title: Preservation of photoreceptor morphology and function in P23H rats using an allele independent ribozyme.
abstract: 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.
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, Box 100266, Gainesville, FL 32610, USA. mari543@ufl.edu
authors.forename: M
authors.initials: M
authors.lastname: Gorbatyuk
authors.forename: V
authors.initials: V
authors.lastname: Justilien
authors.forename: J
authors.initials: J
authors.lastname: Liu
authors.forename: W W
authors.initials: WW
authors.lastname: Hauswirth
authors.forename: A S
authors.initials: AS
authors.lastname: Lewin
mesh_terms: D000483:Alleles; D000818:Animals; D030801:Animals, Genetically Modified; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics; D004596:Electroretinography; D015316:Genetic Therapy / Q000379:methods*; D051379:Mice; D010775:Photic Stimulation; D016337:RNA, Catalytic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D012160:Retina / Q000503:physiopathology; D012174:Retinitis Pigmentosa / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology / Q000628:therapy*; D012243:Rhodopsin / Q000096:biosynthesis / Q000235:genetics; D017299:Rod Opsins / Q000235:genetics; D013045:Species Specificity; D014162:Transfection
chemical_list: D016337:RNA, Catalytic; D012333:RNA, Messenger; D017299:Rod Opsins; D012243:Rhodopsin