Rescue of Vision in P23H Rats With an rAAV Delivered Ribozyme Targeting Mouse Opsin

Abstract

Abstract: : Purpose:To design and test a hammerhead ribozyme (Rz) that would act in a gene specific manner to limit mutated (P23H) mouse rhodopsin (RHO) expression that leads to Autosomal Dominant Retinites Pigmentosa (ADRP). This ribozyme (Rz525) is species specific but is not specific for any mutant allele of rhodospsin. Methods: A ribozyme targeting the dog, mouse, human, but not the rat, RHO mRNA sequence was designed and tested using cell–free kinetic assays. The activity of the ribozyme was verified in tissue culture experiments with dog, mouse or human cDNA inserted into plasmid pcDNA3.1 and expressed under control of the CMV promoter. Rz525 was inserted into an AAV–2 plasmid vector with expression driven by the CBA promoter. HEK 293 cells were co–transfected with both target and ribozyme plasmids in a 1:10 (target:Rz) molar ratio. RHO mRNA reduction was measured by RT–PCR. For in vivo experiments, Rz525 driven by a mouse opsin promoter was inserted into an AAV2 plasmid that was packaged in AAV–5 capsids. 2µl of AAV–5 Rz (2.4x10¹¹ particles) were injected subretinaly into the right eye of P23H transgenic rats at postnatal day 16. These rats contain a mutant mouse transgene that is susceptible to cleavage by Rz525. Left eyes were injected with saline solution. Animals were analyzed at 1, 2 and 3 months post injection by full–field scotopic electroretinography (ERG). After 3 months animals, frozen sections were analyzed from dissected retinas using propidium iodide fluorescence. Quantitative analysis of nuclei in the outer nuclear layer was performed using NIH Image Software. Results: In vitro kinetic analysis revealed that Rz525 cleaved 15% of a target RNA in 2 minutes in physiologic magnesium conditions. Transient transfection of 293 cells showed that RHO mRNA was reduced 40% relative to an unchanged mRNA (b–actin). Following infection of transgenic rats, the AAV expressing Rz 525 led to a significant increase in scotopic a– and b– wave ERG amplitudes. This outcome remained stable for over 3 months. In treated superior retinas the number of photoreceptor nuclei was significantly higher in ribozyme treated eyes compared to contralateral control eyes. Conclusions: AAV–5 delivery of Rz525 to P23H rat retinas led to a significant rescue of vision. Up to 50% increase in a– and b– wave amplitudes was observed. Histological analysis confirmed that vision rescue is due to prevention of photoreceptor degeneration. This result suggests that allele–independent ribozymes delivered by AAV might be therapeutic for ADRP.

Keywords: gene transfer/gene therapy • photoreceptors • gene/expression