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J. Tosi, R. J. Davis, N.-K. Wang, J. M. Kasanuki, C.-S. Lin, S. H. Tsang; shRNA-mediated Targeting of Guanylate Cyclase2e Results in an Functional Rescue of Photoreceptor in a cGMP Phosphodiesterase Mouse Mutant Model of Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2008;49(13):158.
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© ARVO (1962-2015); The Authors (2016-present)
In dark-adapted eyes, phototransduction second messenger, cyclic GMP (cGMP), is synthesized by guanylate cyclases 2e (GUCY2e) and 2f (GUCY2f), increasing cGMP levels and resulting in opening of cGMP-gated (CNG) cation channels and depolarization. In the light, cGMP phosphodiesterase 6 (PDE6) activation hydrolyses cGMP and closures CNG cation channels, producing hyperpolarization. Pde6bH620Q, a mouse model of RP, showed a dramatic elevation of retinal cGMP concomitant with degeneration onset. To test the hypothesis that cGMP is a major contributor to RP pathogenesis, we reduced the expression of Gucy2e in Pde6bH620Q mice and measured this effect on photoreceptor degeneration.
To knockdown Gucy2e expression, we injected Gucy2e-shRNA lentivirus (1µL) subretinally into the right eye of Pde6bH620Q mice at P5 (n=75). shRNA vectors deliver a short 21 nucleotide stem hairpin RNA duplexes, thereby decreasing the expression of retinal Gucy2e. The left eye was used as control and injected subretinally with CMV::EGFP (1µL) lentivirus. Retinal sections were examined and the number and morphology of photoreceptors of lentiviral shRNA injected eyes were compared to control eyes. Electroretinograms (ERGs) were performed weekly from P28 to P56 to assess global retina function in injected and control eyes.
Gucy2e-shRNA virus increased histological survival of photoreceptors in Pde6bH620Q mutants. At P56, the control retinae showed a single row of photoreceptors with scattered outer segments, as is typically observed in Pde6bH620Q mutants. In contrast, shRNA transduced eyes showed, surrounding the injection site, rod outer segments and six rows of photoreceptor nuclei. ERGs performed from P28 through P56 in Pde6bH620Q mutants showed Gucy2e-shRNA virus maintained photoreceptor function for a limited period of time in lentiviral shRNA injected eyes compared to control eyes.
Lentiviral shRNA designed to supress Gucy2e expression produces an increase of photoreceptor function in a cGMP PDE6 mouse mutant model of RP. This finding suggests lentiviral shRNA therapy may decrease the high intracellular levels of cGMP that are observed in Pde6bH620Q mutants. We speculate that reduction of GUCY2e function compensates low PDE6 activity, resulting in an improvement of photoreceptor survival. This innovative strategy tested in mice shows that cGMP modulation maybe a potential novel approach for treating patients with RP occasioned by defects in rod-specific PDE6.
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