gms | German Medical Science

25th Annual Meeting of the German Retina Society

German Retina Society

01.06. - 02.06.2012, Münster

Sleeping Beauty-mediated transposition as safe and efficient methodology for stable non-viral gene transfer

Meeting Abstract

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  • Sandra Johnen - Universitäts-Augenklinik, Aachen
  • G. Thumann - Universitäts-Augenklinik, Aachen

German Retina Society. 25th Annual Conference of the German Retina Society. Münster, 01.-02.06.2012. Düsseldorf: German Medical Science GMS Publishing House; 2012. Doc12rg19

DOI: 10.3205/12rg19, URN: urn:nbn:de:0183-12rg192

This is the translated version of the article.
The original version can be found at: http://www.egms.de/de/meetings/rg2012/12rg19.shtml

Published: May 30, 2012

© 2012 Johnen 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: Efficient delivery of a therapeutic gene into adequate target cells, its long-term expression, and a minimum of side effects are important parameters for an effective gene therapeutic approach. The Sleeping Beauty transposon system (SB100X) meets this criteria and has been shown to provide stable gene transfer in different primary cell types. In the case of neovascular AMD, which is characterized by an imbalance of the pro-vascular VEGF and the anti-angiogenic PEDF, we postulate that vision improvement requires the inhibition of neovascularization by higher levels of PEDF. We have developed a protocol for the subretinal transplantation of primary pigment epithelial cells stably transfected with the PEDF gene using SB100X.

Methods: Pigment epithelial cells were electroporated with two plasmids encoding the SB100X transposase gene and the PEDF gene, respectively. Stability of PEDF expression and its genomic integration were determined by RT-PCR. Consistency of PEDF secretion was analyzed by immunoblotting.

Results: Efficient transposition of 10,000 cells was demonstrated in 5 independent experiments per cell type. The PEDF transgene was integrated into the cell’s genome and recombinant PEDF was continuously secreted for longer than one year.

Conclusions: SB100X ensures stable integration and sustained expression of the PEDF gene in primary pigment epithelial cells. The next steps to improve this protocol will comprise the use of mRNA to avoid the possible genomic integration of the SB100X transposase gene, the targeted insertion of the PEDF transgene into genomic “safe harbors”, and the establishment of autologous cell isolation, transfection and subretinal transplantation during one surgical session.