Characterization of NEB pathogenic variants in patients reveals novel nemaline myopathy disease mechanisms and omecamtiv mecarbil force effects.

Nebulin, a critical protein of the skeletal muscle thin filament, plays important roles in physiological processes such as regulating thin filament length (TFL), cross-bridge cycling, and myofibril alignment. Pathogenic variants in the nebulin gene (NEB) cause NEB-based nemaline myopathy (NEM2), a genetically heterogeneous disorder characterized by hypotonia and muscle weakness, currently lacking curative therapies. In this study, we examined a cohort of ten NEM2 patients, each with unique pathogenic variants, aiming to understand their impact on mRNA, protein, and functional levels. Results show that pathogenic truncation variants affect NEB mRNA stability and lead to nonsense-mediated decay of the mutated transcript. Moreover, a high incidence of cryptic splice site activation was found in patients with pathogenic splicing variants that are expected to disrupt the actin-binding sites of nebulin. Determination of protein levels revealed patients with either relatively normal or markedly reduced nebulin. We observed a positive relation between the reduction in nebulin and a reduction in TFL, or reduction in tension (both maximal and submaximal tension). Interestingly, our study revealed a pathogenic duplication variant in nebulin that resulted in a four-copy gain in the triplicate region of NEB and a much larger nebulin protein and longer TFL. Additionally, we investigated the effect of Omecamtiv mecarbil (OM), a small-molecule activator of cardiac myosin, on force production of type 1 muscle fibers of NEM2 patients. OM treatment substantially increased submaximal tension across all NEM2 patients ranging from 87 to 318%, with the largest effects in patients with the lowest level of nebulin. In summary, this study indicates that post-transcriptional or post-translational mechanisms regulate nebulin expression. Moreover, we propose that the pathomechanism of NEM2 involves not only shortened but also elongated thin filaments, along with the disruption of actin-binding sites resulting from pathogenic splicing variants. Significantly, our findings highlight the potential of OM treatment to improve skeletal muscle function in NEM2 patients, especially those with large reductions in nebulin levels.

AAV8 gene therapy reverses cardiac pathology and prevents early mortality in a mouse model of Friedreich's ataxia.

Friedreich's ataxia (FRDA) is an autosomal-recessive disorder primarily attributed to biallelic GAA repeat expansions that reduce expression of the mitochondrial protein frataxin (FXN). FRDA is characterized by progressive neurodegeneration, with many patients developing cardiomyopathy that progresses to heart failure and death. The potential to reverse or prevent progression of the cardiac phenotype of FRDA was investigated in a mouse model of FRDA, using an adeno-associated viral vector (AAV8) containing the coding sequence of the FXN gene. The Fxnflox/null::MCK-Cre conditional knockout mouse (FXN-MCK) has an FXN gene ablation that prevents FXN expression in cardiac and skeletal muscle, leading to cardiac insufficiency, weight loss, and morbidity. FXN-MCK mice received a single intravenous injection of an AAV8 vector containing human (hFXN) or mouse (mFXN) FXN genes under the control of a phosphoglycerate kinase promoter. Compared to vehicle-treated FXN-MCK control mice, AAV-treated FXN-MCK mice displayed increases in body weight, reversal of cardiac deficits, and increases in survival without apparent toxicity in the heart or liver for up to 12 weeks postdose. FXN protein expression in heart tissue was detected in a dose-dependent manner, exhibiting wide distribution throughout the heart similar to wild type, but more speckled. These results support an AAV8-based approach to treat FRDA-associated cardiomyopathy.

Effects of gene replacement therapy with resamirigene bilparvovec (AT132) on skeletal muscle pathology in X-linked myotubular myopathy: results from a substudy of the ASPIRO open-label clinical trial.

