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Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that disrupt the open reading frame and thus prevent production of functional dystrophin proteins. Recent advances in DMD treatment, notably exon skipping and AAV gene therapy, have achieved some success aimed at alleviating the symptoms related to progressive muscle damage. However, they do not address the brain comorbidities associated with DMD, which remains a critical aspect of the disease. The mdx52 mouse model recapitulates one of the most frequent genetic pathogenic variants associated with brain involvement in DMD. Deletion of exon 52 impedes expression of two brain dystrophins, Dp427 and Dp140, expressed from distinct promoters. Interestingly, this mutation is eligible for exon skipping strategies aimed at excluding exon 51 or 53 from dystrophin mRNA. We previously showed that exon 51 skipping can restore partial expression of internally deleted yet functional Dp427 in the brain following intracerebroventricular (ICV) injection of antisense oligonucleotides (ASO). This was associated with a partial improvement of anxiety traits, unconditioned fear response, and Pavlovian fear learning and memory in the mdx52 mouse model. In the present study, we investigated in the same mouse model the skipping of exon 53 in order to restore expression of both Dp427 and Dp140. However, in contrast to exon 51, we found that exon 53 skipping was particularly difficult in mdx52 mice and a combination of multiple ASOs had to be used simultaneously to reach substantial levels of exon 53 skipping, regardless of their chemistry (tcDNA, PMO, or 2′MOE). Following ICV injection of a combination of ASO sequences, we measured up to 25% of exon 53 skipping in the hippocampus of treated mdx52 mice, but this did not elicit significant protein restoration. These findings indicate that skipping mouse dystrophin exon 53 is challenging. As such, it has not yet been possible to answer the pertinent question whether rescuing both Dp427 and Dp140 in the brain is imperative to more optimal treatment of neurological aspects of dystrophinopathy.

Neuromuscular disorders (NMDs) are a heterogenous group of rare inherited diseases that compromise the function of peripheral nerves and/or muscles. With limited treatment options available, there is a growing need to design effective preclinical studies that can lead to greater success in clinical trials for novel therapeutics. Here, I discuss recent advances in modelling NMDs to improve preclinical studies as well as two articles from this issue that work in parallel to enable a deeper understanding of a particularly rare NMD, known as X-linked myotubular myopathy.

Muscle atrophy is common in patients with increased glucocorticoid exposure. Glucocorticoid effects are often sex-specific, and while different glucocorticoid responses between male and female subjects are reported, it is unclear why this is. In this study, we evaluated the effects of corticosterone and synthetic glucocorticoid treatment on muscle atrophy in male and female mice. We found that corticosterone treatment reduced grip strength in female mice only, whereas muscle mass was reduced in both sexes. Skeletal muscle transcriptional responses to corticosterone treatment were more pronounced and widespread in male mice. Synthetic glucocorticoid treatment reduced grip strength in both sexes, while female mice were more sensitive to muscle atrophy than male mice. To evaluate the role of androgens, chemically-castrated male mice were treated with synthetic glucocorticoids. We observed additively reduced muscle mass, but did not observe any interaction effects. Although sex differences in glucocorticoid responses in skeletal muscle are partly influenced by androgen signaling, further studies are warranted to fully delineate the underlying mechanisms.

Abstract Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disorder caused by loss of dystrophin. This lack also affects cardiac structure and function, and cardiovascular complications are a major cause of death in DMD. Newly developed therapies partially restore dystrophin expression. It is unclear whether this will be sufficient to prevent or ameliorate cardiac involvement in DMD. We here establish the cardiac electrophysiological and structural phenotype in young (2–3 months) and aged (6–13 months) dystrophin-deficient mdx mice expressing 100% human dystrophin (hDMD), 0% human dystrophin (hDMDdel52-null) or low levels (~ 5%) of human dystrophin (hDMDdel52-low). Compared to hDMD, young and aged hDMDdel52-null mice displayed conduction slowing and repolarisation abnormalities, while only aged hDMDdel52-null mice displayed increased myocardial fibrosis. Moreover, ventricular cardiomyocytes from young hDMDdel52-null animals displayed decreased sodium current and action potential (AP) upstroke velocity, and prolonged AP duration at 20% and 50% of repolarisation. Hence, cardiac electrical remodelling in hDMDdel52-null mice preceded development of structural alterations. In contrast to hDMDdel52-null, hDMDdel52-low mice showed similar electrophysiological and structural characteristics as hDMD, indicating prevention of the cardiac DMD phenotype by low levels of human dystrophin. Our findings are potentially relevant for the development of therapeutic strategies aimed at restoring dystrophin expression in DMD.

