CLONING AND CHARACTERIZATION OF HOTSPOT II EXON EDITED DYSTROPHIN RODS
Duchenne muscular dystrophy, DMD, is an X–link recessive disorder with an incidence of 1 in 3500 male births worldwide. This fatal condition has no effective treatment, but due to its high incidence and severity, several strategies are the subject of on-going clinical trials including gene therapy (delivery of replacement genes via viral vector systems) and exon skipping (administration of therapeutic compounds to mask certain exons and so repair the defective gene). Both of these approaches result in the production of modified dystrophin proteins with deletions in the central rod region. It is unknown how such edits will affect protein structure, although it has been shown that the nature of the edit is related to clinical severity. DMD defects are non-randomly distributed along the gene, being clustered in two regions: the so-called hotspot region I (Exons 11 – 22) and the hotspot region II (Exons 45 – 55). We are producing alternative exon skipped proteins in the hotspot II region and are characterizing them with respect to biophysical and biochemical stability by thermal denaturation and proteinase challenge studies in order to determine which of these potential edits are most similar to native, undamaged dystrophin.