X-linked Myotubular Myopathy Dogs Research Articles

RAAV-related therapy fully rescues myonuclear and myofilament function in X-linked myotubular myopathy

X-linked myotubular myopathy (XLMTM) is a life-threatening skeletal muscle disease caused by mutations in the MTM1 gene. XLMTM fibres display a population of nuclei mispositioned in the centre. In the present study, we aimed to explore whether positioning and overall distribution of nuclei affects cellular organization and contractile function, thereby contributing to muscle weakness in this disease. We also assessed whether gene therapy alters nuclear arrangement and function. We used tissue from human patients and animal models, including XLMTM dogs that had received increasing doses of recombinant AAV8 vector restoring MTM1 expression (rAAV8-cMTM1). We then used single isolated muscle fibres to analyze nuclear organization and contractile function. In addition to the expected mislocalization of nuclei in the centre of muscle fibres, a novel form of nuclear mispositioning was observed: irregular spacing between those located at the fibre periphery, and an overall increased number of nuclei, leading to dramatically smaller and inconsistent myonuclear domains. Nuclear mislocalization was associated with decreases in global nuclear synthetic activity, contractile protein content and intrinsic myofilament force production. A contractile deficit originating at the myofilaments, rather than mechanical interference by centrally positioned nuclei, was supported by experiments in regenerated mouse muscle. Systemic administration of rAAV8-cMTM1 at doses higher than 2.5 × 1013 vg kg−1 allowed a full rescue of all these cellular defects in XLMTM dogs. Altogether, these findings identify previously unrecognized pathological mechanisms in human and animal XLMTM, associated with myonuclear defects and contractile filament function. These defects can be reversed by gene therapy restoring MTM1 expression in dogs with XLMTM.

P.252 - High-throughput transcriptome analysis provides new indicators of gene therapy efficacy in XLMTM dogs

Despite years of intensive efforts, inherited muscle disorders still represent a significant challenge for the gene therapy community. In large animal models, high systemic doses of recombinant adeno-associated virus (rAAV) are required to achieve phenotype correction, which can be partial and not sustained over time. Possible causes include the difficulty of achieving robust transduction of all muscle tissues, the disease and/or immune-mediated elimination of transduced cells, and the challenges associated with restoring the expression of large structural proteins. A notable exception is X-linked myotubular myopathy (XLMTM), in which the absence of myotubularin, a relatively small lipid phosphatase, leads to multiple defects in muscle cells, profound hypotonia and reduced lifespan. In this context, rAAV vectors have shown dramatic efficacy in XLMTM dogs, with a complete, long-term, and dose-dependent rescue of the phenotype. This provides us with a powerful model to study the molecular events associated not only with disease development, but also with its reversal after gene transfer. In this study, we use RNA-Seq to explore the transcriptome of canine XLMTM muscles after systemic gene therapy with increasing doses of rAAV vector. In line with cellular hallmarks of XLMTM, untreated dogs show marked dysregulations of genes involved in muscle development, myofibril structure and excitation-contraction coupling. Essentially, our analysis indicates that as vector dose increases, the transcriptome of treated dogs becomes more similar to those of healthy controls and less like untreated XLMTM counterparts. More precisely, we have identified subsets of transcripts whose expression is rescued by, or resistant to, each gene therapy regimen, and studied their evolution with increasing rAAV doses. In conclusion, our study describes an innovative approach for analyzing rAAV-treated muscles, and provides unprecedented indicators of phenotype correction in the context of XLMTM.

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

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. We evaluated gait, strength, respiration, neurological function, muscle pathology, AAV vector copy number (VCN), and transgene expression. 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. 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.

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 therapyin 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 10weeks of age, when signsof 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.

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.

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 to 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.

G.P.41: Canine X-linked myotubular myopathy: A dose-finding study of systemic AAV8-MTM1 effects on muscle strength, gait, respiration, neuromuscular function and survival

Mutations in the myotubularin gene (MTM1) result in X-linked myotubular myopathy (XLMTM), a fatal pediatric disease of skeletal muscle characterized by small centrally nucleated myofibers containing abnormal mitochondrial accumulations. Patients typically present with severe hypotonia and respiratory failure. Previous local studies in Mtm1-mutant mice demonstrated potential efficacy of gene therapy to treat the disease. We recently reported that administration of adeno-associated virus serotype 8 (AAV8) vector expressing myotubularin under the muscle-specific desmin promoter, delivered systemically in myotubularin deficient mice and by regional hind limb perfusion in XLMTM dogs, could prolong life and restore muscle function. Here we report initial results of a larger dose escalation study testing the efficacy of AAV8-MTM1 systemic venous infusion in XLMTM dogs. Four groups were studied in a blinded manner: Wild-type or female carrier controls (N = 6); affected dogs given saline only (N = 4); affected dogs given AAV8-MTM1 (5E12 vg/kg) (N = 3) or (2.5E13 vg/kg) (N = 3). AAV8-MTM1 was administered at 10 weeks of age and dogs were followed for 9 months. Efficacy readouts included limb strength, gait, respiratory and neurological assessments at 13, 15, and 17 weeks-of-age, then monthly to 40 weeks-of-age. Results indicate a dose-dependent response to AAV8-MTM1. At the highest AAV dose, we observed marked improvements to near-normal levels in all physiological and clinical parameters tested along with improved survival, whereas the lowest dose provided only modest improvements. Together, these data indicate a dose-response for AAV8-MTM1 on muscle strength, walking speed, neuromuscular function and survival in dogs with myotubularin deficiency.

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 10weeks and continuing through 17weeks. 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.

P.4.3 Intravenous infusion of AAV8–MTM1 prolongs life and ameliorates severe muscle pathology in mouse and dog models of X-linked myotubular myopathy

Mutations in the myotubularin gene (MTM1) result in X-linked myotubular myopathy (XLMTM), a fatal pediatric disease of skeletal muscle characterized by small myofibers with frequent central nuclei and abnormal mitochondrial accumulations. Patients with XLMTM typically present with severe hypotonia, muscle weakness and respiratory failure. Previous local studies in Mtm1-mutant mice demonstrated potential efficacy of gene therapy to treat the disease. Here we report long-term survival data in mice and dogs following intravenous delivery of an adeno-associated virus serotype 8 (AAV8) vector expressing myotubularin under the muscle-specific desmin promoter. XLMTM dogs (<i>n</i>=3) were treated at 8weeks of age with full-length canine MTM1 cDNA using an AAV8 vector. Affected XLMTM dogs were infused with AAV8–MTM1 (2.5×10 13 vg/kg), IV under high pressure into the saphenous vein while a tourniquet was applied around the upper thigh. While AAV8–MTM1 was administered by hindlimb infusion, effects were observed systemically in dogs. Mice were given intravenous AAV8–MTM1 by tail vein injection. In both murine and canine models, myotubularin transgene was expressed in all limb muscles and this translated into marked improvement of contractile force in mice and dogs. All mutant mice, including those injected at a late stage of the disease, survived until the end of the 6-month study. The lifespan of MTM1 mutant dogs that normally survive to 4months was prolonged beyond 1year in the first of three treated animals. Two additional treated dogs remain robust and continue to thrive beyond 8months-of-age. Our findings in these two species provide proof-of-principle that intravenous AAV-mediated myotubularin replacement can rescue the severe phenotype of both small and large animal models of myotubular myopathy.

Muscle function in A canine model of X‐linked myotubular myopathy

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). We studied hindlimb muscle function in age-matched canine XLMTM genotypes between ages 9 and 18 weeks. 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). 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.