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Oleg E Tolmachov, Tanya Tolmachova, Diogo A. Feleciano, Miguel C Seabra; Enhancement of non-viral gene delivery to primary mouse retinal pigment epithelium cells using paramagnetic nanoparticles. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5890.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigment epithelium (RPE) cells are vital for well-being of photoreceptor cells and, thus, are desired target cells to treat age-related macular degeneration (AMD), choroideremia (CHM) and other ocular degenerative diseases through therapeutic gene delivery. Circulating neutralizing antibodies in patients reduce the efficiency of gene transfer with many viral vectors, such as adeno-associated viral (AAV) vectors. Therefore, gene transfer with non-viral vectors, i.e., transfection in vivo, is a promising gene delivery strategy for clinical trials. However, the efficiency of non-viral gene transfer to RPE cells is low. Thus, the aim of this study was to optimize non-viral gene transfer to RPE cells using magnetofection.
We generated non-viral gene vector complexes with plasmid DNA (pEGFP-C3, Takara-Clontech), Combi-Mag paramagnetic particles (OZ Biosciences) and a range of commercially available transfection reagents: Optifect, Lipofectamine LTX&Plus reagent, FreeStyle Max, 293fectin (all from Life Technologies), Fugene 6 (Roche) and Fugene HD (Roche). These vector complexes were used to transfect primary mouse RPE cells (C57Bl6 mouse strain). Transfection efficiency was analysed by flow cytometry and summarized as the mean and its standard error for biological replicates (mean ± standard error, M ± SE). The transfection enhancement was evaluated as the ratio of two mean transfection efficiencies (M1 ± SE1, M2 ± SE2) with average standard error (ASE) of the ratio of two means calculated using formula ASE = (M1/M2)*(SE1^2/M1^2+SE2^2/M2^2)^(1/2). The significance was evaluated using the unpaired t-test.
Comparison of transfection reagents alone, without paramagnetic particles, showed that Optifect promoted transfection of RPE cells 3.1±0.4 times more efficiently than a gold standard Lipofectamine LTX&Plus reagent and that superiority of Optifect was significant against all tested transfection reagents. Supplementation of Optifect-driven transfection with paramagnetic particles, which were focused on the RPE cells with high-strength permanent magnet, allowed further 3.0±0.4 times enhancement of the transfection efficiency, which reached 31±2%.
The non-viral transfer of the EGFP marker gene into mouse RPE cells in vitro is significantly enhanced when transfection reagent Optifect is used in conjunction with magnetofection.
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