Alternate translational initiation and amelioration of phenotype in the DMD gene

Abstract Nonsense mutations within exon 1 of DMD do not result in severe DMD but instead lead to very mild BMD, due to an alternative initiation of translation at AUG codons within in exon 6. This leads to translation of a nearly full-length but N-terminal truncated dystrophin lacking the first half of the canonical actin binding domain 1 (ABD1). We have identified the motif encoded in exon 5 that recruits ribosomes for alternate translational initiation within exon 6, and using a dual luciferase reporter system we have determined that this motif is selectively activated in muscle cell lines but not fibroblasts or HEK cells. Our data suggest that this motif is an IRES (internal ribosome entry site) as complementary experiments have ruled out any promoter activity or aberrant splicing. The exceedingly mild clinical features of patients with an exon 1 DMD founder allele (p.Trp3X) suggest that the product of this IRES initiation is a highly functional protein. We are therefore exploring different strategies to induce IRES utilisation for therapeutic purposes in patients with mutations in exons 1 through 4. One of them is based on forced synthesis of this IRES protein isoform that can be induced by exon 2 skipping. This leads to a frameshift and premature stop codon in exon 3, which thereby force the use of the IRES. We have developed four different antisense sequences for efficient skipping of exon 2 that are incorporated into a short U7 RNA derivative that was previously used in other studies to induce efficient exon skipping in DMD . We are currently testing these antisense constructs in patient cells carrying mutations in the first exons to evaluate the potential positive benefit of this out of frame skipping strategy.