Validation of RNAi technology for suppression and replacement of rds–peripherin

Abstract

Abstract: : Purpose: A mutation–independent suppression and replacement approach using RNA interference (RNAi) has been developed for mouse rds–peripherin (rds). Small interfering RNA (siRNA) and short hairpin RNA (shRNA) were used to suppress expression of rds mRNA in cell culture. Furthermore, a replacement rds gene (r–rds) resistant to RNAi–suppression but still encoding wild type protein was engineered and assessed. Methods: Target expression vectors and siRNAs were co–transfected in COS–7 cells and mRNA levels determined by real–time RT–PCR. An rds shRNA was endogenously expressed under the control of the human H1 promoter. A replacement r–rds gene containing eight degenerate substitutions in the siRNA target sequence was engineered. Mouse rds, r–rds and human RDS–peripherin (RDS) target vectors were co–transfected with the rds shRNA vector and suppression levels determined by real–time RT–PCR. Results: siRNAs targeting rds decreased rds transcript levels to variable extents, the most efficient molecule suppressed mouse rds expression by approximately 75%. The same siRNA sequence expressed endogenously as shRNA was similarly effective. However, shRNA–based suppression was significantly lower (suppression by 42%) for the human RDS gene, which has one mismatch in the siRNA target sequence. Furthermore, r–rds transcripts containing eight degenerate substitutions in the target sequence avoided suppression completely, while still encoding wild type protein. Conclusions: RNAi technology can significantly silence murine rds gene–expression. When coupled with a replacement r–rds gene, the study provides a proof of principle for the applicability of a mutation–independent suppression and replacement therapeutic strategy. The approach exploits RNAi technology for suppression in combination with the degeneracy of the genetic code to generate replacement genes resistant to RNAi–based suppression but encoding wild type protein. This approach is of particular value for dominantly inherited disorders such as RDS–linked or rhodopsin–linked retinitis pigmentosa where mutational heterogeneity represents a significant obstacle to therapeutic development.

Keywords: gene/expression • gene transfer/gene therapy • retinal degenerations: hereditary