BACKGROUND: X-linked myotubular myopathy (XLMTM) is a rare, life-threatening congenital muscle disease caused by mutations in the MTM1 gene that result in profound muscle weakness, significant respiratory insufficiency, and high infant mortality. There is no approved disease-modifying therapy for XLMTM. Resamirigene bilparvovec (AT132; rAAV8-Des-hMTM1) is an investigational adeno-associated virus (AAV8)-mediated gene replacement therapy designed to deliver MTM1 to skeletal muscle cells and achieve long-term correction of XLMTM-related muscle pathology. The clinical trial ASPIRO (NCT03199469) investigating resamirigene bilparvovec in XLMTM is currently paused while the risk:benefit balance associated with this gene therapy is further investigated. METHODS: Muscle biopsies were taken before treatment and 24 and 48 weeks after treatment from ten boys with XLMTM in a clinical trial of resamirigene bilparvovec (ASPIRO; NCT03199469). Comprehensive histopathological analysis was performed. FINDINGS: Baseline biopsies uniformly showed findings characteristic of XLMTM, including small myofibres, increased internal or central nucleation, and central aggregates of organelles. Biopsies taken at 24 weeks post-treatment showed marked improvement of organelle localisation, without apparent increases in myofibre size in most participants. Biopsies taken at 48 weeks, however, did show statistically significant increases in myofibre size in all nine biopsies evaluated at this timepoint. Histopathological endpoints that did not demonstrate statistically significant changes with treatment included the degree of internal/central nucleation, numbers of triad structures, fibre type distributions, and numbers of satellite cells. Limited (predominantly mild) treatment-associated inflammatory changes were seen in biopsy specimens from five participants. INTERPRETATION: Muscle biopsies from individuals with XLMTM treated with resamirigene bilparvovec display statistically significant improvement in organelle localisation and myofibre size during a period of substantial improvements in muscle strength and respiratory function. This study identifies valuable histological endpoints for tracking treatment-related gains with resamirigene bilparvovec, as well as endpoints that did not show strong correlation with clinical improvement in this human study. FUNDING: Astellas Gene Therapies (formerly Audentes Therapeutics, Inc.).

Safety and efficacy of gene replacement therapy for X-linked myotubular myopathy (ASPIRO): a multinational, open-label, dose-escalation trial.

BACKGROUND: X-linked myotubular myopathy is a rare, life-threatening, congenital muscle disease observed mostly in males, which is caused by mutations in MTM1. No therapies are approved for this disease. We aimed to assess the safety and efficacy of resamirigene bilparvovec, which is an adeno-associated viral vector serotype 8 delivering human MTM1. METHODS: ASPIRO is an open-label, dose-escalation trial at seven academic medical centres in Canada, France, Germany, and the USA. We included boys younger than 5 years with X-linked myotubular myopathy who required mechanical ventilator support. The trial was initially in two parts. Part 1 was planned as a safety and dose-escalation phase in which participants were randomly allocated (2:1) to either the first dose level (1·3 × 1014 vector genomes [vg]/kg bodyweight) of resamirigene bilparvovec or delayed treatment, then, for later participants, to either a higher dose (3·5 × 1014 vg/kg bodyweight) of resamirigene bilparvovec or delayed treatment. Part 2 was intended to confirm the dose selected in part 1. Resamirigene bilparvovec was administered as a single intravenous infusion. An untreated control group comprised boys who participated in a run-in study (INCEPTUS; NCT02704273) or those in the delayed treatment cohort who did not receive any dose. The primary efficacy outcome was the change from baseline to week 24 in hours of daily ventilator support. After three unexpected deaths, dosing at the higher dose was stopped and the two-part feature of the study design was eliminated. Because of changes to the study design during its implementation, analyses were done on an as-treated basis and are deemed exploratory. All treated and control participants were included in the safety analysis. The trial is registered with ClinicalTrials.gov, NCT03199469. Outcomes are reported as of Feb 28, 2022. ASPIRO is currently paused while deaths in dosed participants are investigated. FINDINGS: Between Aug 3, 2017 and June 1, 2021, 30 participants were screened for eligibility, of whom 26 were enrolled; six were allocated to the lower dose, 13 to the higher dose, and seven to delayed treatment. Of the seven children whose treatment was delayed, four later received the higher dose (n=17 total in the higher dose cohort), one received the lower dose (n=7 total in the lower dose cohort), and two received no dose and joined the control group (n=14 total, including 12 children from INCEPTUS). Median age at dosing or enrolment was 12·1 months (IQR 10·0-30·9; range 9·5-49·7) in the lower dose cohort, 31·1 months (16·0-64·7; 6·8-72·7) in the higher dose cohort, and 18·7 months (10·1-31·5; 5·9-39·3) in the control cohort. Median follow-up was 46·1 months (IQR 41·0-49·5; range 2·1-54·7) for lower dose participants, 27·6 months (24·6-29·1; 3·4-41·0) for higher dose participants, and 28·3 months (9·7-46·9; 5·7-32·7) for control participants. At week 24, lower dose participants had an estimated 77·7 percentage point (95% CI 40·22 to 115·24) greater reduction in least squares mean hours per day of ventilator support from baseline versus controls (p=0·0002), and higher dose participants had a 22·8 percentage point (6·15 to 39·37) greater reduction from baseline versus controls (p=0·0077). One participant in the lower dose cohort and three in the higher dose cohort died; at the time of death, all children had cholestatic liver failure following gene therapy (immediate causes of death were sepsis; hepatopathy, severe immune dysfunction, and pseudomonal sepsis; gastrointestinal haemorrhage; and septic shock). Three individuals in the control group died (haemorrhage presumed related to hepatic peliosis; aspiration pneumonia; and cardiopulmonary failure). INTERPRETATION: Most children with X-linked myotubular myopathy who received MTM1 gene replacement therapy had important improvements in ventilator dependence and motor function, with more than half of dosed participants achieving ventilator independence and some attaining the ability to walk independently. Investigations into the risk for underlying hepatobiliary disease in X-linked myotubular myopathy, and the need for monitoring of liver function before gene replacement therapy, are ongoing. FUNDING: Astellas Gene Therapies.

Loss of Mtm1 causes cholestatic liver disease in a model of X-linked myotubular myopathy.

X-linked myotubular myopathy (XLMTM) is a fatal congenital disorder caused by mutations in the MTM1 gene. Currently, there are no approved treatments, although AAV8-mediated gene transfer therapy has shown promise in animal models and preliminarily in patients. However, 4 patients with XLMTM treated with gene therapy have died from progressive liver failure, and hepatobiliary disease has now been recognized more broadly in association with XLMTM. In an attempt to understand whether loss of MTM1 itself is associated with liver pathology, we have characterized what we believe to be a novel liver phenotype in a zebrafish model of this disease. Specifically, we found that loss-of-function mutations in mtm1 led to severe liver abnormalities including impaired bile flux, structural abnormalities of the bile canaliculus, and improper endosome-mediated trafficking of canalicular transporters. Using a reporter-tagged Mtm1 zebrafish line, we established localization of Mtm1 in the liver in association with Rab11, a marker of recycling endosomes, and canalicular transport proteins and demonstrated that hepatocyte-specific reexpression of Mtm1 could rescue the cholestatic phenotype. Last, we completed a targeted chemical screen and found that Dynasore, a dynamin-2 inhibitor, was able to partially restore bile flow and transporter localization to the canalicular membrane. In summary, we demonstrate, for the first time to our knowledge, liver abnormalities that were directly caused by MTM1 mutation in a preclinical model, thus establishing the critical framework for better understanding and comprehensive treatment of the human disease.

Aberrations in Energetic Metabolism and Stress-Related Pathways Contribute to Pathophysiology in the Neb Conditional Knockout Mouse Model of Nemaline Myopathy.

Nemaline myopathy (NM) is a genetically and clinically heterogeneous disease that is diagnosed on the basis of the presence of nemaline rods on skeletal muscle biopsy. Although NM has typically been classified by causative genes, disease severity or prognosis cannot be predicted. The common pathologic end point of nemaline rods (despite diverse genetic causes) and an unexplained range of muscle weakness suggest that shared secondary processes contribute to the pathogenesis of NM. We speculated that these processes could be identified through a proteome-wide interrogation using a mouse model of severe NM in combination with pathway validation and structural/functional analyses. A proteomic analysis was performed using skeletal muscle tissue from the Neb conditional knockout mouse model compared with its wild-type counterpart to identify pathophysiologically relevant biological processes that might impact disease severity or provide new treatment targets. A differential expression analysis and Ingenuity Pathway Core Analysis predicted perturbations in several cellular processes, including mitochondrial dysfunction and changes in energetic metabolism and stress-related pathways. Subsequent structural and functional studies demonstrated abnormal mitochondrial distribution, decreased mitochondrial respiratory function, an increase in mitochondrial transmembrane potential, and extremely low ATP content in Neb conditional knockout muscles relative to wild type. Overall, the findings of these studies support a role for severe mitochondrial dysfunction as a novel contributor to muscle weakness in NM.

