Suppression of mouse rhodopsin expression in vivo by AAV mediated siRNA delivery.

PURPOSE: The purpose of this study is to demonstrate that the expression of rhodopsin can be down regulated in vivo by AAV-delivered siRNA. This is the first step in an RNA replacement strategy for the allele-independent treatment of Autosomal Dominant Retinitis Pigmentosa (ADRP).
METHODS: HEK 293 cells were co-transfected with a plasmid carrying mouse RHO cDNA driven by the CMV promoter and a chemically synthesized siRNA duplex of 21 nucleotides. Reduction of RHO mRNA was confirmed by RT-PCR. One active siRNA and a control siRNA were embedded in a small hairpin RNA (shRNA) and cloned in Adeno-associated virus (AAV) vector under regulation of the H1 promoter and containing a GFP reporter. AAV5 expressing either active siRNA or an irrelevant siRNA were subretinaly injected into the right eyes of wild-type or RHO+/- heterozygote mice at post-natal day 16. At 1 and 2 months post-injection, animals were analyzed by electroretinography (ERG). Animals were then sacrificed, and retinas were examined by Western blot, RT-PCR, histology and immunohistochemistry.
RESULTS: All of the siRNAs tested in HEK 293 cells caused degradation of RHO mRNA, although the efficiency varied from 25% to 80%. In vivo siRNA delivery to the retina led to more than 40% reduction of scotopic a- and b-wave amplitudes in RHO+/- heterozygotes. Although the reduction of RHO mRNA was estimated at 30% compared to control animals, Western blots revealed 60% decrease in rhodopsin content. Histological analysis showed significant reduction in the thickness of the ONL, ranging between 53% and 86%.
CONCLUSIONS: AAV-siRNA delivery into the subretinal space resulted in the reduction of retinal function caused by diminished RHO mRNA and protein content. This level of reduction may permit the replacement of endogenous mRNA with siRNA-resistant mRNA encoding wild-type RHO.