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Find video protocols related to scientific articles indexed in Pubmed.
Copackaging of multiple adeno-associated viral vectors in a single production step.
Hum Gene Ther Methods
PUBLISHED: 09-19-2014
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Abstract Limiting factors in large preclinical and clinical studies utilizing adeno-associated virus (AAV) for gene therapy are focused on the restrictive packaging capacity, the overall yields, and the versatility of the production methods for single AAV vector production. Furthermore, applications where multiple vectors are needed to provide long expression cassettes, whether because of long cDNA sequences or the need of different regulatory elements, require that each vector be packaged and characterized separately, directly affecting labor and cost associated with such manufacturing strategies. To overcome these limitations, we propose a novel method of vector production that allows for the packaging of multiple expression cassettes in a single transfection step. Here we combined two expression cassettes in predetermined ratios before transfection and empirically demonstrate that the output vector recapitulates the predicted ratios. Titration by quantitative polymerase chain reaction of AAV vector genome copies using shared or unique genetic elements allowed for delineation of the individual vector contribution to the total preparation that showed the predicted differential packaging outcomes. By copackaging green fluorescent protein (GFP) and mCherry constructs, we demonstrate that both vector genome and infectious titers reiterated the ratios utilized to produce the constructs by transfection. Copackaged therapeutic constructs that only differ in transcriptional elements produced a heterogeneous vector population of both constructs in the predefined ratios. This study shows feasibility and reproducibility of a method that allows for two constructs, differing in either transgene or transcription elements, to be efficiently copackaged and characterized simultaneously, reducing cost of manufacturing and release testing.
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Peripheral nerve and neuromuscular junction pathology in Pompe disease.
Hum. Mol. Genet.
PUBLISHED: 09-12-2014
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Pompe disease is a systemic metabolic disorder characterized by lack of acid-alpha glucosidase (GAA) resulting in ubiquitous lysosomal glycogen accumulation. Respiratory and ambulatory dysfunction are prominent features in patients with Pompe yet the mechanism defining the development of muscle weakness is currently unclear. Transgenic animal models of Pompe disease mirroring the patient phenotype have been invaluable in mechanistic and therapeutic study. Here, we demonstrate significant pathological alterations at neuromuscular junctions (NMJs) of the diaphragm and tibialis anterior muscle as prominent features of disease pathology in Gaa knockout mice. Postsynaptic defects including increased motor endplate area and fragmentation were readily observed in Gaa(-/-) but not wild-type mice. Presynaptic neuropathic changes were also evident, as demonstrated by significant reduction in the levels of neurofilament proteins, and alterations in axonal fiber diameter and myelin thickness within the sciatic and phrenic nerves. Our data suggest the loss of NMJ integrity is a primary contributor to the decline in respiratory and ambulatory function in Pompe and arises from both pre- and postsynaptic pathology. These observations highlight the importance of systemic phenotype correction, specifically restoration of GAA to skeletal muscle and the nervous system for treatment of Pompe disease.
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Altered activation of the tibialis anterior in individuals with pompe disease: Implications for motor unit dysfunction.
Muscle Nerve
PUBLISHED: 09-03-2014
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Introduction: Pompe disease is a progressive disease that affects skeletal muscles and leads to loss of ambulation. We investigated the activation of the tibialis anterior (TA) in late onset Pompe disease (LOPD) individuals during maximal voluntary contraction (MVC) and evoked involuntary responses. Methods: Four LOPD patients and matched control subjects performed MVC of the TA using dorsiflexion and TA evoked responses. Activation of the TA was recorded with surface EMG. Results: The Pompe patients exhibited greater power at frequencies below 60 Hz and reduced power above 100 Hz. They exhibited reduced increase in M-wave and prolonged M-wave latency and duration in response to stimulation. Discussion: These results provide evidence that LOPD individuals have an altered activation pattern of the TA during maximal contractions. The observed activation pattern may reflect impairments in voluntary command, neuromuscular junction pathology, or compensatory drive due to a reduced number of functional motoneurons. © 2014 Wiley Periodicals, Inc.
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Examination of effects of corticosteroids on skeletal muscles of boys with DMD using MRI and MRS.
Neurology
PUBLISHED: 08-06-2014
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To evaluate the effects of corticosteroids on the lower extremity muscles in boys with Duchenne muscular dystrophy (DMD) using MRI and magnetic resonance spectroscopy (MRS).
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Magnetic resonance imaging and spectroscopy assessment of lower extremity skeletal muscles in boys with Duchenne muscular dystrophy: a multicenter cross sectional study.
