cell therapies

2026-05-01 | Myoblast-seeded alginate tubes exacerbate dystrophic features in a mouse model of dysferlinopathy

Introduction: Physical therapy (PT) alone is insufficient to restore lost skeletal muscle tissue in individuals with genetic muscle diseases, such as limb-girdle muscular dystrophy type 2B (LGMD2B). However, PT delivered within a regenerative rehabilitation framework -- one that combines cellular therapies with movement-based interventions has the potential to change the status quo in the field. Therefore, we developed a novel muscle regeneration strategy, which could be augmented by targeted, dosage-adjusted progressive resistance training. This study examined if alginate tubes containing myogenic cells could support in vivo muscle regeneration in a mouse model of LGMD2B. Methods: The technology to generate and implant myogenic alginate tubes was first developed and validated through in vitro experimentation. Using this workflow, we produced 21-gauge alginate tubes composed of 10% sodium alginate and healthy donor myogenic cells. Our laboratory has previously developed a technique known as minimally-invasive muscle embedding (MIME), which is effective in generating donor-cell-derived myogenesis in the mouse tibialis anterior (TA) muscle. In this study, we implanted cellularized alginate tubes into the left TA muscle of four female mice from a mouse model of LGMD2B (dysferlin-deficient) using MIME. Histological evaluation was performed 28 days post-implantation. No statistical analyses were performed due to the exploratory nature and small sample size. Results: Cellularized alginate tubes did not dissolve within host muscle even 28 days post-implantation – we had predicted that the alginate tubes would dissolve within a few days. Furthermore, there was no evidence of donor-cell-derived myogenesis. Unexpectedly, we found that MIME-treated muscles exhibited aggravated dystrophic features, including increased fibrosis, lipid deposition, and muscle fiber size heterogeneity, relative to contralateral control limbs. Discussion: The persistence of alginate constructs and absence of donor myogenesis suggest that delivery systems requiring faster degradation or better host-cell integration may be needed. Despite the unexpected outcome, the model revealed a more severe phenotype than typical LGMD2B mice, offering potential utility in future preclinical testing. Conclusions: Engineered myogenic constructs using alginate tubes failed to promote donor-cell-derived regeneration but may enhance modeling of LGMD2B for basic and preclinical studies. Improvements in construct design and additional testing in healthy and dystrophic mice are warranted. Acknowledgments: Supported by NIH R03HD091648, NIH P2CHD086843 (AR3T Pitch Award), NIH R01AR079884 (subcontract), Wayne State Warrior Funder, and the Jain Foundation. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

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2024-01-04 | The super-healing MRL strain promotes muscle growth in muscular dystrophy through a regenerative extracellular matrix

The Murphy Roths Large (MRL) mouse strain has "super-healing" properties that enhance recovery from injury. In mice, the DBA/2J strain intensifies many aspects of muscular dystrophy, so we evaluated the ability of the MRL strain to suppress muscular dystrophy in the Sgcg-null mouse model of limb girdle muscular dystrophy. A comparative analysis of Sgcg-null mice in the DBA/2J versus MRL strains showed greater myofiber regeneration, with reduced structural degradation of muscle in the MRL strain. Transcriptomic profiling of dystrophic muscle indicated strain-dependent expression of extracellular matrix (ECM) and TGF-β signaling genes. To investigate the MRL ECM, cellular components were removed from dystrophic muscle sections to generate decellularized myoscaffolds. Decellularized myoscaffolds from dystrophic mice in the protective MRL strain had significantly less deposition of collagen and matrix-bound TGF-β1 and TGF-β3 throughout the matrix. Dystrophic myoscaffolds from the MRL background, but not the DBA/2J background, were enriched in myokines like IGF-1 and IL-6. C2C12 myoblasts seeded onto decellularized matrices from Sgcg-/- MRL and Sgcg-/- DBA/2J muscles showed the MRL background induced greater myoblast differentiation compared with dystrophic DBA/2J myoscaffolds. Thus, the MRL background imparts its effect through a highly regenerative ECM, which is active even in muscular dystrophy.

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2021-06-30 | Endothelial dysfunction in muscular dystrophies

Endotheliocytes are the key elements of the vascular wall and are involved in regulation of vascular tone and permeability, inflammation, hemostasis, angiogenesis etc. Impaired function of endothelial cells universally recognized as endothelial dysfunction is associated with a number of common diseases such as ischemic heart disease, arterial hypertension, atherosclerosis, peripheral arterial disease, septic shock, chronic kidney disease, obesity, oncological and autoimmune diseases. Less is known about the role of endothelial cells in pathogenesis of development and progression of rare diseases, such as muscular dystrophies. Muscular dystrophies involve over 30 genetically determined diseases, which are characterized by the development of a progressive muscular weakness and skeletal muscle degeneration. Presence of a nucleotide variant associated with a certain muscular dystrophy is primarily marked by a limited potential of skeletal muscle regeneration due to the impaired structure and function of myogenic cells. Inherited myopathies include a group of severe neuromuscular diseases caused by a mutation in the dysferlin gene DYSF, which leads to the synthesis of a dysfunctional dysferlin. Complex molecular and cellular interactions involved in skeletal muscle damage and endothelial dysfunction play an important role in the pathogenesis of dysferlinopathies. The possibility to produce an effect on different pathological aspects of dysferlinassociated myopathies such as complement system activation, inflammation, impaired function of endothelial cells, damage and necrosis of myofibrils are extensively studied in vitro and in vivo. This article is dedicated to the current understanding of relationship between the endothelium and its dysfunction in myogenesis and skeletal muscle regeneration in normal and pathological conditions caused by a group of inherited progressive myodystrophies, dysferlinopathies in particular, as well as possible clinical application of endothelial cells in treatment of muscular dystrophies.

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2019-05-09 | (Epi)genetic Modifications in Myogenic Stem Cells: From Novel Insights to Therapeutic Perspectives

The skeletal muscle is considered to be an ideal target for stem cell therapy as it has an inherent regenerative capacity. Upon injury, the satellite cells, muscle stem cells that reside under the basal lamina of the myofibres, start to differentiate in order to reconstitute the myofibres while maintaining the initial stem cell pool. In recent years, it has become more and more evident that epigenetic mechanisms such as histon modifications, DNA methylations and microRNA modulations play a pivatol role in this differentiation process. By understanding the mechanisms behind myogenesis, researchers are able to use this knowledge to enhance the differentiation and engraftment potential of different muscle stem cells. Besides manipulation on an epigenetic level, recent advances in the field of genome-engineering allow site-specific modifications in the genome of these stem cells. Combining epigenetic control of the stem cell fate with the ability to site-specifically correct mutations or add genes for further cell control, can increase the use of stem cells as treatment of muscular dystrophies drastically. In this review, we will discuss the advances that have been made in genome-engineering and the epigenetic regulation of muscle stem cells and how this knowledge can help to get stem cell therapy to its full potential.

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2017-08-08 | Dysferlinopathy Promotes an Intramuscle Expansion of Macrophages with a Cyto-Destructive Phenotype.

Dysferlinopathies are a group of muscular dystrophies resulting from a genetic deficiency in Dysf. Macrophages, highly plastic cells that mediate tissue repair and destruction, are prominent within dystrophic skeletal muscles of dysferlinopathy patients. We hypothesized that Dysf-deficient muscle promotes recruitment, proliferation, and skewing of macrophages toward a cyto-destructive phenotype in dysferlinopathy. To track macrophage dynamics in dysferlinopathy, we adoptively transferred enhanced green fluorescent protein-labeled monocytes into Dysf-deficient BLA/J mice with age-related (2 to 10 months) muscle disease and Dysf-intact (C57BL/6 [B6]) mice. We detected an age- and disease-related increase in monocyte recruitment into Dysf-deficient muscles. Moreover, macrophages recruited into muscle proliferated locally and were skewed toward a cyto-destructive phenotype. By comparing Dysf-deficient and -intact monocytes, our data showed that Dysf in muscle, but not in macrophages, mediate intramuscle macrophage recruitment and proliferation. To further elucidate macrophage mechanisms related to dysferlinopathy, we investigated in vitro macrophage-myogenic cell interactions and found that Dysf-deficient muscle i) promotes macrophage proliferation, ii) skews macrophages toward a cyto-destructive phenotype, and iii) is more vulnerable to macrophage-mediated apoptosis. Taken together, our data suggest that the loss of Dysf expression in muscle, not macrophages, promotes the intramuscle expansion of cyto-destructive macrophages likely to contribute to dysferlinopathy. Identifying pathways within the Dysf-deficient muscle milieu that regulate cyto-destructive macrophages will potentially uncover therapeutic strategies for dysferlinopathies.

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small molecules

2026-06-21 | Consistent MYORG and STRADB Downregulation in DMD and LGMD: Rationale for Deoxygalactonojirimycin Repurposing in Dystrophic and Aging Muscle

Abstract Background MYORG (myogenesis-regulating glycosidase) and STRADB (STE20-related kinase adapter protein beta) were previously identified as activity-mediated skeletal muscle genes with potential roles in frailty and sarcopenia. We hypothesized that, if these genes are sustained by neuromuscular contractile activity, their expression should be consistently downregulated in muscular dystrophies, conditions defined by progressive muscle degeneration and secondary functional disuse. Methods We performed a systematic cross-dataset transcriptomic analysis of five publicly available GEO microarray datasets of human skeletal muscle. Discovery analysis was conducted in GSE3307 (Affymetrix HG-U133A/B; samples spanning DMD, LGMD2A/B/I, BMD, FSHD, JDM, ALS, AQM versus healthy controls). Independent external validation was performed in GSE38417 (HG-U133 Plus 2.0, DMD; n=16/6), GSE11681 (HG-U133A/B, LGMD2A; n=8-10/9-10), GSE465 (HG-U95Av2/B/C, multi-disease), and GSE1007 (HG-U95B/C/E, DMD; n=10-11/11). Raw CEL files underwent array-level quality assessment using NUSE and RLE diagnostics prior to normalization. Seven poor-quality arrays were excluded (none from Control, DMD, or LGMD groups). Remaining arrays were processed by robust multi-array average (RMA) normalization, and differential expression was assessed by limma with Benjamini-Hochberg FDR correction. Results MYORG was significantly downregulated in DMD (log2 fold-change [logFC] = -0.93, adj.P<0.001), LGMD2A (logFC = -0.82, adj.P<0.01), LGMD2B (logFC = -1.01, adj.P<0.01), and LGMD2I (logFC = -1.03, adj.P<0.01) in GSE3307. STRADB was significantly reduced in DMD (logFC = -0.33, adj.P<0.05) and showed a near-significant trend in LGMD2I (logFC = - 0.42, adj.P = 0.061). MYORG downregulation in DMD was independently replicated in GSE38417 (logFC = -1.40, adj.P<0.001) and GSE1007 (logFC = -0.80, adj.P<0.001). STRADB was also significantly downregulated in GSE38417 DMD (logFC = -0.45, adj.P<0.001). Deoxygalactonojirimycin, an iminosugar and an FDA/EMA-approved pharmacological chaperone (migalastat/Galafold) for Fabry disease, has been reported to be a specific molecular interactor that stabilizes MYORG protein in skeletal muscle. Conclusions This multi-dataset study further supports the role of MYORG and STRADB as activity-sensitive muscle genes that are robustly downregulated in DMD and LGMD. The pharmacological interaction between migalastat and MYORG provides a mechanistically grounded rationale for investigating this approved agent as an adjunct therapy in muscular dystrophies, in combination with the existing standard of care. This also supports active investigation of iminosugar analogs to target MYORG as potential therapeutics for improving skeletal muscle function in dystrophies, frailty, and sarcopenia.

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2025-11-03 | Abstract 4359395: Cardiac Function is Preserved in a Tissue-Engineered Model of LGMD2B

Background: Loss of dysferlin results in the rare, currently untreatable muscular dystrophy known as Limb Girdle Muscular Dystrophy 2B (LGMD2B). In LGMD2B mice, skeletal muscle undergoes progressive myopathy, whereas cardiac deficits only arise with advanced age, stress, or injury – suggesting the heart may harbor protective mechanisms that could guide future therapy developments. While dysferlin’s roles in membrane repair, Ca 2+ handling, and metabolism are well characterized in skeletal muscle, its function in the heart is poorly defined. We therefore engineered 3D LGMD2B cardiac and skeletal muscle tissues (“cardio- and myobundles”) to compare dysferlin’s differential roles in cardiac vs. skeletal muscle. Methods: Three healthy (HLT) and three LGMD2B human induced pluripotent stem cell (hiPSC) lines were differentiated into cardiomyocytes (hCMs) and muscle progenitor cells to generate cardio- and myobundles. After 2 weeks of culture, we performed isometric force tests, Ca 2+ transient imaging, and optical mapping of action potential propagation. To probe membrane repair capacity, osmotic shock injury (OSI) was induced with ~30 mOsm medium for 5 min followed by 15 min recovery, with contractile force recorded every minute. Tissues were also immunostained for sarcomere structure and dysferlin localization. Results: Both HLT and LGMD2B cardio- and myobundles exhibited aligned, cross-striated sarcomeric structure with dysferlin predominantly localized at the plasma membrane. Dysferlin-deficiency in myobundles resulted in a ~2-fold decrease in specific force generation and Ca 2+ transient amplitude. In contrast, loss of dysferlin did not affect cardiobundle specific force generation, Ca 2+ transient amplitude, conduction velocity, or action potential duration. Following OSI, cardiobundles lost >60 % of peak force independent of phenotype, while LGMD2B myobundles exhibited significantly greater force loss than HLT controls. Conclusions: We present the first in vitro tissue-engineered model of human LGMD2B cardiac muscle and show that, unlike engineered skeletal muscle and similar to in vivo findings, dysferlin deficiency does not compromise engineered cardiac tissue structure or function. Ongoing transcriptional analysis of HLT and LGMD2B cardiobundles vs. myobundles will probe putative cardioprotective pathways, with subsequent loss- and gain-of-function studies planned to validate novel therapeutic targets transferable to skeletal muscle.

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2025-03-20 | High-throughput screening identifies bazedoxifene as a potential therapeutic for dysferlin-deficient limb girdle muscular dystrophy.

Limb-girdle muscular dystrophy R2 (LGMD R2) is a rare genetic disorder characterised by progressive weakness and wasting of proximal muscles. LGMD R2 is caused by the loss of function of dysferlin, a transmembrane protein crucial for plasma membrane repair in skeletal muscles. This study aimed to identify drugs that could improve the localisation and restore the function of an aggregated mutant form of dysferlin (DYSFL1341P). We developed an in vitro high-throughput assay to monitor the expression and reallocation of aggregated mutant dysferlin (DYSFL1341P) in immortalised myoblasts. After screening 2239 clinically approved drugs and bioactive compounds, the ability of the more promising candidates to improve cell survival following hypo-osmotic shock was assessed. Their protective effects were evaluated on immortalised myoblasts carrying other dysferlin mutations and on dysferlin-deficient muscle fibres from Bla/J mice. We identified two compounds, saracatinib and bazedoxifene, that increase dysferlin content in cells carrying the DYSFL1341P mutation. Both drugs improved cell survival and plasma membrane resistance following osmotic shock. Whereas saracatinib acts specifically on misfolded L1341P dysferlin, bazedoxifene shows an additional protective effect on dysferlin KO immortalised myoblasts and mice muscle fibres. Further analysis revealed that bazedoxifene induces autophagy flux, which may enhance the survival of LGMD R2 myofibres. Our drug screening identified saracatinib and bazedoxifene as potential treatments for LGMD R2, especially for patients with the L1341P mutation. The widespread protective effect of bazedoxifene reveals a new avenue toward genotype-independent treatment of LGMD R2 patients.

