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M.S. Gorbatyuk, J. Pang, W. Hauswirth, A. Lewin; Ribozyme knockdown of endogenous mouse rhodopsin by AAV–delivered ribozymes. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4774.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Our long term goal is to develop ribozymes that may function in a "digest and replace" gene therapy strategy for autosomal dominant retinitis pigmentosa (ADRP) caused by mutations in the gene for rod cell opsin. Previously, we have shown that ribozymes Rz397 and Rz448 designed to cleave canine rhodopsin (RHO) mRNA effectively digest that mRNA in tissue culture experiments. In the present experiments, we test whether these ribozymes would function in vivo in mice. Mouse RHO mRNA matches perfectly with Rz397 but contains a mismatch with Rz448 that should diminish its activity. Methods: Genes for both ribozymes were cloned in an AAV vector under the control of the murine opsin proximal promoter and were packaged in AAV2 capsids. Both wild–type C57–Bl/6 (RHO +/+) and rhodopsin knockout hemizygotes (RHO+/–) were treated. Virus was injected in the right eye at postnatal day 6 (0.5 µl) or at postnatal day 30 (1 µl). In the P30 injections, animals were also injected with lactated Ringers solution in the left eye. At 1 and 2 months following injection, animals were analyzed by electroretinography (ERG) for a– and b–wave amplitudes. Mice were then sacrificed and enucleated. Retinal extracts were then analyzed for RHO protein level by western blot. To visualize rhodopsin reduction, immunocytochemistry and in situ hybridization were also performed. For this purposes, eyes were fixed in 4% paraformaldehyde and 10 µm cryosections were incubated with a primary antibody. Antibody–antigen complexes were detected with Cy3–conjugated anti–mouse IgG antibody. In situ hybridization was performed with an 33P–labeled RNA probe to murine rhodopsin. Selected slides were dipped in photographic emulsion and quantitative analysis of hybridization was made. Results: RHO +/– injected with AAV–Rz397 at P6 showed a 50% reduction in b–wave amplitudes of injected eyes relative to the uninjected contralateral eye. However, injection of RHO (+/–) animals at one month and of RHO (+/+) animals at either age had no impact on ERG. AAV–Rz 448 had a lesser impact on b–wave amplitudes of the hemizygous animals. Western blot indicated a 50% reduction of RHO protein in injected eyes of both RHO (+/+) and RHO (+/–) mice. These results were confirmed by in situ hybridization and imunocytochemistry. Conclusions: AAV–Rz397 could lead to significant (50%) reduction of rhodopsin mRNA and protein, though this affected ERG amplitudes only in hemizygous rhodopsin knockout mice. Since this ribozyme is less catalytically active with the canine mRNA than Rz448, we expect both ribozymes to be useful in treating canine models of ADRP
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