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BackgroundThe lack of dystrophin in cardiomyocytes in Duchenne muscular dystrophy (DMD) is associated with progressive decline in cardiac function eventually leading to death by 20-40 years of age. The aim of this prospective study was to determine rate of progressive decline in left ventricular (LV) function in Duchenne muscular dystrophy (DMD) over 5 years.MethodsShort axis cine and grid tagged images of the LV were acquired in individuals with DMD (n = 59; age = 5.3-18.0 years) yearly, and healthy controls at baseline (n = 16, age = 6.0-18.3 years) on a 3 T MRI scanner. Grid-tagged images were analyzed for composite circumferential strain (ℇcc%) and ℇcc% in six mid LV segments. Cine images were analyzed for left ventricular ejection fraction (LVEF), LV mass (LVM), end-diastolic volume (EDV), end-systolic volume (ESV), LV atrioventricular plane displacement (LVAPD), and circumferential uniformity ratio estimate (CURE). LVM, EDV, and ESV were normalized to body surface area for a normalized index of LVM (LVMI), EDV (EDVI) and ESV (ESVI).ResultsAt baseline, LV ℇcc% was significantly worse in DMD compared to controls and five of the six mid LV segments demonstrated abnormal strain in DMD. Longitudinal measurements revealed that ℇcc% consistently declined in individuals with DMD with the inferior segments being more affected. LVEF progressively declined between 3 to 5 years post baseline visit. In a multivariate analysis, the use of cardioprotective drugs trended towards positively impacting cardiac measures while loss of ambulation and baseline age were associated with negative impact. Eight out of 17 cardiac parameters reached a minimal clinically important difference with a threshold of 1/3 standard deviation.ConclusionThe study shows a worsening of circumferential strain in dystrophic myocardium. The findings emphasize the significance of early and longitudinal assessment of cardiac function in DMD and identify early biomarkers of cardiac dysfunction to help design clinical trials to mitigate cardiac pathology. This study provides valuable non-invasive and non-contrast based natural history data of cardiac changes which can be used to design clinical trials or interpret the results of current trials aimed at mitigating the effects of decreased cardiac function in DMD.

BACKGROUND. Systemic administration of adeno-associated virus (AAV) can trigger life-threatening inflammatory responses, including thrombotic microangiopathy (TMA), acute kidney injury due to atypical hemolytic uremic syndrome- like complement activation, immune-mediated myocardial inflammation, and hepatic toxicity. METHODS. We describe the kinetics of immune activation following systemic AAV serotype 9 (AAV9) administration in 38 individuals following 2 distinct prophylactic immunomodulation regimens. Group 1 received corticosteroids and Group 2 received rituximab plus sirolimus in addition to steroids to prevent anti-AAV antibody formation. RESULTS. Group 1 participants had a rapid increase in immunoglobulin M (IgM) and IgG. Increase in D-dimer, decline in platelet count, and complement activation are indicative of TMA. All Group 1 participants demonstrated activation of both classical and alternative complement pathways, as indicated by depleted C4 and elevated soluble C5b-9, Ba, and Bb antigens. Group 2 patients did not have a significant change in IgM or IgG and had minimal complement activation. CONCLUSIONS. This study demonstrates that TMA in the setting of AAV gene therapy is antibody dependent (classical pathway) and amplified by the alternative complement pathway. Critical time points and interventions are identified to allow for management of immune-mediated events that impact the safety and efficacy of systemic gene therapy., Introduction Adeno-associated virus-mediated (AAV-mediated) gene therapy has emerged as a promising treatment approach for a spectrum of monogenic genetic disorders (1-4). With the approval of the first recombinant AAV vector-based [...]

Adeno-associated virus (AAV) based viral vectors are widely used in human gene therapy and form the basis of approved treatments for several genetic diseases. Immune responses to vector and transgene products, however, substantially complicate these applications in clinical practice. The role of innate immune recognition of AAV vectors was initially unclear, given that inflammatory responses early after vector administration were typically mild in animal models. However, more recent research continues to identify innate immune pathways that are triggered by AAV vectors and that serve to provide activation signals for antigen-presenting cells and initiation of adaptive immune responses. Sensing of the AAV genome by the endosomal DNA receptor toll-like receptor 9 (TLR9) promotes early inflammatory response and interferon expression. Thus, activation of the TLR9>MyD88 pathway in plasmacytoid dendritic cells (pDCs) leads to the conditioning of antigen cross-presenting DCs through type I interferon (IFN-I) and ultimately CD8+ T cell activation. Alternatively, pDCs may also promote CD8+ T cell responses in a TLR9-independent manner by the production of IL-1 cytokines, thereby activating the IL-1R1>MyD88 signaling pathway. AAV can induce cytokine expression in monocyte-derived DCs, which in turn increases antibody formation. Binding of AAV capsid to complement components likely further elevates B cell activation. At high systemic vector doses in humans and in non-human primates, AAV vectors can trigger complement activation, with contributions by classical and alternative pathways, leading to severe toxicities. Finally, evidence for activation of TLR2 by the capsid and of additional innate receptors for nucleic acids has been presented. These observations show that AAV vectors can initiate several and likely redundant innate immune pathways resulting in an exaggerated adaptive immune response. [ABSTRACT FROM AUTHOR]

BackgroundLate-onset Pompe disease is a rare genetic neuromuscular disorder caused by lysosomal acid alpha-glucosidase (GAA) deficiency that ultimately results in mobility loss and respiratory failure. Current enzyme replacement therapy with recombinant human (rh)GAA has demonstrated efficacy in subjects with late-onset Pompe disease. However, long-term effects of rhGAA on pulmonary function have not been observed, likely related to inefficient delivery of rhGAA to skeletal muscle lysosomes and associated deficits in the central nervous system. To address this limitation, reveglucosidase alfa, a novel insulin-like growth factor 2 (IGF2)-tagged GAA analogue with improved lysosomal uptake, was developed. This study evaluated the pharmacokinetics, safety, and exploratory efficacy of reveglucosidase alfa in 22 subjects with late-onset Pompe disease who were previously untreated with rhGAA.ResultsReveglucosidase alfa plasma concentrations increased linearly with dose, and the elimination half-life was

Neuromuscular disorders such as Pompe disease (glycogen storage disease, type II), result in early and potentially irreversible cellular damage with a very limited opportunity for intervention in the newborn period. Pompe disease is due to deficiency in acid α-glucosidase (GAA) leading to lysosomal accumulation of glycogen in all cell types, abnormal myofibrillogenesis, respiratory insufficiency, neurological deficits, and reduced contractile function in striated muscle. Previous studies have shown that fetal delivery of recombinant adeno-associated virus (rAAV) encoding GAA to the peritoneal cavity of Gaa-/- mice resulted in high-level transduction of the diaphragm. While progression of other genetic disorders may occur later in life, the potential of fetal gene delivery to avoid the onset of irreversible damage suggests it is an attractive option for many inherited diseases. In this study, rhesus monkey fetuses were administered 4.5 × 1012 particles of rAAV type 1 expressing human GAA (rAAV1-CMV-hGAA), human α-1-antitrypsin (rAAV1-CBA-hAAT), or human mini-dystrophin (rAAV1-CMV-miniDMD) in the late first trimester using an established intraperitoneal ultrasound-guided approach. Fetuses were monitored sonographically and newborns delivered at term for postnatal studies. All animals remained healthy during the study period (growth, hematology, and clinical chemistry), with no evidence of adverse effects. Tissues were collected at a postnatal age of 3 months (∼7 months post-fetal gene transfer) for immunohistochemistry (IHC) and quantitative PCR. Both the diaphragm and peritoneum from vector-treated animals were strongly positive for expression of human GAA, AAT, or dystrophin by IHC, similar to findings when reporter genes were used. Protein expression in the diaphragm and peritoneum correlated with high vector copy numbers detected by real-time PCR. Other anatomical areas were negative, although the liver showed minimal evidence of human GAA, AAT, and DMD, vector genomes. In summary, delivery of rAAV vectors provided stable transduction of the muscular component of the diaphragm without any evidence of adverse effects.

Glycogen is a biologically essential macromolecule that is directly involved in multiple human diseases. While its primary role in carbohydrate storage and energy metabolism in the liver and muscle is well characterized, recent research has highlighted critical metabolic and non-metabolic roles for glycogen in the brain. In this review, the emerging roles of glycogen homeostasis in the healthy and diseased brain are discussed with a focus on advancing our understanding of the role of glycogen in the brain. Innovative technologies that have led to novel insights into glycogen functions are detailed. Key insights into how cellular localization impacts neuronal and glial function are discussed. Perturbed glycogen functions are observed in multiple disorders of the brain, including where it serves as a disease driver in the emerging category of neurological glycogen storage diseases (n-GSDs). n-GSDs include Lafora disease (LD), adult polyglucosan body disease (APBD), Cori disease, Glucose transporter type 1 deficiency syndrome (G1D), GSD0b, and late-onset Pompe disease (PD). They are neurogenetic disorders characterized by aberrant glycogen which results in devastating neurological and systemic symptoms. In the most severe cases, rapid neurodegeneration coupled with dementia results in death soon after diagnosis. Finally, we discuss current treatment strategies that are currently being developed and have the potential to be of great benefit to patients with n-GSD. Taken together, novel technologies and biological insights have resulted in a renaissance in brain glycogen that dramatically advanced our understanding of both biology and disease. Future studies are needed to expand our understanding and the multifaceted roles of glycogen and effectively apply these insights to human disease. [ABSTRACT FROM AUTHOR]

