ABSTRACT Aged dystrophin-null canines are excellent models for studying experimental therapies for Duchenne muscular dystrophy, a lethal muscle disease caused by dystrophin deficiency. To establish the baseline, we studied the extensor carpi ulnaris (ECU) muscle in 15 terminal age (3-year-old) male affected dogs and 15 age/sex-matched normal dogs. Affected dogs showed histological and anatomical hallmarks of dystrophy, including muscle inflammation and fibrosis, myofiber size variation and centralized myonuclei, as well as a significant reduction of muscle weight, muscle-to-body weight ratio and muscle cross-sectional area. To rigorously characterize the contractile properties of the ECU muscle, we developed a novel in situ assay. Twitch and tetanic force, contraction and relaxation rate, and resistance to eccentric contraction-induced force loss were significantly decreased in affected dogs. Intriguingly, the time-to-peak tension and half-relaxation time were significantly shortened in affected dogs. Contractile kinetics predicted an unforeseen slow-to-fast myofiber-type switch, which we confirmed at the protein and transcript level. Our study establishes a foundation for studying long-term and late-stage therapeutic interventions in dystrophic canines. The unexpected myofiber-type switch highlights the complexity of muscle remodeling in dystrophic large mammals. This article has an associated First Person interview with the first author of the paper.

ABSTRACT Dystrophin, encoded by the DMD gene on the X chromosome, stabilizes the sarcolemma by linking the actin cytoskeleton with the dystrophin-glycoprotein complex (DGC). In-frame mutations in DMD cause a milder form of X-linked muscular dystrophy, called Becker muscular dystrophy (BMD), characterized by the reduced expression of truncated dystrophin. So far, no animal model with in-frame mutations in Dmd has been established. As a result, the effect of in-frame mutations on the dystrophin expression profile and disease progression of BMD remains unclear. In this study, we established a novel rat model carrying in-frame Dmd gene mutations (IF rats) and evaluated the pathology. We found that IF rats exhibited reduced expression of truncated dystrophin in a proteasome-independent manner. This abnormal dystrophin expression caused dystrophic changes in muscle tissues but did not lead to functional deficiency. We also found that the expression of additional dystrophin named dpX, which forms the DGC in the sarcolemma, was associated with the appearance of truncated dystrophin. In conclusion, the outcomes of this study contribute to the further understanding of BMD pathology and help elucidate the efficiency of dystrophin recovery treatments in Duchenne muscular dystrophy, a more severe form of X-linked muscular dystrophy.

ABSTRACT Limb-girdle muscular dystrophy type 2C is caused by autosomal recessive mutations in the γ-sarcoglycan ( SGCG ) gene. The most common SGCG mutation is a single nucleotide deletion from a stretch of five thymine residues in SGCG exon 6 (521ΔT). This founder mutation disrupts the transcript reading frame, abolishing protein expression. An antisense oligonucleotide exon-skipping method to reframe the human 521ΔT transcript requires skipping four exons to generate a functional, internally truncated protein. In vivo evaluation of this multi-exon skipping, antisense-mediated therapy requires a genetically appropriate mouse model. The human and mouse γ-sarcoglycan genes are highly homologous in sequence and gene structure, including the exon 6 region harboring the founder mutation. Herein, we describe a new mouse model of this form of limb-girdle muscular dystrophy generated using CRISPR/Cas9-mediated gene editing to introduce a single thymine deletion in murine exon 6, recreating the 521ΔT point mutation in Sgcg . These mice express the 521ΔT transcript, lack γ-sarcoglycan protein and exhibit a severe dystrophic phenotype. Phenotypic characterization demonstrated reduced muscle mass, increased sarcolemmal leak and fragility, and decreased muscle function, consistent with the human pathological findings. Furthermore, we showed that intramuscular administration of a murine-specific multiple exon-directed antisense oligonucleotide cocktail effectively corrected the 521ΔT reading frame. These data demonstrate a molecularly and pathologically suitable model for in vivo testing of a multi-exon skipping strategy to advance preclinical development of this genetic correction approach.

ABSTRACT Recent emphasis has been placed on the role that cerebellar dysfunctions could have in the genesis of cognitive deficits in Duchenne muscular dystrophy (DMD). However, relevant genotype-phenotype analyses are missing to define whether cerebellar defects underlie the severe cases of intellectual deficiency that have been associated with genetic loss of the smallest product of the dmd gene, the Dp71 dystrophin. To determine for the first time whether Dp71 loss could affect cerebellar physiology and functions, we have used patch-clamp electrophysiological recordings in acute cerebellar slices and a cerebellum-dependent behavioral test battery addressing cerebellum-dependent motor and non-motor functions in Dp71 - null transgenic mice. We found that Dp71 deficiency selectively enhances excitatory transmission at glutamatergic synapses formed by climbing fibers (CFs) on Purkinje neurons, but not at those formed by parallel fibers. Altered basal neurotransmission at CFs was associated with impairments in synaptic plasticity and clustering of the scaffolding postsynaptic density protein PSD-95. At the behavioral level, Dp71 -null mice showed some improvements in motor coordination and were unimpaired for muscle force, static and dynamic equilibrium, motivation in high-motor demand and synchronization learning. Dp71 -null mice displayed altered strategies in goal-oriented navigation tasks, however, suggesting a deficit in the cerebellum-dependent processing of the procedural components of spatial learning, which could contribute to the visuospatial deficits identified in this model. In all, the observed deficits suggest that Dp71 loss alters cerebellar synapse function and cerebellum-dependent navigation strategies without being detrimental for motor functions.

