Systemic AAV8-Mediated Gene Therapy Drives Whole-Body Correction of Myotubular Myopathy in Dogs.

X-linked myotubular myopathy (XLMTM) results from MTM1 gene mutations and myotubularin deficiency. Most XLMTM patients develop severe muscle weakness leading to respiratory failure and death, typically within 2 years of age. Our objective was to evaluate the efficacy and safety of systemic gene therapy in the p.N155K canine model of XLMTM by performing a dose escalation study. A recombinant adeno-associated virus serotype 8 (rAAV8) vector expressing canine myotubularin (cMTM1) under the muscle-specific desmin promoter (rAAV8-cMTM1) was administered by simple peripheral venous infusion in XLMTM dogs at 10 weeks of age, when signs of the disease are already present. A comprehensive analysis of survival, limb strength, gait, respiratory function, neurological assessment, histology, vector biodistribution, transgene expression, and immune response was performed over a 9-month study period. Results indicate that systemic gene therapy was well tolerated, prolonged lifespan, and corrected the skeletal musculature throughout the body in a dose-dependent manner, defining an efficacious dose in this large-animal model of the disease. These results support the development of gene therapy clinical trials for XLMTM.

Long-term effects of systemic gene therapy in a canine model of myotubular myopathy.

INTRODUCTION: X-linked myotubular myopathy (XLMTM), a devastating pediatric disease caused by the absence of the protein myotubularin, results from mutations in the MTM1 gene. While there is no cure for XLMTM, we previously reported effects of MTM1 gene therapy using adeno-associated virus (AAV) vector on muscle weakness and pathology in MTM1-mutant dogs. Here, we followed 2 AAV-infused dogs over 4 years. METHODS: We evaluated gait, strength, respiration, neurological function, muscle pathology, AAV vector copy number (VCN), and transgene expression. RESULTS: Four years following AAV-mediated gene therapy, gait, respiratory performance, neurological function and pathology in AAV-infused XLMTM dogs remained comparable to their healthy littermate controls despite a decline in VCN and muscle strength. CONCLUSIONS: AAV-mediated gene transfer of MTM1 in young XLMTM dogs results in long-term expression of myotubularin transgene with normal muscular performance and neurological function in the absence of muscle pathology. These findings support a clinical trial in patients. Muscle Nerve 56: 943-953, 2017.

Gene therapy in monogenic congenital myopathies.

Current treatment options for patients with monogenetic congenital myopathies (MCM) ameliorate the symptoms of the disorder without resolving the underlying cause. However, gene therapies are being developed where the mutated or deficient gene target is replaced. Preclinical findings in animal models appear promising, as illustrated by gene replacement for X-linked myotubular myopathy (XLMTM) in canine and murine models. Prospective applications and approaches to gene replacement therapy, using these disorders as examples, are discussed in this review.

Respiratory assessment in centronuclear myopathies.

The centronuclear myopathies (CNMs) are a group of inherited neuromuscular disorders classified as congenital myopathies. While several causative genes have been identified, some patients do not harbor any of the currently known mutations. These diverse disorders have common histological features, which include a high proportion of centrally nucleated muscle fibers, and clinical attributes of muscle weakness and respiratory insufficiency. Respiratory problems in CNMs may manifest initially during sleep, but daytime symptoms, ineffective airway clearance, and hypoventilation predominate as more severe respiratory muscle dysfunction evolves. Respiratory muscle capacity can be evaluated using a variety of clinical tests selected with consideration for the age and baseline motor function of the patient. Similar clinical tests of respiratory function can also be incorporated into preclinical CNM canine models to offer insight for clinical trials. Because respiratory problems account for significant morbidity in patients, routine assessments of respiratory muscle function are discussed.

Muscle function in a canine model of X-linked myotubular myopathy.

