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A. Koirala, Z. Han, R. S. Makkia, M. J. Cooper, M. I. Naash; Sustained and Efficient Transduction of Retinal Pigment Epithelial Cells by Nanoparticles Containing Modified Vectors. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2528.
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© ARVO (1962-2015); The Authors (2016-present)
Our objective is to develop an effective non-viral gene delivery method for genetically-based blinding diseases. In this study we evaluated the ability of compacted DNA nanoparticles to generate efficient and stable transgene expression in mouse retinal pigment epithelial (RPE) cells.
Three expression cassettes containing eGFP driven by the vitelliform macular dystrophy-2 (VMD2) promoter were generated. These are linear without bacterial backbone (L-eGFP); circular plasmid with bacterial backbone (C-eGFP) and circular plasmid modified with mammalian sequences (CM-eGFP). The vectors were compacted with CK30PEG10K into rod-like particles with diameters of 8-11 nm. Nanoparticles or naked DNA (1µl, 4.3µg DNA), and saline were injected subretinally into 1-month-old BALB/c mice. Uninjected controls were also included. eGFP expression levels and localization were evaluated by qRT-PCR, Western blot and immunohistochemistry (IHC) over a period of 1-year post-injection (PI).
eGFP expression from all compacted vectors peaked at PI-2, the earliest timepoint evaluated. Expression levels from L-eGFP and C-eGFP nanoparticles decreased after PI-30 and reached baseline levels by PI-60. IHC at PI-2 to PI-30 showed that all nanoparticles directed eGFP expression specifically in RPE cells. By PI-60, the RPE labeling from both linear and circular particles was noticeably reduced. However, eyes injected with the modified CM-eGFP nanoparticles exhibited considerable eGFP fluorescence in RPE cells up to PI-360 (the last timepoint measured). At PI-360, eGFP fluorescence was uniformly distributed throughout the RPE layer with transduction in more than 50% of the cells.
Our results demonstrate the clinical potential of nano-technology based gene therapy for providing a safe, highly efficient and sustained therapeutic delivery of genes to RPE cells. The modified vector surpassed the others in terms of maintaining transgene expression up to one year PI. We are currently evaluating this modified vector for sustained rescue of visual defects in several animal models.
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