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To develop an allele independent ribozyme for the treatment of autosomal dominant retinitis pigmentosa (ADRP) associated with mutations in the rhodopsin (RHO) gene, a ribozyme targeting dog, mouse, human but not rat rhodopsin (RHO) mRNA was designed and tested in vitro. Activity of this ribozyme was tested in tissue culture by co-transfection of HEK 293 cells with plasmids expressing opsin mRNA and ribozyme, followed by quantitative RT-PCR to evaluate the level of RHO mRNA. For experiments in vivo, Rz525 driven by the mouse opsin proximal promoter was inserted in plasmids with AAV 2 terminal repeats (TR) and packaged in AAV serotype 5 capsids. AAV-Rz525 was injected subretinally into the right eyes of P23H rat pups. Left eyes were injected with virus expressing GFP from the identical promoter. Animals were analyzed at 4, 8 and 12 weeks post-injection by full field scotopic electroretinography (ERG). After 12 weeks, animals were sacrificed and retinas were dissected, fixed and sectioned. Rz525 had high catalytic activity in vitro and led to a 50% reduction of RHO mRNA in cells. AAV-Rz525 injection into P23H transgenic rats led to significant preservation (about 50%) of scotopic ERG a- and b-wave amplitudes. Histological analysis showed an increased number of ONL nuclei in the central and superior retina of treated eyes relative to control eyes. RT-PCR analysis revealed 46% reduction of transgenic (mouse) RHO mRNA in right eyes relative to left eyes and no change in rat RHO mRNA. AAV5 delivery of Rz525 resulted in a partial rescue of the light response and structural preservation of photoreceptors in transgenic rats. This ribozyme may be a useful component of an RNA replacement gene therapy for ADRP.