With recent successes in gene therapy trials for hemophilia and retinal diseases, the promise and prospects for gene therapy are once again garnering significant attention. To build on this momentum, the National Institute of Neurological Disorders and Stroke and the Muscular Dystrophy Association jointly hosted a workshop in April 2014 on "Best Practices for Gene Therapy Programs," with a focus on neuromuscular disorders. Workshop participants included researchers from academia and industry as well as representatives from the regulatory, legal, and patient advocacy sectors to cover the gamut from preclinical optimization to intellectual property concerns and regulatory approval. The workshop focused on three key issues in the field: (1) establishing adequate scientific premise for clinical trials in gene therapy, (2) addressing regulatory process issues, and (3) intellectual property and commercialization issues as they relate to gene therapy. The outcomes from the discussions at this workshop are intended to provide guidance for researchers and funders in the gene therapy field.

Purpose While factors leading to hypoventilation have been well studied in Pompe disease, cough effectiveness and airway clearance practices are less understood. We aimed to identify significant factors that influence peak cough flow (PCF) in Pompe, and to detect whether pulmonary hygiene practices were reflective of reduced PCF. Methods This is a prospective observational study of 20 subjects with Pompe disease (infantile-onset: 7, juvenile-onset: 6, adult-onset: 14). Subjects performed spirometry, maximal respiratory pressures, and cough (voluntary: n = 24, spontaneous: n = 3). Subjects or their parents reported airway clearance and secretion management practices. Relationships between disease variables, pulmonary function, and cough parameters as well as group differences in cough parameters were evaluated. Results Subjects with infantile-onset disease had significantly lower PCF (p < 0.05) and tended to require more external ventilatory support (p = 0.07). In juvenile- and adult-onset disease, PCF differed according to external ventilatory requirement [daytime: 83.6 L/min (95% CI 41.2-126.0); nighttime: 224.6 L/min (95% CI 139.1-310.2); none: 340.2 L/min (95% CI 193.3-487.6), p < 0.005]. Cough inspiratory volume also differed significantly by ventilatory requirement [daytime: 5.5 mL/kg (95% CI 3.0-8.0); nighttime: 16.0 mL/kg (95% CI 11.8-20.2); none: 26.8 mL/kg (95% CI 11.9-41.7), p < 0.001]. However, routine airway clearance or secretion management practices were only consistently reported among patients with infantile-onset disease (infantile: 86%, juvenile: 0%, adult: 14%, p < 0.005). Conclusions Cough weakness was detected in the majority of patients with Pompe disease and was influenced by both inspiratory and expiratory muscle function. Patients at risk for problems or with ineffective PCF should be urged to complete routine pulmonary hygiene., Author(s): Teresa Pitts [sup.1] , Rachel Bordelon [sup.2] , Alyssa Huff [sup.3] , Barry J. Byrne [sup.4] , Barbara K. Smith [sup.5] Author Affiliations: (Aff1) 0000 0001 2113 1622, grid.266623.5, [...]

Adeno-associated virus serotype 9 (AAV9) has enhanced capsid-associated tropism for cardiac muscle and the ability to cross the blood-brain barrier compared to other AAV serotypes. To help identify the structural features facilitating these properties, we have used cryo-electron microscopy (cryo-EM) and three-dimensional image reconstruction (cryo-reconstruction) and X-ray crystallography to determine the structure of the AAV9 capsid at 9.7- and 2.8-Å resolutions, respectively. The AAV9 capsid exhibits the surface topology conserved in all AAVs: depressions at each icosahedral two-fold symmetry axis and surrounding each five-fold axis, three separate protrusions surrounding each three-fold axis, and a channel at each five-fold axis. The AAV9 viral protein (VP) has a conserved core structure, consisting of an eight-stranded, β-barrel motif and the αA helix, which are present in all parvovirus structures. The AAV9 VP differs in nine variable surface regions (VR-I to -IX) compared to AAV4, but at only three (VR-I, VR-II, and VR-IV) compared to AAV2 and AAV8. VR-I differences modify the raised region of the capsid surface between the two-fold and five-fold depressions. The VR-IV difference produces smaller three-fold protrusions in AAV9 that are less "pointed" than AAV2 and AAV8. Significantly, residues in the AAV9 VRs have been identified as important determinants of cellular tropism and transduction and dictate its antigenic diversity from AAV2. Hence, the AAV9 VRs likely confer the unique infection phenotypes of this serotype.