Functional repair of a mutant chloride channel using a trans-splicing ribozyme.
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.
Rogers
Christopher S
CS
Division of Genetic Medicine, Department of Medicine, Vanderbilt University, Nashville, Tennessee, USA.
Vanoye
Carlos G
CG
Sullenger
Bruce A
BA
George
Alfred L
AL
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
C086321:CLC-1 channel; D018118:Chloride Channels; D015800:Protozoan Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger