Duchenne Muscular Dystrophy Mouse Research Articles

Angiopoietin 1 Attenuates Dysregulated Angiogenesis in the Gastrocnemius of DMD Mice.

Duchenne muscular dystrophy (DMD) is a degenerative neuromuscular disease caused by a lack of functional dystrophin. Ang 1 paracrine signalling maintains the endothelial barrier of blood vessels, preventing plasma leakage. Chronic inflammation, a consequence of DMD, causes endothelial barrier dysfunction in skeletal muscle. We aim to elucidate changes in the DMD mouse's gastrocnemius microvascular niche following local administration of Ang 1. Gastrocnemii were collected from eight-week-old mdx/utrn+/- and healthy mice. Additional DMD cohort received an intramuscular injection of Ang 1 to gastrocnemius and contralateral control. Gastrocnemii were collected for analysis after two weeks. Using immunohistochemistry and real-time quantitative reverse transcription, we demonstrated an abundance of endothelial cells in DMD mouse's gastrocnemius, but morphology and gene expression were altered. Myofiber perimeters were shorter in DMD mice. Following Ang 1 treatment, fewer endothelial cells were present, and microvessels were more circular. Vegfr1, Vegfr2, and Vegfa expression in Ang 1-treated gastronemii increased, while myofiber size distribution was consistent with vehicle-only gastrocnemii. These results suggest robust angiogenesis in DMD mice, but essential genes were underexpressed-furthermore, exogenous Ang 1 attenuated angiogenesis. Consequentially, gene expression increased. The impact must be investigated further, as Ang 1 therapy may be pivotal in restoring the skeletal muscle microvascular niche.

PTPN1/2 inhibition promotes muscle stem cell differentiation in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal disease caused by mutations in the DMD gene that encodes dystrophin. Dystrophin deficiency also impacts muscle stem cells (MuSCs), resulting in impaired asymmetric stem cell division and myogenic commitment. Using MuSCs from DMD patients and the DMD mouse model mdx, we found that PTPN1 phosphatase expression is up-regulated and STAT3 phosphorylation is concomitantly down-regulated in DMD MuSCs. To restore STAT3-mediated myogenic signaling, we examined the effect of K884, a novel PTPN1/2 inhibitor, on DMD MuSCs. Treatment with K884 enhanced STAT3 phosphorylation and promoted myogenic differentiation of DMD patient-derived MuSCs. In MuSCs from mdx mice, K884 treatment increased the number of asymmetric cell divisions, correlating with enhanced myogenic differentiation. Interestingly, the pro-myogenic effect of K884 is specific to human and murine DMD MuSCs and is absent from control MuSCs. Moreover, PTPN1/2 loss-of-function experiments indicate that the pro-myogenic impact of K884 is mediated mainly through PTPN1. We propose that PTPN1/2 inhibition may serve as a therapeutic strategy to restore the myogenic function of MuSCs in DMD.

297P In-depth behavioral characterization of Duchenne muscular dystrophy mouse models lacking one, multiple or all dystrophin isoforms in the brain

297P In-depth behavioral characterization of Duchenne muscular dystrophy mouse models lacking one, multiple or all dystrophin isoforms in the brain

298P Magnetic resonance imaging analyses of the brain of Duchenne muscular dystrophy mouse models lacking one, multiple or all dystrophin isoforms

298P Magnetic resonance imaging analyses of the brain of Duchenne muscular dystrophy mouse models lacking one, multiple or all dystrophin isoforms

CRISPR-mediated megabase-scale transgene de-duplication to generate a functional single-copy full-length humanized DMD mouse model

