Development and systematic evaluation of decellularization protocols in different application models for diaphragmatic tissue engineering.

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 quantification and biomechanical testing, (2) qualitative and semiquantitative analysis by proteomics, as well as (3) qualitative assessment with macroscopic and microscopic evaluation by histological staining, immunohistochemistry 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 beneficial 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.

Cas9-induced single cut enables highly efficient and template-free repair of a muscular dystrophy causing founder mutation.

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.

Corrigendum: Exercise blood-drop metabolic profiling links metabolism with perceived exertion.

[This corrects the article DOI: 10.3389/fmolb.2022.1042231.].

"suMus," a novel digital system for arm movement metrics and muscle energy expenditure.

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.

Disintegration of the NuRD Complex in Primary Human Muscle Stem Cells in Critical Illness Myopathy.

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.

LMNA Co-Regulated Gene Expression as a Suitable Readout after Precise Gene Correction.

LMNA-related muscular dystrophy is an autosomal-dominant progressive disorder caused by mutations in LMNA. LMNA missense mutations are becoming correctable with CRISPR/Cas9-derived tools. Evaluating the functional recovery of LMNA after gene editing bears challenges as there is no reported direct loss of function of lamin A/C proteins in patient-derived cells. The proteins encoded by LMNA are lamins A/C, important ubiquitous nuclear envelope proteins but absent in pluripotent stem cells. We induced lamin A/C expression in induced pluripotent stem cells (iPSCs) of two patients with LMNA-related muscular dystrophy, NM_170707.4 (LMNA): c.1366A > G, p.(Asn456Asp) and c.1494G > T, p.(Trp498Cys), using a short three-day, serum-induced differentiation protocol and analyzed expression profiles of co-regulated genes, examples being COL1A2 and S100A6. We then performed precise gene editing of LMNA c.1366A > G using the near-PAMless (PAM: protospacer-adjacent motif) cytosine base editor. We show that the mutation can be repaired to 100% efficiency in individual iPSC clones. The fast differentiation protocol provided a functional readout and demonstrated increased lamin A/C expression as well as normalized expression of co-regulated genes. Collectively, our findings demonstrate the power of CRISPR/Cas9-mediated gene correction and effective outcome measures in a disease with, so far, little perspective on therapies.

Human primary muscle stem cells regenerate injured urethral sphincter in athymic rats.

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 × 105 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.

Therapeutic targeting of ATR in alveolar rhabdomyosarcoma.

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.

Generation of hiPSC-Derived Skeletal Muscle Cells: Exploiting the Potential of Skeletal Muscle-Derived hiPSCs.

Cell therapies for muscle wasting disorders are on the verge of becoming a realistic clinical perspective. Muscle precursor cells derived from human induced pluripotent stem cells (hiPSCs) represent the key to unrestricted cell numbers indispensable for the treatment of generalized muscle wasting such as cachexia or intensive care unit (ICU)-acquired weakness. We asked how the cell of origin influences efficacy and molecular properties of hiPSC-derived muscle progenitor cells. We generated hiPSCs from primary muscle stem cells and from peripheral blood mononuclear cells (PBMCs) of the same donors (n = 4) and compared their molecular profiles, myogenic differentiation potential, and ability to generate new muscle fibers in vivo. We show that reprogramming into hiPSCs from primary muscle stem cells was faster and 35 times more efficient than from blood cells. Global transcriptome comparison revealed significant differences, but differentiation into induced myogenic cells using a directed transgene-free approach could be achieved with muscle- and PBMC-derived hiPSCs, and both cell types generated new muscle fibers in vivo. Differences in myogenic differentiation efficiency were identified with hiPSCs generated from individual donors. The generation of muscle-stem-cell-derived hiPSCs is a fast and economic method to obtain unrestricted cell numbers for cell-based therapies in muscle wasting disorders, and in this aspect are superior to blood-derived hiPSCs.

Myofibrillar protein synthesis rates are increased in chronically exercised skeletal muscle despite decreased anabolic signaling.

