Search Results (4)

Duchenne muscular dystrophy (DMD) is the most frequent genetic myopathy in childhood and leads to progressive muscle atrophy, weakness, and premature death. So far, there is no curative treatment available. Therapeutic development from bench to bedside takes time, and promising therapies need to be tested in suitable preclinical animal models prior to clinical trials in DMD patients. Existing mouse and dog models are limited with regard to the comparability of the clinical phenotype and the underlying mutation. Therefore, our group established a tailored large animal model of DMD, the DMD pig, mirroring the human size, anatomy, and physiology. For testing novel approaches, we developed a corresponding in vitro model, facilitating preclinical testing for toxicity, dosing, and efficacy, which we describe here. We first extracted primary muscle cells from wild-type and DMD pigs of different age groups and characterized those cells, then improved their differentiation process for identification of dystrophin and utrophin in myotubes. Our porcine in vitro model represents an important step for the development of novel therapeutic approaches, which should be validated further to minimize the need for living animals for bioassays, and thereby support the ‘3R’ (replace, reduce, refine) principle, as fewer animals have to be raised and treated for preclinical trials. Full article

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the DMD gene and the subsequent lack of dystrophin protein. Recently, phosphorodiamidate morpholino oligomer (PMO)-antisense oligonucleotides (ASOs) targeting exon 51 or 53 to reestablish the DMD reading frame have received regulatory approval as commercially available drugs. However, their applicability and efficacy remain limited to particular patients. Large animal models and exon skipping evaluation are essential to facilitate ASO development together with a deeper understanding of dystrophinopathies. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, we generated a Yucatan miniature pig model of DMD with an exon 52 deletion mutation equivalent to one of the most common mutations seen in patients. Exon 52-deleted mRNA expression and dystrophin deficiency were confirmed in the skeletal and cardiac muscles of DMD pigs. Accordingly, dystrophin-associated proteins failed to be recruited to the sarcolemma. The DMD pigs manifested early disease onset with severe bodywide skeletal muscle degeneration and with poor growth accompanied by a physical abnormality, but with no obvious cardiac phenotype. We also demonstrated that in primary DMD pig skeletal muscle cells, the genetically engineered exon-52 deleted pig DMD gene enables the evaluation of exon 51 or 53 skipping with PMO and its advanced technology, peptide-conjugated PMO. The results show that the DMD pigs developed here can be an appropriate large animal model for evaluating in vivo exon skipping efficacy. Full article

Tenderness is one of the most important meat quality traits and it can be measured through shear force with the Warner–Bratzler test. In the current study, we use the RNA-seq technique to analyze the transcriptome of Longissimus dorsi (LD) muscle in two groups of Iberian pigs (Tough and Tender) divergent for shear force breeding values. We identified 200 annotated differentially expressed genes (DEGs) and 245 newly predicted isoforms. The RNAseq expression results of 10 genes were validated with quantitative PCR (qPCR). Functional analyses showed an enrichment of DE genes in biological processes related to proteolysis (CTSC, RHOD, MYH8, ACTC1, GADD45B, CASQ2, CHRNA9 and ANKRD1), skeletal muscle tissue development (ANKRD1, DMD, FOS and MSTN), lipid metabolism (FABP3 and PPARGC1A) and collagen metabolism (COL14A1). The upstream analysis revealed a total of 11 transcription regulatory factors that could regulate the expression of some DEGs. Among them, IGF1, VGLL3 and PPARG can be highlighted since they regulate the expression of genes involved in biological pathways that could affect tenderness. The experiment revealed a set of candidate genes and regulatory factors suggestive to search polymorphisms that could be incorporated in a breeding program for improving meat tenderness. Full article

Dystrophinopathy, including Duchenne muscle dystrophy (DMD) and Becker muscle dystrophy (BMD) is an incurable X-linked hereditary muscle dystrophy caused by a mutation in the DMD gene in coding dystrophin. Advances in further understanding DMD/BMD for therapy are expected. Studies on mdx mice and dogs with muscle dystrophy provide limited insight into DMD disease mechanisms and therapeutic testing because of the different pathological manifestations. Miniature pigs share similar physiology and anatomy with humans and are thus an excellent animal model of human disease. Here, we successfully achieved precise DMD targeting in Chinese Diannan miniature pigs by co-injecting zygotes with Cas9 mRNA and sgRNA targeting DMD. Two piglets were obtained after embryo transfer, one of piglets was identified as DMD-modified individual via traditional cloning, sequencing and T7EN1 cleavage assay. An examination of targeting rates in the DMD-modified piglet revealed that sgRNA:Cas9-mediated on-target mosaic mutations were 70% and 60% of dystrophin alleles in skeletal and smooth muscle, respectively. Meanwhile, no detectable off-target mutations were found, highlighting the high specificity of genetic modification using CRISPR/Cas9. The DMD-modified piglet exhibited degenerative and disordered phenotypes in skeletal and cardiac muscle, and declining thickness of smooth muscle in the stomach and intestine. In conclusion, we successfully generated myopathy animal model by modifying the DMD via CRISPR/Cas9 system in a miniature pig. Full article