June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
pFAR4 miniplasmids in combination with the Sleeping Beauty transposon system allow efficient transfection of freshly isolated iris pigment epithelial cells
Author Affiliations & Notes
  • Martina Kropp
    service d'ophtalmologie, Hopitaux Universitaires de Genève, Genève, Switzerland
  • Nina Harmening
    service d'ophtalmologie, Hopitaux Universitaires de Genève, Genève, Switzerland
  • Sandra Johnen
    Ophthalmology, University Hospital RWTH Aachen, Aachen, Germany
  • Shuwei Tian
    service d'ophtalmologie, Hopitaux Universitaires de Genève, Genève, Switzerland
  • Daniel Scherman
    Unité de Technologies Chimiques et Biologiques pour la Santé, INSERM U1022 - CNRS UMR8258, Paris, France
  • Corinne Marie
    Unité de Technologies Chimiques et Biologiques pour la Santé, INSERM U1022 - CNRS UMR8258, Paris, France
  • Zsuzsanna Izsvák
    Max Delbrück Center for Molecular Medicine, Berlin, Germany
  • Gabriele Thumann
    service d'ophtalmologie, Hopitaux Universitaires de Genève, Genève, Switzerland
  • Footnotes
    Commercial Relationships Martina Kropp, None; Nina Harmening, None; Sandra Johnen, None; Shuwei Tian, None; Daniel Scherman, None; Corinne Marie, None; Zsuzsanna Izsvák, None; Gabriele Thumann, None
  • Footnotes
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Investigative Ophthalmology & Visual Science June 2015, Vol.56, 2316. doi:
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      Martina Kropp, Nina Harmening, Sandra Johnen, Shuwei Tian, Daniel Scherman, Corinne Marie, Zsuzsanna Izsvák, Gabriele Thumann; pFAR4 miniplasmids in combination with the Sleeping Beauty transposon system allow efficient transfection of freshly isolated iris pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2316.

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

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Abstract

Purpose: To achieve a definite treatment for exudative age-related macular degeneration (AMD), we developed a protocol to transplant autologous iris pigment epithelial (IPE) cells transfected with the anti-angiogenic and neurogenic pigment epithelium-derived factor (PEDF). However, efficient transfection of freshly isolated cells is challenging especially since only low cells numbers can be isolated from an iridectomy. To address this challenge, we have used the non-viral Sleeping Beauty transposon system combined with antibiotic-resistance free pFAR4 miniplasmids to transfect as few as 5x103 IPE cells from iris biopsies. This protocol enables stable and efficient transfection of primary IPE cells and the absence of antibiotic-resistance genes enhances safety for the use in clinical trials.

Methods: IPE cells isolated from whole irises or biopsies (9 mm2 and 2.25 mm2) were immediately electroporated with the pFAR4-ITRs CAGGS Venus or the pFAR4-ITRs CMV PEDF BGH transposon and the pFAR4-CMV SB100X SV40 transposase (ratio 16:1) in a total plasmid concentration of 0.5 µg DNA. Transfection efficiency was evaluated by image-based cytometry. PEDF secretion was analyzed by Western Blot and quantified by ELISA.

Results: 1.1±0.8 x106 cells were isolated from whole iris, 1.0±0.5 x105 from 9 mm2 biopsies and 5.1±2.4 x104 cells from 2.25 mm2 biopsies. Efficiencies of 7±2.6% (whole iris), 11±9.0% (9 mm2 biopsy) and 8±2.0% (2.25 mm2 biopsy) were obtained with mean fluorescence intensity (MFI) ranging from 1077 to 1441 MFI. Western Blot analyses detected a significant and reproducible increase in PEDF secretion after transfection that was stable for the 191 days of follow-up. By ELISA it was determined that PEDF transfected IPE cells secreted 2.9±0.6 ng (whole iris), 1.8±0.6 ng (9 mm2 biopsy), and 0.9±0.7 ng PEDF/h/104 cells (2.25 mm2 biopsy). Only 0.07±0.02 ng PEDF/h/104 cells was detected in non-transfected control cells.<br />

Conclusions: Here we have shown the stable transfection of low numbers of freshly isolated IPE cells by using the Sleeping Beauty Transposon system in combination with pFAR4-miniplasmids. This is an important step for the establishment of a safe and specific gene therapeutic treatment of exudative AMD.<br />

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