Duchenne muscular dystrophy (DMD) is a severe, progressive neuromuscular disorder caused by mutations in the DMD gene resulting in loss of functional dystrophin protein. The muscle dystrophin isoform is essential to protect muscles from contraction-induced damage. However, most dystrophin isoforms are expressed in the brain. In addition to progressive muscle weakness, many DMD patients therefore also exhibit intellectual and behavioral abnormalities. The most commonly used mouse model for DMD, the mdx mouse, lacks only the full-length dystrophin isoforms and has been extensively characterized for muscle pathology. In this study, we assessed behavioral effects of a lack of full-length dystrophins on spontaneous behavior, discrimination and reversal learning, anxiety, and short-term spatial memory and compared performance between male and female mdx mice. In contrast to our previous study using only female mdx mice, we could not reproduce the earlier observed reversal learning deficit. However, we did notice small differences in the number of visits made during the Y-maze and dark-light box. Results indicate that it is advisable to establish standard operating procedures specific to behavioral testing in mdx mice to allow the detection of the subtle phenotypic differences and to eliminate inter and intra laboratory variance.

Neuromuscular disorders (NMDs) encompass a diverse group of genetic diseases characterized by loss of muscle functionality. Despite extensive efforts to develop therapies, no curative treatment exists for any of the NMDs. For multiple disorders, however, therapeutic strategies are currently being tested in clinical settings, and the first successful treatments have now entered clinical practice (e.g. spinraza for spinal muscular atrophy). Successful clinical translation depends on the quality and translatability of preclinical findings and on the predictive value of the experimental models used in their initial development. This Special Issue of Disease Models & Mechanisms has a particular focus on translational research for NMDs. The collection includes original research focusing on advances in the development of novel in vitro and in vivo models, broader understanding of disease pathology and progression, and approaches to modify the disease course in these models. We also present a series of special articles and reviews that highlight our understanding of cellular mechanisms, biomarkers to tract disease pathology, the diversity of mouse models for NMDs, the importance of high-quality preclinical studies and data validation, and the pitfalls of successfully moving a potential therapeutic strategy to the clinic. In this Editorial, we summarize the highlights of these articles and place their findings in the broader context of the NMD research field.

Muscular dystrophies (MDs) encompass a wide variety of inherited disorders that are characterized by loss of muscle tissue associated with a progressive reduction in muscle function. With a cure lacking for MDs, preclinical developments of therapeutic approaches depend on well-characterized animal models that recapitulate the specific pathology in patients. The mouse is the most widely and extensively used model for MDs, and it has played a key role in our understanding of the molecular mechanisms underlying MD pathogenesis. This has enabled the development of therapeutic strategies. Owing to advancements in genetic engineering, a wide variety of mouse models are available for the majority of MDs. Here, we summarize the characteristics of the most commonly used mouse models for a subset of highly studied MDs, collated into a table. Together with references to key publications describing these models, this brief but detailed overview would be useful for those interested in, or working with, mouse models of MD.

Peripheral neuropathy (PN) is a complication of prediabetes and type 2 diabetes (T2D). Increasing evidence suggests that factors besides hyperglycaemia contribute to PN development, including dyslipidaemia. The objective of this study was to determine differential lipid classes and altered gene expression profiles in prediabetes and T2D mouse models in order to identify the dysregulated pathways in PN. Here, we used high-fat diet (HFD)-induced prediabetes and HFD/streptozotocin (STZ)-induced T2D mouse models that develop PN. These models were compared to HFD and HFD-STZ mice that were subjected to dietary reversal. Both untargeted and targeted lipidomic profiling, and gene expression profiling were performed on sciatic nerves. Lipidomic and transcriptomic profiles were then integrated using complex correlation analyses, and biological meaning was inferred from known lipid-gene interactions in the literature. We found an increase in triglycerides (TGs) containing saturated fatty acids. In parallel, transcriptomic analysis confirmed the dysregulation of lipid pathways. Integration of lipidomic and transcriptomic analyses identified an increase in diacylglycerol acyltransferase 2 (DGAT2), the enzyme required for the last and committed step in TG synthesis. Increased DGAT2 expression was present not only in the murine models but also in sural nerve biopsies from hyperlipidaemic diabetic patients with PN. Collectively, these findings support the hypothesis that abnormal nerve-lipid signalling is an important factor in peripheral nerve dysfunction in both prediabetes and T2D. This article has an associated First Person interview with the joint first authors of the paper.

