Restoration of dystrophin expression in mdx muscle cells by chimeraplast-mediated exon skipping
Authors: Thomas A Rando
Carmen Bertoni
Catherine Lau
Journal: Human Molecular Genetics (2003)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Duchenne muscular dystrophy; DMD; dystrophin gene mutations; exon skipping; chimeraplasts
Affiliations: Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305-5235, USA.

Abstract:  The most common types of dystrophin gene mutations that cause Duchenne muscular dystrophy (DMD) are large deletions that result in a shift of the translational reading frame. Such mutations generally lead to a complete absence of dystrophin protein in the muscle cells of affected individuals. Any therapeutic modality that could restore the reading frame would have the potential to substantially reduce the severity of the disease by allowing the production of an internally deleted, but partially functional, dystrophin protein as occurs in Becker muscular dystrophy (BMD). One approach to restoring the reading frame would be to alter the splicing of the pre-mRNA to produce an in-frame transcript. We have tested the ability of chimeric RNA/DNA oligonucleotides (chimeraplasts) to alter key bases in specific splice sequences in the dystrophin gene to induce exon skipping. Using cells from the mdx mouse as a model system, we show that chimeraplast-mediated base conversion in the intron 22/exon 23 splice junction induces alternative splicing and the production of in-frame transcripts. Interestingly, multiple alternative transcripts were induced by this targeted splice site mutation. Direct sequencing indicated that several of these were predicted to produce in-frame dystrophin transcripts with internal deletions. Indeed, multiple forms of dystrophin protein were observed by western blot analysis, and the functionality of the products was demonstrated by the restoration of expression and localization of a dystrophin-associated protein, alpha-dystroglycan, in differentiated cells. These data demonstrate that chimeraplasts can induce exon skipping by altering splice site sequences at the genomic level. As such, chimeraplast-mediated exon skipping has the potential to be used to transform a severe DMD phenotype into a much milder BMD phenotype. 

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 https: science.sciencemag.org content 298 5601 2116.full  Most researchers who tried chimeraplasty failed from the beginning. Science spoke to researchers from over 30 laboratories that had tried the RDOs and failed to produce evidence that they could target and correct dysfunctional genes, either in vitro or in vivo. Researchers at biotech companies such as Epoch Biosciences, Isis Pharmaceuticals, Millennium Pharmaceuticals, and Lexicon Genetics all failed to get chimeraplasty to work in their labs. Experienced gene-targeting researchers at MIT s Whitehead Institute, NIH (including in Blaese s own laboratory), Maine s Jackson Laboratory, and Sweden s Karolinska Institute also saw no effects. Even members of Kimeragen s own scientific advisory panel, such as Baylor s John Wilson, tried it and failed. u201cUnder our conditions,u201d Wilson says, u201cwe found no correction above background.u201d  The great majority of researchers interviewed by Science say they find the negative results, even though unpublished, more persuasive than the positive ones because they come from independent labs with considerably more experience in gene repair and gene therapy than those that succeeded have. u201cThe people I trusted, the ones I polled who are really good,u201d says Neal Copeland, for instance, director of the Mammalian Genetics Laboratory at the National Cancer Institute, u201cinvested a lot of time, and none of them got it to work.u201d ,