Elusive sources of variability of dystrophin rescue by exon skipping

Abstract

BackgroundSystemic delivery of anti-sense oligonucleotides to Duchenne muscular dystrophy (DMD) patients to induce de novo dystrophin protein expression in muscle (exon skipping) is a promising therapy. Treatment with Phosphorodiamidate morpholino oligomers (PMO) lead to shorter de novo dystrophin protein in both animal models and DMD boys who otherwise lack dystrophin; however, restoration of dystrophin has been observed to be highly variable. Understanding the factors causing highly variable induction of dystrophin expression in pre-clinical models would likely lead to more effective means of exon skipping in both pre-clinical studies and human clinical trials.MethodsIn the present study, we investigated possible factors that might lead to the variable success of exon skipping using morpholino drugs in the mdx mouse model. We tested whether specific muscle groups or fiber types showed better success than others and also correlated residual PMO concentration in muscle with the amount of de novo dystrophin protein 1 month after a single high-dose morpholino injection (800 mg/kg). We compared the results from six muscle groups using three different methods of dystrophin quantification: immunostaining, immunoblotting, and mass spectrometry assays.ResultsThe triceps muscle showed the greatest degree of rescue (average 38±28 % by immunostaining). All three dystrophin detection methods were generally concordant for all muscles. We show that dystrophin rescue occurs in a sporadic patchy pattern with high geographic variability across muscle sections. We did not find a correlation between residual morpholino drug in muscle tissue and the degree of dystrophin expression.ConclusionsWhile we found some evidence of muscle group enhancement and successful rescue, our data also suggest that other yet-undefined factors may underlie the observed variability in the success of exon skipping. Our study highlights the challenges associated with quantifying dystrophin in clinical trials where a single small muscle biopsy is taken from a DMD patient.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0070-6) contains supplementary material, which is available to authorized users.

Morpholino‐induced exon skipping stimulates cell‐mediated and humoral responses to dystrophin in mdx mice

“…Exon-skipping efficiency and dystrophin restoration were assessed after chronic PMO administration, in which mdx mice received high-dose intravenous delivery of 800 mg/kg PMO at monthly intervals for 6 months. PMO-induced dystrophin protein expression was verified by Western blotting (WB) in two typically high-responding muscle groups: triceps and gastrocnemius [20]. Dystrophin rescue occurred in all PMO-treated mice, even though the extent of the dystrophin expression varied widely among the individual mice and between the two muscle groups [20].…”

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

“…Ten more characterized duplications of the exon 2 from DMD patients were collected from the literature [27][28][29][30] and represented with a custom track ( Figure 1A). …”

“…18 These tools have been used to create new animal models [19][20][21] and, recently, in vitro and in vivo therapeutic strategies for DMD [22][23][24][25] aimed at restoring disrupted reading frames by deleting instead of skipping mutated/ out-of-frame exons. Compared to AON-mediated exon skipping, which involves repeated injection of the therapeutic molecules with toxicity and variable tissue uptake, 26,27 the exon deletion strategy would require only one administration of the gene-editing system to achieve full exclusion of the targeted exon. Restoration of the reading frame for some duplication by excising one copy of the duplicated exon could permit expression of a normal dystrophin transcript, as recently demonstrated for a multi-exonic duplication of 139 kb 28 and allow synthesis of a normal dystrophin isoform as compared to the restoration of the reading frame around the more common deletions that can generate a less severe, Becker muscular dystrophy (BMD)-like phenotype.…”

Correction of the Exon 2 Duplication in DMD Myoblasts by a Single CRISPR/Cas9 System

“…These tools have been used to create new animal models19, 20, 21 and, recently, in vitro and in vivo therapeutic strategies for DMD22, 23, 24, 25 aimed at restoring disrupted reading frames by deleting instead of skipping mutated/out-of-frame exons. Compared to AON-mediated exon skipping, which involves repeated injection of the therapeutic molecules with toxicity and variable tissue uptake,26, 27 the exon deletion strategy would require only one administration of the gene-editing system to achieve full exclusion of the targeted exon. Restoration of the reading frame for some duplication by excising one copy of the duplicated exon could permit expression of a normal dystrophin transcript, as recently demonstrated for a multi-exonic duplication of 139 kb 28 and allow synthesis of a normal dystrophin isoform as compared to the restoration of the reading frame around the more common deletions that can generate a less severe, Becker muscular dystrophy (BMD)-like phenotype.…”