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2025-03-01 | Metabolic dysregulation contributes to the development of dysferlinopathy.

Dysferlin is a transmembrane protein that plays a prominent role in membrane repair of damaged muscle fibers. Accordingly, mutations in the dysferlin gene cause progressive muscular dystrophies, collectively referred to as dysferlinopathies for which no effective treatment exists. Unexpectedly, experimental approaches that successfully restore membrane repair fail to prevent a dystrophic phenotype, suggesting that additional, hitherto unknown dysferlin-dependent functions contribute to the development of the pathology. Our experiments revealed an altered metabolic phenotype in dysferlin-deficient muscles, characterized by (1) mitochondrial abnormalities and elevated death signaling and (2) increased glucose uptake, reduced glycolytic protein levels, and pronounced glycogen accumulation. Strikingly, elevating mitochondrial volume density and muscle glycogen accelerates disease progression; whereas, improvement of mitochondrial function and recruitment of muscle glycogen with exercise ameliorated functional parameters in a mouse model of dysferlinopathy. Collectively, our results not only shed light on a metabolic function of dysferlin but also imply new therapeutic avenues aimed at promoting mitochondrial function and normalizing muscle glycogen to ameliorate dysferlinopathies, complementing efforts that target membrane repair.

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2024-08-21 | Bioengineered Model of Human LGMD2B Skeletal Muscle Reveals Roles of Intracellular Calcium Overload in Contractile and Metabolic Dysfunction in Dysferlinopathy.

Dysferlin is a multi-functional protein that regulates membrane resealing, calcium homeostasis, and lipid metabolism in skeletal muscle. Genetic loss of dysferlin results in limb girdle muscular dystrophy 2B/2R (LGMD2B/2R) and other dysferlinopathies - rare untreatable muscle diseases that lead to permanent loss of ambulation in humans. The mild disease severity in dysferlin-deficient mice and diverse genotype-phenotype relationships in LGMD2B patients have prompted the development of new in vitro models for personalized studies of dysferlinopathy. Here the first 3-D tissue-engineered hiPSC-derived skeletal muscle ("myobundle") model of LGMD2B is described that exhibits compromised contractile function, calcium-handling, and membrane repair, and transcriptomic changes indicative of impaired oxidative metabolism and mitochondrial dysfunction. In response to the fatty acid (FA) challenge, LGMD2B myobundles display mitochondrial deficits and intracellular lipid droplet (LD) accumulation. Treatment with the ryanodine receptor (RyR) inhibitor dantrolene or the dissociative glucocorticoid vamorolone restores LGMD2B contractility, improves membrane repair, and reduces LD accumulation. Lastly, it is demonstrated that chemically induced chronic RyR leak in healthy myobundles phenocopies LGMD2B contractile and metabolic deficit, but not the loss of membrane repair capacity. Together, these results implicate intramyocellular Ca2+ leak as a critical driver of dysferlinopathic phenotype and validate the myobundle system as a platform to study LGMD2B pathogenesis.

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proteins

2026-09-19 | Galectin-1 therapy recalibrates lipid remodeling in skeletal muscle and enhances muscle function in a model of LGMD2B/R2

Limb-Girdle Muscular Dystrophy type 2B/R2 (LGMD2B/R2) is a progressive myopathy caused by mutations in the DYSF gene, leading to dysferlin deficiency, impaired membrane repair, chronic inflammation, and lipid accumulation. No approved treatments currently exist. Galectin-1, a redox-sensitive lectin with reparative and immunomodulatory properties, has shown therapeutic potential in short-term models of muscular dystrophies. We evaluated the long-term efficacy of recombinant human Galectin-1 (rHsGal-1) in Bla/J mice, a model of LGMD2B/R2. Mice received weekly rHsGal-1 treatment for six months. Functional, histological, and molecular analyses were performed, including mobility assessments, sarcolemmal repair assays, NF-κB activation assays, quantification of fibro-adipogenic progenitors (FAPs), apoptosis and proliferation analyses, lipid infiltration analysis, and targeted lipidomics. Long-term rHsGal-1 therapy preserved mobility, enhanced sarcolemmal repair, and improved muscle architecture, evidenced by increased fiber size and reduced central nucleation. Treatment suppressed NF-κB activation, reduced FAP abundance, increased apoptotic FAPs without detectable changes in proliferating Ki67 + FAPs, and decreased lipid infiltration. Lipidomic profiling revealed treatment-associated lipid remodeling characterized by reduced neutral lipid accumulation, altered sphingolipid and phospholipid signatures, and selective changes in inflammatory lipid classes. These lipid changes were associated with histological and functional improvements. Long-term rHsGal-1 therapy provides durable, multifaceted benefits in LGMD2B/R2, improving muscle structure, membrane repair, and lipid remodeling while reducing fibro-adipogenic remodeling and lipid accumulation. The identification of increased apoptotic FAPs together with reduced FAP abundance suggests a potential mechanism contributing to these therapeutic effects. These findings support the continued development of Gal-1 as a therapeutic strategy for LGMD2B/R2 and other dysferlinopathies.

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2026-03-13 | A Novel Dysferlin-Binding Kinase CK2α Promotes Plasma Membrane Repair in Dysferlinopathy.

Dysferlinopathy is an adult-onset form of muscular dystrophy caused by mutations in the dysferlin gene and is inherited in an autosomal recessive manner. Dysferlin is primarily known for its role in plasma membrane repair. Although several proteins associated with dysferlin have been identified, many aspects of its signaling pathways and protein-protein interactions remain unclear. Here, we focused on the region between the third and fourth C2 domains, where frequent genetic mutations occur and functional domains are concentrated, and identified the protein kinase CK2α (formerly known as casein kinase 2) as a novel dysferlin-binding protein. CK2α was found to accumulate at membrane injury sites along with dysferlin in mouse skeletal muscle, and membrane repair was delayed in CK2α knockout cells. Furthermore, overexpression of CK2α in dysferlin-deficient mouse muscle led to improved membrane repair. Additionally, we revealed that CK2α plays a role in phosphorylating annexin A1, which is known to bind to dysferlin and is involved in plasma membrane repair. Our results indicated that CK2α controls membrane repair by participating in the phosphorylation of annexin A1. The molecular interplay among dysferlin, CK2α, and phosphorylated annexin A1 represents a novel therapeutic target for promoting membrane repair.

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2024-05-08 | The extracellular matrix differentially directs myoblast motility and differentiation in distinct forms of muscular dystrophy: Dystrophic matrices alter myoblast motility.

Extracellular matrix (ECM) pathologic remodeling underlies many disorders, including muscular dystrophy. Tissue decellularization removes cellular components while leaving behind ECM components. We generated "on-slide" decellularized tissue slices from genetically distinct dystrophic mouse models. The ECM of dystrophin- and sarcoglycan-deficient muscles had marked thrombospondin 4 deposition, while dysferlin-deficient muscle had excess decorin. Annexins A2 and A6 were present on all dystrophic decellularized ECMs, but annexin matrix deposition was excessive in dysferlin-deficient muscular dystrophy. Muscle-directed viral expression of annexin A6 resulted in annexin A6 in the ECM. C2C12 myoblasts seeded onto decellularized matrices displayed differential myoblast mobility and fusion. Dystrophin-deficient decellularized matrices inhibited myoblast mobility, while dysferlin-deficient decellularized matrices enhanced myoblast movement and differentiation. Myoblasts treated with recombinant annexin A6 increased mobility and fusion like that seen on dysferlin-deficient decellularized matrix and demonstrated upregulation of ECM and muscle cell differentiation genes. These findings demonstrate specific fibrotic signatures elicit effects on myoblast activity.

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2023-09-05 | Translational implications of targeting annexin A2: From membrane repair to muscular dystrophy, cardiovascular disease and cancer

Abstract Background Annexin A2 contributes to several key cellular functions and processes, including membrane repair. Effective repair prevents cell death and degeneration, especially in skeletal or cardiac muscle, epithelia, and endothelial cells. To maintain cell integrity after damage, mammalian cells activate multiple membrane repair mechanisms. One protein family that facilitates membrane repair processes are the Ca 2+ ‐regulated phospholipid‐binding annexins. Body Annexin A2 facilitates repair in association with S100A10 and related S100 proteins by forming a plug and linking repair to other physiologic functions. Deficiency of annexin A2 enhances cellular degeneration, exacerbating muscular dystrophy and degeneration. Downstream of repair, annexin A2 links the membrane with the cytoskeleton, calcium‐dependent endocytosis, exocytosis, cell proliferation, transcription, and apoptosis to extracellular roles, including vascular fibrinolysis, and angiogenesis. These roles regulate cardiovascular disease progression. Finally, annexin A2 protects cancer cells from membrane damage due to immune cells or chemotherapy. Since these functions are regulated by post‐translational modifications, they represent a therapeutic target for reducing the negative consequences of annexin A2 expression. Conclusion Thus, connecting the roles of annexin A2 in repair to its other physiologic functions represents a new translational approach to treating muscular dystrophy and cardiovascular diseases without enhancing its pro‐tumorigenic activities.

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2023-01-18 | Minimal expression of dysferlin prevents development of dysferlinopathy in dysferlin exon 40a knockout mice

Dysferlin is a Ca2+-activated lipid binding protein implicated in muscle membrane repair. Recessive variants in DYSF result in dysferlinopathy, a progressive muscular dystrophy. We showed previously that calpain cleavage within a motif encoded by alternatively spliced exon 40a releases a 72 kDa C-terminal minidysferlin recruited to injured sarcolemma. Herein we use CRISPR/Cas9 gene editing to knock out murine Dysf exon 40a, to specifically assess its role in membrane repair and development of dysferlinopathy. We created three Dysf exon 40a knockout (40aKO) mouse lines that each express different levels of dysferlin protein ranging from ~ 90%, ~ 50% and ~ 10-20% levels of wild-type. Histopathological analysis of skeletal muscles from all 12-month-old 40aKO lines showed virtual absence of dystrophic features and normal membrane repair capacity for all three 40aKO lines, as compared with dysferlin-null BLAJ mice. Further, lipidomic and proteomic analyses on 18wk old quadriceps show all three 40aKO lines are spared the profound lipidomic/proteomic imbalance that characterises dysferlin-deficient BLAJ muscles. Collective results indicate that membrane repair does not depend upon calpain cleavage within exon 40a and that ~ 10-20% of WT dysferlin protein expression is sufficient to maintain the muscle lipidome, proteome and membrane repair capacity to crucially prevent development of dysferlinopathy.