PLoS ONE
PUBLISHED: 01-01-2014
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Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder that results in functional deficits. However, these functional declines are often not able to be quantified in clinical trials for DMD until after age 7. In this study, we hypothesized that (1)H2O T2 derived using (1)H-MRS and MRI-T2 will be sensitive to muscle involvement at a young age (5-7 years) consistent with increased inflammation and muscle damage in a large cohort of DMD subjects compared to controls.
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Immune responses and hypercoagulation in ERT for Pompe disease are mutation and rhGAA dose dependent.
PLoS ONE
PUBLISHED: 01-01-2014
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Enzyme replacement therapy (ERT) with recombinant human acid-?-glucosidase (rhGAA) is the only FDA approved therapy for Pompe disease. Without ERT, severely affected individuals (early onset) succumb to the disease within 2 years of life. A spectrum of disease severity and progression exists depending upon the type of mutation in the GAA gene (GAA), which in turn determines the amount of defective protein produced and its enzymatic activity. A large percent of the early onset patients are also cross reactive immunological material negative (CRIM-) and develop high titer immune responses to ERT with rhGAA. New insights from our studies in pre-clinical murine models reveal that the type of Gaa mutation has a profound effect on the immune responses mounted against ERT and the associated toxicities, including activation of clotting factors and disseminated intravascular coagulation (DIC). Additionally, the mouse strain affects outcomes, suggesting the influence of additional genetic components or modifiers. High doses of rhGAA (20 mg/kg) are currently required to achieve therapeutic benefit. Our studies indicate that lower enzyme doses reduce the antibody responses to rhGAA, reduce the incidence of immune toxicity and avoid ERT-associated anaphylaxis. Therefore, development of rhGAA with increased efficacy is warranted to limit immunotoxicities.
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Adeno-associated virus-mediated gene therapy for metabolic myopathy.
Hum. Gene Ther.
PUBLISHED: 10-30-2013
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Metabolic myopathies are a diverse group of rare diseases in which impaired breakdown of stored energy leads to profound muscle dysfunction ranging from exercise intolerance to severe muscle wasting. Metabolic myopathies are largely caused by functional deficiency of a single gene and are generally subcategorized into three major types of metabolic disease: mitochondrial, lipid, or glycogen. Treatment varies greatly depending on the biochemical nature of the disease, and unfortunately no definitive treatments exist for metabolic myopathy. Since this group of diseases is inherited, gene therapy is being explored as an approach to personalized medical treatment. Adeno-associated virus-based vectors in particular have shown to be promising in the treatment of several forms of metabolic myopathy. This review will discuss the most recent advances in gene therapy efforts for the treatment of metabolic myopathies.
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Altered glycosylation of platelet-derived von Willebrand factor confers resistance to ADAMTS13 proteolysis.
Blood
PUBLISHED: 10-08-2013
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Platelet-von Willebrand factor (VWF) is stored within ?-granules and accounts for ?20% of total VWF in platelet-rich plasma. This platelet-VWF pool is distinct from plasma-VWF and is enriched in high molecular weight multimers (HMWM). Previous studies have described significant functional discrepancies between platelet-VWF and plasma-VWF; however, the molecular basis of these differences is not well understood. We have characterized terminal glycan expression on platelet-VWF. Our findings demonstrate that platelet-VWF exists as a distinct natural glycoform. In particular, N-linked sialylation is markedly reduced (>50%) compared with plasma-VWF. Moreover, unlike plasma-VWF, platelet-VWF does not express AB blood group determinants, although precursor H antigen expression is similar to that on plasma-VWF. Because of this differential glycosylation, platelet-VWF exhibits specific resistance to ADAMTS13 proteolysis. Thus platelet activation at sites of vascular injury results in the release of high local concentrations of HMWM platelet-VWF that is more resistant to ADAMTS13, thereby facilitating platelet-plug formation.
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Preclinical toxicology and biodistribution studies of recombinant adeno-associated virus 1 human acid ?-glucosidase.
