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J. Tosi, N.-K. Wang, R. Davis, C.-W. Hsu, S. H. Tsang; Lentiviral PDE6β Combined With shRNA-Mediated Knockdown of Cyclic Nucleotide Gated Channel Alpha 1 Results in an Increase of Photoreceptor Survival in a cGMP Phosphodiesterase (Pde6b) Mouse Mutant Model of Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1366.
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We hypothesize that retinal degeneration in Pde6b deficiency is due to excessive Ca2+ and can be slowed by decreasing Ca2+/Na+ cations entry through cGMP-gated cation channels (Cnga1). To test this hypothesis, we used a bipartite expression lentiviral vector to simultaneously reduce the expression ofCnga1 and increase the expression of PDE6β in Pde6bH620Q mice, and measured this effect on photoreceptor degeneration
We tested the lentiviral vector plenti_mRho_Pde6b_mir34b_shRNA-mCnga1. We injected one microliter subretinally into the right eye of Pde6bH620Q mice at P5 (n=35). The left eye was injected subretinally with one microliter of saline, CMV::EGFP or Lenti-LacZ lentivirus and used as control. Mice were sacrificed and retinal sections were stained with H&E. The number and morphology of photoreceptors of lentiviral shRNA injected eyes were compared to control eyes. Immunoblotting of retinal lysates obtained from Pde6bH620Q and C57BL/6 mice were conducted fifteen days after injection and compared with controls. Electroretinograms (ERGs) were performed weekly up to P65 to assess global retinal function in injected and control eyes, using an Espion simulator.
No undesirable side effect due to shRNA knockdown was observed. plenti_mRho_Pde6b_mir34b_shRNA-mCnga1 virus increased histological and functional survival of photoreceptors in Pde6bH620Q mutants. At P62, the control retinae exhibited a single row of photoreceptors with scattered outer segments. In contrast, transduced eyes showed rod outer segments and five rows of photoreceptor nuclei. The retinal protein expression levels of injected Pde6bH620Q and C57BL/6 mice revealed a decreased band of Cnga1 and an increased band of Pde6b compared to controls.ERGs performed up to P65 showed higher b-wave amplitudes in lentiviral injected eyes compared to control eyes.
Simultaneous increase of cGMP breakdown by increasing Pde6b expression and decreased intracellular Ca2+ levels by reducing Cnga1synthesis using a novel rod-specific bipartite shRNA delivery approach produces an increase of photoreceptor survival. This innovative strategy tested in mice shows that Ca2+ modulation maybe a potential novel approach for treating patients with RP with occasioned by defects in rod-specific PDE6.
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