The molecular responses to acute resistance exercise are well characterized. However, how cellular signals change over time to modulate chronic adaptations to more prolonged exercise training is less well understood. We investigated anabolic signaling and muscle protein synthesis rates at several time points after acute and chronic eccentric loading. Adult rat tibialis anterior muscle was stimulated for six sets of ten repetitions, and the muscle was collected at 0 h, 6 h, 18 h and 48 h. In the last group of animals, 48 h after the first exercise bout a second bout was conducted, and the muscle was collected 6 h later (54 h total). In a second experiment, rats were exposed to four exercise sessions over the course of 2 weeks. Anabolic signaling increased robustly 6 h after the first bout returning to baseline between 18 and 48 h. Interestingly, 6 h after the second bout mTORC1 activity was significantly lower than following the first bout. In the chronically exercised rats, we found baseline anabolic signaling was decreased, whereas myofibrillar protein synthesis (MPS) was substantially increased, 48 h after the last bout of exercise. The increase in MPS occurred in the absence of changes to muscle fiber size or mass. In conclusion, we find that anabolic signaling is already diminished after the second bout of acute resistance type exercise. Further, chronic exposure to resistance type exercise training results in decreased basal anabolic signaling but increased overall MPS rates.

Assessing the Relationship of Patient Reported Outcome Measures With Functional Status in Dysferlinopathy: A Rasch Analysis Approach.

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 R 2 0.68). The INQoL performed less well and was poorly associated with the NSAD total score (Pseudo R 2 0.18). EK scores were strongly associated with PUL (Pseudo R 2 0.69). IPAQ was poorly associated with NSAD scores (Pseudo R 2 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.

mRNA-mediated delivery of gene editing tools to human primary muscle stem cells.

Muscular dystrophies are approximately 50 devastating, untreatable monogenic diseases leading to progressive muscle degeneration and atrophy. Gene correction of transplantable cells using CRISPR/Cas9-based tools is a realistic scenario for autologous cell replacement therapies to restore organ function in many genetic disorders. However, muscle stem cells have so far lagged behind due to the absence of methods to isolate and propagate them and their susceptibility to extensive ex vivo manipulations. Here, we show that mRNA-based delivery of SpCas9 and an adenine base editor results in up to >90% efficient genome editing in human muscle stem cells from many donors regardless of age and gender and without any enrichment step. Using NCAM1 as an endogenous reporter locus expressed by all muscle stem cells and whose knockout does not affect cell fitness, we show that cells edited with mRNA fully retain their myogenic marker signature, proliferation capacity, and functional attributes. Moreover, mRNA-based delivery of a base editor led to the highly efficient repair of a muscular dystrophy-causing SGCA mutation in a single selection-free step. In summary, our work establishes mRNA-mediated delivery of CRISPR/Cas9-based tools as a promising and universal approach for taking gene edited muscle stem cells into clinical application to treat muscle disease.

Cardiac and pulmonary findings in dysferlinopathy: A 3-year, longitudinal study.

INTRODUCTION/AIMS: There is debate about whether and to what extent either respiratory or cardiac dysfunction occurs in patients with dysferlinopathy. This study aimed to establish definitively whether dysfunction in either system is part of the dysferlinopathy phenotype. METHODS: As part of the Jain Foundation's International Clinical Outcome Study (COS) for dysferlinopathy, objective measures of respiratory and cardiac function were collected twice, with a 3-y interval between tests, in 188 genetically confirmed patients aged 11-86 y (53% female). Measures included forced vital capacity (FVC), electrocardiogram (ECG), and echocardiogram (echo). RESULTS: Mean FVC was 90% predicted at baseline, decreasing to 88% at year 3. FVC was less than 80% predicted in 44 patients (24%) at baseline and 48 patients (30%) by year 3, including ambulant participants. ECGs showed P-wave abnormalities indicative of delayed trans-atrial conduction in 58% of patients at baseline, representing a risk for developing atrial flutter or fibrillation. The prevalence of impaired left ventricular function or hypertrophy was comparable to that in the general population. DISCUSSION: These results demonstrate clinically significant respiratory impairment and abnormal atrial conduction in some patients with dysferlinopathy. Therefore, we recommend that annual or biannual follow-up should include FVC measurement, enquiry about arrhythmia symptoms and peripheral pulse palpation to assess cardiac rhythm. However, periodic specialist cardiac review is probably not warranted unless prompted by symptoms or abnormal pulse findings.