Hum Gene Ther Clin Dev
PUBLISHED: 09-12-2013
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A biodistribution and toxicology study was performed to test the acute toxicities of intradiaphragmatic injection of a recombinant adeno-associated virus (rAAV) 2/1-human acid alpha-Glucosidase (hGAA) driven by a cytomegalovirus (CMV) promoter (rAAV1-CMV-hGAA) in New Zealand white rabbits and in the rodent Pompe disease model by injecting at the right quadriceps. Studies performed using fluoroscopy and AAV2-GFP demonstrated spread upon intradiaphragmatic injection, and the ability of AAV to infect and express acid ?-glucosidase (GAA) throughout the diaphragm. For the preclinical study, 10 rabbits (5 male, 5 female) were divided into two groups, vehicle control (Lactated Ringers) and test article (1.5×10(12) vector genomes [vg] rAAV1-CMV-hGAA), and euthanized on day 21. After direct visualization, the left hemidiaphragm was injected at three locations. There was up to a 2,500-fold increase in circulating anti-AAV1 antibodies directed to the vector capsids. In addition, up to an 18-fold increase in antibodies against the GAA protein was generated. Injection sites maintained up to 1.0×10(5) vg/?g genomic DNA (gDNA), while uninjected sites had up to 1.0×10(4) vg/?g gDNA. Vector DNA was present in blood at 24 hr postinjection at up to 1.0×10(6) vg/?g gDNA, followed by a decrease to 1.0×10(3) vg/?g gDNA at euthanization on day 21. Nominal amounts of vector DNA were present in peripheral organs, including the brain, spinal cord, gonads, and skeletal muscle. Upon histopathological examination, fibroplasias of the serosal surface were noted at diaphragm injections sites of both groups. In addition, an increase in mononuclear cell infiltration in the diaphragm and esophagus in vector-dosed animals was found. Elevated creatine phosphokinase levels, an indicator of muscle repair, was observed in all animals postprocedure but persisted in vector-injected rabbits until euthanization. A follow-up study suggested that this was directed against the human transgene expression in a foreign species. Overall, this study demonstrates diffusion of vector throughout the diaphragm after localized injections.
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Sustained correction of motoneuron histopathology following intramuscular delivery of AAV in Pompe mice.
Mol. Ther.
PUBLISHED: 07-26-2013
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Pompe disease is an autosomal recessive disorder caused by mutations in the acid-alpha glucosidase (GAA) gene. Lingual dysfunction is prominent but does not respond to conventional enzyme replacement therapy. Using Pompe (Gaa(-/-)) mice, we tested the hypothesis that intralingual delivery of viral vectors encoding GAA results in GAA expression and glycogen clearance in both tongue myofibers and hypoglossal (XII) motoneurons. An intralingual injection of an adeno-associated virus (AAV) vector encoding GAA (serotypes 1 or 9; 1×10(11) vector genomes, CMV promoter) was performed in 2 month old Gaa(-/-) mice, and tissues were harvested 4 months later. Both serotypes robustly transduced tongue myofibers with histological confirmation of GAA expression (immunochemistry) and glycogen clearance (Period acid-Schiff stain). Both vectors also led to medullary transgene expression. GAA-positive motoneurons did not show the histopathologic features which are typical in Pompe disease and animal models Intralingual injection with the AAV9 vector resulted in approximately 3-fold more GAA-positive XII motoneurons (P<0.02 vs.AAV1); the AAV9 group also gained more body weight over the course of the study (P<0.05 vs.AAV1 and sham). We conclude that intralingual injection of AAV1 or AAV9 drives persistent GAA expression in tongue myofibers and motoneurons, but AAV9 may more effectively target motoneurons.Molecular Therapy (2013); doi:10.1038/mt.2013.282.
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Skeletal muscles of ambulant children with Duchenne muscular dystrophy: validation of multicenter study of evaluation with MR imaging and MR spectroscopy.
Radiology
PUBLISHED: 05-21-2013
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To validate a multicenter protocol that examines lower extremity skeletal muscles of children with Duchenne muscular dystrophy (DMD) by using magnetic resonance (MR) imaging and MR spectroscopy in terms of reproducibility of these measurements within and across centers.
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Phase I/II trial of adeno-associated virus-mediated alpha-glucosidase gene therapy to the diaphragm for chronic respiratory failure in Pompe disease: initial safety and ventilatory outcomes.
Hum. Gene Ther.
PUBLISHED: 04-11-2013
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Pompe disease is an inherited neuromuscular disease caused by deficiency of lysosomal acid alpha-glucosidase (GAA) leading to glycogen accumulation in muscle and motoneurons. Cardiopulmonary failure in infancy leads to early mortality, and GAA enzyme replacement therapy (ERT) results in improved survival, reduction of cardiac hypertrophy, and developmental gains. However, many children have progressive ventilatory insufficiency and need additional support. Preclinical work shows that gene transfer restores phrenic neural activity and corrects ventilatory deficits. Here we present 180-day safety and ventilatory outcomes for five ventilator-dependent children in a phase I/II clinical trial of AAV-mediated GAA gene therapy (rAAV1-hGAA) following intradiaphragmatic delivery. We assessed whether rAAV1-hGAA results in acceptable safety outcomes and detectable functional changes, using general safety measures, immunological studies, and pulmonary functional testing. All subjects required chronic, full-time mechanical ventilation because of respiratory failure that was unresponsive to both ERT and preoperative muscle-conditioning exercises. After receiving a dose of either 1×10(12) vg (n=3) or 5×10(12) vg (n=2) of rAAV1-hGAA, the subjects unassisted tidal volume was significantly larger (median [interquartile range] 28.8% increase [15.2-35.2], p<0.05). Further, most patients tolerated appreciably longer periods of unassisted breathing (425% increase [103-851], p=0.08). Gene transfer did not improve maximal inspiratory pressure. Expected levels of circulating antibodies and no T-cell-mediated immune responses to the vector (capsids) were observed. One subject demonstrated a slight increase in anti-GAA antibody that was not considered clinically significant. These results indicate that rAAV1-hGAA was safe and may lead to modest improvements in volitional ventilatory performance measures. Evaluation of the next five patients will determine whether earlier intervention can further enhance the functional benefit.
