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.

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.

Emergency Preparedness Plans and Perceptions Among a Sample of United States Childcare Providers.

OBJECTIVES: Children are at increased risk for experiencing negative physical and mental health outcomes as a result of disasters. Millions of children spend their days in childcare centers or in residential family childcare settings. The purpose of this study was to describe childcare providers' perceived levels of preparedness capabilities and to assess differences in levels of perceived preparedness between different types of childcare providers. METHODS: A national convenience sample of childcare center administrators and residential family childcare administrators completed a brief online survey about their preparedness efforts. RESULTS: Overall, there were few differences in preparedness between childcare centers and residential family childcare providers. However, childcare centers were more likely to report that they had written plans (94.47%) than residential family childcare providers (83.73%) were (χ12=15.62; P<.001). Both types of providers were more likely to report being very prepared/prepared for fires (91.31%) than they were for any other type of emergency (flooding, active shooter, etc.; 45.08% to 79.34%). CONCLUSIONS: Future work should assess how childcare providers respond to and recover from emergencies, as well as explore the types of resources childcare providers need in order to feel comfortable caring for children during such emergency situations. (Disaster Med Public Health Preparedness. 2019;13:704-708).

Insight into the phenotype of infants with Pompe disease identified by newborn screening with the common c.-32-13T>G "late-onset" GAA variant.

OBJECTIVE: Newborn screening (NBS) has led to early diagnosis and early initiation of treatment for infantile onset Pompe Disease (IOPD). However, guidelines for management of late onset Pompe disease (LOPD) via NBS, especially with the IVS c.-32-13T>G are not clear. This IVS variant is noted in 68-90% cases with LOPD and has been presumed to result in "adult" disease in compound heterozygosity, with a few cases with earlier onset and a mild to no phenotype in homozygosity. Our study evaluates newborns with LOPD having IVS variant with a diligent multidisciplinary approach to determine if they have an early presentation. METHODS: Seven children with LOPD identified by NBS with IVS variant (3 compound heterozygous, and 4 homozygous) were evaluated with clinical, biochemical (CK, AST, ALT, and urinary Glc4), cardiac evaluation, physical therapy (PT), occupational, and speech/language therapy. RESULTS: All seven patients demonstrated motor involvement by age 6months; the three patients with c.-32-13 T>G variant in compound heterozygosity had symptoms as neonates. Patients with c.-32-13 T>G variant in compound heterozygosity had more involvement with persistent hyperCKemia, elevated AST and ALT, swallowing difficulties, limb-girdle weakness, delayed motor milestones, and were initiated on ERT. The patients with c.-32-13T>G variant in homozygosity had normal laboratory parameters, and presented with very subtle yet LOPD specific signs, identified only by meticulous assessments. CONCLUSION: This patient cohort represents the first carefully phenotyped cohort of infants with LOPD with the "late-onset" GAA variant c.-32-13T>G detected by NBS in the USA. It emphasizes not only the opportunity for early detection of skeletal and other muscle involvement in infants with c.-32-13T>G variant but also a high probability of overlooking or underestimating the significance of clinically present and detectable features. It can thus serve as a valuable contribution in the development of evaluation and treatment algorithms for infants with LOPD.

Binding to E1 and E3 is mutually exclusive for the human autophagy E2 Atg3.

Ubiquitin-like proteins (UBLs) are activated, transferred and conjugated by E1-E2-E3 enzyme cascades. E2 enzymes for canonical UBLs such as ubiquitin, SUMO, and NEDD8 typically use common surfaces to bind to E1 and E3 enzymes. Thus, canonical E2s are required to disengage from E1 prior to E3-mediated UBL ligation. However, E1, E2, and E3 enzymes in the autophagy pathway are structurally and functionally distinct from canonical enzymes, and it has not been possible to predict whether autophagy UBL cascades are organized according to the same principles. Here, we address this question for the pathway mediating lipidation of the human autophagy UBL, LC3. We utilized bioinformatic and experimental approaches to identify a distinctive region in the autophagy E2, Atg3, that binds to the autophagy E3, Atg12∼Atg5-Atg16. Short peptides corresponding to this Atg3 sequence inhibit LC3 lipidation in vitro. Notably, the E3-binding site on Atg3 overlaps with the binding site for the E1, Atg7. Accordingly, the E3 competes with Atg7 for binding to Atg3, implying that Atg3 likely cycles back and forth between binding to Atg7 for loading with the UBL LC3 and binding to E3 to promote LC3 lipidation. The results show that common organizational principles underlie canonical and noncanonical UBL transfer cascades, but are established through distinct structural features.

