Limb-girdle muscular dystrophy R2-dysferlin related (LGMD-R2) is a progressive muscle condition with marked variability in disease course, making prognosis challenging. Quantitative MRI (qMRI) has emerged as a complementary tool that may detect progression earlier and more precisely. Integrating different data modalities is challenging with conventional approaches, and artificial intelligence (AI) can help overcome this. Our aim is to develop robust models capable of predicting clinical progression in LGMD-R2 by incorporating AI-based techniques into the analysis pipeline. Data from 188 COS 1 participants were analysed. Disease progression was assessed using the North Star Assessment for Limb Girdle type Muscular Dystrophies (NSAD). Ambulatory individuals with a maximum NSAD ≥ 20 were included, and progression trajectories were identified through hierarchical clustering. Feature selection was performed using a machine learning pipeline, and top predictors were entered into stepwise logistic regression to build clinical-only and combined clinical-MRI models. Two stages of progression were identified, a fast one with a mean three-year loss of 14.4 NSAD points, and a moderate one, with a mean loss of 3.8 NSAD points. The combined model achieved better balanced accuracy than the clinical-only one (83.7

BACKGROUND:Limb-girdle muscular dystrophy (MD) type R2 (LGMDR2, formerly LGMD2B) is an autosomal recessive form of MD caused by variants in the dysferlin gene, DYSF. It leads to slow proximal and distal muscle weakening that generally results in loss of ambulation around early adulthood but without the lethal cardiorespiratory dysfunction observed in the more severe Duchenne MD. How loss of dysferlin causes muscle fibre death is poorly understood, but recent evidence suggests a link between muscle wasting and loss of muscle cholesterol homeostasis with circulating lipoprotein abnormalities in many forms of MD. METHODS:Cross-sectional circulating total cholesterol (CHOL), high-density lipoprotein-associated cholesterol (HDL-C), non-HDL-C, creatine kinase (CK), transaminase levels and bilirubin were collected as part of the Jain Clinical Outcomes Study of Dysferlinopathy, a large multicentre LGMDR2 patient cohort (N = 188), along with ambulatory function values. RESULTS:We report that 43%, 49% and 50% of male patients were found to have abnormal circulating CHOL, HDL-C and non-HDL-C levels, respectively, whereas in female patients 39%, 37% and 30% of values were in the abnormal range. Overall, 68% of the total cohort had at least one abnormal cholesterol value (78% of males and 60% of females) and 89% of male CHOL/HDL-C ratios were in the suboptimal range (above 3.5). Although most patients were ambulant, the severity of circulating lipid abnormalities did not correlate with early loss of ambulation. Transaminase levels were lower in late-stage LGMDR2 samples, whereas bilirubin remained unchanged, suggesting a low muscular mass rather than hepatic origin and the absence of major liver damage. CONCLUSIONS:Data from the largest natural history cohort of LGMDR2 patients support the concept that dyslipidemia is a comorbidity of LGMDR2, and the causal role of cholesterol abnormalities in muscle death should be further investigated.

Dystrophy-associated fer-1-like protein (dysferlin) conducts plasma membrane repair. Mutations in the DYSF gene cause a panoply of genetic muscular dystrophies. We targeted a frequent loss-of-function, DYSF exon 44, founder frameshift mutation with mRNA-mediated delivery of SpCas9 in combination with a mutation-specific sgRNA to primary muscle stem cells from two homozygous patients. We observed a consistent >60% exon 44 re-framing, rescuing a full-length and functional dysferlin protein. A new mouse model harboring a humanized Dysf exon 44 with the founder mutation, hEx44mut, recapitulates the patients' phenotype and an identical re-framing outcome in primary muscle stem cells. Finally, gene-edited murine primary muscle stem-cells are able to regenerate muscle and rescue dysferlin when transplanted back into hEx44mut hosts. These findings are the first to show that a CRISPR-mediated therapy can ameliorate dysferlin deficiency. We suggest that gene-edited primary muscle stem cells could exhibit utility, not only in treating dysferlin deficiency syndromes, but also perhaps other forms of muscular dystrophy.