BackgroundThe development of sequence-specific precision treatments like CRISPR gene editing therapies for Duchenne muscular dystrophy (DMD) requires sequence humanized animal models to enable the direct clinical translation of tested strategies. The current available integrated transgenic mouse model containing the full-length human DMD gene, Tg(DMD)72Thoen/J (hDMDTg), has been found to have two copies of the transgene per locus in a tail-to-tail orientation, which does not accurately simulate the true (single) copy number of the DMD gene. This duplication also complicates analysis when testing CRISPR therapy editing outcomes, as large genetic alterations and rearrangements can occur between the cut sites on the two transgenes.ResultsTo address this, we performed long read nanopore sequencing on hDMDTg mice to better understand the structure of the duplicated transgenes. Following that, we performed a megabase-scale deletion of one of the transgenes by CRISPR zygotic microinjection to generate a single-copy, full-length, humanized DMD transgenic mouse model (hDMDTgSc). Functional, molecular, and histological characterisation shows that the single remaining human transgene retains its function and rescues the dystrophic phenotype caused by endogenous murine Dmd knockout.ConclusionsOur unique hDMDTgSc mouse model simulates the true copy number of the DMD gene, and can potentially be used for the further generation of DMD disease models that would be better suited for the pre-clinical assessment and development of sequence specific CRISPR therapies.

Self-Assembled Antibody-Oligonucleotide Conjugates for Targeted Delivery of Complementary Antisense Oligonucleotides.

Antibody-oligonucleotide conjugate (AOC) affords preferential cell targeting and enhanced cellular uptake of antisense oligonucleotide (ASO). Here, we have developed a modular AOC (MAOC) approach based on accurate self-assembly of separately prepared antibody and ASO modules. Homogeneous multimeric AOC with defined ASO-to-antibody ratio were generated by L-DNA scaffold mediated precise self-assembly of antibodies and ASOs. The MAOC approach has been implemented to deliver exon skipping ASOs via transferrin receptor (TfR1) mediated internalization. We discovered an anti-TfR1 sdAb that can greatly enhance nuclear delivery of ASOs. Cryo-EM structure of the sdAb-TfR1 complex showed a new epitope that does not overlap with the binding sites of endogenous TfR1 ligands. In vivo functional analyses of MAOCs with one ASO for single exon skipping and two ASOs for double exon skipping showed that both ASO concentration and exon skipping efficacy of MAOC in cardiac and skeletal muscles are dramatically higher than conventional ASOs in the transgenic Duchenne muscular dystrophy (DMD) mouse model. MAOC treatment was well tolerated in vivo and not associated with any toxicity-related morbidity or mortality. Collectively, our data suggest that the self-assembled MAOC is a viable option for broadening the therapeutic application of ASO via multi-specific targeting and delivery.

Cell transplantation-mediated dystrophin supplementation efficacy in Duchenne muscular dystrophy mouse motor function improvement demonstrated by enhanced skeletal muscle fatigue tolerance

BackgroundDuchenne muscular dystrophy (DMD) is an incurable neuromuscular disease leading to progressive skeletal muscle weakness and fatigue. Cell transplantation in murine models has shown promise in supplementing the lack of the dystrophin protein in DMD muscles. However, the establishment of novel, long-term, relevant methods is needed to assess its efficiency on the DMD motor function. By applying newly developed methods, this study aimed to evaluate the functional and molecular effects of cell therapy-mediated dystrophin supplementation on DMD muscles.MethodsDystrophin was supplemented in the gastrocnemius of a 5-week-old immunodeficient DMD mouse model (Dmd-null/NSG) by intramuscular xenotransplantation of healthy human immortalized myoblasts (Hu5/KD3). A long-term time-course comparative study was conducted between wild-type, untreated DMD, and dystrophin supplemented-DMD mouse muscle functions and histology. A novel GO-ATeam2 transgenic DMD mouse model was also generated to assess in vivo real-time ATP levels in gastrocnemius muscles during repeated contractions.ResultsWe found that 10.6% dystrophin supplementation in DMD muscles was sufficient to prevent low values of gastrocnemius maximal isometric contraction torque (MCT) at rest, while muscle fatigue tolerance, assessed by MCT decline after treadmill running, was fully ameliorated in 21-week-old transplanted mice. None of the dystrophin-supplemented fibers were positive for muscle damage markers after treadmill running, with 85.4% demonstrating the utilization of oxidative metabolism. Furthermore, ATP levels in response to repeated muscle contractions tended to improve, and mitochondrial activity was significantly enhanced in dystrophin supplemented-fibers.ConclusionsCell therapy-mediated dystrophin supplementation efficiently improved DMD muscle functions, as evaluated using newly developed evaluation methods. The enhanced muscle fatigue tolerance in 21-week-old mice was associated with the preferential regeneration of damage-resistant and oxidative fibers, highlighting increased mitochondrial activity, after cell transplantation. These findings significantly contribute to a more in-depth understanding of DMD pathogenesis.

