Andrew R. Findlay

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

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 has been shown to result from alternative translation initiation beginning in DMD exon 6 that leads to expression of a highly functional N-truncated dystrophin. Here we demonstrate that this isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid inducible. We confirmed IRES activity by both peptide sequencing and ribosome profiling in muscle from individuals with minimal symptoms despite the presence of truncating mutations. We generated a truncated reading frame upstream of the IRES by exon skipping, which led to synthesis of a functional N-truncated isoform in both human subject–derived cell lines and in a new DMD mouse model, where expression of the truncated isoform protected muscle from contraction-induced injury and corrected muscle force to the same level as that observed in control mice. These results support a potential therapeutic approach for patients with mutations within the 5′ exons of DMD.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.

Clinical phenotypes as predictors of the outcome of skipping around DMD exon 45

Exon‐skipping therapies aim to convert Duchenne muscular dystrophy (DMD) into less severe Becker muscular dystrophy (BMD) by altering pre‐mRNA splicing to restore an open reading frame, allowing translation of an internally deleted and partially functional dystrophin protein. The most common single exon deletion—exon 45 (Δ45)—may theoretically be treated by skipping of either flanking exon (44 or 46). We sought to predict the impact of these by assessing the clinical severity in dystrophinopathy patients.

Homozygous recessive MYH2 mutation mimicking dominant MYH2 associated myopathy

Mutations in MYH2 that encodes myosin heavy chain IIa cause both dominant and recessively inherited myopathies. Patients with dominantly inherited MYH2 missense mutations present with ophthalmoplegia and progressive proximal limb weakness. Muscle biopsy reveals rimmed vacuoles and inclusions, prompting this entity to initially be described as hereditary inclusion body myopathy 3. In contrast, patients with recessive MYH2 mutations have early onset, non-progressive, diffuse weakness and ophthalmoplegia. Muscle biopsy reveals near or complete absence of type 2A fibers with no vacuole or inclusion pathology. We describe a patient with childhood onset ophthalmoplegia, progressive proximal muscle weakness beginning in adolescence, and muscle biopsy with myopathic changes and rimmed vacuoles. Although this patients disease course and histopathology is consistent with dominant MYH2 mutations, whole exome sequencing revealed a c.737 G>A p.Arg246His homozygous MYH2 variant. These findings expand the clinical and pathologic phenotype of recessive MYH2 myopathies.

Corrigendum: Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice (Nature Medicine (2014))

Nat Med. 20, 992–1000 (2014); doi:10.1038/nm.3628; corrected 25 August 2014; corrected after print 13 March 2015 In the version of this article initially published, three participants of the study were not included as co-authors. Also, one of the individuals mentioned in the Acknowledgments section of the report was incorrectly included and thus has been removed at their request, and the name of another individual mentioned in the Acknowledgments was originally misspelled (“Fabbri” should have been “Fabris”).

Erratum: Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice: (Nature Medicine (2014) 20 (992-1000) DOI:10.1038/nm.3628)

In the version of this article initially published online, the third sentence of the Abstract read “Gene expression analysis identified higher expression of JAK-STAT signaling targets in 3-week-old relative to 18-month-old mice,” when it should have read “Gene expression analysis identified higher expression of JAK-STAT signaling targets in 18-month-old relative to 3-week-old mice.” The error has been corrected for the print, PDF and HTML versions of this article.

P.20.2 Restoration of dystrophin expression after skipping of single and double exon DMD duplications in patient-derived cell lines using antisense oligonucleotide and AAV-U7snRNA approaches

Exon skipping strategies in Duchenne muscular dystrophy (DMD) have largely been directed toward altering splicing of exons flanking out-of-frame deletions or excluding exons carrying a point mutation in order to restore an open mRNA reading frame. Translation of such a transcript results in an internally truncated yet partially functional dystrophin protein, typically associated with the milder Becker muscular dystrophy (BMD). We sought to apply exon skipping to duplication mutations of one or two exons, making use of the inherently limited efficiency of exon skipping in vivo to generate significant amounts of wild-type, full-length DMD mRNA. We developed an improved lentiviral-delivered tet-inducible MyoD construct and used it to generate stably-infected fibroblast cell lines from patients with a variety of singly (exons 2, 12, 18, 21, and 44) or doubly (exons 3–4, and 8–9) duplicated exons. Treatment of each cell line with doxycycline results in trandifferentiation into the myogenic lineage and expression of the DMD gene. Using a variety of 2’O-Me antisense oligonucleotides, significant skipping can be induced for each duplication, leading to a wild-type transcript as a major mRNA product. Furthermore, for two duplications – exon 2 (the most common single exon duplication) and the tandem duplication of exons 8–9 – we have developed AAV-U7snRNA vectors; after efficient transduction of these into the corresponding cell line, differentiation into myotubes results in restoration of dystrophin protein expression. These results confirm that this cell model is a reliable system to assess restoration of dystrophin expression, and suggest that exon skipping if a feasible approach to therapy for single or double exon duplication mutations that can result in expression of full-length protein, suggesting that personalized exon skipping may be expected to be highly beneficial for a subset of DMD patients.

T.P.17 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.