Dystrophin Gene Repair in mdx Muscle Precursor Cells In Vitro and In Vivo Mediated by RNA-DNA Chimeric Oligonucleotides
- 10 April 2002
- journal article
- research article
- Published by Mary Ann Liebert Inc in Human Gene Therapy
- Vol. 13 (6) , 707-718
- https://doi.org/10.1089/104303402317322276
Abstract
Point mutations in the dystrophin gene cause dystrophin deficiency and muscular dystrophy in the mdx mouse and a subset of patients with Duchenne muscular dystrophy. As an approach to gene therapy for muscular dystrophies due to point mutations, we have studied the ability of RNA-DNA chimeric oligonucleotides (chimeraplasts) to induce repair of the dystrophin gene in mdx mice. We have previously demonstrated that targeting chimeraplasts can repair the exon 23 point mutation in differentiated myofibers in vivo after intramuscular injection. For long-term benefit to patients with muscular dystrophy, any gene therapy technology must target not only differentiated myofibers but also undifferentiated muscle precursor cells that are involved in ongoing muscle repair. The focus of the current studies was to test whether chimeraplasts could repair the dystrophin mutation in mdx muscle precursor cells. Initial studies were done by transfecting a targeting chimeraplast into mdx myoblasts in vitro. Gene repair was demonstrated at the DNA, RNA, and protein levels in these cells, whereas treatment of the cells with a control chimeraplast resulted in no gene correction. After differentiation of mdx cells that had been treated with a targeting chimeraplast, immunoblot analysis demonstrated full-length dystrophin expression. By quantitative analysis of independent cultures, the amount of dystrophin expressed ranged from 2 to 15% of that expressed in wild-type cells, providing a measure of the efficacy of gene conversion in vitro. To extend the assessment to muscle precursor cells in vivo, we injected targeting and control chimeraplasts into muscles of mdx mice. When muscle precursor cells were subsequently derived from muscles injected with a targeting chimeraplast, we found that gene repair had occurred in these cells as well. These results, taken together, further demonstrate that chimeraplast-mediated gene repair may be effective as an approach to gene therapy for muscular dystrophies due to point mutations.Keywords
This publication has 34 references indexed in Scilit:
- Failure to correct murine muscular dystrophyNature, 2001
- The DNA strand of chimeric RNA/DNA oligonucleotides can direct gene repair/conversion activity in mammalian and plant cell-free extractsNucleic Acids Research, 2000
- In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotideNature Biotechnology, 2000
- Localized in vivo genotypic and phenotypic correction of the albino mutation in skin by RNA-DNA oligonucleotideNature Biotechnology, 2000
- Nucleotide Exchange in Genomic DNA of Rat Hepatocytes Using RNA/DNA OligonucleotidesPublished by Elsevier ,1999
- Targeted gene repair directed by the chimeric RNA/DNA oligonucleotide in a mammalian cell-free extractNucleic Acids Research, 1999
- Stable and inheritable changes in genotype and phenotype of albino melanocytes induced by an RNA-DNA oligonucleotideNature Biotechnology, 1998
- Muscle Regeneration by Bone Marrow-Derived Myogenic ProgenitorsScience, 1998
- The mdx-amplification-resistant mutation system assay, a simple and rapid polymerase chain reaction-based detection of the mdx alleleMuscle & Nerve, 1996
- Duchenne muscular dystrophy: Deficiency of dystrophin at the muscle cell surfaceCell, 1988