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Intrapleural administration of AAV9 improves neural and cardiorespiratory function in Pompe disease.
Mol. Ther.
PUBLISHED: 04-04-2013
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Pompe disease is a neuromuscular disease resulting from deficiency in acid ?-glucosidase (GAA), results in cardiac, skeletal muscle, and central nervous system (CNS) pathology. Enzyme replacement therapy (ERT) has been shown to partially correct cardiac and skeletal muscle dysfunction. However, ERT does not cross the blood-brain barrier and progressive CNS pathology ensues. We tested the hypothesis that intrapleural administration of recombinant adeno-associated virus (rAAV9)-GAA driven by a cytomegalovirus (CMV) or desmin (DES) promoter would improve cardiac and respiratory function in Gaa(-/-) mice through a direct effect and retrograde transport to motoneurons. Cardiac magnetic resonance imaging revealed significant improvement in ejection fraction in rAAV9-GAA-treated animals. Inspiratory phrenic and diaphragm activity was examined at baseline and during hypercapnic respiratory challenge. Mice treated with AAV9 had greater relative inspiratory burst amplitude during baseline conditions when compared with Gaa(-/-). In addition, efferent phrenic burst amplitude was significantly correlated with diaphragm activity in both AAV9-DES and AAV9-CMV groups but not in Gaa(-/-). This is the first study to indicate improvements in cardiac, skeletal muscle, and respiratory neural output following rAAV administration in Pompe disease. These results further implicate a role for the CNS in Pompe disease pathology and the critical need to target the neurologic aspects in developing therapeutic strategies.
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Gene therapy for rare diseases: summary of a national institutes of health workshop, september 13, 2012.
Hum. Gene Ther.
PUBLISHED: 03-23-2013
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Gene therapy has shown clinical efficacy for several rare diseases, using different approaches and vectors. The Gene Therapy for Rare Diseases workshop, sponsored by the National Institutes of Health (NIH) Office of Biotechnology Activities and Office of Rare Diseases Research, brought together investigators from different disciplines to discuss the challenges and opportunities for advancing the field including means for enhancing data sharing for preclinical and clinical studies, development and utilization of available NIH resources, and interactions with the U.S. Food and Drug Administration.
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The respiratory neuromuscular system in Pompe disease.
Respir Physiol Neurobiol
PUBLISHED: 03-18-2013
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Pompe disease is due to mutations in the gene encoding the lysosomal enzyme acid ?-glucosidase (GAA). Absence of functional GAA typically results in cardiorespiratory failure in the first year; reduced GAA activity is associated with progressive respiratory failure later in life. While skeletal muscle pathology contributes to respiratory insufficiency in Pompe disease, emerging evidence indicates that respiratory neuron dysfunction is also a significant part of dysfunction in motor units. Animal models show profound glycogen accumulation in spinal and medullary respiratory neurons and altered neural activity. Tissues from Pompe patients show central nervous system glycogen accumulation and motoneuron pathology. A neural mechanism raises considerations about the current clinical approach of enzyme replacement since the recombinant protein does not cross the blood-brain-barrier. Indeed, clinical data suggest that enzyme replacement therapy delays symptom progression, but many patients eventually require ventilatory assistance, especially during sleep. We propose that treatments which restore GAA activity to respiratory muscles, neurons and networks will be required to fully correct ventilatory insufficiency in Pompe disease.
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B-Cell depletion and immunomodulation before initiation of enzyme replacement therapy blocks the immune response to acid alpha-glucosidase in infantile-onset Pompe disease.
J. Pediatr.
PUBLISHED: 01-08-2013
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To evaluate whether B-cell depletion before enzyme replacement therapy (ERT) initiation can block acid alpha-glucosidase (GAA) antibody responses and improve clinical outcomes.
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Spinal delivery of AAV vector restores enzyme activity and increases ventilation in Pompe mice.
Mol. Ther.
PUBLISHED: 10-18-2011
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Pompe disease is a form of muscular dystrophy due to lysosomal storage of glycogen caused by deficiency of acid ?-glucosidase (GAA). Respiratory failure in Pompe disease has been attributed to respiratory muscle dysfunction. However, evaluation of spinal tissue from Pompe patients and animal models indicates glycogen accumulation and lower motoneuron pathology. We hypothesized that restoring GAA enzyme activity in the region of the phrenic motor nucleus could lead to improved breathing in a murine Pompe model (the Gaa(-/-) mouse). Adeno-associated virus serotype 5 (AAV5), encoding either GAA or green fluorescent protein (GFP), was delivered at the C(3)-C(4) spinal level of adult Gaa(-/-) mice and the spinal cords were harvested 4 weeks later. AAV5-GAA injection restored spinal GAA enzyme activity and GAA immunostaining was evident throughout the cervical ventral horn. The periodic acid Schiff (PAS) method was used to examine neuronal glycogen accumulation, and spinal PAS staining was attenuated after AAV5-GAA injection. Lastly, plethysmography revealed that minute ventilation was greater in unanesthetized AAV5-GAA versus AAV5-GFP treated Gaa(-/-) mice at 1-4 months postinjection. These results support the hypothesis that spinal cord pathology substantially contributes to ventilatory dysfunction in Gaa(-/-) mice and therefore requires further detailed evaluation in patients with Pompe disease.
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Structural studies of adeno-associated virus serotype 8 capsid transitions associated with endosomal trafficking.
J. Virol.
PUBLISHED: 09-07-2011
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The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pH 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.
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Impaired cardiac reserve and severely diminished skeletal muscle O? utilization mediate exercise intolerance in Barth syndrome.
Am. J. Physiol. Heart Circ. Physiol.
PUBLISHED: 08-26-2011
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Barth syndrome (BTHS) is a mitochondrial myopathy characterized by reports of exercise intolerance. We sought to determine if 1) BTHS leads to abnormalities of skeletal muscle O(2) extraction/utilization and 2) exercise intolerance in BTHS is related to impaired O(2) extraction/utilization, impaired cardiac function, or both. Participants with BTHS (age: 17 ± 5 yr, n = 15) and control participants (age: 13 ± 4 yr, n = 9) underwent graded exercise testing on a cycle ergometer with continuous ECG and metabolic measurements. Echocardiography was performed at rest and at peak exercise. Near-infrared spectroscopy of the vastus lateralis muscle was continuously recorded for measurements of skeletal muscle O(2) extraction. Adjusting for age, peak O(2) consumption (16.5 ± 4.0 vs. 39.5 ± 12.3 ml·kg(-1)·min(-1), P < 0.001) and peak work rate (58 ± 19 vs. 166 ± 60 W, P < 0.001) were significantly lower in BTHS than control participants. The percent increase from rest to peak exercise in ejection fraction (BTHS: 3 ± 10 vs. control: 19 ± 4%, P < 0.01) was blunted in BTHS compared with control participants. The muscle tissue O(2) saturation change from rest to peak exercise was paradoxically opposite (BTHS: 8 ± 16 vs. control: -5 ± 9, P < 0.01), and the deoxyhemoglobin change was blunted (BTHS: 0 ± 12 vs. control: 10 ± 8, P < 0.09) in BTHS compared with control participants, indicating impaired skeletal muscle extraction in BTHS. In conclusion, severe exercise intolerance in BTHS is due to both cardiac and skeletal muscle impairments that are consistent with cardiac and skeletal mitochondrial myopathy. These findings provide further insight to the pathophysiology of BTHS.
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AAV vectors for cardiac gene transfer: experimental tools and clinical opportunities.
Mol. Ther.
PUBLISHED: 07-26-2011
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Since the first demonstration of in vivo gene transfer into myocardium there have been a series of advancements that have driven the evolution of cardiac gene delivery from an experimental tool into a therapy currently at the threshold of becoming a viable clinical option. Innovative methods have been established to address practical challenges related to tissue-type specificity, choice of delivery vehicle, potency of the delivered material, and delivery route. Most importantly for therapeutic purposes, these strategies are being thoroughly tested to ensure safety of the delivery system and the delivered genetic material. This review focuses on the development of recombinant adeno-associated virus (rAAV) as one of the most valuable cardiac gene transfer agents available today. Various forms of rAAV have been used to deliver "pre-event" cardiac protection and to temper the severity of hypertrophy, cardiac ischemia, or infarct size. Adeno-associated virus (AAV) vectors have also been functional delivery tools for cardiac gene expression knockdown studies and successfully improving the cardiac aspects of several metabolic and neuromuscular diseases. Viral capsid manipulations along with the development of tissue-specific and regulated promoters have greatly increased the utility of rAAV-mediated gene transfer. Important clinical studies are currently underway to evaluate AAV-based cardiac gene delivery in humans.
