Mutation Independent Knock Down of Rhodopsin by Short Hairpin RNAs (shRNA) and Replacement by a shRNA–resistant Rhodopsin Transgene

Abstract

Abstract: : Purpose: Mutations in rhodopsin cause a wide variety of diseases including autosomal dominant and recessive retinitis pigmentosa, congenital stationary night blindness and retinitis punctata albescens. Due to a very heterogeneous mutation spectrum present in this gene, it would be ideal to develop therapies that are mutation independent. The purpose of this study was to explore the potential of RNA intereference (RNAi) strategies to ameliorate diseases associated with mutations in rhodopsin. Methods: Several constructs expressing short hairpin RNAs (shRNA) targeted to either green fluorescent protein, human rhodopsin and mouse rhodopsin were designed and constructed. Various promoters including the H1 RNA promoter and minimal CMV promoter with a minimal SV40 poly A were tested. The shRNA constructs were cotransfected into human embryonic retinoblasts with expression cassettes for GFP or human or mouse rhodopsin. Northern and Western blot, FACS and quantitative RT–PCR were used to quantify the levels of mRNA and proteins knocked down in each experiment. In parallel, transgenes were designed and constructed that would code for wild type rhodopsin protein but through an mRNA that is resistant to the shRNA due to an altered codon usage. Results: FACS analysis indicates a greater than 90% reduction of GFP protein by the H1 RNA promoter compared with 33% reduction by the same shRNA expressed from a minimal CMV promoter. This resulted from a 75% knock down of GFP mRNA by the H1 RNA promoter transcript. Using the H1 RNA promoter we were able to achieve a 60% knock down of wild type human rhodopsin mRNA. The shRNA–resistant rhodopsin which contains 7 altered amino acid codons (9 altered nucleotides), is expressed at the same levels as the wild type rhodopsin according to Western blot analysis and has the same cellular localization as it’s wild type counterpart as determined by immunofluorescence. Conclusions:Short hairpin RNAs may have application in the treatment of a wide variety of retinal degenerations associated with mutations in rhodopsin.

Keywords: gene transfer/gene therapy • retina