For many genetic diseases, researchers are developing personalized medicine approaches. These sometimes employ custom genetic interventions such as antisense-mediated exon skipping or genome editing, aiming to restore protein function in a mutation-specific manner. Animal models can facilitate the development of personalized medicine approaches; however, given that they target human mutations and therefore human genetic sequences, scientists rely on the availability of humanized animal models. Here, we outline the usefulness, caveats and potential of such models, using the example of the hDMDdel52/mdx model, a humanized model recently generated for Duchenne muscular dystrophy (DMD).

ATP7A encodes a copper-transporting P-type ATPase and is one of 23 genes in which mutations produce distal hereditary motor neuropathy (dHMN), a group of diseases characterized by length-dependent axonal degeneration of motor neurons. We have generated induced pluripotent stem cell (iPSC)-derived motor neurons from a patient with the p.T994I ATP7A gene mutation as an in vitro model for X-linked dHMN (dHMNX). Patient motor neurons show a marked reduction of ATP7A protein levels in the soma when compared to control motor neurons and failed to upregulate expression of ATP7A under copper-loading conditions. These results recapitulate previous findings obtained in dHMNX patient fibroblasts and in primary cells from a rodent model of dHMNX, indicating that patient iPSC-derived motor neurons will be an important resource for studying the role of copper in the pathogenic processes that lead to axonal degeneration in dHMNX.

The muscular dystrophy X-linked (mdx) mouse is commonly used as a mouse model of Duchenne muscular dystrophy (DMD). Its phenotype is, however, mild, and other mouse models have been explored. The mdx:Cmah−/− mouse carries a human-like mutation in the Cmah gene and has a severe muscle phenotype, but its growth and bone development are unknown. In this study, we compared male mdx, mdx:Utrn+/−, mdx:Cmah−/− and wild-type (WT) mice at 3, 5 and 7 weeks of age to determine the suitability of the mdx:Cmah−/− mouse as a model for assessing growth and skeletal development in DMD. The mdx:Cmah−/− mice were lighter than WT mice at 3 weeks, but heavier at 7 weeks, and showed an increased growth rate at 5 weeks. Cortical bone fraction as assessed by micro-computed tomography was greater in both mdx and mdx:Cmah−/− mice versus WT mice at 7 weeks. Tissue mineral density was also higher in mdx:Cmah−/− mice at 3 and 7 weeks. Gene profiling of mdx:Cmah−/− bone identified increased expression of Igf1, Igf1r and Vegfa. Both the mdx and mdx:Cmah−/− mice showed an increased proportion of regulated bone marrow adipose tissue (BMAT) but a reduction in constitutive BMAT. The mdx:Cmah−/− mice show evidence of catch-up growth and more rapid bone development. This pattern does not mimic the typical DMD growth trajectory and therefore the utility of the mdx:Cmah−/− mouse for studying growth and skeletal development in DMD is limited. Further studies of this model may, however, shed light on the phenomenon of catch-up growth. This article has an associated First Person interview with the first author of the paper.

Clinical trials for rare neuromuscular diseases imply, among other investments, a high emotional burden for the whole disease community. Translation of data from preclinical studies to justify any clinical trial must be carefully pondered in order to minimize the risk of clinical trial withdrawal or failure. A rigorous distinction between proof-of-concept and preclinical efficacy studies using animal models is key to support the rationale of a clinical trial involving patients. This Review evaluates the experience accumulated by the TREAT-NMD Advisory Committee for Therapeutics, which provides detailed constructive feedback on clinical proposals for neuromuscular diseases submitted by researchers in both academia and industry, and emphasizes that a timely critical review of preclinical efficacy data from animal models, including biomarkers for specific diseases, combined with adherence to existing guidelines and standard protocols, can significantly help to de-risk clinical programs and prevent disappointments and costly engagement.