Dystrophy-associated fer-1-like protein (dysferlin) conducts plasma membrane repair. Mutations in the DYSF gene cause a panoply of genetic muscular dystrophies. We targeted a frequent loss-of-function, DYSF exon 44, founder frameshift mutation with mRNA-mediated delivery of SpCas9 in combination with a mutation-specific sgRNA to primary muscle stem cells from two homozygous patients. We observed a consistent >60% exon 44 re-framing, rescuing a full-length and functional dysferlin protein. A new mouse model harboring a humanized Dysf exon 44 with the founder mutation, hEx44mut, recapitulated the patients’ phenotype and an identical re-framing outcome in primary muscle stem cells. Finally, gene-edited murine primary muscle stem-cells were able to regenerate muscle and rescued dysferlin when transplanted back into hEx44mut hosts. These findings are the first to show that a CRISPR-mediated therapy can ameliorate dysferlin deficiency. We suggest that gene-edited primary muscle stem cells could exhibit utility, not only in treating dysferlin deficiency syndromes, but also perhaps other forms of muscular dystrophy.### Competing Interest StatementSi.S. is an inventor on a technology for primary human muscle stem cell isolation and manufacturing (IP: (DE10 2014 216872), 2015 PCT (WO 2016/030371), granted in EU and US). Si.S. and H.E. are co-inventors on a pending patent application on gene editing of human muscle stem cells (European Patent 666 Office 21 160 696.7). Si.S. is co-founder of MyoPax GmbH and MyoPax Denmark ApS.

Background & AimLimb Girdle Muscular Dystrophy type 2A/R1 (LGMD2A/R1), caused by mutations in the Calpain 3 gene, is a severe muscular dystrophy leading to progressive loss of the hip and shoulder girdle musculature with no treatment available. With thousands of patients worldwide, CAPN3 c.550delA is the most frequent mutation in LGMD2A/R1. We genetically corrected this founder mutation in primary human muscle stem cells (PHSats) from patients. PHSats can be isolated highly efficient without contaminating fibroblasts from patient muscles and expanded in in vitro.Methods, Results & ConclusionMutation-specific targeting with a synthetic sgRNA and Cas9 mRNA yielded highly efficient and precise correction of CAPN3 c.550delA. The single cut generated by Cas9 results in a 5’ staggered overhang of one base pair, which triggers an overhang-dependent base replication of an A:T at the mutation site. This precise reframing recovers the open reading frame and the CAPN3 DNA sequence is repaired template-free to wild type, which leads to CAPN3 re-expression and functional recovery. Off-target analysis using amplicon sequencing of 43 targets nominated by GUIDE-Seq and in silico prediction did not show any editing demonstrating the safety of this approach. This is also true for the translocation analysis confirming the safe and precise repair for CAPN3 c.550delA.Additionally, we evaluated the potential of editing two patient mutations in one cell. Most of the patients with CAPN3 c.550delA are compound heterozygous and carry a second mutation in CAPN3. We can target two mutations even with different gene editing tools. In this regard, we show the successful editing of CAPN3 c.550delA with Cas9 mRNA in combination with base editing of the second mutation CAPN3 c.1468C>T using ABE8e mRNA in compound heterozygous patient PHSats.Resulting genetically corrected PHSats are a valuable tool for autologous transplantation back into patients to recover muscle structure and function. To test if corrected PHSats from CAPN3 c.550delA patients have the potential to form functional muscle tissue, we transplanted repaired cells into immunocompromised Capn3-Knock-out mice. Gene-corrected PHSats contributed to mouse muscle, repopulate the stem cell niche and express CAPN3.Currently, we are planning a First-in-human clinical trial to transplant repaired PHSats into patient muscles. The trial is prepared to launch in 2025.

Delandistrogene moxeparvovec received FDA approval after a negative trial, which highlights the many complexities and challenges of drug development in this setting.

(1) Heart transplantation (HTX) improves the overall survival and functional status of end-stage heart failure patients with cardiomyopathies (CMPs). The majority of CMPs have genetic causes, and the overlap between CMPs and inherited myopathies is well documented. However, the long-term outcome in skeletal muscle function and possibility of an undiagnosed underlying genetic cause of both a cardiac and skeletal pathology remain unknown. (2) Thirty-nine patients were assessed using open and standardized interviews on muscle function, a quality-of-life (EuroQol EQ-5D-3L) questionnaire, and a physical examination (Medical Research Council Muscle scale). Whole-exome sequencing was completed in three stages for those with skeletal muscle weakness. (3) Seven patients (17.9%) reported new-onset muscle weakness and motor limitations. Objective muscle weakness in the upper and lower extremities was seen in four patients. In three of them, exome sequencing revealed pathogenic/likely pathogenic variants in the genes encoding nexilin, myosin heavy chain, titin, and SPG7. (4) Our findings support a positive long-term outcome of skeletal muscle function in HTX patients. However, 10% of patients showed clinical signs of myopathy due to a possible genetic cause. The integration of genetic testing and standardized neurological assessment of motor function during the peri-HTX period should be considered.

New promising genetic and cell-based therapeutic approaches are becoming a reality for untreatable neuromuscular disorders. However, their effective translation to clinical practice is impeded by various challenges, including delays in precise genetic diagnosis, heterogeneous guidelines for longitudinal monitoring, inconsistent capture of Patients-Reported Outcome Measures, documentation of data in incompatible formats and disparate data documentation practices across countries. Leveraging methods like machine learning and AI-based algorithms for diagnostics and post-marketing surveillance necessitate organized and standardized data. In this study, we aimed to create a comprehensive, standardized information model for clinical data, facilitating research and international collaboration efforts. The documentation addresses: 1. Easy and coherent access to clinical, genetic, functional, histological data and imaging findings with the use of branching logic for data capture, minimizing documentation effort 2. Data comparability and international application through coding using the international terminology SNOMED CT and HPO terms 3. User-friendly data collection and subsequent analysis regardless of linguistic differences through the development of value sets with frequent values for the individual data elements 4. Documentation of negative findings and exclusions 5. Possibility of implementation in different systems and environments, thus guaranteeing comparability of the data. Embracing digital health technologies paves the way for streamlining data capture, facilitating knowledge sharing, improving interoperability between electronic health record systems. Addressing the challenges requires consideration of the complex nature of neuromuscular diseases, collaboration among stakeholders, iterative refinement of the information model, enhancement of interoperability and incorporation of innovative solutions such as patient engagement strategies and digital health technologies.

With thousands of patients worldwide, CAPN3 c.550delA is the most frequent mutation causing severe, progressive, and untreatable limb girdle muscular dystrophy. We aimed to genetically correct this founder mutation in primary human muscle stem cells. We designed editing strategies providing CRISPR-Cas9 as plasmid and mRNA first in patient-derived induced pluripotent stem cells and applied this strategy then in primary human muscle stem cells from patients. Mutation-specific targeting yielded highly efficient and precise correction of CAPN3 c.550delA to wild type for both cell types. Most likely a single cut generated by SpCas9 resulted in a 5' staggered overhang of one base pair, which triggered an overhang-dependent base replication of an A:T at the mutation site. This recovered the open reading frame and the CAPN3 DNA sequence was repaired template-free to wild type, which led to CAPN3 mRNA and protein expression. Off-target analysis using amplicon sequencing of 43 in silico predicted sites demonstrates the safety of this approach. Our study extends previous usage of single cut DNA modification since our gene product has been repaired into the wild-type CAPN3 sequence with the perspective of a real cure.

Background: Tissue engineered bioscaffolds based on decellularized composites have gained increasing interest for treatment of various diaphragmatic impairments, including muscular atrophies and diaphragmatic hernias. Detergent-enzymatic treatment (DET) constitutes a standard strategy for diaphragmatic decellularization. However, there is scarce data on comparing DET protocols with different substances in distinct application models in their ability to maximize cellular removal while minimizing extracellular matrix (ECM) damage. Methods: We decellularized diaphragms of male Sprague Dawley rats with 1 % or 0.1 % sodium dodecyl sulfate (SDS) and 4 % sodium deoxycholate (SDC) by orbital shaking (OS) or retrograde perfusion (RP) through the vena cava. We evaluated decellularized diaphragmatic samples by (1) quantitative analysis including DNA quantifi-cation and biomechanical testing, (2) qualitative and semiquantitative analysis by proteomics, as well as (3) qualitative assessment with macroscopic and microscopic evaluation by histological staining, immunohisto-chemistry and scanning electron microscopy. Results: All protocols produced decellularized matrices with micro-and ultramorphologically intact architecture and adequate biomechanical performance with gradual differences. The proteomic profile of decellularized matrices contained a broad range of primal core and ECM-associated proteins similar to native muscle. While no outstanding preference for one singular protocol was determinable, SDS-treated samples showed slightly bene-ficial properties in comparison to SDC-processed counterparts. Both application modalities proved suitable for DET. Conclusion: DET with SDS or SDC via orbital shaking or retrograde perfusion constitute suitable methods to produce adequately decellularized matrices with characteristically preserved proteomic composition. Exposing compositional and functional specifics of variously treated grafts may enable establishing an ideal processing strategy to sustain valuable tissue characteristics and optimize consecutive recellularization. This aims to design an optimal bioscaffold for future transplantation in quantitative and qualitative diaphragmatic defects.

Critical illness myopathy (CIM) is an acquired, devastating, multifactorial muscle-wasting disease with incomplete recovery. The impact on hospital costs and permanent loss of quality of life is enormous. Incomplete recovery might imply that the function of muscle stem cells (MuSC) is impaired. We tested whether epigenetic alterations could be in part responsible. We characterized human muscle stem cells (MuSC) isolated from early CIM and analyzed epigenetic alterations (CIM n = 15, controls n = 21) by RNA-Seq, immunofluorescence, analysis of DNA repair, and ATAC-Seq. CIM-MuSC were transplanted into immunodeficient NOG mice to assess their regenerative potential. CIM-MuSC exhibited significant growth deficits, reduced ability to differentiate into myotubes, and impaired DNA repair. The chromatin structure was damaged, as characterized by alterations in mRNA of histone 1, depletion or dislocation of core proteins of nucleosome remodeling and deacetylase complex, and loosening of multiple nucleosome-spanning sites. Functionally, CIM-MuSC had a defect in building new muscle fibers. Further, MuSC obtained from the electrically stimulated muscle of CIM patients was very similar to control MuSC, indicating the impact of muscle contraction in the onset of CIM. CIM not only affects working skeletal muscle but has a lasting and severe epigenetic impact on MuSC.

Objective: In the field of non-treatable muscular dystrophies, promising new gene and cell therapies are being developed and are entering clinical trials. Objective assessment of therapeutic effects on motor function is mandatory for economical and ethical reasons. Main shortcomings of existing measurements are discontinuous data collection in artificial settings as well as a major focus on walking, neglecting the importance of hand and arm movements for patients' independence. We aimed to create a digital tool to measure muscle function with an emphasis on upper limb motility. Methods: suMus provides a custom-made App running on smartwatches. Movement data are sent to the backend of a suMus web-based platform, from which they can be extracted as CSV data. Fifty patients with neuromuscular diseases assessed the pool of suMus activities in a first orientation phase. suMus performance was hence validated in four upper extremity exercises based on the feedback of the orientation phase. We monitored the arm metrics in a cohort of healthy volunteers using the suMus application, while completing each exercise at low frequency in a metabolic chamber. Collected movement data encompassed average acceleration, rotation rate as well as activity counts. Spearman rank tests correlated movement data with energy expenditure from the metabolic chamber. Results: Our novel application "suMus," sum of muscle activity, collects muscle movement data plus Patient-Related-Outcome-Measures, sends real-time feedback to patients and caregivers and provides, while ensuring data protection, a long-term follow-up of disease course. The application was well received from the patients during the orientation phase. In our pilot study, energy expenditure did not differ between overnight fasted and non-fasted participants. Acceleration ranged from 1.7 ± 0.7 to 3.2 ± 0.5 m/sec2 with rotation rates between 0.9 ± 0.5 and 2.0 ± 3.4 rad/sec. Acceleration and rotation rate as well as derived activity counts correlated with energy expenditure values measured in the metabolic chamber for one exercise (r = 0.58, p < 0.03). Conclusion: In the analysis of slow frequency movements of upper extremities, the integration of the suMus application with smartwatch sensors characterized motion parameters, thus supporting a use in clinical trial outcome measures. Alternative methodologies need to complement indirect calorimetry in validating accelerometer-derived energy expenditure data.

Diaphragmatic impairments exhibit high morbidity as well as mortality while current treatment options remain unsatisfactory. Tissue engineering (TE) approaches have explored the generation of an optimal biocompatible scaffold for diaphragmatic repair through tissue decellularization or de novo construction, with or without the addition of cells. The authors conducted a systematic review on the current state of the art in diaphragmatic tissue engineering (DTE) and found 24 articles eligible for final synthesis. The included approaches studied decellularization‐based graft generation (9) and de novo bioscaffold construction (9). Three studies focused on in vitro host‐scaffold interaction with synthesized, recellularized grafts (2) and decellularized extracellular matrix scaffolds (1). Another three studies investigated evaluation tools for decellularization efficacy. Among all studies, recellularization is performed in both decellularization‐based (4) and de novo generated scaffolds (4). De novo constructed biocomposites as well as decellularized and recellularized scaffolds induced pro‐regenerative remodeling and recovery of diaphragmatic function in all examined animal models. Potential therapeutic applications comprise substance defects requiring patch repair, such as congenital diaphragmatic hernia, and functional diseases demanding an entire organ transplant, like muscular dystrophies or dysfunction after prolonged artificial respiration.

Dysferlinopathy is a muscular dystrophy with a highly variable functional disease progression in which the relationship of function to some patient reported outcome measures (PROMs) has not been previously reported. This analysis aims to identify the suitability of PROMs and their association with motor performance.Two-hundred and four patients with dysferlinopathy were identified in the Jain Foundation's Clinical Outcome Study in Dysferlinopathy from 14 sites in 8 countries. All patients completed the following PROMs: Individualized Neuromuscular Quality of Life Questionnaire (INQoL), International Physical Activity Questionnaire (IPAQ), and activity limitations for patients with upper and/or lower limb impairments (ACTIVLIMs). In addition, nonambulant patients completed the Egen Klassifikation Scale (EK). Assessments were conducted annually at baseline, years 1, 2, 3, and 4. Data were also collected on the North Star Assessment for Limb Girdle Type Muscular Dystrophies (NSAD) and Performance of Upper Limb (PUL) at these time points from year 2. Data were analyzed using descriptive statistics and Rasch analysis was conducted on ACTIVLIM, EK, INQoL. For associations, graphs (NSAD with ACTIVLIM, IPAQ and INQoL and EK with PUL) were generated from generalized estimating equations (GEE). The ACTIVLIM appeared robust psychometrically and was strongly associated with the NSAD total score (Pseudo R2 0.68). The INQoL performed less well and was poorly associated with the NSAD total score (Pseudo R2 0.18). EK scores were strongly associated with PUL (Pseudo R2 0.69). IPAQ was poorly associated with NSAD scores (Pseudo R2 0.09). This study showed that several of the chosen PROMs demonstrated change over time and a good association with functional outcomes. An alternative quality of life measure and method of collecting data on physical activity may need to be selected for assessing dysferlinopathy.