INTRODUCTION: We established a colony of dogs that harbor an X-linked MTM1 missense mutation.Muscle from affected male dogs exhibits reduction and altered localization of the MTM1 gene product, myotubularin, and provides a model analogous to X-linked myotubular myopathy (XLMTM). METHODS: We studied hindlimb muscle function in age-matched canine XLMTM genotypes between ages 9 and 18 weeks. RESULTS: By the end of the study, affected dogs produce only ∼15% of the torque generated by normals or carriers (0.023 ± 0.005 vs. 0.152 ± 0.007 and 0.154 ± 0.003 N-m/kg body mass, respectively, P < 0.05) and are too weak to stand unassisted. At this age, XLMTM dogs also demonstrate an abnormally low twitch:tetanus ratio, a right-shifted torque-frequency relationship and an increase in torque during repetitive stimulation (P < 0.05). CONCLUSIONS: We hypothesize that muscle weakness results from impaired excitation-contraction (E-C) coupling. Interventions that improve E-C coupling might be translated from the XLMTM dog model to patients.

Duchenne muscular dystrophy trajectory in R-DMDdel52 preclinical rat model identifies COMP as biomarker of fibrosis.

Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disorder caused by mutations in the Dystrophin gene and for which there is currently no cure. To bridge the gap between preclinical and therapeutic evaluation studies, we have generated a rat model for DMD that carries an exon 52 deletion (R-DMDdel52) causing a complete lack of dystrophin protein. Here we show that R-DMDdel52 animals recapitulated human DMD pathophysiological trajectory more faithfully than the mdx mouse model. We report that R-DMDdel52 rats displayed progressive and severe skeletal muscle loss associated with fibrotic deposition, fat infiltration and fibre type switch. Early fibrosis was also apparent in the cardiac muscle. These histological modifications led to severe muscle, respiratory and cardiac functional impairments leading to premature death around 1 year. Moreover, DMD muscle exhibited systemic inflammation with a mixed M1/M2 phenotype. A comparative single cell RNAseq analysis of the diaphragm muscle was performed, revealing cellular populations alteration and molecular modifications in all muscle cell types. We show that DMD fibroadipogenic progenitors produced elevated levels of cartilage oligomeric matrix protein, a glycoprotein responsible for modulating homeostasis of extracellular matrix, and whose increased concentration correlated with muscle fibrosis both in R-DMDdel52 rats and human patients. Fibrosis is a component of tissue remodelling impacting the whole musculature of DMD patients, at the tissue level but most importantly at the functional level. We therefore propose that this specific biomarker can optimize the prognostic monitoring of functional improvement of patients included in clinical trials.

Validity of a Neurological Scoring System for Canine X-Linked Myotubular Myopathy.

A simple clinical neurological test was developed to evaluate response to gene therapy in a preclinical canine model of X-linked myotubular myopathy (XLMTM). This devastating congenital myopathy is caused by mutation in the myotubularin (MTM1) gene. Clinical signs include muscle weakness, early respiratory failure, and ventilator dependence. A spontaneously occurring canine model has a similar clinical picture and histological abnormalities on muscle biopsy compared with patients. We developed a neuromuscular assessment score, graded on a scale from 10 (normal) to 1 (unable to maintain sternal recumbency). We hypothesize that this neurological assessment score correlates with genotype and established measures of disease severity and is reliable when performed by an independent observer. At 17 weeks of age, there was strong correlation between neurological assessment scores and established methods of severity testing. The neurological severity score correctly differentiated between XLMTM and wild-type dogs with good interobserver reliability, on the basis of strong agreement between neurological scores assigned by independent observers. Together, these data indicate that the neurological scoring system developed for this canine congenital neuromuscular disorder is reliable and valid. This scoring system may be helpful in evaluating response to therapy in preclinical testing in this disease model, such as response to gene therapy.

Muscle pathology, limb strength, walking gait, respiratory function and neurological impairment establish disease progression in the p.N155K canine model of X-linked myotubular myopathy.