Mutations affecting ryanodine receptor (RyR) calcium release channels commonly underlie congenital myopathies. Although these channels are known principally for their essential roles in muscle contractility, mutations in the human RYR1 gene result in a broad spectrum of phenotypes, including muscle weakness, altered proportions of fiber types, anomalous muscle fibers with cores or centrally placed nuclei, and dysmorphic craniofacial features. Currently, it is unknown which phenotypes directly reflect requirements for RyRs and which result secondarily to aberrant muscle function. To identify biological processes requiring RyR function, skeletal muscle development was analyzed in zebrafish embryos harboring protein-null mutations. RyR channels contribute to both muscle fiber development and function. Loss of some RyRs had modest effects, altering muscle fiber-type specification in the embryo without compromising viability. In addition, each RyR-encoding gene contributed to normal swimming behavior and muscle function. The RyR channels do not function in a simple additive manner. For example, although isoform RyR1a is sufficient for muscle contraction in the absence of RyR1b, RyR1a normally attenuates the activity of the co-expressed RyR1b channel in slow muscle. RyR3 also acts to modify the functions of other RyR channels. Furthermore, diminished RyR-dependent contractility affects both muscle fiber maturation and craniofacial development. These findings help to explain some of the heterogeneity of phenotypes that accompany RyR1 mutations in humans.

Louis Kunkel has devoted his career to understanding the causes, mechanisms and treatment of muscular dystrophies. Dr Kunkel is the past Director of the Genomics Program at Boston Children's Hospital and Professor of Genetics and Pediatrics at Harvard Medical School. In this interview, he talks about his discovery of dystrophin, including patients in preclinical research, and bearded irises.

Elizabeth McNally is a human geneticist and a cardiologist whose research has been instrumental in understanding the mechanisms of inherited heart and skeletal muscle diseases. She is the Director of the Center for Genetic Medicine at Northwestern University's Feinberg School of Medicine. In this interview, Elizabeth talks about her first experiences in science, the importance of understanding complex genetic interactions and the effort we all need to make to facilitate diversity in science.

X-linked myopathy with excessive autophagy (XMEA) is a genetic disease associated with weakness of the proximal muscles. It is caused by mutations in the VMA21 gene, coding for a chaperone that functions in the vacuolar ATPase (v-ATPase) assembly. Mutations associated with lower content of assembled v-ATPases lead to an increase in lysosomal pH, culminating in partial blockage of macroautophagy, with accumulation of vacuoles of undigested content. Here, we studied a 5-year-old boy affected by XMEA, caused by a small indel in the VMA21 gene. Detection of sarcoplasmic Lc3 (also known as MAP1LC3B)-positive vacuoles in his muscle biopsy confirmed an autophagy defect. To understand how autophagy is regulated in XMEA myogenesis, we used patient-derived muscle cells to evaluate autophagy during in vitro muscle differentiation. An increase in lysosomal pH was observed in the patient's cells, compatible with predicted functional defect of his mutation. Additionally, there was an increase in autophagic flux in XMEA myotubes. Interestingly, we observed that differentiation of XMEA myoblasts was altered, with increased myotube formation observed through a higher fusion index, which was not dependent on lysosomal acidification. Moreover, no variation in the expression of myogenic factors nor the presence of regenerating fibers in the patient's muscle were observed. Myoblast fusion is a tightly regulated process; therefore, the uncontrolled fusion of XMEA myoblasts might generate cells that are not as functional as normal muscle cells. Our data provide new evidence on the reason for predominant muscle involvement in the context of the XMEA phenotype. This article has an associated First Person interview with the first author of the paper.

Most Duchenne muscular dystrophy (DMD) cases are caused by deletions or duplications of one or more exons that disrupt the reading frame of DMD mRNA. Restoring the reading frame allows the production of partially functional dystrophin proteins, and result in less severe symptoms. Antisense oligonucleotide mediated exon skipping has been approved for DMD, but this strategy needs repeated treatment. CRISPR/Cas9 can also restore dystrophin reading frame. Although recent in vivo studies showed the efficacy of the single-cut reframing/exon skipping strategy, methods to find the most efficient single-cut sgRNAs for a specific mutation are lacking. Here we show that the insertion/deletion (INDEL) generating efficiency and the INDEL profiles both contribute to the reading frame restoring efficiency of a single-cut sgRNA, thus assays only examining INDEL frequency are not able to find the best sgRNAs. We therefore developed a GFP-reporter assay to evaluate single-cut reframing efficiency, reporting the combined effects of both aspects. We show that the GFP-reporter assay can reliably predict the performance of sgRNAs in myoblasts. This GFP-reporter assay makes it possible to efficiently and reliably find the most efficient single-cut sgRNA for restoring dystrophin expression.

