RNAi-Based Suppression of Rhodopsin in vivo

Abstract

Purpose:: Retinitis Pigmentosa is the most prevalent cause of registered blindness in those of working age in the developed world, affecting approximately 1 in 3,000 people. The autosomal dominant forms of the disease are genetically and mutationally extremely heterogeneous. We previously described strategies requiring mutation-independent suppression of the mutant allele and replacement with a gene construct altered such that it escapes suppression in order to overcome mutational heterogeneity (Millington Ward et al, Hum Mol Genet. 1997 Sep;6(9):1415-26). The present study was designed to see if efficient RNAi-based suppression of rhodopsin could be achieved in vivo.

Methods:: Adult mice carrying a normal human rhodopsin (NHR) transgene on a murine rhodopsin negative (rho^-/-) background were sub-retinally injected with either an eGFP-tagged AAV2/5 construct incorporating a H1 promoter driven short hairpin RNA (shRNA) targeting NHR (N = 14 eyes) or an eGFP-tagged non-targeting construct (N = 12 eyes). Retinas were harvested two weeks post injection, dissociated, and FACS sorted to select for cells expressing eGFP. NHR mRNA levels in these cells were analysed using real-time PCR (rt-PCR).

Results:: rt-PCR analysis of NHR mRNA levels indicated that suppression of the order of 90% was achieved in eGPF positive retinal cells from eyes which received the targeting vector compared with eGFP positive retinal cells from eyes which received the non-targeting vector.

Conclusions:: We have previously shown that shRNAs can efficiently reduce murine rhodopsin and rds mRNA expression levels in COS 7 cells and in electroporated retinal explants by up to 80% and can discriminate, in a mutation independent manner, between wild-type and mutant genes (Kiang et al, Mol Ther. 2005 Sep;12(3):555-61; Palfi et al, Hum Mutat. 2006 Mar;27(3):260-8;). In this study we demonstrate, in vivo, similar suppression levels following sub-retinal injection of AAV-shRNA targeting normal human rhodopsin.

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