Down–regulation of rhodopsin gene expression by small interfering RNA

Abstract

Abstract: : Purpose: RNA interference (RNAi), wherein the introduction of double–stranded RNA leads to gene silencing, exhibits gene silencing with great specificity and potency. A 21–nucleotide duplex RNA, termed small interfering RNA (siRNA), can initiate RNAi in mammalian cells and down–regulate targeted gene expression more effectively than alternative silencing methods, such as anti–sense and ribozyme–based strategies. The purpose of this work was to demonstrate down–regulation of rhodopsin mRNA using siRNA in both in vitro and in vivo models. Methods: Four siRNA duplexes that target different regions of the bovine rhodopsin gene were chemically synthesized. Three of these siRNAs also target corresponding regions in human rhodopsin, but all were specifically designed to have low identity to mouse rhodopsin and other known genes in the NCBI database. 21–bp siRNAs were first tested in a stable HEK 293 cell line that expresses mutant P23H bovine rhodopsin upon induction by tetracycline. One to two days after transfection, P23H rhodopsin mRNA and protein expression was evaluated by real time PCR and Western blotting, respectively. Results: Despite preliminary Western blotting indicating no reduction of target protein, real–time PCR analysis showed that the tetracycline–induced rhodopsin mRNA levels in the HEK 293 cells were significantly down–regulated by two of the four siRNA duplexes compared to untransfected controls. This indicates that although these two siRNAs have a gene silencing effect, a large reservoir of stable protein may still exist in the cell. In order to more effectively deplete protein levels and obtain longer–term suppression of the P23H bovine rhodopsin gene, these siRNAs have now been cloned as stem–looped inserts into the pSilencer expression vector for stable mammalian cell transfection. Based on in vitro observations, we will also package effective siRNA hairpins into AAV vectors for in vivo studies. Conclusions: Careful in vitro screening of candidate siRNAs has identified two that effectively silence rhodopsin mRNA. In vivo analysis of these reagents is underway in a transgenic mouse line that expresses human P23H rhodopsin. It is foreseeable that siRNA technology, coupled to AAV delivery vectors already validated for efficient, long–term retinal gene expression, has potential in the treatment of a variety of autosomal dominant retinal diseases requiring that the defective gene be silenced.

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