Engineering TadA ortholog-derived cytosine base editor without motif preference and adenosine activity limitation

The engineered TadA variants used in cytosine base editors (CBEs) present distinctive advantages, including a smaller size and fewer off-target effects compared to cytosine base editors that rely on natural deaminases. However, the current TadA variants demonstrate a preference for base editing in DNA with specific motif sequences and possess dual deaminase activity, acting on both cytosine and adenosine in adjacent positions, limiting their application scope. To address these issues, we employ TadA orthologs screening and multi sequence alignment (MSA)-guided protein engineering techniques to create a highly effective cytosine base editor (aTdCBE) without motif and adenosine deaminase activity limitations. Notably, the delivery of aTdCBE to a humanized mouse model of Duchenne muscular dystrophy (DMD) mice achieves robust exon 55 skipping and restoration of dystrophin expression. Our advancement in engineering TadA ortholog for cytosine editing enriches the base editing toolkits for gene-editing therapy and other potential applications.

Systemic delivery of full-length dystrophin in Duchenne muscular dystrophy mice

Current gene therapy for Duchenne muscular dystrophy (DMD) utilizes adeno-associated virus (AAV) to deliver micro-dystrophin (µDys), which does not provide full protection for striated muscles as it lacks many important functional domains of full-length (FL) dystrophin. Here we develop a triple vector system to deliver FL-dystrophin into skeletal and cardiac muscles. We split FL-dystrophin into three fragments linked to two orthogonal pairs of split intein, allowing efficient assembly of FL-dystrophin. The three fragments packaged in myotropic AAV (MyoAAV4A) restore FL-dystrophin expression in both skeletal and cardiac muscles in male mdx4cv mice. Dystrophin-glycoprotein complex components are also restored at the sarcolemma of dystrophic muscles. MyoAAV4A-delivered FL-dystrophin significantly improves muscle histopathology, contractility, and overall strength comparable to µDys, but unlike µDys, it also restores defective cavin 4 localization and associated signaling in mdx4cv heart. Therefore, our data support the feasibility of a mutation-independent FL-dystrophin gene therapy for DMD, warranting further clinical development.

Adenine base editing-mediated exon skipping restores dystrophin in humanized Duchenne mouse model

Duchenne muscular dystrophy (DMD) affecting 1 in 3500–5000 live male newborns is the frequently fatal genetic disease resulted from various mutations in DMD gene encoding dystrophin protein. About 70% of DMD-causing mutations are exon deletion leading to frameshift of open reading frame and dystrophin deficiency. To facilitate translating human DMD-targeting CRISPR therapeutics into patients, we herein establish a genetically humanized mouse model of DMD by replacing exon 50 and 51 of mouse Dmd gene with human exon 50 sequence. This humanized mouse model recapitulats patient’s DMD phenotypes of dystrophin deficiency and muscle dysfunction. Furthermore, we target splicing sites in human exon 50 with adenine base editor to induce exon skipping and robustly restored dystrophin expression in heart, tibialis anterior and diaphragm muscles. Importantly, systemic delivery of base editor via adeno-associated virus in the humanized male mouse model improves the muscle function of DMD mice to the similar level of wildtype ones, indicating the therapeutic efficacy of base editing strategy in treating most of DMD types with exon deletion or point mutations via exon-skipping induction.

Learning, memory and blood-brain barrier pathology in Duchenne muscular dystrophy mice lacking Dp427, or Dp427 and Dp140.

Duchenne muscular dystrophy is a severe neuromuscular disorder that is caused by mutations in the DMD gene, resulting in a disruption of dystrophin production. Next to dystrophin expression in the muscle, different isoforms of the protein are also expressed in the brain and lack of these isoforms leads to cognitive and behavioral deficits in patients. It remains unclear how the loss of the shorter dystrophin isoform Dp140 affects these processes. Using a variety of behavioral tests, we found that mdx and mdx4cv mice (which lack Dp427 or Dp427 + Dp140, respectively) exhibit similar deficits in working memory, movement patterns and blood-brain barrier integrity. Neither model showed deficits in spatial learning and memory, learning flexibility, anxiety or spontaneous behavior, nor did we observe differences in aquaporin 4 and glial fibrillary acidic protein. These results indicate that in contrast to Dp427, Dp140 does not play a crucial role in processes of learning, memory and spontaneous behavior.

