Erin Mitchell

Gene Therapy Prolongs Survival and Restores Function in Murine and Canine Models of Myotubular Myopathy

Intravenous injection of an adeno-associated viral vector expressing the myotubularin (MTM1) gene improves survival and rescues skeletal muscle function in mice and dogs affected by myotubular myopathy. Restoring Skeletal Muscle Function X-linked myotubular myopathy is a fatal disease of skeletal muscle that affects about 1 in 50,000 male births. Patients harbor mutations in the MTM1 gene and are typically born floppy, with severely weak limb and respiratory muscles. Survival requires intensive support, often including tube feeding and mechanical ventilation, but effective therapy is not available for patients. Gene replacement therapy using adeno-associated viral (AAV) vectors has potential for the treatment of inherited diseases like myotubular myopathy. Therefore, Childers et al. tested the effects of a recombinant AAV vector expressing myotubularin in two animal models of myotubularin deficiency: Mtm1 knockout mice and dogs carrying a naturally occurring MTM1 gene mutation. Results in both mice and dogs showed that a single intravascular injection of AAV strengthened severely weak muscles, corrected muscle pathology, and prolonged survival. No toxicity or immune response was observed in dogs. These results demonstrate the efficacy of gene replacement therapy for myotubular myopathy in animal models and pave the way to a clinical trial in patients. 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.

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. Muscle Nerve 46: 588–591, 2012

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.

Ultrasound assessment of the diaphragm: Preliminary study of a canine model of X-linked myotubular myopathy

Introduction: We tested the feasibility of using neuromuscular ultrasound for non‐invasive real‐time assessment of diaphragmatic structure and function in a canine model of X‐linked myotubular myopathy (XLMTM). Methods: Ultrasound images in 3 dogs [wild‐type (WT), n = 1; XLMTM untreated, n = 1; XLMTM post–AAV8‐mediated MTM1 gene replacement, n = 1] were analyzed for diaphragm thickness, change in thickness with respiration, muscle echogenicity, and diaphragm excursion amplitude during spontaneous breathing. Results: Quantitative parameters of diaphragm structure were different among the animals. WT diaphragm was thicker and less echogenic than the XLMTM control, whereas the diaphragm measurements of the MTM1‐treated XLMTM dog were comparable to those of the WT dog. Conclusions: This pilot study demonstrates the feasibility of using ultrasound for quantitative assessment of the diaphragm in a canine model. In the future, ultrasonography may replace invasive measures of diaphragm function in canine models and in humans for non‐invasive respiratory monitoring and evaluation of neuromuscular disease. Muscle Nerve 50: 607–609, 2014

GUIDING INTRAMUSCULAR DIAPHRAGM INJECTIONS USING REAL-TIME ULTRASOUND AND ELECTROMYOGRAPHY

Introduction: We describe a unique method that combines ultrasound and electromyography to guide intramuscular diaphragm injections in anesthetized large animals. Methods: Ultrasound was used to visualize the diaphragm on each side of spontaneously breathing, anesthetized beagle dogs and cynomolgus macaques. An electromyography (EMG) needle was introduced and directed by ultrasound to confirm that the needle entered the muscular portion of the diaphragm, and methylene blue was injected. Injection accuracy was confirmed upon necropsy by tracking the spread of methylene blue. Results: All methylene blue injections were confirmed to have been placed appropriately into the diaphragm. Conclusions: This study demonstrates the feasibility and accuracy of using ultrasound and EMG to guide injections and to reduce complications associated with conventional blind techniques. Ultrasound guidance can be used for clinical EMG of the diaphragm. Future applications may include targeted diaphragm injections with gene replacement therapy in neuromuscular diseases. Muscle Nerve 51: 287–289, 2015

Ultrasound assessment of the diaphragm

Introduction: We tested the feasibility of using neuromuscular ultrasound for non‐invasive real‐time assessment of diaphragmatic structure and function in a canine model of X‐linked myotubular myopathy (XLMTM). Methods: Ultrasound images in 3 dogs [wild‐type (WT), n = 1; XLMTM untreated, n = 1; XLMTM post–AAV8‐mediated MTM1 gene replacement, n = 1] were analyzed for diaphragm thickness, change in thickness with respiration, muscle echogenicity, and diaphragm excursion amplitude during spontaneous breathing. Results: Quantitative parameters of diaphragm structure were different among the animals. WT diaphragm was thicker and less echogenic than the XLMTM control, whereas the diaphragm measurements of the MTM1‐treated XLMTM dog were comparable to those of the WT dog. Conclusions: This pilot study demonstrates the feasibility of using ultrasound for quantitative assessment of the diaphragm in a canine model. In the future, ultrasonography may replace invasive measures of diaphragm function in canine models and in humans for non‐invasive respiratory monitoring and evaluation of neuromuscular disease. Muscle Nerve 50: 607–609, 2014

Ultrasound assessment of the diaphragm: Preliminary study of a canine model of X-linked myotubular myopathy: Short Reports

Introduction: We tested the feasibility of using neuromuscular ultrasound for non‐invasive real‐time assessment of diaphragmatic structure and function in a canine model of X‐linked myotubular myopathy (XLMTM). Methods: Ultrasound images in 3 dogs [wild‐type (WT), n = 1; XLMTM untreated, n = 1; XLMTM post–AAV8‐mediated MTM1 gene replacement, n = 1] were analyzed for diaphragm thickness, change in thickness with respiration, muscle echogenicity, and diaphragm excursion amplitude during spontaneous breathing. Results: Quantitative parameters of diaphragm structure were different among the animals. WT diaphragm was thicker and less echogenic than the XLMTM control, whereas the diaphragm measurements of the MTM1‐treated XLMTM dog were comparable to those of the WT dog. Conclusions: This pilot study demonstrates the feasibility of using ultrasound for quantitative assessment of the diaphragm in a canine model. In the future, ultrasonography may replace invasive measures of diaphragm function in canine models and in humans for non‐invasive respiratory monitoring and evaluation of neuromuscular disease. Muscle Nerve 50: 607–609, 2014

A novel antireflux procedure: gastroplasty with restricted antrum to control emesis (GRACE)

PURPOSE Nissen fundoplication is the most commonly performed operation to treat gastroesophageal reflux disease and vomiting in children with neurologic impairment. However, failure rates of Nissen fundoplication in this population are higher, and alternatives to Nissen fundoplication have technical and functional disadvantages. We hypothesize that the novel gastroplasty with restricted antrum to control emesis (GRACE) would be more effective than Nissen fundoplication at reducing emetic reflux. METHODS To compare the GRACE with Nissen fundoplication, 15 canine subjects were randomized to Nissen fundoplication or GRACE. All subjects underwent gastrostomy tube placement. Baseline gastric emptying, electrogastrography, and induced vomiting studies were performed. Nissen fundoplication or GRACE was then performed. Postoperatively, gastric emptying and vomiting studies were repeated. RESULTS Gastric emptying before and after antireflux procedures was not significantly different between groups. Both Nissen fundoplication (38%, P = .04) and GRACE (69%, P < .01) procedures prevented reflux compared with baseline. However, the GRACE procedure significantly reduced reflux when compared with Nissen fundoplication (P = .03). CONCLUSIONS In this canine model, GRACE appears to be significantly more effective than Nissen fundoplication at reducing emetic reflux. This novel procedure preserves gastric function and is well tolerated. The GRACE procedure may provide an alternative to Nissen fundoplication as a primary or repeat antireflux procedure for children with neurologic impairment.