title: Functional repair of a mutant chloride channel using a trans-splicing ribozyme.
abstract: RNA repair has been proposed as a novel gene-based therapeutic strategy. Modified Tetrahymena group I intron ribozymes have been used to mediate trans-splicing of therapeutically relevant RNA transcripts, but the efficiency of the ribozyme-mediated RNA repair process has not been determined precisely and subsequent restoration of protein function has been demonstrated only by indirect means. We engineered a ribozyme that targets the mRNA of a mutant canine skeletal muscle chloride channel (cClC-1) (mutation T268M in ClC-1 causing myotonia congenita) and replaces the mutant-containing 3' portion by trans-splicing the corresponding 4-kb wild-type sequence. Repair efficiency assessed by quantitative RT-PCR was 1.2% +/- 0.1% in a population of treated cells. However, when chloride channel function was examined in single cells, a wide range of electrophysiological activity was observed, with 18% of cells exhibiting significant functional restoration and some cells exhibiting complete rescue of the biophysical phenotype. These results indicate that RNA repair can restore wild-type protein activity and reveal considerable cell-to-cell variability in ribozyme-mediated trans-splicing reaction efficiency.
authors.affiliation: Division of Genetic Medicine, Department of Medicine, Vanderbilt University, Nashville, Tennessee, USA.
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Rogers
authors.forename: Carlos G
authors.initials: CG
authors.lastname: Vanoye
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Sullenger
authors.forename: Alfred L
authors.initials: AL
authors.lastname: George
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D018118:Chloride Channels / Q000235:genetics* / Q000378:metabolism*; D004285:Dogs; D005091:Exons; D006801:Humans; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D009154:Mutation; D009224:Myotonia Congenita / Q000235:genetics; D018408:Patch-Clamp Techniques; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D016337:RNA, Catalytic / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013768:Tetrahymena / Q000235:genetics; D020040:Trans-Splicing
chemical_list: C086321:CLC-1 channel; D018118:Chloride Channels; D015800:Protozoan Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger