Tissue distribution of renadirsen sodium, a dystrophin exon-skipping antisense oligonucleotide, in heart and diaphragm after subcutaneous administration to cynomolgus monkeys.

The pharmacokinetics and tissue distribution of renadirsen sodium, a dystrophin exon-skipping phosphorothioate-modified antisense oligonucleotide with 2'-O,4'-C-ethylene-bridged nucleic acid (ENA), after subcutaneous or intravenous administration to cynomolgus monkeys were investigated. The plasma concentration of renadirsen after subcutaneous administration at 1, 3, and 10 mg/kg increased with the dose. The absolute bioavailability at 3 mg/kg after subcutaneous administration was calculated as 88.6%, and the time to reach maximum plasma concentration of renadirsen was within 4 h, indicating the efficient and rapid absorption following subcutaneous administration. The exposure of muscle tissues to renadirsen was found to increase with repeated dosing at 6 mg/kg, and higher exposure was observed in the diaphragm and heart than in the quadriceps femoris and anterior tibialis muscles. Renadirsen achieved more exon 45-skipped dystrophin mRNA in the diaphragm and heart than in the quadriceps femoris and anterior tibialis muscles. Renadirsen also showed a cumulative skipping effect in a repeated-dose study. The findings on exon 45-skipped dystrophin mRNA in these muscle tissues were consistent with the concentration of renadirsen in these tissues. Because it is not feasible to directly evaluate drug concentration and exon skipping in the heart and diaphragm in humans, the pharmacokinetics and pharmacodynamics of renadirsen in these tissues in monkeys are crucial for the design and interpretation of clinical settings.

Duchenne muscular dystrophy skeletal muscle cells derived from human induced pluripotent stem cells recapitulate various calcium dysregulation pathways.

Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle degenerative disease, caused by mutations in the dystrophin gene and resulting in premature death. As a major secondary event, an abnormal elevation of the intracellular calcium concentration in the dystrophin-deficient muscle contributes to disease progression in DMD. In this study, we investigated the specific functional features of induced pluripotent stem cell-derived muscle cells (hiPSC-skMCs) generated from DMD patients to regulate intracellular calcium concentration. As compared to healthy hiPSC-skMCs, DMD hiPSC-skMCs displayed specific spontaneous calcium signatures with high levels of intracellular calcium concentration. Furthermore, stimulations with electrical field or with acetylcholine perfusion induced higher calcium response in DMD hiPSC-skMCs as compared to healthy cells. Finally, Mn2+ quenching experiments demonstrated high levels of constitutive calcium entries in DMD hiPSC-skMCs as compared to healthy cells. Our findings converge on the fact that DMD hiPSC-skMCs display intracellular calcium dysregulation as demonstrated in several other models. Observed calcium disorders associated with RNAseq analysis on these DMD cells highlighted some mechanisms, such as spontaneous and activated sarcoplasmic reticulum (SR) releases or constitutive calcium entries, known to be disturbed in other dystrophin-deficient models. However, store operated calcium entries (SOCEs) were not found to be dysregulated in our DMD hiPSC-skMCs model. These results suggest that all the mechanisms of calcium impairment observed in other animal models may not be as pronounced in humans and could point to a preference for certain mechanisms that could correspond to major molecular targets for DMD therapies.

Exonisation of an intronic L1 element in the dystrophin gene associated with X-linked muscular dystrophy in a Border Collie dog.

X-linked recessive dystrophinopathies are the most common muscular dystrophies (MDs) in humans and dogs. To date, 20 breed-specific MD-associated variants are described in the canine dystrophin gene (DMD), including one associated with dystrophin-deficient MD in the Border Collie mixed breed. Here, we report the diagnosis and follow-up of mild dystrophin-deficient MD in a 5-month-old male Border Collie, associated with a novel DMD variant. Diagnosis was based on neurological examination and laboratory evaluations including creatine kinase activity, electromyography and muscle biopsies with immunofluorescent staining. Inspection of the Sashimi plots of the RNA-seq data from the affected muscle biopsy led to the discovery of a 162-bp L1 pseudoexon in DMD intron 63, introducing a frameshift and a premature stop codon (NM_001003343.1: c.9271_9272insN[162] p.(Ala3091fs*21)). Reduced DMD mRNA levels were detected for both the non-pseudoexon (50× less) and pseudoexon (3× less) containing transcripts in the affected muscle, compared with the level of the non-pseudoexon containing transcript in a control muscle, resulting in very low dystrophin protein levels and the upregulation of utrophin. Because the variant was only found in the affected dog, not in the healthy mother and grandmother, or in 108 unrelated Border Collies from the Belgian population (46 males and 62 females), it was considered a de novo variant. Although the prognosis for dystrophinopathy is generally regarded as poor, the dog stabilised at the age of 6 months and is still clinically stable at the age of 2 years.

Feline dystrophin-deficient muscular dystrophy misdiagnosed as Toxoplasma myositis.

Case summary: A 6-month-old male entire domestic shorthair cat presented for presumptive Toxoplasma myopathy that was non-responsive to antiprotozoal therapy. Clinical features included marked macroglossia, dysphagia, regurgitation, truncal muscle hypertrophy, pelvic limb gait abnormalities and megaoesophagus. Relevant diagnostics included serial creatine kinase activity, cardiac troponin I, fluoroscopic swallow study and routine muscle histopathology. Ultimately, post-mortem histopathology with immunostaining demonstrated markedly decreased or absent staining for the rod and carboxy terminus of dystrophin, confirming a dystrophin-deficient muscular dystrophy (MD). The misdiagnosis of toxoplasmosis was based on an increased IgG titre and muscle histopathology submitted to a local laboratory. Treatment for megaoesophagus included vertical feeding of wet food only, sildenafil and omeprazole. Dysphagia and regurgitation improved moderately. Presumptive hyperaesthesia and muscle pain were managed with anti-inflammatory doses of prednisolone. The patient was ultimately euthanased as a result of progressive MD signs and uraemia at 2 years of age. Relevance and novel information: This case report highlights the collective clinical features of MD, as they could be considered pathognomonic for this rare condition and must be differentiated from other myopathies via specific immunostaining of muscle biopsies. This is crucial to obtain a correct and early diagnosis, allowing instigation of potentially valuable treatments. Megaoesophagus is an inconsistent feature in feline MD in addition to the more commonly observed oropharyngeal dysphagia. Management with a canned diet, sildenafil, omeprazole and upright feeding was beneficial with moderate improvement in the frequency of regurgitation. Prednisolone was thought to minimise the presumptive myalgia.

Cognitive and emotional-behavioural outcomes of Turkish Duchenne muscular dystrophy population and its association with motor function.

PURPOSE: The aim of this study was to examine the cognitive and emotional-behavioural outcomes of Turkish children with Duchenne muscular dystrophy (DMD) in comparison with healthy peers, to determine its relationship with motor functions, and to analyse the difference of cognitive and emotional-behavioural outcomes according to the site of mutations. METHOD: Children aged 7-16 years with DMD (n = 68) and age-matched typically developing children (n = 33) were included in the study. The cognitive and emotional-behavioural status and the motor functions were assessed in detail. Children with DMD also divided into two groups as "proximal" and "distal" site mutation groups to compare the cognitive and emotional-behavioural outcomes. RESULTS: The children with DMD and typically developing children were similar in terms of age and body mass index (p > 0.05). Significant differences were found between children with DMD and typically developing peers in almost all subtests of both cognitive and emotional-behavioural assessments (p < 0.05). Cognitive and emotional-behavioural parameters were weakly correlated with specific motor parameters responsive to cognitive functioning (p < 0.05). Children with distal site mutation performed significantly worser than those with proximal site mutation in particular cognitive subtest (p < 0.05). CONCLUSIONS: It is concluded that comprehensive and detailed evaluation of cognitive and emotional-behavioural features of children with DMD is essential for better implementation of rehabilitation programs to maintain motor function which especially requires cognitive ability, since a Turkish cohort represented challenges in particular domains of cognitive and emotional-behavioural areas. CLINICAL TRIAL REGISTRATION NUMBER: NCT05661071.

Social and emotional alterations in mice lacking the short dystrophin-gene product, Dp71.

BACKGROUND: The Duchenne and Becker muscular dystrophies (DMD, BMD) are neuromuscular disorders commonly associated with diverse cognitive and behavioral comorbidities. Genotype-phenotype studies suggest that severity and risk of central defects in DMD patients increase with cumulative loss of different dystrophins produced in CNS from independent promoters of the DMD gene. Mutations affecting all dystrophins are nevertheless rare and therefore the clinical evidence on the contribution of the shortest Dp71 isoform to cognitive and behavioral dysfunctions is limited. In this study, we evaluated social, emotional and locomotor functions, and fear-related learning in the Dp71-null mouse model specifically lacking this short dystrophin. RESULTS: We demonstrate the presence of abnormal social behavior and ultrasonic vocalization in Dp71-null mice, accompanied by slight changes in exploratory activity and anxiety-related behaviors, in the absence of myopathy and alterations of learning and memory of aversive cue-outcome associations. CONCLUSIONS: These results support the hypothesis that distal DMD gene mutations affecting Dp71 may contribute to the emergence of social and emotional problems that may relate to the autistic traits and executive dysfunctions reported in DMD. The present alterations in Dp71-null mice may possibly add to the subtle social behavior problems previously associated with the loss of the Dp427 dystrophin, in line with the current hypothesis that risk and severity of behavioral problems in patients increase with cumulative loss of several brain dystrophin isoforms.

Exploring lipin1 as a promising therapeutic target for the treatment of Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive and devastating muscle disease, resulting from the absence of dystrophin. This leads to cell membrane instability, susceptibility to contraction-induced muscle damage, subsequent muscle degeneration, and eventually disability and early death of patients. Currently, there is no cure for DMD. Our recent studies identified that lipin1 plays a critical role in maintaining myofiber stability and integrity. However, lipin1 gene expression levels are dramatically reduced in the skeletal muscles of DMD patients and mdx mice. METHODS: To identify whether increased lipin1 expression could prevent dystrophic pathology, we employed unique muscle-specific mdx:lipin1 transgenic (mdx:lipin1Tg/0) mice in which lipin1 was restored in the dystrophic muscle of mdx mice, intramuscular gene delivery, as well as cell culture system. RESULTS: We found that increased lipin1 expression suppressed muscle degeneration and inflammation, reduced fibrosis, strengthened membrane integrity, and resulted in improved muscle contractile and lengthening force, and muscle performance in mdx:lipin1Tg/0 compared to mdx mice. To confirm the role of lipin1 in dystrophic muscle, we then administered AAV1-lipin1 via intramuscular injection in mdx mice. Consistently, lipin1 restoration inhibited myofiber necroptosis and lessened muscle degeneration. Using a cell culture system, we further found that differentiated primary mdx myoblasts had elevated expression levels of necroptotic markers and medium creatine kinase (CK), which could be a result of sarcolemmal damage. Most importantly, increased lipin1 expression levels in differentiated myoblasts from mdx:lipin1Tg/0 mice substantially inhibited the elevation of necroptotic markers and medium CK levels. CONCLUSIONS: Overall, our data suggest that lipin1 is a promising therapeutic target for the treatment of dystrophic muscles.

Pharmacological Treatments and Therapeutic Targets in Muscle Dystrophies Generated by Alterations in Dystrophin-Associated Proteins.

Muscular dystrophies (MDs) are a heterogeneous group of diseases of genetic origin characterized by progressive skeletal muscle degeneration and weakness. There are several types of MDs, varying in terms of age of onset, severity, and pattern of the affected muscles. However, all of them worsen over time, and many patients will eventually lose their ability to walk. In addition to skeletal muscle effects, patients with MDs may present cardiac and respiratory disorders, generating complications that could lead to death. Interdisciplinary management is required to improve the surveillance and quality of life of patients with an MD. At present, pharmacological therapy is only available for Duchene muscular dystrophy (DMD)-the most common type of MD-and is mainly based on the use of corticosteroids. Other MDs caused by alterations in dystrophin-associated proteins (DAPs) are less frequent but represent an important group within these diseases. Pharmacological alternatives with clinical potential in patients with MDs and other proteins associated with dystrophin have been scarcely explored. This review focuses on drugs and molecules that have shown beneficial effects, mainly in experimental models involving alterations in DAPs. The mechanisms associated with the effects leading to promising results regarding the recovery or maintenance of muscle strength and reduction in fibrosis in the less-common MDs (i.e., with respect to DMD) are explored, and other therapeutic targets that could contribute to maintaining the homeostasis of muscle fibers, involving different pathways, such as calcium regulation, hypertrophy, and maintenance of satellite cell function, are also examined. It is possible that some of the drugs explored here could be used to affordably improve the muscular function of patients until a definitive treatment for MDs is developed.

Dystrophin S3059 phosphorylation partially attenuates denervation atrophy in mouse tibialis anterior muscles.

The dystrophin protein has well-characterized roles in force transmission and maintaining membrane integrity during muscle contraction. Studies have reported decreased expression of dystrophin in atrophying muscles during wasting conditions, and that restoration of dystrophin can attenuate atrophy, suggesting a role in maintaining muscle mass. Phosphorylation of S3059 within the cysteine-rich region of dystrophin enhances binding between dystrophin and ß-dystroglycan, and mimicking phosphorylation at this site by site-directed mutagenesis attenuates myotube atrophy in vitro. To determine whether dystrophin phosphorylation can attenuate muscle wasting in vivo, CRISPR-Cas9 was used to generate mice with whole body mutations of S3059 to either alanine (DmdS3059A) or glutamate (DmdS3059E), to mimic a loss of, or constitutive phosphorylation of S3059, on all endogenous dystrophin isoforms, respectively. Sciatic nerve transection was performed on these mice to determine whether phosphorylation of dystrophin S3059 could attenuate denervation atrophy. At 14 days post denervation, atrophy of tibialis anterior (TA) but not gastrocnemius or soleus muscles, was partially attenuated in DmdS3059E mice relative to WT mice. Attenuation of atrophy was associated with increased expression of ß-dystroglycan in TA muscles of DmdS3059E mice. Dystrophin S3059 phosphorylation can partially attenuate denervation-induced atrophy, but may have more significant impact in less severe modes of muscle wasting.

Chimeric Cell Therapy Transfers Healthy Donor Mitochondria in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a severe X-linked disorder characterized by dystrophin gene mutations and mitochondrial dysfunction, leading to progressive muscle weakness and premature death of DMD patients. We developed human Dystrophin Expressing Chimeric (DEC) cells, created by the fusion of myoblasts from normal donors and DMD patients, as a foundation for DT-DEC01 therapy for DMD. Our preclinical studies on mdx mouse models of DMD revealed enhanced dystrophin expression and functional improvements in cardiac, respiratory, and skeletal muscles after systemic intraosseous DEC administration. The current study explored the feasibility of mitochondrial transfer and fusion within the created DEC cells, which is crucial for developing new therapeutic strategies for DMD. Following mitochondrial staining with MitoTracker Deep Red and MitoTracker Green dyes, mitochondrial fusion and transfer was assessed by Flow cytometry (FACS) and confocal microscopy. The PEG-mediated fusion of myoblasts from normal healthy donors (MBN/MBN) and normal and DMD-affected donors (MBN/MBDMD), confirmed the feasibility of myoblast and mitochondrial fusion and transfer. The colocalization of the mitochondrial dyes MitoTracker Deep Red and MitoTracker Green confirmed the mitochondrial chimeric state and the creation of chimeric mitochondria, as well as the transfer of healthy donor mitochondria within the created DEC cells. These findings are unique and significant, introducing the potential of DT-DEC01 therapy to restore mitochondrial function in DMD patients and in other diseases where mitochondrial dysfunction plays a critical role.

Deletion of miR-146a enhances therapeutic protein restoration in model of dystrophin exon skipping.

Duchenne muscular dystrophy (DMD) is a progressive muscle disease caused by the absence of dystrophin protein. One current DMD therapeutic strategy, exon skipping, produces a truncated dystrophin isoform using phosphorodiamidate morpholino oligomers (PMOs). However, the potential of exon skipping therapeutics has not been fully realized as increases in dystrophin protein have been minimal in clinical trials. Here, we investigate how miR-146a-5p, which is highly elevated in dystrophic muscle, impacts dystrophin protein levels. We find inflammation strongly induces miR-146a in dystrophic, but not wild-type myotubes. Bioinformatics analysis reveals that the dystrophin 3' UTR harbors a miR-146a binding site, and subsequent luciferase assays demonstrate miR-146a binding inhibits dystrophin translation. In dystrophin-null mdx52 mice, co-injection of miR-146a reduces dystrophin restoration by an exon 51 skipping PMO. To directly investigate how miR-146a impacts therapeutic dystrophin rescue, we generated mdx52 with body-wide miR-146a deletion (146aX). Administration of an exon skipping PMO via intramuscular or intravenous injection markedly increases dystrophin protein levels in 146aX vs. mdx52 muscles while skipped dystrophin transcript levels are unchanged supporting a post-transcriptional mechanism of action. Together, these data show that miR-146a expression opposes therapeutic dystrophin restoration, suggesting miR-146a inhibition warrants further research as a potential DMD exon skipping co-therapy.

Dystrophin 71 deficiency causes impaired aquaporin-4 polarization contributing to glymphatic dysfunction and brain edema in cerebral ischemia.

OBJECTIVE: The glymphatic system serves as a perivascular pathway that aids in clearing liquid and solute waste from the brain, thereby enhancing neurological function. Disorders in glymphatic drainage contribute to the development of vasogenic edema following cerebral ischemia, although the molecular mechanisms involved remain poorly understood. This study aims to determine whether a deficiency in dystrophin 71 (DP71) leads to aquaporin-4 (AQP4) depolarization, contributing to glymphatic dysfunction in cerebral ischemia and resulting in brain edema. METHODS: A mice model of middle cerebral artery occlusion and reperfusion was used. A fluorescence tracer was injected into the cortex and evaluated glymphatic clearance. To investigate the role of DP71 in maintaining AQP4 polarization, an adeno-associated virus with the astrocyte promoter was used to overexpress Dp71. The expression and distribution of DP71 and AQP4 were analyzed using immunoblotting, immunofluorescence, and co-immunoprecipitation techniques. The behavior ability of mice was evaluated by open field test. Open-access transcriptome sequencing data were used to analyze the functional changes of astrocytes after cerebral ischemia. MG132 was used to inhibit the ubiquitin-proteasome system. The ubiquitination of DP71 was detected by immunoblotting and co-immunoprecipitation. RESULTS: During the vasogenic edema stage following cerebral ischemia, a decline in the efflux of interstitial fluid tracer was observed. DP71 and AQP4 were co-localized and interacted with each other in the perivascular astrocyte endfeet. After cerebral ischemia, there was a notable reduction in DP71 protein expression, accompanied by AQP4 depolarization and proliferation of reactive astrocytes. Increased DP71 expression restored glymphatic drainage and reduced brain edema. AQP4 depolarization, reactive astrocyte proliferation, and the behavior of mice were improved. After cerebral ischemia, DP71 was degraded by ubiquitination, and MG132 inhibited the decrease of DP71 protein level. CONCLUSION: AQP4 depolarization after cerebral ischemia leads to glymphatic clearance disorder and aggravates cerebral edema. DP71 plays a pivotal role in regulating AQP4 polarization and consequently influences glymphatic function. Changes in DP71 expression are associated with the ubiquitin-proteasome system. This study offers a novel perspective on the pathogenesis of brain edema following cerebral ischemia.

Dual FKRP/FST gene therapy normalizes ambulation, increases strength, decreases pathology, and amplifies gene expression in LGMDR9 mice.

Recent clinical studies of single gene replacement therapy for neuromuscular disorders have shown they can slow or stop disease progression, but such therapies have had little impact on reversing muscle disease that was already present. To reverse disease in patients with muscular dystrophy, new muscle mass and strength must be rebuilt at the same time that gene replacement prevents subsequent disease. Here, we show that treatment of FKRPP448L mice with a dual FKRP/FST gene therapy packaged into a single adeno-associated virus (AAV) vector can build muscle strength and mass that exceed levels found in wild-type mice and can induce normal ambulation endurance in a 1-h walk test. Dual FKRP/FST therapy also showed more even increases in muscle mass and amplified muscle expression of both genes relative to either single gene therapy alone. These data suggest that treatment with single AAV-bearing dual FKRP/FST gene therapies can overcome loss of ambulation by improving muscle strength at the same time it prevents subsequent muscle damage. This design platform could be used to create therapies for other forms of muscular dystrophy that may improve patient outcomes.

The Development of Robust Antibodies to Sarcospan, a Dystrophin- and Integrin-Associated Protein, for Basic and Translational Research.

Sarcospan (SSPN) is a 25-kDa transmembrane protein that is broadly expressed at the cell surface of many tissues, including, but not limited to, the myofibers from skeletal and smooth muscles, cardiomyocytes, adipocytes, kidney epithelial cells, and neurons. SSPN is a core component of the dystrophin-glycoprotein complex (DGC) that links the intracellular actin cytoskeleton with the extracellular matrix. It is also associated with integrin α7ß1, the predominant integrin expressed in skeletal muscle. As a tetraspanin-like protein with four transmembrane spanning domains, SSPN functions as a scaffold to facilitate protein-protein interactions at the cell membrane. Duchenne muscular dystrophy, Becker muscular dystrophy, and X-linked dilated cardiomyopathy are caused by the loss of dystrophin at the muscle cell surface and a concomitant loss of the entire DGC, including SSPN. SSPN overexpression ameliorates Duchenne muscular dystrophy in the mdx murine model, which supports SSPN being a viable therapeutic target. Other rescue studies support SSPN as a biomarker for the proper assembly and membrane expression of the DGC. Highly specific and robust antibodies to SSPN are needed for basic research on the molecular mechanisms of SSPN rescue, pre-clinical studies, and biomarker evaluations in human samples. The development of SSPN antibodies is challenged by the presence of its four transmembrane domains and limited antigenic epitopes. To address the significant barrier presented by limited commercially available antibodies, we aimed to generate a panel of robust SSPN-specific antibodies that can serve as a resource for the research community. We created antibodies to three SSPN protein epitopes, including the intracellular N- and C-termini as well as the large extracellular loop (LEL) between transmembrane domains 3 and 4. We developed a panel of rabbit antibodies (poly- and monoclonal) against an N-terminal peptide fragment of SSPN. We used several assays to show that the rabbit antibodies recognize mouse SSPN with a high functional affinity and specificity. We developed mouse monoclonal antibodies against the C-terminal peptide and the large extracellular loop of human SSPN. These antibodies are superior to commercially available antibodies and outperform them in various applications, including immunoblotting, indirect immunofluorescence analysis, immunoprecipitation, and an ELISA. These newly developed antibodies will significantly improve the quality and ease of SSPN detection for basic and translational research.

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.

Loss of endogenous estrogen alters mitochondrial metabolism and muscle clock-related protein Rbm20 in female mdx mice.

Female carriers of a Duchenne muscular dystrophy (DMD) gene mutation manifest exercise intolerance and metabolic anomalies that may be exacerbated following menopause due to the loss of estrogen, a known regulator of skeletal muscle function and metabolism. Here, we studied the impact of estrogen depletion (via ovariectomy) on exercise tolerance and muscle mitochondrial metabolism in female mdx mice and the potential of estrogen replacement therapy (using estradiol) to protect against functional and metabolic perturbations. We also investigated the effect of estrogen depletion, and replacement, on the skeletal muscle proteome through an untargeted proteomic approach with TMT-labelling. Our study confirms that loss of estrogen in female mdx mice reduces exercise capacity, tricarboxylic acid cycle intermediates, and citrate synthase activity but that these deficits are offset through estrogen replacement therapy. Furthermore, ovariectomy downregulated protein expression of RNA-binding motif factor 20 (Rbm20), a critical regulator of sarcomeric and muscle homeostasis gene splicing, which impacted pathways involving ribosomal and mitochondrial translation. Estrogen replacement modulated Rbm20 protein expression and promoted metabolic processes and the upregulation of proteins involved in mitochondrial dynamics and metabolism. Our data suggest that estrogen mitigates dystrophinopathic features in female mdx mice and that estrogen replacement may be a potential therapy for post-menopausal DMD carriers.

A Novel CRISPR-Cas9 Strategy to Target DYSTROPHIN Mutations Downstream of Exon 44 in Patient-Specific DMD iPSCs.

Mutations in the DMD gene cause fatal Duchenne Muscular Dystrophy (DMD). An attractive therapeutic approach is autologous cell transplantation utilizing myogenic progenitors derived from induced pluripotent stem cells (iPSCs). Given that a significant number of DMD mutations occur between exons 45 and 55, we developed a gene knock-in approach to correct any mutations downstream of exon 44. We applied this approach to two DMD patient-specific iPSC lines carrying mutations in exons 45 and 51 and confirmed mini-DYSTROPHIN (mini-DYS) protein expression in corrected myotubes by western blot and immunofluorescence staining. Transplantation of gene-edited DMD iPSC-derived myogenic progenitors into NSG/mdx4Cv mice produced donor-derived myofibers, as shown by the dual expression of human DYSTROPHIN and LAMIN A/C. These findings further provide proof-of-concept for the use of programmable nucleases for the development of autologous iPSC-based therapy for muscular dystrophies.