Effects of a Ketogenic Diet on the Assessment of Biochemical and Clinical Parameters in Duchenne Muscular Dystrophy: A Preclinical Investigation.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder characterized by progressive skeletal muscle degeneration and systemic effects, including the central nervous system (CNS). This study aimed to assess the impact of a 14-day ketogenic diet (DCet) on biochemical and clinical parameters in a DMD mouse model. Young adult mice (50 days old) were fed DCet, while control groups received a standard diet. On the 14th day, memory and behavior tests were conducted, followed by biochemical evaluations of oxidative stress, inflammatory biomarkers, body weight, feed intake, and brain-derived neurotrophic factor (BDNF) levels. mdx + DCet mice showed reduced mass (0.2 g ± 2.49) and improved memory retention (p < 0.05) compared to controls. Oxidative damage in muscle tissue and CNS decreased, along with a significant cytokine level reduction (p <0.05). The protocol led to an increase in hippocampal BDNF and mitochondrial respiratory complex activity in muscle tissue and the central nervous system (CNS), while also decreasing creatine kinase activity only in the striatum. Overall, a 14-day DCet showed protective effects by improving spatial learning and memory through reductions in oxidative stress and immune response, as well as increases in BDNF levels, consistent with our study's findings.

Expression of Dystrophin Dp71 Splice Variants Is Temporally Regulated During Rodent Brain Development.

Dystrophin Dp71 is the major product of the Duchenne muscular dystrophy (DMD) gene in the brain, and its loss in DMD patients and mouse models leads to cognitive impairments. Dp71 is expressed as a range of proteins generated by alternative splicing of exons 71 to 74 and 78, classified in the main Dp71d and Dp71f groups that contain specific C-terminal ends. However, it is unknown whether each isoform has a specific role in distinct cell types, brain regions, and/or stages of brain development. In the present study, we characterized the expression of Dp71 isoforms during fetal (E10.5, E15.5) and postnatal (P1, P7, P14, P21 and P60) mouse and rat brain development. We finely quantified the expression of several Dp71 transcripts by RT-PCR and cloning assays in samples from whole-brain and distinct brain structures. The following Dp71 transcripts were detected: Dp71d, Dp71d∆71, Dp71d∆74, Dp71d∆71,74, Dp71d∆71-74, Dp71f, Dp71f∆71, Dp71f∆74, Dp71f∆71,74, and Dp71fΔ71-74. We found that the Dp71f isoform is the main transcript expressed at E10.5 (> 80%), while its expression is then progressively reduced and replaced by the expression of isoforms of the Dp71d group from E15.5 to postnatal and adult ages. This major finding was confirmed by third-generation nanopore sequencing. In addition, we found that the level of expression of specific Dp71 isoforms varies as a function of postnatal stages and brain structure. Our results suggest that Dp71 isoforms have different and complementary roles during embryonic and postnatal brain development, likely taking part in a variety of maturation processes in distinct cell types.

Respiratory characterization of a humanized Duchenne muscular dystrophy mouse model

Duchenne muscular dystrophy (DMD) is the most common X-linked disease. DMD is caused by a lack of dystrophin, a critical structural protein in striated muscle. Dystrophin deficiency leads to inflammation, fibrosis, and muscle atrophy. Boys with DMD have progressive muscle weakness within the diaphragm that results in respiratory failure in the 2nd or 3rd decade of life. The most common DMD mouse model – the mdx mouse – is not sufficient for evaluating genetic medicines that specifically target the human DMD (hDMD) gene sequence. Therefore, a novel transgenic mouse carrying the hDMD gene with an exon 52 deletion was created (hDMDΔ52;mdx). We characterized the respiratory function and pathology in this model using whole body plethysmography, histology, and immunohistochemistry. At 6-months-old, hDMDΔ52;mdx mice have reduced maximal respiration, neuromuscular junction pathology, and fibrosis throughout the diaphragm, which worsens at 12-months-old. In conclusion, the hDMDΔ52;mdx exhibits moderate respiratory pathology, and serves as a relevant animal model to study the impact of novel genetic therapies, including gene editing, on respiratory function.

