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R. Baid, R. Scheinman, T. Shinohara, U. B. Kompella; LEDGF Decreases Damage Mediated by Rhodopsin P23H Aggregation in Human ARPE-19 Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):699.
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© ARVO (1962-2015); The Authors (2016-present)
To validate lens epithelium derived growth factor (LEDGF) as a therapeutic strategy for the treatment of retinal diseases caused by protein aggregation and to formulate a nanoparticulate delivery system encapsulating a LEDGF expression plasmid.
LEDGF efficacy was tested in human ARPE-19 cultures. Protein aggregation was modeled using the human rhodopsin mutant rhoP23H tagged with CFP. LEDGF was tagged with GFP. ARPE-19 cultures were transfected using lipofectamine with either lipofectamine alone, LEDGF alone, rhoP23H alone, or cotransfected with LEDGF plus rhoP23H, and allowed to express for either 24 or 48 hours. Cells were then qualitatively assessed by confocal microscopy and quantitatively assessed by flow cytometry. LEDGF expression plasmid was encapsulated in PLGA nanoparticles using an emulsion solvent evaporation method. The size and polydispersity of these nanoparticles were characterized using a dynamic light scattering (DLS) method.
Confocal microscopy established that ARPE-19 cells expressing rhoP23H showed evidence of damage including distended nuclei and the presence of material that resembled cell debris. Much less damage was seen in mock transfected cells (lipofectamine alone) or cells expressing LEDGF alone. More intact nuclei were observed in cells coexpressing LEDGF and rhoP23H as compared to rhoP23H alone. Additionally, rhoP23H fluorescence appeared more diffuse in the cotransfected group. The absolute fluorescence of rhoP23H (mean fluorescence intensity) remained constant as measured by flow cytometry in the absence or presence of co-expressed LEDGF. PLGA nanoparticles with a mean diameter of 275 nm, a polydispersity index of 0.164, and a plasmid loading of 6 µg/mg were prepared.
The data suggests that LEDGF decreases the aggregation of rhoP23H in AREP-19 cells leading to decreased aggregation mediated damage. LEDGF plasmid loaded nanoparticles might provide a useful therapeutic strategy for the treatment of degenerative retinal diseases.
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