Beside their beneficial effects on cardiovascular events, statins are thought to contribute to insulin resistance and type-2 diabetes. It is not known whether these effects are long-term events from statin-treatment or already triggered with the first statin-intake. Skeletal muscle is considered the main site for insulin-stimulated glucose uptake and therefore, a primary target for insulin resistance in the human body. We analyzed localization and expression of proteins related to GLUT4 mediated glucose uptake via AMPKα or AKT in human skeletal muscle tissue from patients with statin-intake >6 months and in primary human myotubes after 96 h statin treatment. The ratio for AMPKα activity significantly increased in human skeletal muscle cells treated with statins for long- and short-term. Furthermore, the insulin-stimulated counterpart, AKT, significantly decreased in activity and protein level, while GSK3ß and mTOR protein expression reduced in statin-treated primary human myotubes, only. However, GLUT4 was normally distributed whereas CAV3 was internalized from plasma membrane around the nucleus in statin-treated primary human myotubes. Statin-treatment activates AMPKα-dependent glucose uptake and remains active after long-term statin treatment. Permanent blocking of its insulin-dependent counterpart AKT activation may lead to metabolic inflexibility and insulin resistance in the long run and may be a direct consequence of statin-treatment.

Despite advances in multi-modal treatment approaches, clinical outcomes of patients suffering from PAX3-FOXO1 fusion oncogene-expressing alveolar rhabdomyosarcoma (ARMS) remain dismal. Here we show that PAX3-FOXO1-expressing ARMS cells are sensitive to pharmacological ataxia telangiectasia and Rad3 related protein (ATR) inhibition. Expression of PAX3-FOXO1 in muscle progenitor cells is not only sufficient to increase sensitivity to ATR inhibition, but PAX3-FOXO1-expressing rhabdomyosarcoma cells also exhibit increased sensitivity to structurally diverse inhibitors of ATR. Mechanistically, ATR inhibition leads to replication stress exacerbation, decreased BRCA1 phosphorylation and reduced homologous recombination-mediated DNA repair pathway activity. Consequently, ATR inhibitor treatment increases sensitivity of ARMS cells to PARP1 inhibition in vitro, and combined treatment with ATR and PARP1 inhibitors induces complete regression of primary patient-derived ARMS xenografts in vivo. Lastly, a genome-wide CRISPR activation screen (CRISPRa) in combination with transcriptional analyses of ATR inhibitor resistant ARMS cells identifies the RAS-MAPK pathway and its targets, the FOS gene family, as inducers of resistance to ATR inhibition. Our findings provide a rationale for upcoming biomarker-driven clinical trials of ATR inhibitors in patients suffering from ARMS.

Background The aim of the study was to demonstrate the efficacy of human muscle stem cells (MuSCs) isolated using innovative technology in restoring internal urinary sphincter function in a preclinical animal model. Methods Colonies of pure human MuSCs were obtained from muscle biopsy specimens. Athymic rats were subjected to internal urethral sphincter damage by electrocauterization. Five days after injury, 2 × 10 5 muscle stem cells or medium as control were injected into the area of sphincter damage ( n = 5 in each group). Peak bladder pressure and rise in pressure were chosen as outcome measures. To repeatedly obtain the necessary pressure values, telemetry sensors had been implanted into the rat bladders 10 days prior to injury. Results There was a highly significant improvement in the ability to build up peak pressure as well as a pressure rise in animals that had received muscle stem cells as compared to control ( p = 0.007) 3 weeks after the cells had been injected. Only minimal histologic evidence of scarring was observed in treated rats. Conclusion Primary human muscle stem cells obtained using innovative technology functionally restore internal urethral sphincter function after injury. Translation into use in clinical settings is foreseeable.

Hintergrund Tissue engineered bioscaffolds based on decellularized composites have gained increasing interest for treatment of various diaphragmatic impairments, including patch repair of diaphragmatic hernias, and defects due to trauma or oncologic resections (e.g. lung cancer, pleural metastasis).The in vivo performance of decellularized diaphragmatic tissues depends on bioengineering parameters such as applied agents and processing techniques, which vary in their efficacy to maximize cellular removal and minimize extracellular matrix (ECM) damage.