623. Dystrophin Exon 52-Deleted Pigs as a New Animal Model of Duchenne Muscular Dystrophy: Its Characterization and Potential as a Tool for Developing Exon Skipping Therapy

Abstract

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.