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oligonucleotides

2026-07-14 | PPMO therapy for dysferlinopathy induces pseudoexon skipping and restoration of functional protein.

The dysferlinopathies are a spectrum of autosomal recessive muscle diseases caused by mutations in the dysferlin gene (DYSF) gene. Clinical manifestations vary from asymptomatic hyperCKemia to severe muscle pathology and loss of muscle function. These are designated limb-girdle muscular dystrophy type 2R or LGMDR2 (formerly LGMD2B or Miyoshi myopathy). Among other functions, dysferlin is crucial for plasma membrane repair and maintenance of intracellular calcium homeostasis. In previous studies, we identified in two independent point mutations deep within introns that cause aberrant DYSF mRNA splicing and the inclusion of pseudoexons within transcripts that disrupt protein expression. In this study, we generated and characterized a novel mouse model for one of these mutations (within DYSF intron 44). In these mice, a segment of human DYSF DNA containing the mutant intronic sequence flanked by surrounding human exon sequences replaces the normal homologous mouse DNA. These mice exhibit aberrant Dysf pre-mRNA splicing, pseudoexon inclusion, loss of DYSF protein expression, and muscle pathology similar to that observed in patients. Using this new model, we identified antisense oligonucleotides and then a PPMO that blocks the mouse Dysf pre-mRNA splicing complexes from binding the mutant pre-mRNA, thereby restoring nearly normal muscle histology and function.

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2025-07-28 | An Overview of Recent Advances and Clinical Applications of Exon Skipping and Splice Modulation for Muscular Dystrophy and Various Genetic Diseases.

Exon skipping and splice modulation using synthetic antisense oligonucleotides (AONs) are therapeutic approaches to delay or arrest the progression of genetic diseases. AON-mediated exon skipping modifies pre-mRNA splicing to correct the reading frame of genes responsible for disease and modulate a protein structure, providing therapeutic benefits. Studied for over two decades, exon skipping AONs have received conditional marketing authorization for Duchenne muscular dystrophy (DMD). The US Food and Drug Administration (FDA) has granted accelerated approval for four AONs to skip a single DMD exon for treating patients with DMD: eteplirsen for exon 51 skipping, golodirsen and viltolarsen for exon 53 skipping, and casimersen for exon 45 skipping. Various preclinical and clinical trials are underway with AONs targeting other exons and ones loading bioconjugates, such as cell-penetrating peptides and antibodies to enhance the exon skipping ability. Besides AONs targeting pre-mRNA, genomic DNA target exon skipping using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology, which holds promise in current preclinical trials for DMD. In hopes of achieving clinical success parallel to DMD, exon skipping and splice modulation are studied in other muscular dystrophies, such as Fukuyama congenital muscular dystrophy (FCMD), dysferlinopathy including limb-girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy (MM), and distal anterior compartment myopathy (DMAT), myotonic dystrophy, and merosin-deficient congenital muscular dystrophy type 1A (MDC1A). Splice-modulating AONs have also been extensively tested in other inherited diseases, such as Usher syndrome, dystrophic epidermolysis bullosa (DEB), fibrodysplasia ossificans progressiva (FOP), and allergic diseases. This chapter will introduce the developmental status of exon skipping and splice-modulating therapies for genetic diseases.

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2024-06-19 | Limb Girdle Muscular Dystrophy Type 2B (LGMD2B): Diagnosis and Therapeutic Possibilities.

Dysferlin is a large transmembrane protein involved in critical cellular processes including membrane repair and vesicle fusion. Mutations in the dysferlin gene (DYSF) can result in rare forms of muscular dystrophy; Miyoshi myopathy; limb girdle muscular dystrophy type 2B (LGMD2B); and distal myopathy. These conditions are collectively known as dysferlinopathies and are caused by more than 600 mutations that have been identified across the DYSF gene to date. In this review, we discuss the key molecular and clinical features of LGMD2B, the causative gene DYSF, and the associated dysferlin protein structure. We also provide an update on current approaches to LGMD2B diagnosis and advances in drug development, including splice switching antisense oligonucleotides. We give a brief update on clinical trials involving adeno-associated viral gene therapy and the current progress on CRISPR/Cas9 mediated therapy for LGMD2B, and then conclude by discussing the prospects of antisense oligomer-based intervention to treat selected mutations causing dysferlinopathies.

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2023-09-16 | Portrait of Dysferlinopathy: Diagnosis and Development of Therapy

Dysferlinopathy is a disease caused by a dysferlin deficiency due to mutations in the DYSF gene. Dysferlin is a membrane protein in the sarcolemma and is involved in different functions, such as membrane repair and vesicle fusion, T-tubule development and maintenance, Ca2+ signalling, and the regulation of various molecules. Miyoshi Myopathy type 1 (MMD1) and Limb–Girdle Muscular Dystrophy 2B/R2 (LGMD2B/LGMDR2) are two possible clinical presentations, yet the same mutations can cause both presentations in the same family. They are therefore grouped under the name dysferlinopathy. Onset is typically during the teenage years or young adulthood and is characterized by a loss of Achilles tendon reflexes and difficulty in standing on tiptoes or climbing stairs, followed by a slow progressive loss of strength in limb muscles. The MRI pattern of patient muscles and their biopsies show various fibre sizes, necrotic and regenerative fibres, and fat and connective tissue accumulation. Recent tools were developed for diagnosis and research, especially to evaluate the evolution of the patient condition and to prevent misdiagnosis caused by similarities with polymyositis and Charcot–Marie–Tooth disease. The specific characteristic of dysferlinopathy is dysferlin deficiency. Recently, mouse models with patient mutations were developed to study genetic approaches to treat dysferlinopathy. The research fields for dysferlinopathy therapy include symptomatic treatments, as well as antisense-mediated exon skipping, myoblast transplantation, and gene editing.