Structures of Atg7-Atg3 and Atg7-Atg10 reveal noncanonical mechanisms of E2 recruitment by the autophagy E1.

Central to most forms of autophagy are two ubiquitin-like proteins (UBLs), Atg8 and Atg12, which play important roles in autophagosome biogenesis, substrate recruitment to autophagosomes, and other aspects of autophagy. Typically, UBLs are activated by an E1 enzyme that (1) catalyzes adenylation of the UBL C terminus, (2) transiently covalently captures the UBL through a reactive thioester bond between the E1 active site cysteine and the UBL C terminus, and (3) promotes transfer of the UBL C terminus to the catalytic cysteine of an E2 conjugating enzyme. The E2, and often an E3 ligase enzyme, catalyzes attachment of the UBL C terminus to a primary amine group on a substrate. Here, we summarize our recent work reporting the structural and mechanistic basis for E1-E2 protein interactions in autophagy.

Single-molecule FRET analysis of DNA binding and bending by yeast HMGB protein Nhp6A.

High-mobility group B (HMGB) proteins bind duplex DNA without sequence specificity, facilitating the formation of compact nucleoprotein structures by increasing the apparent flexibility of DNA through the introduction of DNA kinks. It has remained unclear whether HMGB binding and DNA kinking are simultaneous and whether the induced kink is rigid (static) or flexible. The detailed molecular mechanism of HMGB-induced DNA 'softening' is explored here by single-molecule fluorescence resonance energy transfer studies of single yeast Nhp6A (yNhp6A) proteins binding to short DNA duplexes. We show that the local effect of yNhp6A protein binding to DNA is consistent with formation of a single static kink that is short lived (lifetimes of a few seconds) under physiological buffer conditions. Within the time resolution of our experiments, this static kink occurs at the instant the protein binds to the DNA, and the DNA straightens at the instant the protein dissociates from the DNA. Our observations support a model in which HMGB proteins soften DNA through random dynamic binding and dissociation, accompanied by DNA kinking and straightening, respectively.

Conformational trapping of mismatch recognition complex MSH2/MSH3 on repair-resistant DNA loops.

Insertion and deletion of small heteroduplex loops are common mutations in DNA, but why some loops are prone to mutation and others are efficiently repaired is unknown. Here we report that the mismatch recognition complex, MSH2/MSH3, discriminates between a repair-competent and a repair-resistant loop by sensing the conformational dynamics of their junctions. MSH2/MSH3 binds, bends, and dissociates from repair-competent loops to signal downstream repair. Repair-resistant Cytosine-Adenine-Guanine (CAG) loops adopt a unique DNA junction that traps nucleotide-bound MSH2/MSH3, and inhibits its dissociation from the DNA. We envision that junction dynamics is an active participant and a conformational regulator of repair signaling, and governs whether a loop is removed by MSH2/MSH3 or escapes to become a precursor for mutation.

Fibrillogenesis in continuously spun synthetic collagen fiber.

The universal structural role of collagen fiber networks has motivated the development of collagen gels, films, coatings, injectables, and other formulations. However, reported synthetic collagen fiber fabrication schemes have either culminated in short, discontinuous fiber segments at unsuitably low production rates, or have incompletely replicated the internal fibrillar structure that dictates fiber mechanical and biological properties. We report a continuous extrusion system with an off-line phosphate buffer incubation step for the manufacture of synthetic collagen fiber. Fiber with a cross-section of 53+ or - 14 by 21 + or - 3 microm and an ultimate tensile strength of 94 + or - 19 MPa was continuously produced at 60 m/hr from an ultrafiltered monomeric collagen solution. The effect of collagen solution concentration, flow rate, and spinneret size on fiber size was investigated. The fiber was further characterized by microdifferential scanning calorimetry, transmission electron microscopy (TEM), second harmonic generation (SHG) analysis, and in a subcutaneous murine implant model. Calorimetry demonstrated stabilization of the collagen triple helical structure, while TEM and SHG revealed a dense, axially aligned D-periodic fibril structure throughout the fiber cross-section. Implantation of glutaraldehyde crosslinked and noncrosslinked fiber in the subcutaneous tissue of mice demonstrated limited inflammatory response and biodegradation after a 6-week implant period.

Results of the veterinary enalapril trial to prove reduction in onset of heart failure in dogs chronically treated with enalapril alone for compensated, naturally occurring mitral valve insufficiency.

