Lentiviral PDE6β Combined With shRNA-Mediated Targeting of Guanylate Cyclase2e Results in an Increase of Photoreceptor Survival in a cGMP Phosphodiesterase Mouse Mutant Model of Retinitis Pigmentosa

Abstract

Purpose: : The phototransduction second messenger cyclic GMP (cGMP) is synthesized by guanylate cyclases 2e (GUCY2e) and 2f (GUCY2f). In the dark, GUCY2 activity increases cGMP levels, opening cGMP-gated (CNG) cation channels that permit Na⁺ and Ca²⁺ ions to flow and depolarize the cell. In the light, cGMP phosphodiesterase 6 (PDE6) activation leads to the cGMP hydrolysis and closure of CNG cation channels, producing hyperpolarization. Studies conducted in mouse models of RP, such as Pde6b^rd1 mice, showed a dramatic elevation of retinal cGMP concomitant with degeneration. Presumably, defective PDE6 can not hydrolyze cGMP, which is continuous synthesized by guanylate cyclases at the basal rate, producing an accumulation of cGMP. To test the hypothesis that cGMP is a major contributor to RP pathogenesis, we reduced the expression of guanylate cyclase 2e and we increased the expression of PDE6β in Pde6b^H620Q mice, and measured this effect on photoreceptor degeneration.

Methods: : We tested two lentiviral vectors: a) pRho-Pde6b-shGucy2e, b) pLenti-Rho::Pde6b-H1::shRNA-EF1a-GFP. We injected one microliter subretinally into the right eye of Pde6b^H620Q mice at P5 (n=50 per vector). 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 hematoxilin and eosin. The number and morphology of photoreceptors of lentiviral shRNA injected eyes were compared to control eyes. Electroretinograms (ERGs) were performed weekly from P28 to P110 to assess global retinal function in injected and control eyes, using an Espion simulator.

Results: : pRho-Pde6b-shGucy2e and pLenti-Rho::Pde6b-H1::ShRNA-EF1a-GFP virus increased histological and functional survival of photoreceptors in Pde6b^H620Q mutants. At P62, the control retinae exhibited a single row of photoreceptors with scattered outer segments. In contrast, shRNA transduced eyes showed, surrounding the injection site, rod outer segments and seven rows of photoreceptor nuclei. ERGs performed up to P80 showed higher b-wave amplitudes in lentiviral shRNA injected eyes compared to control eyes.

Conclusions: : Simultaneous increase of cGMP breakdown and diminish cGMP synthesis using a novel rod-specific bipartite shRNA delivery approach produces an increase of photoreceptor survival in a cGMP phosphodiesterase mouse mutant model of RP. This innovative strategy tested in mice shows that cGMP modulation maybe a potential novel approach for treating patients with RP with occasioned by defects in rod-specific PDE6.

Keywords: retinal degenerations: hereditary • gene transfer/gene therapy • photoreceptors