A single c.1715G>C calpain 3 gene variant causes dominant calpainopathy with loss of calpain 3 expression and activity.

Recessively inherited limb girdle muscular dystrophy (LGMD) type 2A is the most common LGMD worldwide. Here, we report the first single missense variant in CAPN3 causing dominantly inherited calpainopathy. A 43-year-old proband, his father and two sons were heterozygous for a c.1715G>C p.(Arg572Pro) variant in CAPN3. Affected family members had at least three of the following; muscle pain, a LGMD2A pattern of muscle weakness and wasting, muscle fat replacement on magnetic resonance imaging, myopathic muscle biopsy, and elevated creatine kinase. Total calpain 3 protein expression was 4 ± 3% of normal. In vitro analysis of c.1715G>C and the previously described c.643_663del variant indicated that the mutant proteins lack autolytic and proteolytic activity and decrease the quantity of wild-type CAPN3 protein. Our findings suggest that dominantly inherited calpainopathy is not unique to the previously reported c.643_663del mutation of CAPN3, and that dominantly inherited calpainopathy should be considered for other single variations in CAPN3.

Serum proteomic profiling reveals fragments of MYOM3 as potential biomarkers for monitoring the outcome of therapeutic interventions in muscular dystrophies.

Therapy-responsive biomarkers are an important and unmet need in the muscular dystrophy field where new treatments are currently in clinical trials. By using a comprehensive high-resolution mass spectrometry approach and western blot validation, we found that two fragments of the myofibrillar structural protein myomesin-3 (MYOM3) are abnormally present in sera of Duchenne muscular dystrophy (DMD) patients, limb-girdle muscular dystrophy type 2D (LGMD2D) and their respective animal models. Levels of MYOM3 fragments were assayed in therapeutic model systems: (1) restoration of dystrophin expression by antisense oligonucleotide-mediated exon-skipping in mdx mice and (2) stable restoration of α-sarcoglycan expression in KO-SGCA mice by systemic injection of a viral vector. Following administration of the therapeutic agents MYOM3 was restored toward wild-type levels. In the LGMD model, where different doses of vector were used, MYOM3 restoration was dose-dependent. MYOM3 fragments showed lower inter-individual variability compared with the commonly used creatine kinase assay, and correlated better with the restoration of the dystrophin-associated protein complex and muscle force. These data suggest that the MYOM3 fragments hold promise for minimally invasive assessment of experimental therapies for DMD and other neuromuscular disorders.

Restriction of calpain3 expression to the skeletal muscle prevents cardiac toxicity and corrects pathology in a murine model of limb-girdle muscular dystrophy.

BACKGROUND: Genetic defects in calpain3 (CAPN3) lead to limb-girdle muscular dystrophy type 2A, a disease of the skeletal muscle that affects predominantly the proximal limb muscles. We previously demonstrated the potential of adeno-associated virus-mediated transfer of the CAPN3 gene to correct the pathological signs in a murine model for limb-girdle muscular dystrophy type 2A after intramuscular and locoregional administrations. METHODS AND RESULTS: Here, we showed that intravenous injection of calpain3-expressing vector in mice can induce mortality in a dose-dependent manner. An anatomopathological investigation revealed large areas of fibrosis in the heart that we related to unregulated proteolytic activity of calpain3. To circumvent this toxicity, we developed new adeno-associated virus vectors with skeletal muscle-restricted expression by using new muscle-specific promoters that include the CAPN3 promoter itself and by introducing a target sequence of the cardiac-specific microRNA-208a in the cassette. Our results show that CAPN3 transgene expression can be successfully suppressed in the cardiac tissue, preventing the cardiac toxicity, whereas expression of the transgene in skeletal muscle reverts the pathological signs of calpain3 deficiency. CONCLUSIONS: The molecular strategies used in this study may be useful for any gene transfer strategy with potential toxicity in the heart.

