gms | German Medical Science

24th Annual Meeting of the German Retina Society

German Retina Society

17.06. - 18.06.2011, Aachen

Efficient and stable PEDF gene delivery into primary pigment epithelial cells mediated by the Sleeping Beauty Transposition

Meeting Abstract

  • Sandra Johnen - IZKF der RWTH Aachen
  • Z. Izsvak - Max-Delbrück-Centrum für Molekulare Medizin, Berlin
  • P. Walter - Universitäts-Augenklinik der RWTH Aachen
  • G. Thumann - IZKF der RWTH Aachen; Universitäts-Augenklinik 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. Doc11rg59

doi: 10.3205/11rg59, urn:nbn:de:0183-11rg592

This is the translated version of the article.
The original version can be found at: http://www.egms.de/de/meetings/rg2011/11rg59.shtml

Published: June 15, 2011

© 2011 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

Text

Purpose: The treatment of retinal degenerations using subretinal pigment epithelial cell transplantation has resulted in very limited success. One reason may be that the transplanted cells did not secrete necessary factors to maintain a healthy environment. Here, we detail a protocol for the efficient and stable transfection of primary pigment epithelial cells with pigment epithelium-derived factor (PEDF) using the hyperactive Sleeping Beauty (SB100X) transposon system. Transplantation of these cells would engender an anti-angiogenic and neuroprotective subretinal microenvironment.

Methods: Using electroporation, ARPE-19 and primary pigment epithelial cells were transfected with a control or a PEDF encoding plasmid, controlled either by a CMV or a CAG promoter. Transfection efficiency and protein expression stability were evaluated by flow cytometry and Western blot analysis.

Results: SB100X-based gene delivery resulted in efficiencies of 100% with the control and 40% with the PEDF encoding plasmid. Subsequent sorting enabled establishment of populations containing 99% PEDF-transfected cells. PEDF secretion was maintained in all cell types for the 8 months the cells have been followed. However, PEDF secretion was higher under the control of the CMV than the CAG promoter.

Conclusions: We have shown that SB100X-mediated gene transfer ensured the stable expression and continuous secretion of PEDF in primary pigment epithelial cells. Transfection with the SB100X system results in the transgene integration into the host cell’s genome without potential complications of retroviral mediated gene delivery and is therefore an important step in the development of a cell-based, non-viral gene addition therapy for retinal degenerative diseases.