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2022-11-22 | Morpholino-Mediated Exons 28-29 Skipping of Dysferlin and Characterization of Multiexon-skipped Dysferlin using RT-PCR, Immunoblotting, and Membrane Wounding Assay.

Dysferlinopathies are a group of disabling muscular dystrophies  that includes limb girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy, and distal myopathy with anterior tibial onset (DMAT) as the main phenotypes. They are associated with molecular defects in DYSF, which encodes dysferlin, a key player in sarcolemmal homeostasis. Previous investigations have suggested that exon skipping may be a promising therapy for many patients with dysferlinopathies. It was reported that exons 28-29 of DYSF are dispensable for dysferlin functions. Here, we present a method for multiexon skipping of DYSF exons 28-29 using a cocktail of two phosphorodiamidate morpholino oligomers (PMOs) on cells derived from a dystrophinopathy patient. Also, we describe assays to characterize the multiexon skipped dysferlin at several levels by using one-step RT-PCR, immunoblotting, and a membrane wounding assay.

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2026-04-09 | Dysferlinopathies: phenotypic study of a Moroccan series of 28 cases.

Dysferlinopathies are a spectrum of autosomal recessive muscular dystrophies caused by mutations in the dysferlin gene (DYSF), leading to heterogeneous phenotypes, mainly limb-girdle muscular dystrophy type 2R (LGMDR2) and Miyoshi distal myopathy (MMD1). In Morocco, dysferlinopathies accounted for 18% of all limb-girdle muscular dystrophies, ranking second after sarcoglycanopathies. We retrospectively analyzed 28 confirmed cases, diagnosed either by dysferlin deficiency or DYSF mutation. The median age at examination was 25,5 years (21-33). Twelve patients (43%) were female, and 19 (68%) had consanguineous parents. The proximodistal phenotype was predominant (50%), followed by LGMDR2 (39%) and MMD1 (11%). Selective biceps brachii involvement was observed in 68% of patients. Seven patients (25%) showed hypertrophy of the extensor digitorum brevis muscle, and two (7%) presented with severe genu recurvatum. In addition to dystrophic lesions, muscle biopsy revealed inflammatory infiltrates in 15 cases (58%), leading to an initial misdiagnosis of inflammatory myopathy in two cases. Dysferlin deficiency confirmed the diagnosis in 26 cases, while two others had homozygous DYSF variants. Four patients received corticosteroids, with clear improvement in only one. Overall, disease progression was slow, although two patients lost ambulation at the ages of 26 and 32. Regular physical activity appeared to have a beneficial effect in most patients.

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2026-03-23 | DAB2 in LGMD R2: a molecular link between disease progression and lipid dysregulation.

Limb-girdle muscular dystrophy R2 (LGMD R2) is an autosomal recessive disorder caused by dysferlin deficiency, leading to progressive muscle weakness and wasting. The lack of reliable clinical biomarkers has limited disease monitoring and therapeutic evaluation. Here, we identified Disabled-2 (DAB2) as a molecular and clinical indicator of disease state in LGMD R2. Transcriptomic profiling revealed a significant upregulation of DAB2 in induced pluripotent stem cell-derived (iPSC-derived) myotubes from patients, a finding validated in muscle biopsies from 14 dysferlin-deficient individuals and in dysferlin-deficient Bla/J mice, where DAB2 levels increased with disease progression. Importantly, AAV-mediated expression of full-length dysferlin restored DAB2 levels, supporting its value as a dynamic readout of disease activity for both disease monitoring and therapeutic response. Given the established role of DAB2 in clathrin-mediated endocytosis, particularly in LDL receptor internalization and cholesterol homeostasis, and the pathological lipid accumulation reported in LGMD R2, we investigated its contribution to lipid dysregulation. High DAB2 expression paralleled lipid deposition in patient muscles, iPSC-derived myotubes, and mouse tissue, whereas siRNA-mediated DAB2 knockdown reduced lipid accumulation in LGMD R2 myotubes. Collectively, these findings suggest that DAB2 functions as a mechanistic link between dysferlin deficiency, altered lipid handling, and disease severity, and they highlight its potential as a prognostic marker and therapeutic response measure for LGMD R2.

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2026-01-13 | Complement C5 Inhibitor Ameliorates a Case of Dysferlinopathy.

