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Shannon Conley, Adarsha Koirala, Rasha Makkia, Zhao Liu, Mark Cooper, Janet Sparrow, Muna Naash; S/MAR containing nanoparticles mediate long-term therapeutic gene targeting in the RPE and rescue the rpe65-/- LCA model. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1960. doi: https://doi.org/.
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Clinically relevant genetic therapies for chronic ocular diseases must exhibit long-term gene expression. Our goal was to test the ability of engineered plasmid DNA vectors, either naked or compacted into high capacity DNA nanoparticles (NP), to mediate long-term improvement in genetic diseases of the RPE.
Plasmid or NP vectors carrying the hRPE65 cDNA and the RPE-specific vitelliform macular dystrophy 2 promoter were generated (VMD2-hRPE65-S/MAR) and subretinally delivered to the rpe65-/- mouse model of Leber’s congenital amaurosis at post natal day 16. Outcomes were assessed at post-injection (PI)-15 months and included gene expression and distribution, retinal structure and function, and assessment of retinyl ester levels.
At PI-15 months, hRPE65 message levels in treated eyes were 32% of wild-type (WT) for NPs and 44% of WT for naked DNA, and no reduction was seen from PI-6 months. Importantly, spectral ERG demonstrated significant improvement in cone ERG amplitudes in treated vs. untreated animals (green photopic b-waves were 76% of WT for NPs and 79% of WT for naked DNA, vs. 39% of WT for untreated, Fig. 1A). We also observed a reduction in the fundus albipunctatus phenotype in NP or naked DNA treated eyes compared to untreated (Fig. 1B). Biochemical studies showed a significant reduction in the accumulation of retinyl esters in treated mice (Fig. 1C), suggesting the transferred hRPE65 was functional.
These results indicate that both NP and uncompacted plasmid VMD2-hRPE65-S/MAR can mediate appreciable gene expression and improvement in an RPE-associated disease phenotype for the life of the animal. They further highlight that nanocompaction may not be essential for effective expression in the RPE, although additional safety studies for uncompacted DNA will be an essential step. These outcomes establish proof-of-concept for effective non-viral gene therapy in the RPE, and provides an excellent platform for delivery of large genes.
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