Functional repair of a mutant chloride channel using a trans-splicing ribozyme.

Journal Article (Journal Article)

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.

Full Text

Duke Authors

Cited Authors

  • Rogers, CS; Vanoye, CG; Sullenger, BA; George, AL

Published Date

  • December 2002

Published In

Volume / Issue

  • 110 / 12

Start / End Page

  • 1783 - 1789

PubMed ID

  • 12488428

Pubmed Central ID

  • PMC151654

International Standard Serial Number (ISSN)

  • 0021-9738

Digital Object Identifier (DOI)

  • 10.1172/JCI16481


  • eng

Conference Location

  • United States