Statins Aggravate the Risk of Insulin Resistance in Human Muscle.

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.

4-Phenylbutyrate restores localization and membrane repair to human dysferlin mutations.

Dysferlinopathies are muscular dystrophies caused by recessive loss-of-function mutations in dysferlin (DYSF), a membrane protein involved in skeletal muscle membrane repair. We describe a cell-based assay in which human DYSF proteins bearing missense mutations are quantitatively assayed for membrane localization by flow cytometry and identified 64 localization-defective DYSF mutations. Using this platform, we show that the clinically approved drug 4-phenylbutryric acid (4-PBA) partially restores membrane localization to 25 mutations, as well as membrane repair to cultured myotubes expressing 2 different mutations. Two-day oral administration of 4-PBA to mice homozygous for one of these mutations restored myofiber membrane repair. 4-PBA may hold therapeutic potential for treating a subset of humans with muscular dystrophy due to dysferlin deficiency.

Exercise blood-drop metabolic profiling links metabolism with perceived exertion.

Background: Assessing detailed metabolism in exercising persons minute-to-minute has not been possible. We developed a "drop-of-blood" platform to fulfill that need. Our study aimed not only to demonstrate the utility of our methodology, but also to give insights into unknown mechanisms and new directions. Methods: We developed a platform, based on gas chromatography and mass spectrometry, to assess metabolism from a blood-drop. We first observed a single volunteer who ran 13 km in 60 min. We particularly monitored relative perceived exertion (RPE). We observed that 2,3-bisphosphoglycerate peaked at RPE in this subject. We next expanded these findings to women and men volunteers who performed an RPE-based exercise protocol to RPE at Fi O 2 20.9% or Fi O 2 14.5% in random order. Results: At 6 km, our subject reached his maximum relative perceived exertion (RPE); however, he continued running, felt better, and finished his run. Lactate levels had stably increased by 2 km, ketoacids increased gradually until the run's end, while the hypoxia marker, 2,3 bisphosphoglycerate, peaked at maximum relative perceived exertion. In our normal volunteers, the changes in lactate, pyruvate, ß hydroxybutyrate and a hydroxybutyrate were not identical, but similar to our model proband runner. Conclusion: Glucose availability was not the limiting factor, as glucose availability increased towards exercise end in highly exerted subjects. Instead, the tricarboxylic acid→oxphos pathway, lactate clearance, and thus and the oxidative capacity appeared to be the defining elements in confronting maximal exertion. These ideas must be tested further in more definitive studies. Our preliminary work suggests that our single-drop methodology could be of great utility in studying exercise physiology.

Progressive myocardial injury in myotonic dystrophy type II and facioscapulohumeral muscular dystrophy 1: a cardiovascular magnetic resonance follow-up study.

AIM: Muscular dystrophy (MD) is a progressive disease with predominantly muscular symptoms. Myotonic dystrophy type II (MD2) and facioscapulohumeral muscular dystrophy type 1 (FSHD1) are gaining an increasing awareness, but data on cardiac involvement are conflicting. The aim of this study was to determine a progression of cardiac remodeling in both entities by applying cardiovascular magnetic resonance (CMR) and evaluate its potential relation to arrhythmias as well as to conduction abnormalities. METHODS AND RESULTS: 83 MD2 and FSHD1 patients were followed. The participation was 87% in MD2 and 80% in FSHD1. 1.5 T CMR was performed to assess functional parameters as well as myocardial tissue characterization applying T1 and T2 mapping, fat/water-separated imaging and late gadolinium enhancement. Focal fibrosis was detected in 23% of MD2) and 33% of FSHD1 subjects and fat infiltration in 32% of MD2 and 28% of FSHD1 subjects, respectively. The incidence of all focal findings was higher at follow-up. T2 decreased, whereas native T1 remained stable. Global extracellular volume fraction (ECV) decreased similarly to the fibrosis volume while the total cell volume remained unchanged. All patients with focal fibrosis showed a significant increase in left ventricular (LV) and right ventricular (RV) volumes. An increase of arrhythmic events was observed. All patients with ventricular arrhythmias had focal myocardial changes and an increased volume of both ventricles (LV end-diastolic volume (EDV) p = 0.003, RVEDV p = 0.031). Patients with supraventricular tachycardias had a significantly higher left atrial volume (p = 0.047). CONCLUSION: We observed a remarkably fast and progressive decline of cardiac morphology and function as well as a progression of rhythm disturbances, even in asymptomatic patients with a potential association between an increase in arrhythmias and progression of myocardial tissue damage, such as focal fibrosis and fat infiltration, exists. These results suggest that MD2 and FSHD1 patients should be carefully followed-up to identify early development of remodeling and potential risks for the development of further cardiac events even in the absence of symptoms. Trial registration ISRCTN, ID ISRCTN16491505. Registered 29 November 2017 - Retrospectively registered, http://www.isrctn.com/ISRCTN16491505.

