Rescue of dystrophin expression in mdx mouse muscle by RNA/DNA oligonucleotides

Chimeric RNA/DNA oligonucleotides ("chimeraplasts") have been shown to indu ce single base alterations in genomic DNA both in vitro and in vivo. The md x mouse strain has a point mutation in the dystrophin gene, the consequence of which is a muscular dystrophy resulting from deficiency of the dystroph in protein in skeletal muscle. To test the feasibility of chimeraplast-medi ated gene therapy for muscular dystrophies, we used a chimeraplast (designa ted "MDX1") designed to correct the point mutation in the dystrophin gene i n mdx mice. After direct injection of MDX1 into muscles of mdx mice, immuno histochemical analysis revealed dystrophin-positive fibers clustered around the injection site. Two weeks after single injections into tibialis anteri or muscles, the maximum number of dystrophin-positive fibers (approximately 30) in any muscle represented 1-2% of the total number of fibers in that m uscle, Ten weeks after single injections, the range of the number of dystro phin-positive fibers was similar to that seen after 2 wk, suggesting that t he expression was stable, as would be predicted for a gene-conversion event . Staining with exon-specific antibodies showed that none of these were "re vertant fibers." Furthermore, dystrophin from MDX1-injected muscles was ful l length by immunoblot analysis. No dystrophin was detectable by immunohist ochemical or immunoblot analysis after control chimeraplast injections. Fin ally, reverse transcription-PCR analysis demonstrated the presence of trans cripts with the wild-type dystrophin sequence only in mdx muscles injected with MDX1 chimeraplasts. These results provide the foundation for further s tudies of chimeraplast-mediated gene therapy as a therapeutic approach to m uscular dystrophies and other genetic disorders of muscle.