Dlk1-Dio3 cluster miRNAs regulate mitochondrial functions in the dystrophic muscle in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe muscle disease caused by impaired expression of dystrophin. Whereas mitochondrial dysfunction is thought to play an important role in DMD, the mechanism of this dysfunction remains to be clarified. Here we demonstrate that in DMD and other muscular dystrophies, a large number of Dlk1-Dio3 clustered miRNAs (DD-miRNAs) are coordinately up-regulated in regenerating myofibers and in the serum. To characterize the biological effect of this dysregulation, 14 DD-miRNAs were simultaneously overexpressed in vivo in mouse muscle. Transcriptomic analysis revealed highly similar changes between the muscle ectopically overexpressing 14 DD-miRNAs and the mdx diaphragm, with naturally up-regulated DD-miRNAs. Among the commonly dysregulated pathway we found repressed mitochondrial metabolism, and oxidative phosphorylation (OxPhos) in particular. Knocking down the DD-miRNAs in iPS-derived skeletal myotubes resulted in increased OxPhos activities. The data suggest that (1) DD-miRNAs are important mediators of dystrophic changes in DMD muscle, (2) mitochondrial metabolism and OxPhos in particular are targeted in DMD by coordinately up-regulated DD-miRNAs. These findings provide insight into the mechanism of mitochondrial dysfunction in muscular dystrophy.

Rescue of sarcoglycan mutations by inhibition of endoplasmic reticulum quality control is associated with minimal structural modifications.

Sarcoglycanopathies (SGP) are a group of autosomal recessive muscle disorders caused by primary mutations in one of the four sarcoglycan genes. The sarcoglycans (α-, ß-, γ-, and δ-sarcoglycan) form a tetrameric complex at the muscle membrane that is part of the dystrophin-glycoprotein complex and plays an essential role for membrane integrity during muscle contractions. We previously showed that the most frequent missense mutation in α-sarcoglycan (p.R77C) leads to the absence of the protein at the cell membrane due to its blockade by the endoplasmic reticulum (ER) quality control. Moreover, we demonstrated that inhibition of the ER α-mannosidase I activity using kifunensine could rescue the mutant protein localization at the cell membrane. Here, we investigate 25 additional disease-causing missense mutations in the sarcoglycan genes with respect to intracellular fate and localization rescue of the mutated proteins by kifunensine. Our studies demonstrate that, similarly to p.R77C, 22 of 25 of the selected mutations lead to defective intracellular trafficking of the SGs proteins. Six of these were saved from ER retention upon kifunensine treatment. The trafficking of SGs mutants rescued by kifunensine was associated with mutations that have moderate structural impact on the protein.

Late-onset camptocormia caused by a heterozygous in-frame CAPN3 deletion.

Camptocormia is defined by a pathological involuntary flexion of the thoracic and lumbar spine that is fully reducible in the supine position. Although originally described as a manifestation of conversion disorder, it is more commonly caused by a wide range of neurological diseases, in particular movement and neuromuscular disorders. We describe here a rare case of late onset camptocormia caused by autosomal dominant calpainopathy due to a heterozygous in-frame deletion in CAPN3 leading to loss of a single lysin amino acid in the catalytic domain of calpain-3. Creatine kinase levels, electromyography, and thigh muscle MRI were normal. Muscle biopsy did not show lobulated fibers and calpain-3 protein expression was not decreased, but in vitro functional assays showed impaired proteolytic function of. Lys254del CAPN3. Autosomal dominant calpainopathy should be considered in the differential diagnosis of late onset camptocormia and unexplained paravertebral myopathies even in presence of normal creatine kinase levels, and in absence of lobulated fibers, of decreased calpain-3 protein expression, and of muscle limb involvement.

An AAV-SGCG Dose-Response Study in a γ-Sarcoglycanopathy Mouse Model in the Context of Mechanical Stress.

Sarcoglycanopathies are rare autosomic limb girdle muscular dystrophies caused by mutations in one of the genes coding for sarcoglycans. Sarcoglycans form a complex, which is an important part of the dystrophin-associated glycoprotein complex and which protects the sarcolemma against muscle contraction-induced damage. Absence of one of the sarcoglycans on the plasma membrane reduces the stability of the whole complex and perturbs muscle fiber membrane integrity. There is currently no curative treatment for any of the sarcoglycanopathies. A first clinical trial to evaluate the safety of a recombinant AAV2/1 vector expressing γ-sarcoglycan using an intramuscular route of administration showed limited expression of the transgene and good tolerance of the approach. In this report, we undertook a dose-effect study in mice to evaluate the efficiency of an AAV2/8-expressing γ-sarcoglycan controlled by a muscle-specific promoter with a systemic mode of administration. We observed a dose-related efficiency with a nearly complete restoration of gamma sarcoglycan (SGCG) expression, histological appearance, biomarker level, and whole-body strength at the highest dose tested. In addition, our data suggest that a high expression threshold level must be achieved for effective protection of the transduced muscle, while a suboptimal transgene expression level might be less protective in the context of mechanical stress.

Temporary Reduction of Membrane CD4 with the Antioxidant MnTBAP Is Sufficient to Prevent Immune Responses Induced by Gene Transfer.

Unexpectedly, the synthetic antioxidant MnTBAP was found to cause a rapid and reversible downregulation of CD4 on T cells in vitro and in vivo. This effect resulted from the internalization of membrane CD4 T cell molecules into clathrin-coated pits and involved disruption of the CD4/p56Lck complex. The CD4 deprivation induced by MnTBAP had functional consequences on CD4-dependent infectious processes or immunological responses as shown in various models, including gene therapy. In cultured human T cells, MnTBAP-induced downregulation of CD4 functionally suppressed gp120- mediated lentiviral transduction in a model relevant for HIV infection. The injection of MnTBAP in mice reduced membrane CD4 on lymphocytes in vivo within 5 days of treatment, preventing OVA peptide T cell immunization while allowing subsequent immunization once treatment was stopped. In a mouse gene therapy model, MnTBAP treatment at the time of adenovirus-associated virus (AAV) vector administration, successfully controlled the induction of anti-transgene and anti-capsid immune responses mediated by CD4+ T cells, enabling the redosing mice with the same vector. These functional data provide new avenues to develop alternative therapeutic immunomodulatory strategies based on temporary regulation of CD4. These could be particularly useful for AAV gene therapy in which novel strategies for redosing are needed.

Role of Regulatory T Cell and Effector T Cell Exhaustion in Liver-Mediated Transgene Tolerance in Muscle.

The pro-tolerogenic environment of the liver makes this tissue an ideal target for gene replacement strategies. In other peripheral tissues such as the skeletal muscle, anti-transgene immune response can result in partial or complete clearance of the transduced fibers. Here, we characterized liver-induced transgene tolerance after simultaneous transduction of liver and muscle. A clinically relevant transgene, α-sarcoglycan, mutated in limb-girdle muscular dystrophy type 2D, was fused with the SIINFEKL epitope (hSGCA-SIIN) and expressed with adeno-associated virus vectors (AAV-hSGCA-SIIN). Intramuscular delivery of AAV-hSGCA-SIIN resulted in a strong inflammatory response, which could be prevented and reversed by concomitant liver expression of the same antigen. Regulatory T cells and upregulation of checkpoint inhibitor receptors were required to establish and maintain liver-mediated peripheral tolerance. This study identifies the fundamental role of the synergy between Tregs and upregulation of checkpoint inhibitor receptors in the liver-mediated control of anti-transgene immunity triggered by muscle-directed gene transfer.

Phenotypic correction of alpha-sarcoglycan deficiency by intra-arterial injection of a muscle-specific serotype 1 rAAV vector.

alpha-Sarcoglycanopathy (limb-girdle muscular dystrophy type 2D, LGMD2D) is a recessive muscular disorder caused by deficiency in alpha-sarcoglycan, a transmembrane protein part of the dystrophin-associated complex. To date, no treatment exists for this disease. We constructed recombinant pseudotype-1 adeno-associated virus (rAAV) vectors expressing the human alpha-sarcoglycan cDNA from a ubiquitous or a muscle-specific promoter. Evidence of specific immune response leading to disappearance of the vector was observed with the ubiquitous promoter. In contrast, efficient and sustained transgene expression with correct sarcolemmal localization and without evident toxicity was obtained with the muscle-specific promoter after intra-arterial injection into the limbs of an LGMD2D murine model. Transgene expression resulted in restoration of the sarcoglycan complex, histological improvement, membrane stabilization, and correction of pseudohypertrophy. More importantly, alpha-sarcoglycan transfer produced full rescue of the contractile force deficits and stretch sensibility and led to an increase of the global activity of the animals when both posterior limbs are injected. Our results establish the feasibility for AAV-mediated alpha-sarcoglycan gene transfer as a therapeutic approach.

Phenotypic Correction of α-Sarcoglycan Deficiency by Intra-arterial Injection of a Muscle-specific Serotype 1 rAAV Vector.

α-Sarcoglycanopathy (limb-girdle muscular dystrophy type 2D, LGMD2D) is a recessive muscular disorder caused by deficiency in α-sarcoglycan, a transmembrane protein part of the dystrophin-associated complex. To date, no treatment exists for this disease. We constructed recombinant pseudotype-1 adeno-associated virus (rAAV) vectors expressing the human α-sarcoglycan cDNA from a ubiquitous or a muscle-specific promoter. Evidence of specific immune response leading to disappearance of the vector was observed with the ubiquitous promoter. In contrast, efficient and sustained transgene expression with correct sarcolemmal localization and without evident toxicity was obtained with the muscle-specific promoter after intra-arterial injection into the limbs of an LGMD2D murine model. Transgene expression resulted in restoration of the sarcoglycan complex, histological improvement, membrane stabilization, and correction of pseudohypertrophy. More importantly, α-sarcoglycan transfer produced full rescue of the contractile force deficits and stretch sensibility and led to an increase of the global activity of the animals when both posterior limbs are injected. Our results establish the feasibility for AAV-mediated α-sarcoglycan gene transfer as a therapeutic approach.

A third of LGMD2A biopsies have normal calpain 3 proteolytic activity as determined by an in vitro assay.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive muscular disorder caused by mutations in the gene coding for calpain 3, a calcium-dependent protease. We developed an in vitro assay that can detect the proteolytic activity of calpain 3 in a muscle sample. This assay is based on the use of an inactive calpain 3 as a substrate for active calpain 3 molecules. A total of 79 human biopsies have been analysed using an unbiased single blind method. Results were confronted with the molecular diagnosis for confirmation. Proteolytic activity was either reduced or absent in 68% of LGMD2A biopsies. In the remaining 32%, normal proteolytic activity was found despite the presence of calpain 3 mutation(s), suggesting that other calpain 3 properties might be impaired to give rise to the LGMD2A phenotype. Our assay is easily adaptable to routine and appears to be more sensitive than common analysis by immunodetection.

Circulating miRNAs are generic and versatile therapeutic monitoring biomarkers in muscular dystrophies.

The development of medical approaches requires preclinical and clinical trials for assessment of therapeutic efficacy. Such evaluation entails the use of biomarkers, which provide information on the response to the therapeutic intervention. One newly-proposed class of biomarkers is the microRNA (miRNA) molecules. In muscular dystrophies (MD), the dysregulation of miRNAs was initially observed in muscle biopsy and later extended to plasma samples, suggesting that they may be of interest as biomarkers. First, we demonstrated that dystromiRs dysregulation occurs in MD with either preserved or disrupted expression of the dystrophin-associated glycoprotein complex, supporting the utilization of dystromiRs as generic biomarkers in MD. Then, we aimed at evaluation of the capacity of miRNAs as monitoring biomarkers for experimental therapeutic approach in MD. To this end, we took advantage of our previously characterized gene therapy approach in a mouse model for α-sarcoglycanopathy. We identified a dose-response correlation between the expression of miRNAs on both muscle tissue and blood serum and the therapeutic benefit as evaluated by a set of new and classically-used evaluation methods. This study supports the utility of profiling circulating miRNAs for the evaluation of therapeutic outcome in medical approaches for MD.

Prolonged gene expression in muscle is achieved without active immune tolerance using microrRNA 142.3p-regulated rAAV gene transfer.

Gene transfer efficacy is limited by unwanted immunization against transgene products. In some models, immunization may be avoided by regulating transgene expression with mir142.3p target sequences. Yet, it is unclear if such a strategy controls T-cell responses following recombinant adeno-associated viral vector (rAAV)-mediated gene transfer, particularly in muscle. In mice, intramuscular rAAV1 gene delivery of a tagged human sarcoglycan muscle protein is robustly immunogenic and leads to muscle destruction. In this model, the simple insertion of mir142.3p-target sequences in the transgene expression cassette modifies the outcome of gene transfer, providing high and persistent levels of muscle transduction in C57Bl/6 mice. Such regulated vector fails to prime specific CD4 and CD8 T cells; although, transgene tolerance seems to result from ignorance and could be broken by a robust antigenic challenge. While effective in normal mice, the mir142.3p-regulated transgene remains immunogenic in sarcoglycan-deficient dystrophic mice. In these mice, transgene expression is only prolonged but does not persist as effector CD4 and CD8 T-cell responses develop. Thus, using a mir142.3p-regulated transgene can improve rAAV muscle gene transfer results, but the level of efficacy depends on the context of application. In normal muscle, this strategy is sufficient to prevent immunization and functions even more effectively than tissue-specific promoters. In dystrophic models, additional strategies are required to fully control T-cell responses.