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Prevention and Reversal of Antibody Responses Against Factor IX in Gene Therapy for Hemophilia B.
Front Microbiol
PUBLISHED: 06-20-2011
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Intramuscular (IM) administration of an adeno-associated viral (AAV) vector represents a simple and safe method of gene transfer for treatment of the X-linked bleeding disorder hemophilia B (factor IX, F.IX, deficiency). However, the approach is hampered by an increased risk of immune responses against F.IX. Previously, we demonstrated that the drug cocktail of immune suppressants rapamycin, IL-10, and a specific peptide (encoding a dominant CD4(+) T cell epitope) caused an induction of regulatory T cells (Treg) with a concomitant apoptosis of antigen-specific effector T cells (Nayak et al., 2009). This protocol was effective in preventing inhibitory antibody formation against human F.IX (hF.IX) in muscle gene transfer to C3H/HeJ hemophilia B mice (with targeted F9 gene deletion). Here, we show that this protocol can also be used to reverse inhibitor formation. IM injection of AAV1-hF.IX vector resulted in inhibitors of on average 8-10?BU within 1?month. Subsequent treatment with the tolerogenic cocktail accomplished a rapid reduction of hF.IX-specific antibodies to <2?BU, which lasted for >4.5?months. Systemic hF.IX expression increased from undetectable to >200?ng/ml, and coagulation times improved. In addition, we developed an alternative prophylactic protocol against inhibitor formation that did not require knowledge of T cell epitopes, consisting of daily oral administration of rapamycin for 1-month combined with frequent, low-dose intravenous injection of hF.IX protein. Experiments in T cell receptor transgenic mice showed that the route and dosing schedule of drug administration substantially affected Treg induction. When combined with intravenous antigen administration, oral delivery of rapamycin had to be performed daily in order to induce Treg, which were suppressive and phenotypically comparable to natural Treg.
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Characterization of a transgenic short hairpin RNA-induced murine model of Tafazzin deficiency.
Hum. Gene Ther.
PUBLISHED: 05-19-2011
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Barths syndrome (BTHS) is an X-linked mitochondrial disease that is due to a mutation in the Tafazzin (TAZ) gene. Based on sequence homology, TAZ has been characterized as an acyltransferase involved in the metabolism of cardiolipin (CL), a unique phospholipid almost exclusively located in the mitochondrial inner membrane. Yeast, Drosophila, and zebrafish models have been invaluable in elucidating the role of TAZ in BTHS, but until recently a mammalian model to study the disease has been lacking. Based on in vitro evidence of RNA-mediated TAZ depletion, an inducible short hairpin RNA (shRNA)-mediated TAZ knockdown (TAZKD) mouse model has been developed (TaconicArtemis GmbH, Cologne, Germany), and herein we describe the assessment of this mouse line as a model of BTHS. Upon induction of the TAZ-specific shRNA in vivo, transgenic mouse TAZ mRNA levels were reduced by >89% in cardiac and skeletal muscle. TAZ deficiency led to the absence of tetralineoyl-CL and accumulation of monolyso-CL in cardiac muscle. Furthermore, mitochondrial morphology from cardiac and skeletal muscle was altered. Skeletal muscle mitochondria demonstrated disrupted cristae, and cardiac mitochondria were significantly enlarged and displace neighboring myofibrils. Physiological measurements demonstrated a reduction in isometric contractile strength of the soleus and a reduction in cardiac left ventricular ejection fraction of TAZKD mice compared with control animals. Therefore, the inducible TAZ-deficient model exhibits some of the molecular and clinical characteristics of BTHS patients and may ultimately help to improve our understanding of BTHS-related cardioskeletal myopathy as well as serve as an important tool in developing therapeutic strategies for BTHS.
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Pompe disease gene therapy.
Hum. Mol. Genet.