Different Mouse Models of Nemaline Myopathy Harboring Acta1 Mutations Display Differing Abnormalities Related to Mitochondrial Biology.

ACTA1 encodes skeletal muscle-specific α-actin, which polymerizes to form the thin filament of the sarcomere. Mutations in ACTA1 are responsible for approximately 30% of nemaline myopathy (NM) cases. Previous studies of weakness in NM have focused on muscle structure and contractility, but genetic issues alone do not explain the phenotypic heterogeneity observed in patients with NM or NM mouse models. To identify additional biological processes related to NM phenotypic severity, proteomic analysis was performed using muscle protein isolates from wild-type mice in comparison to moderately affected knock-in (KI) Acta1H40Y and the minimally affected transgenic (Tg) ACTA1D286G NM mice. This analysis revealed abnormalities in mitochondrial function and stress-related pathways in both mouse models, supporting an in-depth assessment of mitochondrial biology. Interestingly, evaluating each model in comparison to its wild-type counterpart identified different degrees of mitochondrial abnormality that correlated well with the phenotypic severity of the mouse model. Muscle histology, mitochondrial respiration, electron transport chain function, and mitochondrial transmembrane potential were all normal or minimally affected in the TgACTA1D286G mouse model. In contrast, the more severely affected KI.Acta1H40Y mice displayed significant abnormalities in relation to muscle histology, mitochondrial respirometry, ATP, ADP, and phosphate content, and mitochondrial transmembrane potential. These findings suggest that abnormal energy metabolism is related to symptomatic severity in NM and may constitute a contributor to phenotypic variability and a novel treatment target.

Spinal muscular atrophy-like phenotype in a mouse model of acid ceramidase deficiency.

Mutations in ASAH1 have been linked to two allegedly distinct disorders: Farber disease (FD) and spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME). We have previously reported FD-like phenotypes in mice harboring a single amino acid substitution in acid ceramidase (ACDase), P361R, known to be pathogenic in humans (P361R-Farber). Here we describe a mouse model with an SMA-PME-like phenotype (P361R-SMA). P361R-SMA mice live 2-3-times longer than P361R-Farber mice and have different phenotypes including progressive ataxia and bladder dysfunction, which suggests neurological dysfunction. We found profound demyelination, loss of axons, and altered sphingolipid levels in P361R-SMA spinal cords; severe pathology was restricted to the white matter. Our model can serve as a tool to study the pathological effects of ACDase deficiency on the central nervous system and to evaluate potential therapies for SMA-PME.

Effects of Regulatory T Cell Depletion in BALB/c Mice Infected with Low Doses of Borrelia burgdorferi.

We previously demonstrated that a depletion of regulatory T (Treg) cells in Lyme arthritis-resistant C57BL/6 mice leads to pathological changes in the tibiotarsal joints following infection with Borrelia burgdorferi. Here, we assessed the effects of Treg cells on the response to B. burgdorferi infection in BALB/c mice, which exhibit infection-dose-dependent disease and a different sequence of immune events than C57BL/6 mice. The depletion of Treg cells prior to infection with 1 × 102, but not 5 × 103, organisms led to increased swelling of the tibiotarsal joints. However, Treg cell depletion did not significantly affect the development of histopathology at these low doses of infection. BALB/c mice depleted of Treg cells before infection with 1 × 103 spirochetes harbored a higher borrelial load in the hearts and exhibited higher levels of serum interleukin-10 five weeks later. These results indicate that Treg cells regulate certain aspects of the response to B. burgdorferi in a mouse strain that may display a range of disease severities. As the presentation of Lyme disease may vary among humans, it is necessary to consider multiple animal models to obtain a complete picture of the various means by which Treg cells affect the host response to B. burgdorferi.

ACTA1 H40Y mutant iPSC-derived skeletal myocytes display mitochondrial defects in an in vitro model of nemaline myopathy.

Nemaline myopathies (NM) are a group of congenital myopathies that lead to muscle weakness and dysfunction. While 13 genes have been identified to cause NM, over 50% of these genetic defects are due to mutations in nebulin (NEB) and skeletal muscle actin (ACTA1), which are genes required for normal assembly and function of the thin filament. NM can be distinguished on muscle biopsies due to the presence of nemaline rods, which are thought to be aggregates of the dysfunctional protein. Mutations in ACTA1 have been associated with more severe clinical disease and muscle weakness. However, the cellular pathogenesis linking ACTA1 gene mutations to muscle weakness are unclear To evaluate cellular disease phenotypes, iPSC-derived skeletal myocytes (iSkM) harboring an ACTA1 H40Y point mutation were used to model NM in skeletal muscle. These were generated by Crispr-Cas9, and include one non-affected healthy control (C) and 2 NM iPSC clone lines, therefore representing isogenic controls. Fully differentiated iSkM were characterized to confirm myogenic status and subject to assays to evaluate nemaline rod formation, mitochondrial membrane potential, mitochondrial permeability transition pore (mPTP) formation, superoxide production, ATP/ADP/phosphate levels and lactate dehydrogenase release. C- and NM-iSkM demonstrated myogenic commitment as evidenced by mRNA expression of Pax3, Pax7, MyoD, Myf5 and Myogenin; and protein expression of Pax4, Pax7, MyoD and MF20. No nemaline rods were observed with immunofluorescent staining of NM-iSkM for ACTA1 or ACTN2, and these mRNA transcript and protein levels were comparable to C-iSkM. Mitochondrial function was altered in NM, as evidenced by decreased cellular ATP levels and altered mitochondrial membrane potential. Oxidative stress induction revealed the mitochondrial phenotype, as evidenced by collapsed mitochondrial membrane potential, early formation of the mPTP and increased superoxide production. Early mPTP formation was rescued with the addition of ATP to media. Together, these findings suggest that mitochondrial dysfunction and oxidative stress are disease phenotypes in the in vitro model of ACTA1 nemaline myopathy, and that modulation of ATP levels was sufficient to protect NM-iSkM mitochondria from stress-induced injury. Importantly, the nemaline rod phenotype was absent in our in vitro model of NM. We conclude that this in vitro model has the potential to recapitulate human NM disease phenotypes, and warrants further study.

Biodistribution of Adeno-Associated Virus Gene Therapy Following Cerebrospinal Fluid-Directed Administration.

Adeno-associated virus (AAV)-based gene therapies, exemplified by the approved therapy for spinal muscular atrophy, have the potential to deliver disease-course-altering treatments for central nervous system (CNS) indications. However, several clinical trials have reported severe adverse events, including patient deaths following high-dose systemic administration for muscle-directed gene transfer, highlighting the need to explore approaches utilizing lower doses when targeting the CNS. Animal models of disease provide insight into the response to new AAV therapies. However, translation from small to larger animals and eventually to humans is hampered by anatomical and biological differences across the species and their impact on AAV delivery. We performed a literature review of preclinical studies of AAV gene therapy biodistribution following cerebrospinal fluid (CSF) delivery (intracerebroventricular, intra-cisterna magna, and intrathecal lumbar). The reviewed literature varies greatly in the reported biodistribution of AAV following administration into the CSF. Differences between studies, including animal model, vector serotype used, method used to assess biodistribution, and route of administration, among other variables, contribute to differing outcomes and difficulties in translating these preclinical results. For example, only half of the published AAV-based gene therapy studies report vector copy number, the most direct readout following administration of a vector; none of these studies reported details such as the empty:full capsid ratio and quality of encapsidated genome. Analysis of the last decade's literature focusing on AAV-based gene therapies targeting the CNS underscores limitations of the body of knowledge and room for continued research. In particular, there is a need to understand the biodistribution achieved by different CSF-directed routes of administration and determining if specific cell types/structures of interest will be transduced. Our findings point to a clear need for a more systematic approach across the field to align the assessments and elements reported in preclinical research to enable more reliable translation across animal models and into human studies.