GDE5/Gpcpd1 activity determines phosphatidylcholine composition in skeletal muscle and regulates contractile force in mice

Glycerophosphocholine (GPC) is an important precursor for intracellular choline supply in phosphatidylcholine (PC) metabolism. GDE5/Gpcpd1 hydrolyzes GPC into choline and glycerol 3-phosphate; this study aimed to elucidate its physiological function in vivo. Heterozygous whole-body GDE5-deficient mice reveal a significant GPC accumulation across tissues, while homozygous whole-body knockout results in embryonic lethality. Skeletal muscle-specific GDE5 deletion (Gde5 skKO) exhibits reduced passive force and improved fatigue resistance in electrically stimulated gastrocnemius muscles in vivo. GDE5 deficiency also results in higher glycolytic metabolites and glycogen levels, and glycerophospholipids alteration, including reduced levels of phospholipids that bind polyunsaturated fatty acids (PUFAs), such as DHA. Interestingly, this PC fatty acid compositional change is similar to that observed in skeletal muscles of denervated and Duchenne muscular dystrophy mouse models. These are accompanied by decrease of GDE5 expression, suggesting a regulatory role of GDE5 activity for glycerophospholipid profiles. Furthermore, a DHA-rich diet enhances contractile force and lowers fatigue resistance, suggesting a functional relationship between PC fatty acid composition and muscle function. Finally, skinned fiber experiments show that GDE5 loss increases the probability of the ryanodine receptor opening and lowers the maximum Ca2+-activated force. Collectively, GDE5 activity plays roles in PC and glucose/glycogen metabolism in skeletal muscle.

Leucyl-tRNA Synthetase Contributes to Muscle Weakness through Mammalian Target of Rapamycin Complex 1 Activation and Autophagy Suppression in a Mouse Model of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD), caused by loss-of-function mutations in the dystrophin gene, results in progressive muscle weakness and early fatality. Impaired autophagy is one of the cellular hallmarks of DMD, contributing to the disease progression. Molecular mechanisms underlying the inhibition of autophagy in DMD are not well understood. In the current study, the DMD mouse model mdx was used for the investigation of signaling pathways leading to suppression of autophagy. Mammalian target of rapamycin complex 1 (mTORC1) was hyperactive in the DMD muscles, accompanying muscle weakness and autophagy impairment. Surprisingly, Akt, a well-known upstream regulator of mTORC1, was not responsible for mTORC1 activation or the dystrophic muscle phenotypes. Instead, leucyl-tRNA synthetase (LeuRS) was overexpressed in mdx muscles compared with the wild type. LeuRS activates mTORC1 in a noncanonical mechanism that involves interaction with RagD, an activator of mTORC1. Disrupting LeuRS interaction with RagD by the small-molecule inhibitor BC-LI-0186 reduced mTORC1 activity, restored autophagy, and ameliorated myofiber damage in the mdx muscles. Furthermore, inhibition of LeuRS by BC-LI-0186 improved dystrophic muscle strength in an autophagy-dependent manner. Taken together, our findings uncovered a noncanonical function of the housekeeping protein LeuRS as a potential therapeutic target in the treatment of DMD.

Generation of allogeneic and xenogeneic functional muscle stem cells for intramuscular transplantation.

Satellite cells, the stem cells of skeletal muscle tissue, hold a remarkable regeneration capacity and therapeutic potential in regenerative medicine. However, low satellite cell yield from autologous or donor-derived muscles hinders the adoption of satellite cell transplantation for the treatment of muscle diseases, including Duchenne muscular dystrophy (DMD). To address this limitation, here we investigated whether satellite cells can be derived in allogeneic or xenogeneic animal hosts. First, injection of CRISPR/Cas9-corrected Dmdmdx mouse induced pluripotent stem cells (iPSCs) into mouse blastocysts carrying an ablation system of host satellite cells gave rise to intraspecies chimeras exclusively carrying iPSC-derived satellite cells. Furthermore, injection of genetically corrected DMD iPSCs into rat blastocysts resulted in the formation of interspecies rat-mouse chimeras harboring mouse satellite cells. Notably, iPSC-derived satellite cells or derivative myoblasts produced in intraspecies or interspecies chimeras restored dystrophin expression in DMD mice following intramuscular transplantation and contributed to the satellite cell pool. Collectively, this study demonstrates the feasibility of producing therapeutically competent stem cells across divergent animal species, raising the possibility of generating human muscle stem cells in large animals for regenerative medicine purposes.

