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Marta Czugala, Olga Mykhaylyk, Anja Katharina Gruenert, Friedrich E Kruse, Sebastian Wesselborg, Christian Plank, Thomas Armin Fuchsluger; Functional gene transfer for antiapoptotic therapy of corneal endothelial cells – a magnetofection approach.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):No Pagination Specified. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Preventing corneal endothelial cell death still remains one of the important challenges in ocular research. We have previously shown the advantages of magnetofection for plasmid DNA delivery to corneal endothelial cells (CEC). Magnetofection has proven to yield high transfection efficiency in CEC, while causing no negative effects on endothelial cell function and presenting outstanding bio- and immunocompatibility. We further combined these features of silica iron-oxide nanoparticles (SO-Mag5) with the antiapoptotic genes Bcl2 and p35 to test their capacity of protecting CEC from apoptotic cell death.
Gene transfer into CEC was performed using SO-Mag5/XtremeGeneHP/pDNA complexes carrying Bcl2 or p35 at a pDNA dose of 3.0 pg/cell. Transfection efficiency and viability in CEC were assessed using flow cytometry. Expression of p35 protein as well as of apoptosis-related proteins was evaluated using western blot and in-cell western. At 24 h after magnetofection, induction of apoptosis was performed using 1 µM staurosporine for 3 h. The protective effect of magnetofection-assisted antiapoptotic gene transfer was studied by means of Caspase-3 activity assay using the fluorogenic Caspase-3 substrate DEVD-AMC.
Magnetofection with the Bcl2 and p35 gene achieved a transfection efficiency of 29.8 ± 1.8% and 31.0 ± 2.3%, respectively, as measured by flow cytometry. Expression of Bcl2 and p35 protein was also confirmed by western blot. Reduction of apoptosis as measured by Caspase-3 activity was 35.5 ± 0.6% (p < 0.01) for Bcl2 and 56.1 ± 0.6% (p < 0.001) for p35 in comparison to empty vector control.
We could show that magnetofection allows efficient transfer of plasmid DNA carrying antiapoptotic genes to human CEC. The applied method significantly reduced staurosporine-induced apoptosis. Our results indicate also ex vivo cornea therapy potential. When combined with a specifically shaped magnetic field, magnetofection serves as a promising tool for targeted antiapoptotic gene therapy in the treatment of corneal endothelial pathologies resulting from endothelial cell death.
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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