Duchenne Muscular Dystrophy, DMD, one of the most common fatal genetic diseases, is caused by the absence of dystrophin protein. This protein... Show moreDuchenne Muscular Dystrophy, DMD, one of the most common fatal genetic diseases, is caused by the absence of dystrophin protein. This protein is coded by the largest gene in the human genome, which has 79 exons and spans 2.4 Mbp. DMD affects 1 in 3600 boys and the average life expectancy of patients is 25 years. The most common type of defects leading to DMD are large exonic deletions that juxtapose remaining exons of incompatible phases, causing a frameshift. Inevitably this introduces a nonsense mutation (i.e. a stop codon) in the frame shifted exons and thus protein truncation. A related but milder condition called Becker’s Muscular Dystrophy, BMD, results when deletions are in-frame and so allow for the production of some, albeit modified, dystrophin protein. While there is no treatment available for DMD the recent clinical trials involving exon skipping suggest this will be highly promising treatment option. Exon skipping therapy is a strategy that uses anti-sense oligonucleotide analogs, AONs, to induce skipping of additional exons during mRNA maturation and thus restore the reading frame. This allows dystrophin translation to continue to its normal C-terminus, albeit with an internal deletion edit corresponding to both the original deletion, and newly skipped exon, which results in expression of protein thus converting the severe form of DMD to a milder form BMD. Frameshift causing deletions require exons that begin and end in different phases, and DMD deletions thus are clustered where such exons are located in, two so called ‘hotspots’ in the gene. The first of these occur in the region from exon 11-22 and is known as Hotspot 1. In many cases, DMD defects that are amenable to exon skip repair can be repaired in two or more alternative fashions by skipping of alternative exons. It is thought that the differences in severity of BMD, can range from being nearly as debilitating as DMD, to xi nearly benign, are at least in part related to the nature of the defect and its impact on the protein’s structure. Thus we are producing 5 different exon edited proteins from hotspot 1 rod region and assessing them for structure and stability as compared to unskipped fully functional dystrophin, to determine which edits hold the most promise of producing a well formed and stable edit. M.S. in Biology, December 2013 Show less