OBJECTIVE: To determine the efficacy of long-term enalapril administration in delaying the onset of congestive heart failure (CHF). DESIGN: Placebo-controlled, double-blind, multicenter, randomized trial. ANIMALS: 124 dogs with compensated mitral valve regurgitation (MR). PROCEDURES: Dogs randomly assigned to receive enalapril or placebo were monitored for the primary endpoint of onset of CHF for < or = 58 months. Secondary endpoints included time from study entry to the combined endpoint of CHF-all-cause death; number of dogs free of CHF at 500, 1,000, and 1,500 days; and mean number of CHF-free days. RESULTS: Kaplan-Meier estimates of the effect of enalapril on the primary endpoint did not reveal a significant treatment benefit. Chronic enalapril administration did have a significant benefit on the combined endpoint of CHF-all-cause death (benefit was 317 days [10.6 months]). Dogs receiving enalapril remained free of CHF for a significantly longer time than those receiving placebo and were significantly more likely to be free of CHF at day 500 and at study end. CONCLUSIONS AND CLINICAL RELEVANCE: Chronic enalapril treatment of dogs with naturally occurring, moderate to severe MR significantly delayed onset of CHF, compared with placebo, on the basis of number of CHF-free days, number of dogs free of CHF at days 500 and study end, and increased time to a combined secondary endpoint of CHF-all-cause death. Improvement in the primary endpoint, CHF-free survival, was not significant. Results suggest that enalapril modestly delays the onset of CHF in dogs with moderate to severe MR.

Prospective clinical evaluation of serum cardiac troponin T in dogs admitted to a veterinary teaching hospital.

The purpose of this study was to measure serum cardiac troponin T (cTnT) with a commercially available human enzyme-linked immunoassay (ELISA) test in various groups of dogs, including those undergoing doxorubicin chemotherapy. Serum samples were obtained from 6 groups of dogs: (1) normal adult dogs (n = 15); (2) dogs with asymptomatic dilated cardiomyopathy (n = 5); (3) dogs with congestive heart failure (n = 10); (4) dogs with untreated neoplasia (n = 20); (5) dogs with skeletal muscle trauma (n = 10); and (6) dogs with neoplasia receiving doxorubicin chemotherapy (n = 4). One serum sample was obtained from each of the normal dogs, those with asymptomatic cardiomyopathy, those with congestive heart failure, and those with untreated neoplasia. Serum samples were obtained serially from the dogs that were undergoing doxorubicin chemotherapy; samples were collected before doxorubicin (30 mg/m2) administration and then 1, 5, 7, and 14 days after administration throughout 6 cycles for a cumulative total dose of 180 mg/m2. All normal dogs, dogs with untreated neoplasia, and dogs with asymptomatic dilated cardiomyopathy had cTnT concentrations below the lower limits of detection for the assay used (<0.05 ng/mL). Detectable concentrations of cTnT were found in 3 dogs with congestive heart failure and in 2 dogs with skeletal muscle trauma. Detectable concentrations also were found in both dogs that had received 180 mg/m2 of doxorubicin. We conclude that dogs with congestive heart failure and those with skeletal muscle trauma and dogs with neoplasia receiving high-dose doxorubicin chemotherapy may have increased serum cTnT concentration, which may be suggestive of myocardial damage.

Effects of long-term administration of enalapril on clinical indicators of renal function in dogs with compensated mitral regurgitation.

OBJECTIVE: To determine the effect of long-term administration of enalapril on renal function in dogs with severe, compensated mitral regurgitation. DESIGN: Randomized controlled trial. ANIMALS: 139 dogs with mitral regurgitation but without overt signs of heart failure. PROCEDURE: Dogs were randomly assigned to be treated with enalapril (0.5 mg/kg [0.23 mg/lb], PO, q 24 h) or placebo, and serum creatinine and urea nitrogen concentrations were measured at regular intervals for up to 26 months. RESULTS: Adequate information on renal function was obtained from 132 dogs; follow-up time ranged from 0.5 to 26 months (median, 12 months). Mean serum creatinine and urea nitrogen concentrations were not significantly different between dogs receiving enalapril and dogs receiving the placebo at any time, nor were concentrations significantly different from baseline concentrations. Proportions of dogs that developed azotemia or that had a +/- 35% increase in serum creatinine or urea nitrogen concentration were also not significantly different between groups. CONCLUSIONS: And Clinical Relevance: Results suggest that administration of enalapril for up to 2 years did not have any demonstrable adverse effects on renal function in dogs with severe, compensated mitral regurgitation.