September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Human PEDF optimized gene for transposon-based gene therapy to treat age-related macular degeneration
Author Affiliations & Notes
  • Cécile Prat-Souteyrand
    Ophthalmology, University of Geneva, GENEVA, GENEVA, Switzerland
  • Nina Harmening
    Ophthalmology, University of Geneva, GENEVA, GENEVA, Switzerland
  • Martina Kropp
    Ophthalmology, University of Geneva, GENEVA, GENEVA, Switzerland
  • Gregg Sealy
    Ophthalmology, University of Geneva, GENEVA, GENEVA, Switzerland
  • Zsuzsanna Izsvák
    Max-Delbrück-Center for Molecular Medicine (MDC), Berlin, Germany
  • Daniel Scherman
    Centre National de Recherche Scientifique (CNRS), Paris, France
  • Corinne Marie
    Centre National de Recherche Scientifique (CNRS), Paris, France
  • Sandra Johnen
    Universitätsklinikum Aachen (UKA), Aachen, Germany
  • Gabriele Thumann
    Ophthalmology, University of Geneva, GENEVA, GENEVA, Switzerland
  • Footnotes
    Commercial Relationships   Cécile Prat-Souteyrand, None; Nina Harmening, None; Martina Kropp, None; Gregg Sealy, None; Zsuzsanna Izsvák, None; Daniel Scherman, None; Corinne Marie, None; Sandra Johnen, None; Gabriele Thumann, None
  • Footnotes
    Support  EU FP7 - 305134
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 1172. doi:
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      Cécile Prat-Souteyrand, Nina Harmening, Martina Kropp, Gregg Sealy, Zsuzsanna Izsvák, Daniel Scherman, Corinne Marie, Sandra Johnen, Gabriele Thumann; Human PEDF optimized gene for transposon-based gene therapy to treat age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1172.

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

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Abstract

Purpose : An alternative treatment for neovascular age-related macular degeneration (nvAMD) would be the transplantation of genetically modified autologous pigment epithelial cells. Since autologous retinal pigment epithelial (RPE) cells may carry transformations that underlie nvAMD, we have developed a protocol for the transplantation of autologous iris pigment epithelial (IPE) cells transfected ex vivo with recombinant human pigment epithelium-derived factor (rhPEDF) to inhibit choroidal neovascularization. To increase and ensure stable and continuous rhPEDF expression and secretion, we have modified the human PEDF gene sequence and transfected IPE cells using the hyperactive Sleeping Beauty (SB100X) transposon delivered in Free of Antibiotic Resistance (pFAR4) miniplasmids. Here, we compare endogenous PEDF and rhPEDF expression from 5’000 and 10’000 IPE cells transfected with the native human PEDF (rhPEDF) and the optimized human PEDF gene (rhPEDFo).

Methods : Using bioinformatics analysis, the rhPEDF gene sequence was optimized to theoretically result in an elevated expression. 10’000 and 5’000 primary bovine (b)IPE cells were electroporated with pFAR4 miniplasmids encoding SB100X transposase, rhPEDF gene or rhPEDFo gene. rhPEDF and rhPEDFo gene expression was quantified by qRT-PCR; protein secretion was analyzed by Western Blot and quantified by ELISA.

Results : After 21 days of culture, transfected bIPE cells with either rhPEDF or rhPEDFo gene showed overexpression of PEDF. Transfection of 10’000 IPE cells with hPEDF and hPEDFo gene showed an about 1’000-fold and 900-fold increase in PEDF secretion, respectively, and transfection of 5’000 cells showed an increase of about 1’000-fold and 1’400 fold, respectively, compared to non-transfected cells. A stability of expression appeared in cells transfected with the native rhPEDF gene whatever the number of transfected cells. Both rhPEDF and rhPEDFo showed a stable secretion for up to 300 days in culture.

Conclusions : The present work demonstrates that small numbers of primary IPE cells can be efficiently transfected by pFAR4 miniplasmidscarrying the PEDF gene within the SB transposon. Bioinformatics-based optimization of the PEDF gene does not consistently increase the level of expression in bIPE cells, while the transfection of the rhPEDF gene revealed a constant expression in both 5’000 and 10’000 transfected cells.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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