Assessment of systemic AAV-microdystrophin gene therapy in the GRMD model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by the absence of dystrophin, a membrane-stabilizing protein encoded by the DMD gene. Although mouse models of DMD provide insight into the potential of a corrective therapy, data from genetically homologous large animals, such as the dystrophin-deficient golden retriever muscular dystrophy (GRMD) model, may more readily translate to humans. To evaluate the clinical translatability of an adeno-associated virus serotype 9 vector (AAV9)-microdystrophin (µDys5) construct, we performed a blinded, placebo-controlled study in which 12 GRMD dogs were divided among four dose groups [control, 1 × 1013 vector genomes per kilogram (vg/kg), 1 × 1014 vg/kg, and 2 × 1014 vg/kg; n = 3 each], treated intravenously at 3 months of age with a canine codon-optimized microdystrophin construct, rAAV9-CK8e-c-µDys5, and followed for 90 days after dosing. All dogs received prednisone (1 milligram/kilogram) for a total of 5 weeks from day -7 through day 28. We observed dose-dependent increases in tissue vector genome copy numbers; µDys5 protein in multiple appendicular muscles, the diaphragm, and heart; limb and respiratory muscle functional improvement; and reduction of histopathologic lesions. As expected, given that a truncated dystrophin protein was generated, phenotypic test results and histopathologic lesions did not fully normalize. All administrations were well tolerated, and adverse events were not seen. These data suggest that systemically administered AAV-microdystrophin may be dosed safely and could provide therapeutic benefit for patients with DMD.

Clinical potential of microdystrophin as a surrogate endpoint.

Accelerated approval based on a likely surrogate endpoint can be life-changing for patients suffering from a rare progressive disease with unmet medical need, as it substantially hastens access to potentially lifesaving therapies. In one such example, antisense morpholinos were approved to treat Duchenne muscular dystrophy (DMD) based on measurement of shortened dystrophin in skeletal muscle biopsies as a surrogate biomarker. New, promising therapeutics for DMD include AAV gene therapy to restore another form of dystrophin termed mini- or microdystrophin. AAV-microdystrophins are currently in clinical trials but have yet to be accepted by regulatory agencies as reasonably likely surrogate endpoints. To evaluate microdystrophin expression as a reasonably likely surrogate endpoint for DMD, this review highlights dystrophin biology in the context of functional and clinical benefit to support the argument that microdystrophin proteins have a high probability of providing clinical benefit based on their rational design. Unlike exon-skipping based strategies, the approach of rational design allows for functional capabilities (i.e. quality) of the protein to be maximized with every patient receiving the same optimized microdystrophin. Therefore, the presence of rationally designed microdystrophin in a muscle biopsy is likely to predict clinical benefit and is consequently a strong candidate for a surrogate endpoint analysis to support accelerated approval.

Characterization of NEB mutations in patients reveals novel nemaline myopathy disease mechanisms and omecamtiv mecarbil force effects.

