title: RNAi-based suppression and replacement of rds-peripherin in retinal organotypic culture.
abstract: Extensive mutational heterogeneity presents a significant barrier to the development of therapeutics for RDS-peripherin-linked autosomal-dominant retinitis pigmentosa (RP), for which more than 50 disease-related mutations have been identified to date. Mutation-independent suppression, using RNA interference (RNAi), together with simultaneous expression of a replacement rds gene (r-rds, which has been altered to escape suppression but nevertheless encodes wild-type protein) has been explored in COS-7 cells and mouse retinal explants. The efficacy of small interfering and short hairpin RNAs (si/shRNAs) silencing mouse rds, and the function of r-rds (containing degenerate substitutions in the RNAi target sequence) were analyzed at transcript (RT-PCR) and protein (ELISA) levels in COS-7 cells. "Dual-" and "triple-expression" constructs carrying the shRNA suppressor and the marker EGFP with or without the r-rds cassette were electroporated in vitro into retinal explants from 1-day-old pups. The retinae were dissociated at day 14, and transduced cells were FACS-sorted using the coexpressed EGFP marker and analyzed by RT-PCR. si/shRNAs decreased rds mRNA and protein expression by up to 82%, while r-rds was protected from suppression in COS-7 cells. Similarly, efficient RNAi-mediated suppression of endogenous rds was detected in retinal explants, while concomitant rescue of r-rds was also achieved. These data validate the concept of RNAi-based suppression coupled with replacement technology for the development of therapies targeting RDS-linked autosomal-dominant RP, and suggest that such approaches could potentially be used for other autosomal-dominant diseases with similarly extensive intragenic heterogeneity.
authors.affiliation: Department of Genetics, Trinity College Dublin, Dublin, Ireland. palfia@tcd.ie
authors.forename: Arpad
authors.initials: A
authors.lastname: Palfi
authors.forename: Marius
authors.initials: M
authors.lastname: Ader
authors.forename: Anna-Sophia
authors.initials: AS
authors.lastname: Kiang
authors.forename: Sophia
authors.initials: S
authors.lastname: Millington-Ward
authors.forename: Gerry
authors.initials: G
authors.lastname: Clark
authors.forename: Mary
authors.initials: M
authors.lastname: O'Reilly
authors.forename: Helena P
authors.initials: HP
authors.lastname: McMahon
authors.forename: Paul F
authors.initials: PF
authors.lastname: Kenna
authors.forename: Peter
authors.initials: P
authors.lastname: Humphries
authors.forename: G Jane
authors.initials: GJ
authors.lastname: Farrar
mesh_terms: D000818:Animals; D019556:COS Cells; D002469:Cell Separation; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D020868:Gene Silencing; D007381:Intermediate Filament Proteins / Q000235:genetics* / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000502:physiology*; D051379:Mice; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000502:physiology*; D064531:Peripherins; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D012160:Retina / Q000378:metabolism*; D012174:Retinitis Pigmentosa / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D007381:Intermediate Filament Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D064531:Peripherins; C578996:Prph2 protein, mouse; D034741:RNA, Small Interfering