Duchenne muscular dystrophy (DMD) is a severe, progressive neuromuscular disorder caused by reading frame disrupting mutations in the DMD gene leading to absence of functional dystrophin. Antisense oligonucleotide (AON)-mediated exon skipping is a therapeutic approach aimed at restoring the reading frame at the pre-mRNA level, allowing the production of internally truncated partly functional dystrophin proteins. AONs work in a sequence specific manner, which warrants generating humanized mouse models for preclinical tests. To address this, we previously generated the hDMDdel52/mdx mouse model using transcription activator like effector nuclease (TALEN) technology. This model contains mutated murine and human DMD genes, and therefore lacks mouse and human dystrophin resulting in a dystrophic phenotype. It allows preclinical evaluation of AONs inducing the skipping of human DMD exons 51 and 53 and resulting in restoration of dystrophin synthesis. Here, we have further characterized this model genetically and functionally. We discovered that the hDMD and hDMDdel52 transgene is present twice per locus, in a tail-to-tail-orientation. Long-read sequencing revealed a partial deletion of exon 52 (first 25 bp), and a 2.3 kb inversion in intron 51 in both copies. These new findings on the genomic make-up of the hDMD and hDMDdel52 transgene do not affect exon 51 and/or 53 skipping, but do underline the need for extensive genetic analysis of mice generated with genome editing techniques to elucidate additional genetic changes that might have occurred. The hDMDdel52/mdx mice were also evaluated functionally using kinematic gait analysis. This revealed a clear and highly significant difference in overall gait between hDMDdel52/mdx mice and C57BL6/J controls. The motor deficit detected in the model confirms its suitability for preclinical testing of exon skipping AONs for human DMD at both the functional and molecular level.

Duchenne muscular dystrophy (DMD) is caused by a lack of dystrophin protein. Next to direct effects on the muscles, this has also metabolic consequences. The influence of nutrition on disease progression becomes more and more recognized. Protein intake by DMD patients may be insufficient to meet the increased demand of the constantly regenerating muscle fibers. This led to the hypothesis that improving protein uptake by the muscles could have therapeutic effects. The present study examined the effects of a modified diet, which composition might stimulate muscle growth, on disease pathology in the D2-mdx mouse model. D2-mdx males were fed with either a control diet or modified diet, containing high amounts of branched-chain amino acids, vitamin D3 and ursolic acid, for six weeks. Our study indicates that the modified diet could not ameliorate the muscle pathology. No effects on bodyweight or weight of individual muscles were observed. Neither did the diet affect severity of fibrosis or calcification of the muscles.

Limb girdle muscular dystrophy (LGMD) types 2D and 2F are caused by mutations in the genes encoding for α- and δ-sarcoglycan, respectively, leading to progressive muscle weakness. Mouse models exist for LGMD2D (Sgca-/-) and 2F (Sgcd-/-). In a previous natural history study, we described the pathology in these mice at 34 weeks of age. However, the development of muscle pathology at younger ages has not been fully characterised yet. We therefore performed a study into age-related changes in muscle function and pathology by examining mice at different ages. From 4 weeks of age onwards, male mice were subjected to functional tests and sacrificed at respectively 8, 16 or 24 weeks of age. Muscle histopathology and expression of genes involved in muscle pathology were analysed for several skeletal muscles, while miRNA levels were assessed in serum. In addition, for Sgcd-/- mice heart pathology was assessed. Muscle function showed a gradual decline in both Sgca-/- and Sgcd-/- mice. Respiratory function was also impaired at all examined timepoints. Already at 8 weeks of age, muscle pathology was prominent, and fibrotic, inflammatory and regenerative markers were elevated, which remained relatively constant with age. In addition, Sgcd-/- mice showed signs of cardiomyopathy from 16 weeks of age onwards. These results indicate that Sgca-/- and Sgcd-/- are relevant disease models for LGMD2D and 2F.