ABSTRACT Duchenne muscular dystrophy (DMD) is an X-linked muscle-wasting disorder caused by mutations in the dystrophin gene, with an incidence of 1 in 3500 in new male births. Mdx mice are widely used as an animal model for DMD. However, these mice do not faithfully recapitulate DMD patients in many aspects, rendering the preclinical findings in this model questionable. Although larger animal models of DMD, such as dogs and pigs, have been generated, usage of these animals is expensive and only limited to several facilities in the world. Here, we report the generation of a rabbit model of DMD by co-injection of Cas9 mRNA and sgRNA targeting exon 51 into rabbit zygotes. The DMD knockout (KO) rabbits exhibit the typical phenotypes of DMD, including severely impaired physical activity, elevated serum creatine kinase levels, and progressive muscle necrosis and fibrosis. Moreover, clear pathology was also observed in the diaphragm and heart at 5 months of age, similar to DMD patients. Echocardiography recording showed that the DMD KO rabbits had chamber dilation with decreased ejection fraction and fraction shortening. In conclusion, this novel rabbit DMD model generated with the CRISPR/Cas9 system mimics the histopathological and functional defects in DMD patients, and could be valuable for preclinical studies. This article has an associated First Person interview with the first author of the paper .

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder. It is caused by loss-of-function mutations in the dystrophin gene. Currently, there is no cure. A highly promising therapeutic strategy is to replace or repair the defective dystrophin gene by gene therapy. Numerous animal models of DMD have been developed over the last 30 years, ranging from invertebrate to large mammalian models. mdx mice are the most commonly employed models in DMD research and have been used to lay the groundwork for DMD gene therapy. After ~30 years of development, the field has reached the stage at which the results in mdx mice can be validated and scaled-up in symptomatic large animals. The canine DMD (cDMD) model will be excellent for these studies. In this article, we review the animal models for DMD, the pros and cons of each model system, and the history and progress of preclinical DMD gene therapy research in the animal models. We also discuss the current and emerging challenges in this field and ways to address these challenges using animal models, in particular cDMD dogs.

In the translational process of developing innovative therapies for DMD (Duchenne muscular dystrophy), the last preclinical validation step is often carried out in the most relevant animal model of this human disease, namely the GRMD (Golden Retriever muscular dystrophy) dog. The disease in GRMD dogs mimics human DMD in many aspects, including the inter-individual heterogeneity. This last point can be seen as a drawback for an animal model but is inherently related to the disease in GRMD dogs closely resembling that of individuals with DMD. In order to improve the management of this inter-individual heterogeneity, we have screened a combination of biomarkers in sixty-one 2-month-old GRMD dogs at the onset of the disease and a posteriori we addressed their predictive value on the severity of the disease. Three non-invasive biomarkers obtained at early stages of the disease were found to be highly predictive for the loss of ambulation before 6 months of age. An elevation in the number of circulating CD4 + CD49d hi T cells and a decreased stride frequency resulting in a reduced spontaneous speed were found to be strongly associated with the severe clinical form of the disease. These factors can be used as predictive tests to screen dogs to separate them into groups with slow or fast disease progression before their inclusion into a therapeutic preclinical trial, and therefore improve the reliability and translational value of the trials carried out on this invaluable large animal model. These same biomarkers have also been described to be predictive for the time to loss of ambulation in boys with DMD, strengthening the relevance of GRMD dogs as preclinical models of this devastating muscle disease.

Duchenne muscular dystrophy (DMD) is a lethal muscle-wasting disease. Studies in Drosophila showed that genetic increase of the levels of the bioactive sphingolipid sphingosine-1-phosphate (S1P) or delivery of 2-acetyl-5-tetrahydroxybutyl imidazole (THI), an S1P lyase inhibitor, suppresses dystrophic muscle degeneration. In the dystrophic mouse ( mdx ), upregulation of S1P by THI increases regeneration and muscle force. S1P can act as a ligand for S1P receptors and as a histone deacetylase (HDAC) inhibitor. Because Drosophila has no identified S1P receptors and DMD correlates with increased HDAC2 levels, we tested whether S1P action in muscle involves HDAC inhibition. Here we show that beneficial effects of THI treatment in mdx mice correlate with significantly increased nuclear S1P, decreased HDAC activity and increased acetylation of specific histone residues. Importantly, the HDAC2 target microRNA genes miR-29 and miR-1 are significantly upregulated, correlating with the downregulation of the miR-29 target Col1a1 in the diaphragm of THI-treated mdx mice. Further gene expression analysis revealed a significant THI-dependent decrease in inflammatory genes and increase in metabolic genes. Accordingly, S1P levels and functional mitochondrial activity are increased after THI treatment of differentiating C2C12 cells. S1P increases the capacity of the muscle cell to use fatty acids as an energy source, suggesting that THI treatment could be beneficial for the maintenance of energy metabolism in mdx muscles.