Myospreader improves gene editing in skeletal muscle by myonuclear propagation

Successful CRISPR/Cas9-based gene editing in skeletal muscle is dependent on efficient propagation of Cas9 to all myonuclei in the myofiber. However, nuclear-targeted gene therapy cargos are strongly restricted to their myonuclear domain of origin. By screening nuclear localization signals and nuclear export signals, we identify "Myospreader," a combination of short peptide sequences that promotes myonuclear propagation. Appending Myospreader to Cas9 enhances protein stability and myonuclear propagation in myoblasts and myofibers. AAV-delivered Myospreader dCas9 better inhibits transcription of toxic RNA in a myotonic dystrophy mouse model. Furthermore, Myospreader Cas9 achieves higher rates of gene editing in CRISPR reporter and Duchenne muscular dystrophy mouse models. Myospreader reveals design principles relevant to all nuclear-targeted gene therapies and highlights the importance of the spatial dimension in therapeutic development.

Differential metabolic secretion between muscular dystrophy mouse-derived spindle cell sarcomas and rhabdomyosarcomas drives tumor type development.

The dystrophin gene (Dmd) is recognized for its significance in Duchenne muscular dystrophy (DMD), a lethal and progressive skeletal muscle disease. Some patients with DMD and model mice with muscular dystrophy (mdx) spontaneously develop various types of tumors, among which rhabdomyosarcoma (RMS) is the most prominent. By contrast, spindle cell sarcoma (SCS) has rarely been reported in patients or mdx mice. In this study, we aimed to use metabolomics to better understand the rarity of SCS development in mdx mice. Gas chromatography-mass spectrometry was used to compare the metabolic profiles of spontaneously developed SCS and RMS tumors from mdx mice, and metabolite supplementation assays and silencing experiments were used to assess the effects of metabolic differences in SCS tumor-derived cells. The levels of 75 metabolites exhibited differences between RMS and SCS, 25 of which were significantly altered. Further characterization revealed downregulation of nonessential amino acids, including alanine, in SCS tumors. Alanine supplementation enhanced the growth, epithelial mesenchymal transition, and invasion of SCS cells. Reduction of intracellular alanine via knockdown of the alanine transporter Slc1a5 reduced the growth of SCS cells. Lower metabolite secretion and reduced proliferation of SCS tumors may explain the lower detection rate of SCS in mdx mice. Targeting of alanine depletion pathways may have potential as a novel treatment strategy.NEW & NOTEWORTHY To the best of our knowledge, SCS has rarely been identified in patients with DMD or mdx mice. We observed that RMS and SCS tumors that spontaneously developed from mdx mice with the same Dmd genetic background exhibited differences in metabolic secretion. We proposed that, in addition to dystrophin deficiency, the levels of secreted metabolites may play a role in the determination of tumor-type development in a Dmd-deficient background.

The BALB/c.mdx62 mouse exhibits a dystrophic muscle pathology and is a model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a devastating monogenic skeletal muscle-wasting disorder. Although many pharmacological and genetic interventions have been reported in preclinical studies, few have progressed to clinical trials with meaningful benefit. Identifying therapeutic potential can be limited by availability of suitable preclinical mouse models. More rigorous testing across models with varied background strains and mutations can identify treatments for clinical success. Here, we report the generation of a DMD mouse model with a CRISPR-induced deletion within exon 62 of the dystrophin gene (Dmd) and the first generated in BALB/c mice. Analysis of mice at 3, 6 and 12 months of age confirmed loss of expression of the dystrophin protein isoform Dp427 and resultant dystrophic pathology in limb muscles and the diaphragm, with evidence of centrally nucleated fibers, increased inflammatory markers and fibrosis, progressive decline in muscle function, and compromised trabecular bone development. The BALB/c.mdx62 mouse is a novel model of DMD with associated variations in the immune response and muscle phenotype, compared with those of existing models. It represents an important addition to the preclinical model toolbox for developing therapeutic strategies.