Distinctive serum miRNA profile in mouse models of striated muscular pathologies.

Biomarkers are critically important for disease diagnosis and monitoring. In particular, close monitoring of disease evolution is eminently required for the evaluation of therapeutic treatments. Classical monitoring methods in muscular dystrophies are largely based on histological and molecular analyses of muscle biopsies. Such biopsies are invasive and therefore difficult to obtain. The serum protein creatine kinase is a useful biomarker, which is however not specific for a given pathology and correlates poorly with the severity or course of the muscular pathology. The aim of the present study was the systematic evaluation of serum microRNAs (miRNAs) as biomarkers in striated muscle pathologies. Mouse models for five striated muscle pathologies were investigated: Duchenne muscular dystrophy (DMD), limb-girdle muscular dystrophy type 2D (LGMD2D), limb-girdle muscular dystrophy type 2C (LGMD2C), Emery-Dreifuss muscular dystrophy (EDMD) and hypertrophic cardiomyopathy (HCM). Two-step RT-qPCR methodology was elaborated, using two different RT-qPCR miRNA quantification technologies. We identified miRNA modulation in the serum of all the five mouse models. The most highly dysregulated serum miRNAs were found to be commonly upregulated in DMD, LGMD2D and LGMD2C mouse models, which all exhibit massive destruction of striated muscle tissues. Some of these miRNAs were down rather than upregulated in the EDMD mice, a model without massive myofiber destruction. The dysregulated miRNAs identified in the HCM model were different, with the exception of one dysregulated miRNA common to all pathologies. Importantly, a specific and distinctive circulating miRNA profile was identified for each studied pathological mouse model. The differential expression of a few dysregulated miRNAs in the DMD mice was further evaluated in DMD patients, providing new candidates of circulating miRNA biomarkers for DMD.

Methods for noninvasive monitoring of muscle fiber survival with an AAV vector encoding the mSEAP reporter gene.

Muscular dystrophies (MD) are a group of genetically and phenotypically heterogeneous inherited disorders characterized by the progressive degeneration of the skeletal muscle tissue. In the last decade, a tremendous amount of studies were performed to test therapeutic strategies in animal models. Evaluation of such strategies requires the use of criteria predictive of their therapeutic relevance. Here we describe a simple, noninvasive assay to monitor muscle degenerative process. An adeno-associated vector encoding a secreted form of murine embryonic alkaline phosphatase (mSEAP) reporter gene is administrated at the time of treatment. The amount of circulating mSEAP will reflect the level of myofiber survival. We tested this assay with therapeutic gene transfer. We found a strong correlation between therapeutic gene expression/muscle disease amelioration and the circulating levels of mSEAP. The assay will be very useful for monitoring muscle cell survival after therapeutic intervention.

Safety and efficacy of AAV-mediated calpain 3 gene transfer in a mouse model of limb-girdle muscular dystrophy type 2A.

Calpainopathy (limb-girdle muscular dystrophy type 2A, LGMD2A) is a recessive muscular disorder caused by deficiency in the calcium-dependent cysteine protease calpain 3. To date, no treatment exists for this disease. We evaluated the potential of recombinant adeno-associated virus (rAAV) vectors for gene therapy in a murine model for LGMD2A. To drive the expression of calpain 3, we used rAAV2/1 pseudotyped vectors and muscle-specific promoters to avoid calpain 3 cell toxicity. We report efficient and stable transgene expression in muscle with restoration of the proteolytic activity and without evident toxicity. In addition, calpain 3 was correctly targeted to the sarcomere. Moreover, its presence resulted in improvement of the histological features and in therapeutic efficacy at the physiological levels, including correction of atrophy and full rescue of the contractile force deficits. Our results establish the feasibility of AAV-mediated calpain 3 gene transfer as a therapeutic approach.