial cells with the PEDF gene using mRNA as source of transposase

Meeting Abstract

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  • Nina Harmening - Universitäts-Augenklinik der RWTH Aachen
  • S. Johnen - IZKF der RWTH Aachen
  • Z. Izsvak - Max-Delbrück Center for Molecular Medicine, Berlin
  • P. Walter - Universitäts-Augenklinik der RWTH Aachen
  • G. Thumann - Universitäts-Augenklinik der RWTH Aachen; IZKF der RWTH Aachen

German Retina Society. 24th Annual Conference of the German Retina Society. Aachen, 17.-18.06.2011. Düsseldorf: German Medical Science GMS Publishing House; 2011. Doc11rg42

doi: 10.3205/11rg42, urn:nbn:de:0183-11rg421

This is the English version of the article.
The German version can be found at: http://www.egms.de/de/meetings/rg2011/11rg42.shtml

Published: June 15, 2011

© 2011 Harmening et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en). You are free: to Share – to copy, distribute and transmit the work, provided the original author and source are credited.

Outline

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Purpose: Transplantation of autologous pigment epithelial cells expressing recombinant pigment epithelium-derived factor (PEDF) into the subretinal space is a promising treatment modality for AMD. The Sleeping Beauty transposase system ensures stable transgene insertion and continuous protein secretion. However, the transposase-encoding DNA molecule has the potential to integrate spontaneously into the genome, potentially leading to transposon re-mobilization and re-integration. To avoid such risk, we have used transposase-encoding transient mRNA and showed that mRNA as the source of transposase is effective and efficient at mediating gene delivery and integration in pigment epithelial cells.

Methods: Using microporation, ARPE-19 cells were co-transfected either with a control or a PEDF encoding plasmid plus SB100X mRNA. Transfection efficiency and protein expression stability were evaluated by flow cytometry and Western Blot analysis.

Results: Using in vitro-transcribed mRNA as a source of SB100X ARPE-19 were transfected with an efficiency similar to transfection using encoding SB100X DNA: specifically 100% of ARPE19 were transfected with the control plasmid and 60% with the PEDF-encoding plasmid. The control-transfected cells showed fluorescence and PEDF-transfected cells have continuously secreted PEDF into the media for the 50 days (7 passages) since transfection.

Conclusions: The use of mRNA is an effective alternative to DNA as source of SB100X transposase to deliver a transgene into the genome of pigment epithelial cells. Stable transfection and long term expression of PEDF is a critical step for a gene therapy approach to the treatment of AMD.