PUBLISHED: 04-25-2011
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Pompe disease is an autosomal recessive metabolic myopathy caused by the deficiency of the lysosomal enzyme acid alpha-glucosidase and results in cellular lysosomal and cytoplasmic glycogen accumulation. A wide spectrum of disease exists from hypotonia and severe cardiac hypertrophy in the first few months of life due to severe mutations to a milder form with the onset of symptoms in adulthood. In either condition, the involvement of several systems leads to progressive weakness and disability. In early-onset severe cases, the natural history is characteristically cardiorespiratory failure and death in the first year of life. Since the advent of enzyme replacement therapy (ERT), the clinical outcomes have improved. However, it has become apparent that a new natural history is being defined in which some patients have substantial improvement following ERT, while others develop chronic disability reminiscent of the late-onset disease. In order to improve on the current clinical outcomes in Pompe patients with diminished clinical response to ERT, we sought to address the cause and potential for the treatment of disease manifestations which are not amenable to ERT. In this review, we will focus on the preclinical studies that are relevant to the development of a gene therapy strategy for Pompe disease, and have led to the first clinical trial of recombinant adeno-associated virus-mediated gene-based therapy for Pompe disease. We will cover the preliminary laboratory studies and rationale for a clinical trial, which is based on the treatment of the high rate of respiratory failure in the early-onset patients receiving ERT.
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Hypoglossal neuropathology and respiratory activity in pompe mice.
Front Physiol
PUBLISHED: 03-11-2011
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Pompe disease is a lysosomal storage disorder associated with systemic deficiency of acid ?-glucosidase (GAA). Respiratory-related problems in Pompe disease include hypoventilation and upper airway dysfunction. Although these problems have generally been attributed to muscular pathology, recent work has highlighted the potential role of central nervous system (CNS) neuropathology in Pompe motor deficiencies. We used a murine model of Pompe disease to test the hypothesis that systemic GAA deficiency is associated with hypoglossal (XII) motoneuron pathology and altered XII motor output during breathing. Brainstem tissue was harvested from adult Gaa(-/-) mice and the periodic acid Schiff method was used to examine neuronal glycogen accumulation. Semi-thin (2??m) plastic sections showed widespread medullary neuropathology with extensive cytoplasmic glycogen accumulation in XII motoneuron soma. We next recorded efferent XII bursting in anesthetized and ventilated Gaa(-/-) and B6/129 mice both before and after bilateral vagotomy. The coefficient of variation of respiratory cycle duration was greater in Gaa(-/-) compared to B6/129 mice (p??0.05). We conclude that CNS GAA deficiency results in substantial glycogen accumulation in XII motoneuron cell bodies and altered XII motor output. Therapeutic strategies targeting the CNS may be required to fully correct respiratory-related deficits in Pompe disease.
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Pompe disease: design, methodology, and early findings from the Pompe Registry.
Mol. Genet. Metab.
PUBLISHED: 02-04-2011
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Pompe disease is an autosomal recessive, progressive, debilitating, and often fatal neuromuscular disorder caused by deficiency of lysosomal acid ?-glucosidase (GAA). It is characterized by the accumulation of glycogen in muscle tissue that leads to progressive muscle weakness and loss of function. It presents as a broad spectrum of clinical phenotypes, with varying rates of progression, symptom onset, degree of organ involvement, and severity. The Pompe Registry represents worldwide data collection on this rare and clinically heterogeneous disease. This report describes the design, methodology, and early findings from the Registry and presents an overview of the Registry population over a five-year period from its inception in September 2004 through September 2009. Among the 742 patients from 28 countries in the Registry, 70% (517/742) reported symptom onset >12 months of age and 23% (170/742) reported symptom onset ?12 months of age. Seventy-eight percent (582/742) of registry patients have received enzyme replacement therapy. Overall, Registry data appear to be consistent with smaller natural history studies in terms of symptoms and disease course in classical infantile Pompe disease (?12 months of age with cardiomyopathy) and late-onset Pompe disease (>12 months of age). In addition, a subset of patients with symptom onset ?12 months of age do not have cardiomyopathy (14.7%); these patients appear to have a later age at first symptoms and diagnosis than their peers with cardiomyopathy. As the largest dataset on Pompe disease to date, the Pompe Registry will serve to improve recognition of the disease, enhance understanding of the variable disease course, and offer insights into treated and untreated disease course.
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A simple method to increase the transduction efficiency of single-stranded adeno-associated virus vectors in vitro and in vivo.
Hum. Gene Ther.
PUBLISHED: 01-12-2011
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We have recently shown that co-administration of conventional single-stranded adeno-associated virus 2 (ssAAV2) vectors with self-complementary (sc) AAV2-protein phosphatase 5 (PP5) vectors leads to a significant increase in the transduction efficiency of ssAAV2 vectors in human cells in vitro as well as in murine hepatocytes in vivo. In the present study, this strategy has been further optimized by generating a mixed population of ssAAV2-EGFP and scAAV2-PP5 vectors at a 10:1 ratio to achieve enhanced green fluorescent protein (EGFP) transgene expression at approximately 5- to 10-fold higher efficiency, both in vitro and in vivo. This simple coproduction method should be adaptable to any ssAAV serotype vector containing transgene cassettes that are too large to be encapsidated in scAAV vectors.