BACKGROUND: Loss-of-function mutations in the myotubularin (MTM1) gene cause X-linked myotubular myopathy (XLMTM), a fatal, inherited pediatric disease that affects the entire skeletal musculature. Labrador retriever dogs carrying an MTM1 missense mutation exhibit strongly reduced synthesis of myotubularin, the founder member of a lipid phosphatase required for normal skeletal muscle function. The resulting canine phenotype resembles that of human patients with comparably severe mutations, and survival does not normally exceed 4 months. METHODS: We studied MTM1 mutant dogs (n=7) and their age-matched control littermates (n=6) between the ages of 10 and 25 weeks. Investigators blinded to the animal identities sequentially measured limb muscle pathology, fore- and hind limb strength, walking gait, respiratory function and neurological impairment. RESULTS: MTM1-mutant puppies display centrally-nucleated myofibers of reduced size and disrupted sarcotubular architecture progressing until the end of life, an average of 17 weeks. In-life measures of fore- and hind limb strength establish the rate at which XLMTM muscles weaken, and their corresponding decrease in gait velocity and stride length. Pulmonary function tests in affected dogs reveal a right-shifted relationship between peak inspiratory flow (PIF) and inspiratory time (TI); neurological assessments indicate that affected puppies as young as 10 weeks show early signs of neurological impairment (neurological severity score, NSS =8.6±0.9) with progressive decline (NSS =5.6±1.7 at 17 weeks-of-age). CONCLUSIONS: Our findings document the rate of disease progression in a large animal model of XLMTM and lay a foundation for preclinical studies.

Gait characteristics in a canine model of X-linked myotubular myopathy.

X-linked myotubular myopathy (XLMTM) is a fatal pediatric disease where affected boys display profound weakness of the skeletal muscles. Possible therapies are under development but robust outcome measures in animal models are required for effective translation to human patients. We established a naturally-occurring canine model, where XLMTM dogs display clinical symptoms similar to those observed in humans. The aim of this study was to determine potential endpoints for the assessment of future treatments in this model. Video-based gait analysis was selected, as it is a well-established method of assessing limb function in neuromuscular disease and measures have been correlated to the patient's quality of life. XLMTM dogs (N = 3) and their true littermate wild type controls (N = 3) were assessed at 4-5 time points, beginning at 10 weeks and continuing through 17 weeks. Motion capture and an instrumented carpet were used separately to evaluate spatiotemporal and kinematic changes over time. XLMTM dogs walk more slowly and with shorter stride lengths than wild type dogs, and these differences became greater over time. However, there was no clear difference in angular measures between affected and unaffected dogs. These data demonstrate that spatiotemporal parameters capture functional changes in gait in an XLMTM canine model and support their utility in future therapeutic trials.

Gene therapy prolongs survival and restores function in murine and canine models of myotubular myopathy.

Loss-of-function mutations in the myotubularin gene (MTM1) cause X-linked myotubular myopathy (XLMTM), a fatal, congenital pediatric disease that affects the entire skeletal musculature. Systemic administration of a single dose of a recombinant serotype 8 adeno-associated virus (AAV8) vector expressing murine myotubularin to Mtm1-deficient knockout mice at the onset or at late stages of the disease resulted in robust improvement in motor activity and contractile force, corrected muscle pathology, and prolonged survival throughout a 6-month study. Similarly, single-dose intravascular delivery of a canine AAV8-MTM1 vector in XLMTM dogs markedly improved severe muscle weakness and respiratory impairment, and prolonged life span to more than 1 year in the absence of toxicity or a humoral or cell-mediated immune response. These results demonstrate the therapeutic efficacy of AAV-mediated gene therapy for myotubular myopathy in small- and large-animal models, and provide proof of concept for future clinical trials in XLMTM patients.

Establishing clinical end points of respiratory function in large animals for clinical translation.

Respiratory dysfunction due progressive weakness of the respiratory muscles, particularly the diaphragm, is a major cause of death in the neuromuscular disease (NMD) X-linked myotubular myopathy (XLMTM). Methods of respiratory assessment in patients are often difficult, especially in those who are mechanically ventilated. The naturally occuring XLMTM dog model exhibits a phenotype similar to that in patients and can be used to determine quantitative descriptions of dysfunction as clinical endpoints for treatment and the development of new therapies. In experiments using respiratory impedance plethysmography (RIP), XLMTM dogs challenged with the respiratory stimulant doxapram displayed significant changes indicative of diaphragmatic weakness.