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease generally caused by reading frame disrupting mutations in the DMD gene resulting in loss of functional dystrophin protein. The reading frame can be restored by antisense oligonucleotide (AON)-mediated exon skipping, allowing production of internally deleted, but partially functional dystrophin proteins as found in the less severe Becker muscular dystrophy. Due to genetic variation between species, mouse models with mutations in the murine genes are of limited use to test and further optimize human specific AONs in vivo. To address this we have generated the del52hDMD/mdx mouse. This model carries both murine and human DMD genes. However, mouse dystrophin expression is abolished due to a stop mutation in exon 23, while the expression of human dystrophin is abolished due to a deletion of exon 52. The del52hDMD/mdx model, like mdx, shows signs of muscle dystrophy on a histological level and phenotypically mild functional impairment. Local administration of human specific vivo morpholinos induces exon skipping and dystrophin restoration in these mice. Depending on the number of mismatches, occasional skipping of the murine Dmd gene, albeit at low levels, could be observed. Unlike previous models, the del52hDMD/mdx model enables the in vivo analysis of human specific AONs targeting exon 51 or exon 53 on RNA and protein level and muscle quality and function. Therefore, it will be a valuable tool for optimizing human specific AONs and genome editing approaches for DMD.

Duchenne muscular dystrophy (DMD) affects besides muscle also the brain, resulting in memory and behavioral problems. The consequences of dystrophinopathy on gross macroscopic alterations are unclear. To elucidate the effect of full-length dystrophin expression on brain morphology, we used high-resolution post-mortem MRI in mouse models that either express 0% (mdx), 100% (BL10) or a low amount of full-length dystrophin (mdx-XistΔhs). While absence or low amounts of full-length dystrophin did not significantly affect whole brain volume and skull morphology, we found differences in volume of individual brain structures. The results are in line with observations in humans, where whole brain volume was found to be reduced only in patients lacking both full-length dystrophin and the shorter isoform Dp140.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Poly(A) Binding Protein Nuclear 1 (PABPN1) is a multifunctional regulator of mRNA processing, and its expression levels specifically decline in aging muscles. An expansion mutation in PABPN1 is the genetic cause of oculopharyngeal muscle dystrophy (OPMD), a late onset and rare myopathy. Moreover, reduced PABPN1 expression correlates with symptom manifestation in OPMD. PABPN1 regulates alternative polyadenylation site (PAS) utilization. However, the impact of PAS utilization on cell and tissue function is poorly understood. We hypothesized that altered PABPN1 expression levels is an underlying cause of muscle wasting. To test this, we stably down-regulated PABPN1 in mouse tibialis anterior (TA) muscles by localized injection of adeno-associated viruses expressing shRNA to PABPN1 (shPab). We found that a mild reduction in PABPN1 levels causes muscle pathology including myofiber atrophy, thickening of extracellular matrix and myofiber-type transition. Moreover, reduced PABPN1 levels caused a consistent decline in distal PAS utilization in the 3'-UTR of a subset of OPMD-dysregulated genes. This alternative PAS utilization led to up-regulation of Atrogin-1, a key muscle atrophy regulator, but down regulation of proteasomal genes. Additionally reduced PABPN1 levels caused a reduction in proteasomal activity, and transition in MyHC isotope expression pattern in myofibers. We suggest that PABPN1-mediated alternative PAS utilization plays a central role in aging-associated muscle wasting.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder typically caused by frame-shifting mutations in the DMD gene. Restoration of the reading frame would allow the production of a shorter but partly functional dystrophin protein as seen in Becker muscular dystrophy patients. This can be achieved with antisense oligonucleotides (AONs) that induce skipping of specific exons during pre-mRNA splicing. Different chemical modifications have been developed to improve AON properties. The 2’-deoxy-2’-fluoro (2F) RNA modification is attractive for exon skipping due to its ability to recruit ILF2/3 proteins to the 2F/pre-mRNA duplex, which resulted in enhanced exon skipping in spinal muscular atrophy models. In this study, we examined the effect of two different 2’-substituted AONs (2’-F phosphorothioate (2FPS) and 2’-O-Me phosphorothioate (2OMePS)) on exon skipping in DMD cell and animal models. In human cell cultures, 2FPS AONs showed higher exon skipping levels than their isosequential 2OMePS counterparts. Interestingly, in the mdx mouse model, 2FPS was less efficient than 2OMePS and suggested safety issues as evidenced by increased spleen size and weight loss. Our results do not support a clinical application for 2FPS AON.

