Secreted acid sphingomyelinase as a potential gene therapy for limb girdle muscular dystrophy 2B.
ABSTRACT: Efficient sarcolemmal repair is required for muscle cell survival, with deficits in this process leading to muscle degeneration. Lack of the sarcolemmal protein dysferlin impairs sarcolemmal repair by reducing secretion of the enzyme acid sphingomyelinase (ASM), and causes limb girdle muscular dystrophy 2B (LGMD2B). The large size of the dysferlin gene poses a challenge for LGMD2B gene therapy efforts aimed at restoring dysferlin expression in skeletal muscle fibers. Here, we present an alternative gene therapy approach targeting reduced ASM secretion, the consequence of dysferlin deficit. We showed that the bulk endocytic ability is compromised in LGMD2B patient cells, which was addressed by extracellularly treating cells with ASM. Expression of secreted human ASM (hASM) using a liver-specific adeno-associated virus (AAV) vector restored membrane repair capacity of patient cells to healthy levels. A single in vivo dose of hASM-AAV in the LGMD2B mouse model restored myofiber repair capacity, enabling efficient recovery of myofibers from focal or lengthening contraction-induced injury. hASM-AAV treatment was safe, attenuated fibro-fatty muscle degeneration, increased myofiber size, and restored muscle strength, similar to dysferlin gene therapy. These findings elucidate the role of ASM in dysferlin-mediated plasma membrane repair and to our knowledge offer the first non-muscle-targeted gene therapy for LGMD2B.
Project description:Mutations of the DYSF gene leading to reduced dysferlin protein level causes limb girdle muscular dystrophy type 2B (LGMD2B). Dysferlin facilitates sarcolemmal membrane repair in healthy myofibers, thus its deficit compromises myofiber repair and leads to chronic muscle inflammation. An experimental therapeutic approach for LGMD2B is to protect damage or improve repair of myofiber sarcolemma. Here, we compared the effects of prednisolone and vamorolone (a dissociative steroid; VBP15) on dysferlin-deficient myofiber repair. Vamorolone, but not prednisolone, stabilized dysferlin-deficient muscle cell membrane and improved repair of dysferlin-deficient mouse (B6A/J) myofibers injured by focal sarcolemmal damage, eccentric contraction-induced injury or injury due to spontaneous in vivo activity. Vamorolone decreased sarcolemmal lipid mobility, increased muscle strength, and decreased late-stage myofiber loss due to adipogenic infiltration. In contrast, the conventional glucocorticoid prednisolone failed to stabilize dysferlin deficient muscle cell membrane or improve repair of dysferlinopathic patient myoblasts and mouse myofibers. Instead, prednisolone treatment increased muscle weakness and myofiber atrophy in B6A/J mice-findings that correlate with reports of prednisolone worsening symptoms of LGMD2B patients. Our findings showing improved cellular and pre-clinical efficacy of vamorolone compared to prednisolone and better safety profile of vamorolone indicates the suitability of vamorolone for clinical trials in LGMD2B.
Project description:Limb girdle muscular dystrophy type 2B (LGMD2B) and other dysferlinopathies are degenerative muscle diseases that result from mutations in the dysferlin gene and have limited treatment options. The dysferlin protein has been linked to multiple cellular functions including a Ca<sup>2+</sup>-dependent membrane repair process that reseals disruptions in the sarcolemmal membrane. Recombinant human MG53 protein (rhMG53) can increase the membrane repair process in multiple cell types both in vitro and in vivo. Here, we tested whether rhMG53 protein can improve membrane repair in a dysferlin-deficient mouse model of LGMD2B (B6.129-Dysf<sup>tm1Kcam</sup>/J). We found that rhMG53 can increase the integrity of the sarcolemmal membrane of isolated muscle fibers and whole muscles in a Ca<sup>2+</sup>-independent fashion when assayed by a multi-photon laser wounding assay. Intraperitoneal injection of rhMG53 into mice before acute eccentric treadmill exercise can decrease the release of intracellular enzymes from skeletal muscle and decrease the entry of immunoglobulin G and Evans blue dye into muscle fibers in vivo. These results indicate that short-term rhMG53 treatment can ameliorate one of the underlying defects in dysferlin-deficient muscle by increasing sarcolemmal membrane integrity. We also provide evidence that rhMG53 protein increases membrane integrity independently of the canonical dysferlin-mediated, Ca<sup>2+</sup>-dependent pathway known to be important for sarcolemmal membrane repair.
Project description:Mutations in the dysferlin gene are the cause of Limb-girdle Muscular Dystrophy type 2B and Miyoshi Myopathy. The dysferlin protein has been implicated in sarcolemmal resealing, leading to the idea that the pathophysiology of dysferlin deficiencies is due to a deficit in membrane repair. Here, we show using two different approaches that fulfilling membrane repair as asseyed by laser wounding assay is not sufficient for alleviating the dysferlin deficient pathology. First, we generated a transgenic mouse overexpressing myoferlin to test the hypothesis that myoferlin, which is homologous to dysferlin, can compensate for the absence of dysferlin. The myoferlin overexpressors show no skeletal muscle abnormalities, and crossing them with a dysferlin-deficient model rescues the membrane fusion defect present in dysferlin-deficient mice in vitro. However, myoferlin overexpression does not correct muscle histology in vivo. Second, we report that AAV-mediated transfer of a minidysferlin, previously shown to correct the membrane repair deficit in vitro, also fails to improve muscle histology. Furthermore, neither myoferlin nor the minidysferlin prevented myofiber degeneration following eccentric exercise. Our data suggest that the pathogenicity of dysferlin deficiency is not solely related to impairment in sarcolemmal repair and highlight the care needed in selecting assays to assess potential therapies for dysferlinopathies.
Project description:Repair of skeletal muscle after sarcolemmal damage involves dysferlin and dysferlin-interacting proteins such as annexins. Mice and patient lacking dysferlin exhibit chronic muscle inflammation and adipogenic replacement of the myofibers. Here, we show that similar to dysferlin, lack of annexin A2 (AnxA2) also results in poor myofiber repair and progressive muscle weakening with age. By longitudinal analysis of AnxA2-deficient muscle we find that poor myofiber repair due to the lack of AnxA2 does not result in chronic inflammation or adipogenic replacement of the myofibers. Further, deletion of AnxA2 in dysferlin deficient mice reduced muscle inflammation, adipogenic replacement of myofibers, and improved muscle function. These results identify multiple roles of AnxA2 in muscle repair, which includes facilitating myofiber repair, chronic muscle inflammation and adipogenic replacement of dysferlinopathic muscle. It also identifies inhibition of AnxA2-mediated inflammation as a novel therapeutic avenue for treating muscle loss in dysferlinopathy.
Project description:Mutations in the dysferlin gene cause limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy. Dysferlin-deficient cells show abnormalities in vesicular traffic and membrane repair although onset of symptoms is not commonly seen until the late teenage years and is often associated with subacute onset and marked muscle inflammation. To identify molecular networks specific to dysferlin-deficient muscle that might explain disease pathogenesis, muscle mRNA profiles from 10 mutation-positive LGMD2B/MM patients were compared with a disease control [LGMD2I; (n = 9)], and normal muscle samples (n = 11). Query of inflammatory pathways suggested LGMD2B-specific increases in co-stimulatory signaling between dendritic cells and T cells (CD86, CD28, and CTLA4), associated with localized expression of both versican and tenascin. LGMD2B muscle also showed an increase in vesicular trafficking pathway proteins not normally observed in muscle (synaptotagmin-like protein Slp2a/SYTL2 and the small GTPase Rab27A). We propose that Rab27A/Slp2a expression in LGMD2B muscle provides a compensatory vesicular trafficking pathway that is able to repair membrane damage in the absence of dysferlin. However, this same pathway may release endocytotic vesicle contents, resulting in an inflammatory microenvironment. As dysferlin deficiency has been shown to enhance phagocytosis by macrophages, together with our findings of abnormal myofiber endocytosis pathways and dendritic-T cell activation markers, these results suggest a model of immune and inflammatory network over-stimulation that may explain the subacute inflammatory presentation.
Project description:Dysferlin deficiency causes limb-girdle muscular dystrophy type 2B (LGMD2B; proximal weakness) and Miyoshi myopathy (distal weakness). Muscle inflammation is often present in dysferlin deficiency, and patients are frequently misdiagnosed as having polymyositis. Because monocytes normally express dysferlin, we hypothesized that monocyte/macrophage dysfunction in dysferlin-deficient patients might contribute to disease onset and progression. We therefore examined phagocytic activity, in the presence and absence of cytokines, in freshly isolated peripheral blood monocytes from LGMD2B patients and in the SJL dysferlin-deficient mouse model. Dysferlin-deficient monocytes showed increased phagocytic activity compared with control cells. siRNA-mediated inhibition of dysferlin expression in the J774 macrophage cell line resulted in significantly enhanced phagocytosis, both at baseline and in response to tumor necrosis factor-alpha. Immunohistochemical analysis revealed positive staining for several mononuclear cell activation markers in LGMD2B human muscle and SJL mouse muscle. SJL muscle showed strong up-regulation of endocytic proteins CIMPR, clathrin, and adaptin-alpha, and LGMD2B muscle exhibited decreased expression of decay accelerating factor, which was not dysferlin-specific. We further showed that expression levels of small Rho family GTPases RhoA, Rac1, and Cdc 42 were increased in dysferlin-deficient murine immune cells compared with control cells. Therefore, we hypothesize that mild myofiber damage in dysferlin-deficient muscle stimulates an inflammatory cascade that may initiate, exacerbate, and possibly perpetuate the underlying myofiber-specific dystrophic process.
Project description:Dystrophin deficiency is the genetic basis for Duchenne muscular dystrophy (DMD), but the cellular basis of progressive myofiber death in DMD is not fully understood. Using two dystrophin-deficient mdx mouse models, we find that the mitochondrial dysfunction is among the earliest cellular deficits of mdx muscles. Mitochondria in dystrophic myofibers also respond poorly to sarcolemmal injury. These mitochondrial deficits reduce the ability of dystrophic muscle cell membranes to repair and are associated with a compensatory increase in dysferlin-mediated membrane repair proteins. Dysferlin deficit in mdx mice further compromises myofiber cell membrane repair and enhances the muscle pathology at an asymptomatic age for dysferlin-deficient mice. Restoring partial dystrophin expression by exon skipping improves mitochondrial function and offers potential to improve myofiber repair. These findings identify that mitochondrial deficit in muscular dystrophy compromises the repair of injured myofibers and show that this repair mechanism is distinct from and complimentary to the dysferlin-mediated repair of injured myofibers.
Project description:Muscle loss due to fibrotic or adipogenic replacement of myofibers is common in muscle diseases and muscle-resident fibro/adipogenic precursors (FAPs) are implicated in this process. While FAP-mediated muscle fibrosis is widely studied in muscle diseases, the role of FAPs in adipogenic muscle loss is not well understood. Adipogenic muscle loss is a feature of limb girdle muscular dystrophy 2B (LGMD2B) - a disease caused by mutations in dysferlin. Here we show that FAPs cause the adipogenic loss of dysferlin deficient muscle. Progressive accumulation of Annexin A2 (AnxA2) in the myofiber matrix causes FAP differentiation into adipocytes. Lack of AnxA2 prevents FAP adipogenesis, protecting against adipogenic loss of dysferlinopathic muscle while exogenous AnxA2 enhances muscle loss. Pharmacological inhibition of FAP adipogenesis arrests adipogenic replacement and degeneration of dysferlin-deficient muscle. These results demonstrate the pathogenic role of FAPs in LGMD2B and establish these cells as therapeutic targets to ameliorate muscle loss in patients.
Project description:Limb girdle muscular dystrophy 2B (LGMD2B) is without treatment and caused by mutations in the dysferlin gene (DYSF). One-third is missense mutations leading to dysferlin aggregation and amyloid formation, in addition to defects in sarcolemmal repair and progressive muscle wasting. Dysferlin-null mouse models do not allow study of the consequences of missense mutations. We generated a new mouse model (MMex38) carrying a missense mutation in exon 38 in analogy to a clinically relevant human DYSF variant (DYSF p.Leu1341Pro). The targeted mutation induces all characteristics of missense mutant dysferlinopathy, including a progressive dystrophic pattern, amyloid formation, and defects in membrane repair. We chose U7 small nuclear RNA (snRNA)-based splice switching to demonstrate a possible exon-skipping strategy in this new animal model. We show that Dysf exons 37 and 38 can successfully be skipped in vivo. Overall, the MMex38 mouse model provides an ideal tool for preclinical development of treatment strategies for dysferlinopathy.
Project description:Dysferlin deficiency compromises the repair of injured muscle, but the underlying cellular mechanism remains elusive. To study this phenomenon, we have developed mouse and human myoblast models for dysferlinopathy. These dysferlinopathic myoblasts undergo normal differentiation but have a deficit in their ability to repair focal injury to their cell membrane. Imaging cells undergoing repair showed that dysferlin-deficit decreased the number of lysosomes present at the cell membrane, resulting in a delay and reduction in injury-triggered lysosomal exocytosis. We find repair of injured cells does not involve formation of intracellular membrane patch through lysosome-lysosome fusion; instead, individual lysosomes fuse with the injured cell membrane, releasing acid sphingomyelinase (ASM). ASM secretion was reduced in injured dysferlinopathic cells, and acute treatment with sphingomyelinase restored the repair ability of dysferlinopathic myoblasts and myofibers. Our results provide the mechanism for dysferlin-mediated repair of skeletal muscle sarcolemma and identify ASM as a potential therapy for dysferlinopathy.