title: Rescue of dystrophin expression in mdx mouse muscle by RNA/DNA oligonucleotides.
abstract: Chimeric RNA/DNA oligonucleotides ("chimeraplasts") have been shown to induce single base alterations in genomic DNA both in vitro and in vivo. The mdx mouse strain has a point mutation in the dystrophin gene, the consequence of which is a muscular dystrophy resulting from deficiency of the dystrophin protein in skeletal muscle. To test the feasibility of chimeraplast-mediated gene therapy for muscular dystrophies, we used a chimeraplast (designated "MDX1") designed to correct the point mutation in the dystrophin gene in mdx mice. After direct injection of MDX1 into muscles of mdx mice, immunohistochemical analysis revealed dystrophin-positive fibers clustered around the injection site. Two weeks after single injections into tibialis anterior muscles, the maximum number of dystrophin-positive fibers (approximately 30) in any muscle represented 1-2% of the total number of fibers in that muscle. Ten weeks after single injections, the range of the number of dystrophin-positive fibers was similar to that seen after 2 wk, suggesting that the expression was stable, as would be predicted for a gene-conversion event. Staining with exon-specific antibodies showed that none of these were "revertant fibers." Furthermore, dystrophin from MDX1-injected muscles was full length by immunoblot analysis. No dystrophin was detectable by immunohistochemical or immunoblot analysis after control chimeraplast injections. Finally, reverse transcription-PCR analysis demonstrated the presence of transcripts with the wild-type dystrophin sequence only in mdx muscles injected with MDX1 chimeraplasts. These results provide the foundation for further studies of chimeraplast-mediated gene therapy as a therapeutic approach to muscular dystrophies and other genetic disorders of muscle.
authors.affiliation: Department of Neurology and Neurological Sciences, Palo Alto Veterans Affairs Medical Center and Stanford University School of Medicine, Palo Alto, CA 94305, USA. rando@stanford.edu
authors.forename: T A
authors.initials: TA
authors.lastname: Rando
authors.forename: M H
authors.initials: MH
authors.lastname: Disatnik
authors.forename: L Z
authors.initials: LZ
authors.lastname: Zhou
mesh_terms: D000818:Animals; D001483:Base Sequence; D002678:Chimera; D016189:Dystrophin / Q000235:genetics*; D005785:Gene Conversion; D005786:Gene Expression Regulation / Q000187:drug effects; D015316:Genetic Therapy / Q000379:methods; D051379:Mice; D008810:Mice, Inbred C57BL; D018101:Mice, Inbred mdx; D008969:Molecular Sequence Data; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism*; D009137:Muscular Dystrophy, Animal / Q000235:genetics* / Q000628:therapy; D009841:Oligonucleotides / Q000737:chemistry / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D016189:Dystrophin; D009841:Oligonucleotides