Antisense oligonucleotides (AONs) used to reframe dystrophin mRNA transcripts for Duchenne muscular dystrophy (DMD) patients are tested in clinical trials. Here, AONs are administered subcutaneously and intravenously, while the less invasive oral route would be preferred. Oral delivery of encapsulated AONs supplemented with a permeation enhancer, sodium caprate, has been successfully used to target tumor necrosis factor (TNF)-α expression in liver. To test the feasibility of orally delivered AONs for DMD, we applied 2′-O-methyl phosphorothioate AONs (with or without sodium caprate supplementation) directly to the intestine of mdx mice and compared pharmacokinetics and -dynamics with intravenous, intraperitoneal, and subcutaneous delivery. Intestinally infused AONs were taken up, but resulted in lower plasma levels compared to other delivery routes, although bioavailability could be largely improved by supplementation of sodium caprate. After intestinal infusion, AON levels in all tissues were lower than for other administration routes, as were the ratios of target versus nontarget organ levels, except for diaphragm and heart where comparable levels and ratios were observed. For each administration route, low levels of exon skipping in triceps was observed 3 hours post-AON administration. These data suggest that oral administration of naked 2′-O-methyl phosphorothioate AONs may be feasible, but only when high AON concentrations are used in combination with sodium caprate.

Antisense-mediated exon skipping for Duchenne muscular dystrophy (DMD) is currently tested in phase 3 clinical trials. The aim of this approach is to modulate splicing by skipping a specific exon to reframe disrupted dystrophin transcripts, allowing the synthesis of a partly functional dystrophin protein. Studies in animal models allow detailed analysis of the pharmacokinetic and pharmacodynamic profile of antisense oligonucleotides (AONs). Here, we tested the safety and efficacy of subcutaneously administered 2′-O-methyl phosphorothioate AON at 200 mg/kg/week for up to 6 months in mouse models with varying levels of disease severity: mdx mice (mild phenotype) and mdx mice with one utrophin allele (mdx/utrn+/−; more severe phenotype). Long-term treatment was well tolerated and exon skipping and dystrophin restoration confirmed for all animals. Notably, in the more severely affected mdx/utrn+/− mice the therapeutic effect was larger: creatine kinase (CK) levels were more decreased and rotarod running time was more increased. This suggests that the mdx/utrn+/− model may be a more suitable model to test potential therapies than the regular mdx mouse. Our results also indicate that long-term subcutaneous treatment in dystrophic mouse models with these AONs is safe and beneficial.

Duchenne muscular dystrophy (DMD) is a severe progressive muscular disorder caused by reading frame disrupting mutations in the DMD gene, preventing the synthesis of functional dystrophin. As dystrophin provides muscle fiber stability during contractions, dystrophin negative fibers are prone to exercise-induced damage. Upon exhaustion of the regenerative capacity, fibers will be replaced by fibrotic and fat tissue resulting in a progressive loss of function eventually leading to death in the early thirties. With several promising approaches for the treatment of DMD aiming at dystrophin restoration in clinical trials, there is an increasing need to determine more precisely which dystrophin levels are sufficient to restore muscle fiber integrity, protect against muscle damage and improve muscle function.To address this we generated a new mouse model (mdx-Xist(Δhs)) with varying, low dystrophin levels (3-47%, mean 22.7%, stdev 12.1, n = 24) due to skewed X-inactivation. Longitudinal sections revealed that within individual fibers, some nuclei did and some did not express dystrophin, resulting in a random, mosaic pattern of dystrophin expression within fibers.Mdx-Xist(Δhs), mdx and wild type females underwent a 12 week functional test regime consisting of different tests to assess muscle function at base line, or after chronic treadmill running exercise. Overall, mdx-Xist(Δhs) mice with 3-14% dystrophin outperformed mdx mice in the functional tests. Improved histopathology was observed in mice with 15-29% dystrophin and these levels also resulted in normalized expression of pro-inflammatory biomarker genes, while for other parameters >30% of dystrophin was needed. Chronic exercise clearly worsened pathology, which needed dystrophin levels >20% for protection. Based on these findings, we conclude that while even dystrophin levels below 15% can improve pathology and performance, levels of >20% are needed to fully protect muscle fibers from exercise-induced damage.