Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice

Abstract

Most mutations that truncate the reading frame of the DMD gene cause loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity can result from alternate translation initiation beginning in DMD exon 6 that leads to expression of a highly functional N-truncated dystrophin. This novel isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid-inducible. IRES activity is confirmed in patient muscle by both peptide sequencing and ribosome profiling. Generation of a truncated reading frame upstream of the IRES by exon skipping leads to synthesis of a functional N-truncated isoform in both patient-derived cell lines and in a new DMD mouse model, where expression protects muscle from contraction-induced injury and corrects muscle force to the same level as control mice. These results support a novel therapeutic approach for patients with mutations within the 5’ exons of DMD.

Neuronal SH-SY5Y cells use the C-dystrophin promoter coupled with exon 78 skipping and display multiple patterns of alternative splicing including two intronic insertion events

“…2). Although cryptic exon 1a introduces a premature stop codon, a recent study showed that a dystrophin isoform is produced by usage of an internal ribosome entry site within exon 5 (Wein et al 2014). Therefore, we expect that the exon 1a-inserted transcript will produce a form of dystrophin protein that harbors different N-terminal domains from those of C-dystrophin.…”

Correction of the exon 2 duplication in DMD myoblasts by a single CRISPR/Cas9 system

“…We also designed two gRNAs (one of which was 19 nt) targeting the coding sequence of the exon 2 in order to confirm the evidence that shifting the reading frame in the early codons should trigger the translational restart at exon 6 as previously demonstrated by an exon skipping approach ( Figure 1B). 12 gRNAs were named according to the following code: cr (crispr), DMD, int (intron), or ex (exon) 2 and numbered from 1 to 6 (the crDMD notation have been omitted in Figures 2E, 2F, and 3 to limit redundancy). Repeated regions and functional splicing sequences were excluded from the design.…”

“…10 Among the reported DMD mutations, duplications represent a distinct group accounting for $10%-15% of those reported in the Leiden database, 2 although their incidence may be higher, 11 Duplications have been generally neglected by therapeutic approaches. Wein et al 12 recently reported a successful attempt at inducing out-offrame skipping of exon 2, causing an alternative translation initiation in exon 6 and leading to expression of a functional N-truncated dystrophin. These results support a potential therapeutic approach for patients with mutations within the 5 0 exons of DMD, but it would be ineffective for all the other duplications.…”

The emperor's new dystrophin: finding sense in the noise

“…This is an unacceptable situation. In a forwardlooking approach, some groups have developed AOs for multi-exon skipping and to address uncommon deletions, duplications, and a range of other non-deletion DMD mutations [22,[109][110][111][112][113][114].…”