Nebulin, a critical protein of the skeletal muscle thin filament, plays important roles in physiological processes such as regulating thin filament length (TFL), cross-bridge cycling, and myofibril alignment. Mutations in the nebulin gene ( NEB ) cause NEB-based nemaline myopathy (NEM2), a genetically heterogeneous disorder characterized by hypotonia and muscle weakness, currently lacking therapies targeting the underlying pathological mechanisms. In this study, we examined a cohort of ten NEM2 patients, each with unique mutations, aiming to understand their impact on mRNA, protein, and functional levels. Results show that truncation mutations affect NEB mRNA stability and lead to nonsense-mediated decay of the mutated transcript. Moreover, a high incidence of cryptic splice site activation was found in patients with splicing mutations which is expected to disrupt the actin-binding sites of nebulin. Determination of protein levels revealed patients with relatively normal nebulin levels and others with markedly reduced nebulin. We observed a positive relation between the reduction in nebulin and a reduction in TFL, and a positive relation between the reduction in nebulin level and the reduction in tension (both maximal and submaximal tension). Interestingly, our study revealed a duplication mutation in nebulin that resulted in a larger nebulin protein and longer TFL. Additionally, we investigated the effect of Omecamtiv mecarbil (OM), a small-molecule activator of cardiac myosin, on force production of type I muscle fibers of NEM2 patients. OM treatment substantially increased submaximal tension across all NEM2 patients ranging from 87-318%, with the largest effects in patients with the lowest level of nebulin. In summary, this study indicates that post-transcriptional or post-translational mechanisms regulate nebulin expression. Moreover, we propose that the pathomechanism of NEM2 involves not only shortened but also elongated thin filaments, along with the disruption of actin-binding sites resulting from splicing mutations. Significantly, our findings highlight the potential of OM treatment to improve skeletal muscle function in NEM2 patients, especially those with large reductions in nebulin levels.

INCEPTUS Natural History, Run-in Study for Gene Replacement Clinical Trial in X-Linked Myotubular Myopathy.

BACKGROUND: X-linked myotubular myopathy (XLMTM) is a life-threatening congenital myopathy that, in most cases, is characterized by profound muscle weakness, respiratory failure, need for mechanical ventilation and gastrostomy feeding, and early death. OBJECTIVE: We aimed to characterize the neuromuscular, respiratory, and extramuscular burden of XLMTM in a prospective, longitudinal study. METHODS: Thirty-four participants < 4 years old with XLMTM and receiving ventilator support enrolled in INCEPTUS, a prospective, multicenter, non-interventional study. Disease-related adverse events, respiratory and motor function, feeding, secretions, and quality of life were assessed. RESULTS: During median (range) follow-up of 13.0 (0.5, 32.9) months, there were 3 deaths (aspiration pneumonia; cardiopulmonary failure; hepatic hemorrhage with peliosis) and 61 serious disease-related events in 20 (59%) participants, mostly respiratory (52 events, 18 participants). Most participants (80%) required permanent invasive ventilation (>16 hours/day); 20% required non-invasive support (6-16 hours/day). Median age at tracheostomy was 3.5 months (95% CI: 2.5, 9.0). Thirty-three participants (97%) required gastrostomy. Thirty-one (91%) participants had histories of hepatic disease and/or prospectively experienced related adverse events or laboratory or imaging abnormalities. CHOP INTEND scores ranged from 19-52 (mean: 35.1). Seven participants (21%) could sit unsupported for≥30 seconds (one later lost this ability); none could pull to stand or walk with or without support. These parameters remained static over time across the INCEPTUS cohort. CONCLUSIONS: INCEPTUS confirmed high medical impact, static respiratory, motor and feeding difficulties, and early death in boys with XLMTM. Hepatobiliary disease was identified as an under-recognized comorbidity. There are currently no approved disease-modifying treatments.

Adherence to and changes in mental and physiological health during an 8-week yoga intervention: A pilot study.

BACKGROUND: Participating in yoga may be ideal for college students to increase physical activity and improve mental health. PURPOSE: To investigate the feasibility and impact of an 8-week yoga intervention within a university setting on mental and physiologic heath. METHODS: This 8-week yoga intervention included twelve yoga-naïve adults, (23.8 ± 4.6 years; 71% female). Participants attended two 60-min yoga classes/week in addition to baseline, mid- and post-lab visits. RESULTS: 83% of participants attended ≥75% of yoga classes. Stress and depression symptoms decreased by 11% and 25%, respectively and erythrocyte sedimentation rate (ESR) reduced by 28%. Participants who did not meet physical activity recommendations observed greater improvements in stress, depression symptoms, ESR, and C-reactive protein compared to participants who met recommendations. CONCLUSION: The majority of participants attended ≥12 of 16 yoga classes. Exploratory analyses provide preliminary support for the impact of yoga on reducing stress, symptoms of depression, and ESR. Participants who were not meeting physical activity guidelines prior to starting the intervention received greater benefits.

