Limb-girdle Muscular Dystrophy Type 2E Research Articles

Safety, β-sarcoglycan Expression and Functional Outcomes from Systemic Gene Transfer of rAAVrh74.MHCK7.SGCB in Patients with Limb-Girdle Muscular Dystrophy Type 2E (LGMD2E) (4330)

Safety, β-sarcoglycan Expression and Functional Outcomes from Systemic Gene Transfer of rAAVrh74.MHCK7.SGCB in Patients with Limb-Girdle Muscular Dystrophy Type 2E (LGMD2E) (4330)

LIMB GIRDLE MUSCULAR DYSTROPHIES: P.140 Systemic gene transfer with rAAVrh74.MHCK7.SGCB increased Beta-sarcoglycan expression in patients with Limb girdle muscular dystrophy type 2E (LGMD2E)

We present findings of a phase 1 multiple ascending-dose gene transfer trial of ≤9 patients with LGMD2E (β-sarcoglycan [SGCB] deficiency) who received rAAVrh74.MHCK7.SGCB (NCT03652259). Participants: age 4-15y, SGCB mutation (both alleles), no rAAVrh74 antibodies, >40% on 100-meter timed test. Cohort 1 (n=3) received single IV infusion of 5 × 1013 vg/kg rAAVrh74.MHCK7.SGCB. For all patients, prednisone 1 mg/kg/day was initiated 1d before gene delivery (30-d taper). Primary endpoints: ≥20% SGCB-positive fibers (Day 60 muscle biopsy); safety.

LIMB GIRDLE MUSCULAR DYSTROPHIES: P.139 Expression-functional correlation and validation of a surrogate marker for DAPC restoration in LGMD2E mouse model

Limb-girdle muscular dystrophy type 2E (LGMD2E) is caused by mutations in β-sarcoglycan (SGCB) leading to protein deficiency, loss of formation of the sarcoglycan complex, and loss of stabilization of the dystrophin-associated protein complex (DAPC). The sarcoglycans and sarcospan are integral proteins critical for stabilizing the DAPC and providing mechanical support to the sarcolemma. SGCB−/− mice have been shown to concurrently display loss of additional sarcoglycans (α, γ, and δ). Evidence suggests sarcospan may also be lost in the absence of SGCB.

Morphological and functional analyses of skeletal muscles from an immunodeficient animal model of limb-girdle muscular dystrophy type 2E.

ABSTRACT Introduction: Limb‐girdle muscular dystrophy type 2E (LGMD2E) is caused by mutations in the β‐sarcoglycan gene, which is expressed in skeletal, cardiac, and smooth muscles. β‐Sarcoglycan‐deficient (Sgcb‐null) mice develop severe muscular dystrophy and cardiomyopathy with focal areas of necrosis. Methods: In this study we performed morphological (histological and cellular characterization) and functional (isometric tetanic force and fatigue) analyses in dystrophic mice. Comparison studies were carried out in 1‐month‐old (clinical onset of the disease) and 7‐month‐old control mice (C57Bl/6J, Rag2/γc‐null) and immunocompetent and immunodeficient dystrophic mice (Sgcb‐null and Sgcb/Rag2/γc‐null, respectively). Results: We found that the lack of an immunological system resulted in an increase of calcification in striated muscles without impairing extensor digitorum longus muscle performance. Sgcb/Rag2/γc‐null muscles showed a significant reduction of alkaline phosphate‐positive mesoangioblasts. Discussion: The immunological system counteracts skeletal muscle degeneration in the murine model of LGMD2E. Muscle Nerve 58: 133–144, 2018

Systemic AAV-Mediated β-Sarcoglycan Delivery Targeting Cardiac and Skeletal Muscle Ameliorates Histological and Functional Deficits in LGMD2E Mice

Limb-girdle muscular dystrophy type 2E (LGMD2E), resulting from mutations in β-sarcoglycan (SGCB), is a progressive dystrophy with deteriorating muscle function, respiratory failure, and cardiomyopathy in 50% or more of LGMD2E patients. SGCB knockout mice share many of the phenotypicdeficiencies of LGMD2E patients. To investigate systemic SGCB gene transfer to treat skeletal and cardiac muscledeficits, we designed a self-complementary AAVrh74 vector containing a codon-optimized human SGCB transgenedriven by a muscle-specific promoter. We delivered scAAV.MHCK7.hSGCB through the tail vein of SGCB-/- mice to provide a rationale for a clinical trial that would lead to clinically meaningful results. This led to 98.1% transgene expression across all muscles that was accompanied by improvements in histopathology. Serum creatine kinase (CK) levels were reduced following treatment by 85.5%. Diaphragm force production increased by 94.4%, kyphoscoliosis of the spine was significantly reduced by 48.1%, overall ambulation increased by 57%, and vertical rearing increased dramatically by 132% following treatment. Importantly, no adverse effects were seen in muscle of wild-type mice injected systemically with scAAV.hSGCB. In this well-defined model of LGMD2E, we have demonstrated the efficacy and safety of systemic scAAV.hSGCB delivery, and these findings have established a path for clinically beneficial AAV-mediated gene therapy for LGMD2E.

622. Systemic β-Sarcoglycan Gene Therapy for Treatment of Cardiac and Skeletal Muscle Deficits in LGMD2E

Limb-girdle muscular dystrophy type 2E (LGMD2E) results from mutations in the beta-sarcoglycan (SGCB) gene causing loss of a sarcolemmal structural protein component of the dystrophin-associated protein complex (DAPC). This leads to a progressive dystrophy with numerous histopathological features, resulting in deteriorating muscle function. This occurs not only in limb muscle but also in the diaphragm and the heart. Consequences include respiratory failure and cardiomyopathy in 50% or more of LGMD2E patients. SGCB knockout mice share many of the phenotypic deficiencies of LGMD2E patients. In this mouse model we quantified dystrophic histopathology, fibrosis, and functional outcomes in lower limb, upper limb, and torso skeletal muscles, as well as the diaphragm and cardiac muscles. Diaphragms of SGCB-/- mice demonstrated reduced specific force output (116.24 mN/mm2) compared to wild-type (WT) mice (236.67 mN/mm2), and hearts from SGCB-/- mice had lower ejection fraction (58.19%) compared to WT mice (75.04%) as determined by MRI. Additionally, radiographic imaging defined the degree of kyphoscoliosis in SGCB-/- mice. Laser monitoring of open-field cage activity showed a reduction of ~55% in both total ambulation as well as hindlimb vertical rearing in SGCB-/- mice compared to WT. For treatment, we designed a self-complementary AAVrh74 vector containing a codon optimized human SGCB transgene driven by a muscle specific promoter. We next demonstrated efficacy of vector delivery by intramuscular (IM) injection to the tibialis anterior muscle, as well as isolated-limb perfusion (ILP) to the lower limb muscles of SGCB-/- mice. Along with restoration of SGCB expression in treated muscle, we saw histological and functional improvements and a reduction in fibrosis as indicated by reduced collagen deposition. These regional gene delivery studies were followed by systemic delivery of scAAV.hSGCB through the tail vein of SGCB-/- mice to provide a potential rationale for gene delivery in clinical trial that would lead to clinically meaningful results. Tail vein injection of scAAV.hSGCB resulted in nearly 100% transgene expression in numerous muscles throughout the hindlimbs, forelimbs, torso, and the heart, that was accompanied by improvements in histopathology including reduction in central nucleation and increased fiber diameter. Kyphoscoliosis of the spine was also improved, and total ambulation increased in scAAV.hSGCB treated mice by ~22% while hindlimb vertical rearing increased by ~77% in treated mice compared to KO. We also saw complete restoration of diaphragm function following treatment with specific force output improved to 226.07 mN/mm2. In this well-defined mode of LGMD2E, we have demonstrated that systemic delivery of scAAV.hSGCB normalizes histologic and functional outcome measures in limb, diaphragm, and heart. These findings have established a path for AAV mediated gene therapy for LGMD2E that we are currently pursuing.

β-Sarcoglycan gene transfer decreases fibrosis and restores force in LGMD2E mice.

Limb-girdle muscular dystrophy type 2E (LGMD2E) results from mutations in the β-sarcoglycan (SGCB) gene causing loss of functional protein and concomitant loss of dystrophin-associated proteins. The disease phenotype is characterized by muscle weakness and wasting, and dystrophic features including muscle fiber necrosis, inflammation and fibrosis. The Sgcb-null mouse recapitulates the clinical phenotype with significant endomysial fibrosis providing a relevant model to test whether gene replacement will be efficacious. We directly addressed this question using a codon optimized human β-sarcoglycan gene (hSGCB) driven by a muscle-specific tMCK promoter (scAAVrh74.tMCK.hSGCB). Following isolated limb delivery (5 × 10(11) vector genome (vg)), 91.2% of muscle fibers in the lower limb expressed β-sarcoglycan, restoring assembly of the sarcoglycan complex and protecting the membrane from Evans blue dye leakage. Histological outcomes were significantly improved including decreased central nucleation, normalization of muscle fiber size, decreased macrophages and inflammatory mononuclear cells, and an average of a 43% reduction in collagen deposition in treated muscle compared with untreated muscle at end point. These measures correlated with improvement of tetanic force and resistance to eccentric contraction. In 6-month-old mice, as indicated by collagen staining, scAAVrh74.tMCK.hSGCB treatment reduced fibrosis by 42%. This study demonstrates the potential for gene replacement to reverse debilitating fibrosis, typical of muscular dystrophy, thereby providing compelling evidence for movement to clinical gene replacement for LGMD2E.

506. β-Sarcoglycan Gene Transfer Prevents Muscle Fibrosis and Inflammation in an Aged LGMD2E Mouse Model

Limb-girdle muscular dystrophy type 2E (LGMD2E) results from mutations in the β-sarcoglycan (SGCB) gene causing loss of a structural protein component of the dystrophin-associated protein complex (DAPC) located at the sarcolemma. This loss results in histopathological features including chronic muscle fiber necrosis, inflammation, fat replacement and fibrosis, accompanied by deteriorating muscle strength and function. To date, no effective therapy exists to treat this debilitating disease. A suitable model for translational studies is the murine model of LGMD2E that completely lacks β-sarcoglycan (Sgcb-null mouse), and has clinical-pathological features in skeletal and cardiac muscle that replicate the human disease. Considering that a major question unanswered by gene replacement therapy is the potential efficacy of gene replacement once significant degrees of connective tissue have infiltrated dystrophic muscle, the studies described here in the β-sarcoglycan knock out mouse have particular relevance for planning future clinical trials. On the clinical side, fibrosis presents a major impasse for functional recovery. In this study, we directly addressed this question using a codon optimized human β-sarcoglycan gene (hSGCB) driven by a muscle specific tMCK promoter and the AAVrh.74 serotype (scAAVrh74.tMCK.hSGCB). We first showed restoration of expression 12 weeks post treatment following a direct intramuscular injection which was accompanied by improvements in histological parameters. Furthermore, following a clinically relevant isolated limb vascular delivery (5X10^11 vg) we found that >90% of muscle fibers expressed β-sarcoglycan in lower limb muscles. Histopathology showed a decrease in central nucleation and normalization of muscle fiber size. Immunohistochemical staining for various immune components including CD3, CD4, CD8, and macrophages showed a reduction in the numbers of immune cells following treatment. The restoration of β-sarcoglycan expression and improvement in overall histology also correlated with improvement in functional outcomes assessed by absolute and specific force generation and resistance to contraction induced injury. To measure fibrosis, quantification of picrosirius stained sections revealed a reduction of collagen deposition in TA (from 40.72 ± 1.40% to 21.22 ± 1.09%, p 80% of muscle fibers were transduced which was accompanied by a significant reduction of fibrosis similar to what was achieved following vascular delivery in younger mice. This pre-clinical study addresses the potential for gene replacement to reverse the debilitating fibrosis, typical of many of the muscular dystrophies providing momentum for movement to a clinical gene replacement for LGMD2E.

Spotlight on the April 28 Issue

#### Notable in Neurology This issue features articles on GRID2 mutations in congenital to mild adult-onset cerebellar ataxia and on how novel RNF216 mutations cause a Huntington-like disorder phenotype with pure monogenic recessive inheritance. Other featured articles focus on how skeletal muscle metabolism is impaired during exercise in glycogen storage disease type III and the clinical and genetic spectrum of limb-girdle muscular dystrophy type 2E. ### Poststroke suicide attempts and completed suicides: A socioeconomic and nationwide perspective![Graphic][1]</img>![Graphic][2]</img> Suicide rates are doubled in the first 2 years after stroke; risk factors include living alone, having a lower level of education, and poststroke depression. In high-risk groups, … [1]: /embed/inline-graphic-1.gif [2]: /embed/inline-graphic-2.gif

Clinical and genetic spectrum in limb-girdle muscular dystrophy type 2E.

To determine the clinical spectrum of limb-girdle muscular dystrophy 2E (LGMD2E) and to investigate whether genetic or biochemical features can predict the phenotype of the disease. All LGMD2E patients followed in participating centers were included. A specific clinical protocol was created, including quantitative evaluation of motor, respiratory, and cardiac function. Phenotype was defined as severe or mild if the age at loss of ambulation occurred before or after 18 years. Molecular analysis of SGCB gene and biochemical features of muscle biopsies were reviewed. Thirty-two patients were included (16 male, 16 female; age 7-67 years; 15 severe, 12 mild, and 5 unknown). Neurologic examination showed proximal muscle weakness in all patients, but distal involvement was also observed in patients with severe disease early in the disease course. Cardiac involvement was observed in 20 patients (63%) even before overt muscle involvement. Six patients had restrictive respiratory insufficiency requiring assisted ventilation (19%). Seventeen different mutations were identified, and 3 were recurrent. The c.377_384dup (13 alleles) was associated with the severe form, the c.-22_10dup (10) with the milder form, and the c.341C>T (9) with both. The entire sarcoglycan complex was undetectable by muscle immunohistochemistry or Western blot in 9/10 severe cases and reduced in 7/7 mild cases. The residual amount of sarcoglycan in muscle resulted a predictor of age at loss of ambulation. This study expands the spectrum of phenotype in β-sarcoglycanopathy and provides strong evidence that severity of clinical involvement may be predicted by SGCB gene mutation and sarcoglycan protein expression.

G.P.226: Sarcolemmal deficiency of sarcoglycan complex in an eighteen months old Turkish boy with a huge deletion in the beta sarcoglycan gene

Limb-girdle muscular dystrophy type 2E (LGMD-2E) is caused by autosomal recessive defects in the beta-sarcoglycan gene located on chromosome 4q12. In this study, the clinical findings, histopathological features and molecular genetic data in a boy with beta-sarcoglycanopathy are presented. An eighteen-month-old boy had a very high serum creatinine phosphokinase level, which was determined incidentally. He had consanguineous parents. The result of molecular analyses for dystrophin gene was found normal. He underwent a muscle biopsy which showed dystrophic features. Immunohistochemistry showed that, there was a total loss of sarcolemmal sarcoglycan complex (alpha, beta, delta and gamma sarcoglycans) while sarcolemmal dystrophin expression was normal. DNA analysis revealed a huge deletion in the beta-sarcoglycan gene. The deletion was homozygous and occurred from exon 1 to 6. This study demonstrated that the total absence or abnormal expressions of all 4 sarcoglycans are more likely to indicate a primary defect in the beta sarcoglycan gene.

Homozygous microdeletion of chromosome 4q11-q12 causes severe limb-girdle muscular dystrophy type 2E with joint hyperlaxity and contractures

Microdeletion syndromes are commonly transmitted as dominant traits and are frequently associated with variably expressed pleiotropic phenotypes. Nonlethal homozygous microdeletions, on the other hand, are very rare. Here, we delineate the fifth and so far largest homozygous microdeletion in nonmalignancies of approximately 400 kb on chromosome 4q11-q12 in a large consanguineous East-Anatolian family with six affected patients. The deleted region contains the beta-sarcoglycan gene (SGCB), the predicted gene SPATA18 (spermatogenesis associated 18 homolog) and several expressed sequence tags. Patients presented with a severe and progressive Duchenne-like muscular dystrophy phenotype, a combination of hyperlaxity and joint contractures, chest pain, palpitations, and dyspnea.

Disruption of the β-Sarcoglycan Gene Reveals Pathogenetic Complexity of Limb-Girdle Muscular Dystrophy Type 2E

Limb-girdle muscular dystrophy type 2E (LGMD 2E) is caused by mutations in the β-sarcoglycan gene, which is expressed in skeletal, cardiac, and smooth muscle. β-sarcoglycan-deficient ( Sgcb-null) mice developed severe muscular dystrophy and cardiomyopathy with focal areas of necrosis. The sarcoglycan–sarcospan and dystroglycan complexes were disrupted in skeletal, cardiac, and smooth muscle membranes. ε-sarcoglycan was also reduced in membrane preparations of striated and smooth muscle. Loss of the sarcoglycan–sarcospan complex in vascular smooth muscle resulted in vascular irregularities in heart, diaphragm, and kidneys. Further biochemical characterization suggested the presence of a distinct ε-sarcoglycan complex in skeletal muscle that was disrupted in Sgcb-null mice. Thus, perturbation of vascular function together with disruption of the ε-sarcoglycan-containing complex represents a novel mechanism in the pathogenesis of LGMD 2E.