Investigation of genotype-phenotype and familial features of Turkish dystrophinopathy patients.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive allelic muscle diseases caused by dystrophin gene mutations. Eight hundred thirty-seven patients admitted between 1997 and 2022 were included in the study. Two hundred twenty patients were analyzed by multiplex PCR (mPCR) alone. Five hundred ninety-five patients were investigated by multiplex ligation-dependent probe amplification (MLPA), and 54 patients were examined by sequencing. Deletion was detected in 60% (132/220) of the cases in the mPCR group only and in 58.3% (347/595) of the cases with MLPA analysis. The rates of deletion and duplication were 87.7% and 12.3%, respectively, in the MLPA analysis. Single exon deletions were the most common mutation type. The introns 43-55 (81.8%) and exons 2-21 (13.1%) regions were detected as hot spots in deletions. It was determined that 89% of the mutations were suitable for exon skipping therapy. The reading frame rule did not hold in 7.6% of D/BMD cases (17/224). We detected twenty-five pathogenic/likely pathogenic variants in sequencing, five of which were novel variants. Nonsense mutation was the most common small mutation (44%). 21% of DMD patients were familial. We detected germline mosaicism in four families (4.3%) in the large rearrangement group and one gonosomal mosaicism in a family with a nonsense mutation. This is the largest study examining genotype and phenotype data in Turkish D/BMD families investigated by MLPA analysis. The reading frame hypothesis is not valid in all cases. Sharing the genotype and phenotype characteristics of these cases in the literature will shed light on the molecular structure of DMD and guide gene therapy research. In genetic counseling, carrier screening in the family and possible gonadal mosaicism should be emphasized.

Extraction method combining saponin and trehalose useful for analyzing fragile intermolecular association.

Immunoprecipitation (IP) and co-immunoprecipitation (co-IP) are well-established methodologies to analyze protein expression and intermolecular interaction. Composition of extraction and washing buffer for preparing protein is important to accomplish experimental purpose. Various kinds of detergents are included in buffer to adjust extraction efficiency and washing effect. Among them, Triton X-100 (Tx-100), Nonidet P-40 (NP40), deoxycholic acid (DOC) and SDS are generally used according to experimental purpose and characteristic features of protein of interest. In some cases, general detergents disrupt intermolecular interaction and make it impossible to analyze molecular relation of protein of interest with its binding partners. In this study, we propose saponin, a natural detergent, is useful for co-immunoprecipitation when analyzing fragile intermolecular interactions, in which dystrophin and dystroglycan are used as a representative interaction. One of the most notable findings in this report is that intermolecular association between dystrophin and dystroglycan is maintained in saponin buffer whereas general detergents, such as Tx-100, NP40 and DOC, dissociate its binding. Furthermore, supplementation of trehalose, which has been shown to act as a molecular chaperone, facilitates efficient detection of dystrophin-dystroglycan macromolecular complex in co-IP assay. Importantly, the extraction buffer comprising 3 % saponin, 0.5 M trehalose and 0.05 % Tx-100 (we named it STX buffer) is applicable to co-IP for another molecular interaction, N-cadherin and ß-catenin, indicating that this methodology can be used for versatile proteins of interest. Thus, STX buffer emerges as an alternative extraction method useful for analyzing fragile intermolecular associations and provides opportunity to identify complex interactomes, which may facilitate proteome-research and functional analysis of proteins of interest.

Dmd mdx mice have defective oligodendrogenesis, delayed myelin compaction and persistent hypomyelination.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, resulting in the loss of dystrophin, a large cytosolic protein that links the cytoskeleton to extracellular matrix receptors in skeletal muscle. Aside from progressive muscle damage, many patients with DMD also have neurological deficits of unknown etiology. To investigate potential mechanisms for DMD neurological deficits, we assessed postnatal oligodendrogenesis and myelination in the Dmdmdx mouse model. In the ventricular-subventricular zone (V-SVZ) stem cell niche, we found that oligodendrocyte progenitor cell (OPC) production was deficient, with reduced OPC densities and proliferation, despite a normal stem cell niche organization. In the Dmdmdx corpus callosum, a large white matter tract adjacent to the V-SVZ, we also observed reduced OPC proliferation and fewer oligodendrocytes. Transmission electron microscopy further revealed significantly thinner myelin, an increased number of abnormal myelin structures and delayed myelin compaction, with hypomyelination persisting into adulthood. Our findings reveal alterations in oligodendrocyte development and myelination that support the hypothesis that changes in diffusion tensor imaging seen in patients with DMD reflect developmental changes in myelin architecture.