A Long-Term Study Evaluating the Effects of Nicorandil Treatment on Duchenne Muscular Dystrophy-Associated Cardiomyopathy in mdx Mice.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by dystrophin gene mutations affecting striated muscle. Due to advances in skeletal muscle treatment, cardiomyopathy has emerged as a leading cause of death. Previously, nicorandil, a drug with antioxidant and nitrate-like properties, ameliorated cardiac damage and improved cardiac function in young, injured mdx mice. Nicorandil mitigated damage by stimulating antioxidant activity and limiting pro-oxidant expression. Here, we examined whether nicorandil was similarly cardioprotective in aged mdx mice. METHODS AND RESULTS: Nicorandil (6 mg/kg) was given over 15 months. Echocardiography of mdx mice showed some functional defects at 12 months compared to wild-type (WT) mice, but not at 15 months. Disease manifestation was evident in mdx mice via treadmill assays and survival, but not open field and grip strength assays. Cardiac levels of SOD2 and NOX4 were decreased in mdx vs. WT. Nicorandil increased survival in mdx but did not alter cardiac function, fibrosis, diaphragm function or muscle fatigue. CONCLUSIONS: In contrast to our prior work in young, injured mdx mice, nicorandil did not exert cardioprotective effects in 15 month aged mdx mice. Discordant findings may be explained by the lack of cardiac disease manifestation in aged mdx mice compared to WT, whereas significant cardiac dysfunction was previously seen with the sub-acute injury in young mice. Therefore, we are not able to conclude any cardioprotective effects with long-term nicorandil treatment in aging mdx mice.

Intrahepatic Cholestasis Is a Clinically Significant Feature Associated with Natural History of X-Linked Myotubular Myopathy (XLMTM): A Case Series and Biopsy Report.

X-linked myotubular myopathy (XLMTM) is a rare, life-threatening congenital myopathy characterized by profound skeletal muscle weakness, respiratory distress, and motor dysfunction. However, pathology is not limited to muscle and can be associated with life-threatening hepatic peliosis. Hepatobiliary disease has been reported in up to 17% of XLMTM patients but has not been extensively characterized. We report on five XLMTM patients who experienced intrahepatic cholestasis in their disease natural history, illustrating the need to further investigate these manifestations. These patients shared presentations that included pruritus, hypertransaminemia, and hyperbilirubinemia with normal gamma-glutamyl transferase, following infection or vaccination. Three patients who had genetic testing showed no evidence of genetic mutations associated with familial cholestasis. In one patient, progression to cirrhotic, decompensated liver disease occurred. Further investigations into the molecular pathomechanism underpinning these clinical observations in XLMTM patients will be important for informing patient care.

Muscle-directed gene therapy corrects Pompe disease and uncovers species-specific GAA immunogenicity.

Pompe disease is a severe disorder caused by loss of acid α-glucosidase (GAA), leading to glycogen accumulation in tissues and neuromuscular and cardiac dysfunction. Enzyme replacement therapy is the only available treatment. AT845 is an adeno-associated viral vector designed to express human GAA specifically in skeletal muscle and heart. Systemic administration of AT845 in Gaa-/- mice led to a dose-dependent increase in GAA activity, glycogen clearance in muscles and heart, and functional improvement. AT845 was tolerated in cynomolgus macaques at low doses, while high doses caused anti-GAA immune response, inflammation, and cardiac abnormalities resulting in unscheduled euthanasia of two animals. Conversely, a vector expressing the macaque GAA caused no detectable pathology, indicating that the toxicity observed with AT845 was an anti-GAA xenogeneic immune response. Western blot analysis showed abnormal processing of human GAA in cynomolgus muscle, adding to the species-specific effects of enzyme expression. Overall, these studies show that AAV-mediated GAA delivery to muscle is efficacious in Gaa-/- mice and highlight limitations in predicting the toxicity of AAV vectors encoding human proteins in non-human species.

Erratum: Voluntary wheel running complements microdystrophin gene therapy to improve muscle function in mdx mice.

[This corrects the article DOI: 10.1016/j.omtm.2021.02.024.].