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Mutations in the dystrophin (DMD) gene and consequent loss of dystrophin cause Duchenne muscular dystrophy (DMD). A promising therapy for DMD, single-exon skipping using antisense phosphorodiamidate morpholino oligomers (PMOs), currently confronts major issues in that an antisense drug induces the production of functionally undefined dystrophin and may not be similarly efficacious among patients with different mutations. Accordingly, the applicability of this approach is limited to out-of-frame mutations. Here, using an exon-skipping efficiency predictive tool, we designed three different PMO cocktail sets for exons 45–55 skipping aiming to produce a dystrophin variant with preserved functionality as seen in milder or asymptomatic individuals with an in-frame exons 45–55 deletion. Of them, the most effective set was composed of select PMOs that each efficiently skips an assigned exon in cell-based screening. These combinational PMOs fitted to different deletions of immortalized DMD patient muscle cells significantly induced exons 45–55 skipping with removing 3, 8, or 10 exons and dystrophin restoration as represented by western blotting. In vivo skipping of the maximum 11 human DMD exons was confirmed in humanized mice. The finding indicates that our PMO set can be used to create mutation-tailored cocktails for exons 45–55 skipping and treat over 65% of DMD patients carrying out-of-frame or in-frame deletions.

International audience; Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder, is caused by mutations in the dystrophin (DMD) gene. Exon skipping is a therapeutic approach that uses antisense oligonucleotides (AOs) to modulate splicing and restore the reading frame, leading to truncated, yet functional protein expression. In 2016, the US Food and Drug Administration (FDA) conditionally approved the first phosphorodiamidate morpholino oligomer (morpholino)-based AO drug, eteplirsen, developed for DMD exon 51 skipping. Eteplirsen remains controversial with insufficient evidence of its therapeutic effect in patients. We recently developed an in silico tool to design antisense morpholino sequences for exon skipping. Here, we designed morpholino AOs targeting DMD exon 51 using the in silico tool and quantitatively evaluated the effects in immortalized DMD muscle cells in vitro. To our surprise, most of the newly designed morpholinos induced exon 51 skipping more efficiently compared with the eteplirsen sequence. The efficacy of exon 51 skipping and rescue of dystrophin protein expression were increased by up to more than 12-fold and 7-fold, respectively, compared with the eteplirsen sequence. Significant in vivo efficacy of the most effective morpholino, determined in vitro, was confirmed in mice carrying the human DMD gene. These findings underscore the importance of AO sequence optimization for exon skipping.

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder primarily affecting boys, which is caused by mutations in the dystrophin (DMD) gene and the subsequent lack of dystrophin protein. Patients exhibit progressive degeneration and weakness in bodywide muscles, leading to death due to respiratory or cardiac failure. Currently, a most promising therapeutic approach is exon skipping with antisense oligonucleotides (AOs), which can correct the reading frame of mutant dystrophin mRNA and restore truncated-yet-functional dystrophin protein. Dystrophic mouse and dog models have been widely used for developing exon skipping therapies, as well as for improving scientific understanding of DMD pathogenesis; however, currently available animal models have a few limitations: First, they do not always share similar disease phenotypes with DMD patients (e.g., milder symptoms). Second, their available mutation patterns are limited, reducing applicability for research (importantly, AO drugs for exon skipping need to be developed according to mutation patterns). Lastly, mice and dogs are far from humans in terms of their anatomy, physiology, and genetics, which could prove a hurdle to interpreting and extrapolating treatment effects from animal to human. Although the wide variety of currently available animal models may partially compensate for some drawbacks, a more suitable animal model is most desirable to overcome them. Here, we generated a new DMD animal model of miniature pigs. Exon 52 deletion, one of the most common mutation patterns in the human DMD gene, was created in the pig dystrophin gene using a combination technique involving somatic cell nuclear transfer and gene targeting. The pig model systemically produced out-of-frame dystrophin mRNA transcripts lacking exon 52. Accordingly, no expression of dystrophin protein was observed in bodywide muscles, including the heart. Serum creatine kinase levels were dramatically increased, accompanied with histological deterioration in the muscles as observed in patients with DMD. We also tested AO-mediated exon skipping targeting exon 51 or 53 in primary skeletal muscle cells derived from the dystrophic pig model. The exon 52 deletion mutation is correctable by either exon 51 or 53 skipping which approach is theoretically applicable to the largest proportion of DMD patients (14% and 10%, respectively). AO sequences for skipping porcine exon 51 or 53 were designed as analogs of human AO sequences identified with our in silico AO design tool (Echigoya et al, PLoS One 2015 Mar 27;10(3):e0120058) and current antisense drugs under clinical trials. We successfully induced exon 51 or 53-excised transcripts in vitro with primary skeletal muscle cells derived from the exon 52-deleted transgenic pigs. We are currently planning in vivo exon skipping in the new pig model. Therapeutic outcomes derived from the DMD pig model could be more reliably extrapolated to human patients, facilitating development of novel AO drugs and translation into human clinical trials.