Dysferlinopathy is an autosomal recessive muscular dystrophy caused by pathogenic variants of the DYSF gene. Currently, no clinical effective treatment is available. Given the myopathologic and animal model evidence on complement activation in dysferlinopathy, we explored the potential therapeutic effect of complement inhibition. We reported a case of teenager-onset dysferlinopathy with progressive proximal weakness, markedly elevated serum creatine kinase levels, and assistance requirement for ambulation when admission. Muscle biopsy showed dysferlin deficiency and marked deposition of complement C5b-9 on nonnecrotic sarcolemma. Based on these findings and previous preclinical studies, the patient received eculizumab (900 mg weekly for 4 weeks) with informed consent. Clinical improvement was observed following complement inhibition therapy. By week 5, all tested muscle groups reached Medical Research Council grade 5/5, and the North Star Assessment for Dysferlinopathy score increased from 28 to 39. The 6-minute walk test (6MWT) improved from 220 m to 363 m. The muscle MRI revealed reduced muscle edema after eculizumab treatment. These benefits were sustained at 13-month follow-up. Complement inhibition showed promising clinical improvement in this single case of dysferlinopathy. Further studies with larger sample sizes are needed to investigate the efficacy and safety of complement inhibitors in dysferlinopathy.

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2025-09-14 | DAB2 as a biomarker and mechanistic link between lipid dysregulation and disease progression in LGMD R2

Limb-girdle muscular dystrophy R2 (LGMD R2) is an autosomal recessive disorder caused by dysferlin deficiency, leading to progressive muscle weakness and wasting. Despite advances in understanding the mechanisms linking dysferlin loss to membrane fragility and muscle degeneration, the lack of robust clinical biomarkers has limited disease monitoring and therapeutic evaluation. Here, we identify Disabled-2 (DAB2) as a molecular and clinical biomarker for LGMD R2. Transcriptomic profiling revealed a significant upregulation of DAB2 in induced pluripotent stem cell (iPSC)-derived myotubes from patients with LGMD R2. Its expression correlated with disease severity in muscle biopsies from a cohort of 14 dysferlin-deficient individuals and in dysferlin knockout Bla/J mice, where levels increased with disease progression. Crucially, we demonstrate that DAB2 upregulation in muscle is normalized following treatment with AAV gene therapy expressing full-length dysferlin, positioning DAB2 as a dynamic biomarker for both disease monitoring and therapeutic response. Based on the role of DAB2 in lipid trafficking and the reported pathological lipid accumulation in LGMD R2, we then investigated its contribution to disease-associated lipid dysregulation. Consistent with this hypothesis, we show that high DAB2 levels paralleled lipid deposition in affected patients, iPSC-derived myotubes and mouse muscles, while siRNA-mediated DAB2 knockdown reduced lipid accumulation in LGMD R2 myotubes. Together, our findings establish DAB2 as a mechanistic link between disease severity and lipid dysregulation, and highlight its potential as a key prognostic marker, opening new avenues for precision medicine approaches in LGMD R2 and other related muscular dystrophies.

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2025-06-23 | Dysferlinopathy as cause of long-term hyperCKemia with preserved strength.

Dysferlin (DYSF) has a crucial role in sarcolemmal repair. While DYSF mutations commonly manifest as limb-girdle muscular dystrophy (LGMDR2) or distal Miyoshi myopathy, atypical manifestations, such as asymptomatic hyperCKemia and pseudometabolic myopathy, are rarely reported. We describe clinical, serologic, radiologic, genetic, and muscle pathology findings of three patients with rare dysferlinopathy phenotypes and long-term follow up in one of them. We also review the literature pertinent to uncommon forms of dysferlinopathy presenting with hyperCKemia and pseudometabolic phenotype. Patient 1 is a 51-year-old female with exercise-induced myalgia predominantly affecting calf muscles for 7 years. She had a 22-year history of asymptomatic hyperCKemia (CK 812-2,223 U/L). Neurologic exam showed mild calf enlargement without weakness. CT of the lower limb revealed fatty infiltration of distal peroneal and calf muscles. Genetic testing showed two DYSF variants, c.2163-2A > G (pathogenic) and c.866C > G, p.Ser289Cys (VUS), unknown if heteroallelic. Muscle biopsy demonstrated nuclei internalization and absent dysferlin immunoreactivity. Patient 2 is a 20-year-old male, football player, with an episode of exercise-induced myalgia followed by asymptomatic persistent hyperCKemia (729-2,645 U/L). He had normal strength but mild calf muscle atrophy. Muscle MRI demonstrated subtle T2 hyperintensity in the posterior leg compartment musculature. He has two heteroallelic DYSF variants, c.6008G > A, p.Gly2003Asp (pathogenic) and c.854C > T, p.Thr285Met (VUS). Muscle biopsy showed no myopathic changes but reduced dysferlin immunoreactivity. Patient 3 is a 58-year-old female with incidentally detected asymptomatic hyperCKemia (CK: 249-2,096 U/L) for 2 years. She had normal strength and normal lower limb muscle MRI. She carries two heteroallelic DYSF variants, c.2517del, p.Met840Trpfs*108 (pathogenic) and c.6058C > T, p.Arg2020Cys (VUS). Muscle biopsy showed minimal myopathic changes and attenuated dysferlin immunoreactivity. Reduced dysferlin expression was confirmed by western blot in patients 2 and 3. Needle EMG was normal in all patients. Dysferlinopathy should be considered in the differential diagnosis of metabolic myopathies and asymptomatic hyperCKemia. Patient 1's long history of hyperCKemia without weakness over two decades suggests that CK elevation in dysferlinopathy does not necessarily predict development of weakness. Additionally, the lack of dystrophic changes on muscle biopsy of patients with asymptomatic or minimally symptomatic hyperCKemia should not discourage the search for dysferlin deficiency in muscle, particularly in the setting of DYSF variants.

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