Myogenic Cell Transplantation in Genetic and Acquired Diseases of Skeletal Muscle.

This article will review myogenic cell transplantation for congenital and acquired diseases of skeletal muscle. There are already a number of excellent reviews on this topic, but they are mostly focused on a specific disease, muscular dystrophies and in particular Duchenne Muscular Dystrophy. There are also recent reviews on cell transplantation for inflammatory myopathies, volumetric muscle loss (VML) (this usually with biomaterials), sarcopenia and sphincter incontinence, mainly urinary but also fecal. We believe it would be useful at this stage, to compare the same strategy as adopted in all these different diseases, in order to outline similarities and differences in cell source, pre-clinical models, administration route, and outcome measures. This in turn may help to understand which common or disease-specific problems have so far limited clinical success of cell transplantation in this area, especially when compared to other fields, such as epithelial cell transplantation. We also hope that this may be useful to people outside the field to get a comprehensive view in a single review. As for any cell transplantation procedure, the choice between autologous and heterologous cells is dictated by a number of criteria, such as cell availability, possibility of in vitro expansion to reach the number required, need for genetic correction for many but not necessarily all muscular dystrophies, and immune reaction, mainly to a heterologous, even if HLA-matched cells and, to a minor extent, to the therapeutic gene product, a possible antigen for the patient. Finally, induced pluripotent stem cell derivatives, that have entered clinical experimentation for other diseases, may in the future offer a bank of immune-privileged cells, available for all patients and after a genetic correction for muscular dystrophies and other myopathies.

[Diagnostics and treatment of statin-associated muscle symptoms]. / Diagnostik und Therapie von statinassoziierten Muskelsymptomen.

Statins are among the most frequently prescribed drugs in Germany. Their benefits in lowering cardiovascular risk are beyond dispute. Nevertheless, many patients complain of side effects from statin therapy, including statin-associated muscle symptoms (SAMS) in particular. Despite their relative frequency, it is difficult to objectively diagnose them, as the time until appearance of first symptoms, the nature of the complaints and the severity of muscle problems vary widely. This narrative review summarizes the causes of SAMS as well as new possibilities regarding their diagnosis and therapy.

Base editing repairs an SGCA mutation in human primary muscle stem cells.

Skeletal muscle can regenerate from muscle stem cells and their myogenic precursor cell progeny, myoblasts. However, precise gene editing in human muscle stem cells for autologous cell replacement therapies of untreatable genetic muscle diseases has not yet been reported. Loss-of-function mutations in SGCA, encoding α-sarcoglycan, cause limb-girdle muscular dystrophy 2D/R3, an early-onset, severe, and rapidly progressive form of muscular dystrophy affecting both male and female patients. Patients suffer from muscle degeneration and atrophy affecting the limbs, respiratory muscles, and heart. We isolated human muscle stem cells from 2 donors, with the common SGCA c.157G>A mutation affecting the last coding nucleotide of exon 2. We found that c.157G>A is an exonic splicing mutation that induces skipping of 2 coregulated exons. Using adenine base editing, we corrected the mutation in the cells from both donors with > 90% efficiency, thereby rescuing the splicing defect and α-sarcoglycan expression. Base-edited patient cells regenerated muscle and contributed to the Pax7+ satellite cell compartment in vivo in mouse xenografts. Here, we provide the first evidence to our knowledge that autologous gene-repaired human muscle stem cells can be harnessed for cell replacement therapies of muscular dystrophies.