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V. Justilien, X. Qi, J. Guy, A.S. Lewin; Ribozyme Knockdown of Manganese Superoxide Dismutase in the Mouse Outer Retina . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2501.
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Purpose: Age–related macular degeneration (AMD) is a degenerative condition of the macular photoreceptors, retinal pigment epithelium (RPE), Bruch’s membrane and the choriocapillaris that accounts for the majority of irreversible severe visual loss in the elderly population of industrialized countries. Biochemical and epidemiological evidence suggests that generation of toxic oxidants in the retina/RPE/choroid complex results in its damage, and to the eventual manifestation of AMD. We propose to test this theory in the mouse by using ribozymes to knock down the levels of enzymes such as manganese superoxide dismutase (MnSOD) that mediate the levels of reactive oxygen species (ROS). Methods: A ribozyme that targets MnSOD, SOD2 RZ432, was packaged in AAV2 under the control of the CMV–beta actin (CBA) promoter, which leads to expression in the RPE and in photoreceptors following subretinal injection, the mouse opsin proximal promoter (MOPS500), which directs synthesis only in photoreceptors, or the RPE65.8 promoter, which leads to gene expression specifically in the RPE. Seven–week old DBAJ/1 mice were injected subretinally in the right eye with 1µL of CBA–SOD2 RZ432 (2x1013p/mL), RPE65.8–SOD2 RZ432 (4x1013p/mL) or MOPS500–SOD2 RZ432 (1x1013p/mL). The left eyes were left uninjected to serve as controls. Electroretinogram (ERG) analysis was performed at 6, 15, and 22 weeks post injection and eyes were also processed to examine retinal histology. Results: An average of 42 and 50 percent reduction was observed in a– and b–wave amplitudes, respectively, of those mice injected with the CBA–SOD2 RZ432 virus. Mice injected with the RPE65.8–SOD2 RZ432 virus showed a progressive loss of both a– and b–wave amplitudes from 6 weeks to 22 weeks with 22 weeks ERG measurements showing an average of 52 and 38 percent reduction in a– and b–waves respectively. The Mops500–SOD2 RZ432 virus did not cause significant reductions in erg amplitudes. Retinas of mice injected with the CBA–SOD2 RZ432 virus at 22 weeks post injection showed a loss of photoreceptor nuclei, outer and inner segments compared to uninjected eyes. The spillage of pigment granules from the RPE into the photoreceptor layer was also observed in eyes injected with virus. Conclusions: The CBA and RPE65.8 promoters led to a significant reduction in ERG amplitudes while the MOPS500 promoter did not. The CBA promoter also led to the most significant histological damage to the retina. These results suggest that the primary effect of SOD2 RZ432 is on the RPE and not on the rod photoreceptors. Thus oxidative damage to the RPE may contribute to AMD–related pathogenesis.
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