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Glycogen storage disease type Ia in canines: a model for human metabolic and genetic liver disease.
J. Biomed. Biotechnol.
PUBLISHED: 01-03-2011
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A canine model of Glycogen storage disease type Ia (GSDIa) is described. Affected dogs are homozygous for a previously described M121I mutation resulting in a deficiency of glucose-6-phosphatase-?. Metabolic, clinicopathologic, pathologic, and clinical manifestations of GSDIa observed in this model are described and compared to those observed in humans. The canine model shows more complete recapitulation of the clinical manifestations seen in humans including "lactic acidosis", larger size, and longer lifespan compared to other animal models. Use of this model in preclinical trials of gene therapy is described and briefly compared to the murine model. Although the canine model offers a number of advantages for evaluating potential therapies for GSDIa, there are also some significant challenges involved in its use. Despite these challenges, the canine model of GSDIa should continue to provide valuable information about the potential for generating curative therapies for GSDIa as well as other genetic hepatic diseases.
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Sustained alpha-sarcoglycan gene expression after gene transfer in limb-girdle muscular dystrophy, type 2D.
Ann. Neurol.
PUBLISHED: 10-30-2010
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The aim of this study was to attain long-lasting alpha-sarcoglycan gene expression in limb-girdle muscular dystrophy, type 2D (LGMD2D) subjects mediated by adeno-associated virus (AAV) gene transfer under control of a muscle specific promoter (tMCK).
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Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material.
Hum. Gene Ther.
PUBLISHED: 09-16-2010
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A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors. Production and purification of the reference material were carried out by helper virus-free transient transfection and chromatographic purification. The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide. Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ({X}, 9.18?x?10¹¹ particles/ml; 95% confidence interval [CI], 7.89?x?10¹¹ to 1.05?x?10¹² particles/ml), vector genomes ({X}, 3.28?x?10¹? vector genomes/ml; 95% CI, 2.70?x?10¹? to 4.75?x?10¹? vector genomes/ml), transducing units ({X}, 5.09?x?10? transducing units/ml; 95% CI, 2.00?x?10? to 9.60?x?10? transducing units/ml), and infectious units ({X}, 4.37?x?10? TCID?? IU/ml; 95% CI, 2.06?x?10? to 9.26?x?10? TCID?? IU/ml). Further analysis confirmed the identity of the reference material as AAV2 and the purity relative to nonvector proteins as greater than 94%. One obvious trend in the quantitative data was the degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This relatively poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents. This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards. The rAAV2 RSM has been deposited with the American Type Culture Collection and is available to the scientific community to calibrate laboratory-specific internal titer standards. Anticipated uses of the rAAV2 RSM are discussed.
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The effect of DNA-dependent protein kinase on adeno-associated virus replication.
PLoS ONE
PUBLISHED: 07-26-2010
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DNA-dependent protein kinase (DNA-PK) is a DNA repair enzyme and plays an important role in determining the molecular fate of the rAAV genome. However, the effect this cellular enzyme on rAAV DNA replication remains elusive.
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Surface plasmon resonance for vaccine design and efficacy studies: recent applications and future trends.
Expert Rev Vaccines
PUBLISHED: 06-04-2010
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The lack of a clear correlation between design and protection continues to present a barrier to progress in vaccine research. In this article, we outline how surface plasmon resonance (SPR) biosensors are emerging as tools to help resolve some of the key biophysical determinants of protection and, thereby, facilitate more rational vaccine design campaigns. SPR technology has contributed significantly to our understanding of the complex biophysical determinants of HIV neutralization and offers a platform for preclinical evaluation of vaccine candidates. In particular, the concept of reverse-engineering HIV vaccine targets based on known broadly neutralizing antibody modalities is explored and extended to include other infectious diseases, such as malaria and influenza, and other diseases such as cancer. The analytical capacity afforded by SPR includes serum screening to monitor immune responses and highly efficient quality-control surveillance measures. These are discussed alongside key technological advances, such as developments in sample throughput, and a perspective predicting continued growth and diversification of the role of SPR in vaccine development is proposed.
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PRX-08066, a novel 5-hydroxytryptamine receptor 2B antagonist, reduces monocrotaline-induced pulmonary arterial hypertension and right ventricular hypertrophy in rats.
J. Pharmacol. Exp. Ther.
PUBLISHED: 04-29-2010
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Pulmonary arterial hypertension (PAH) is a life-threatening disease that results in right ventricular failure. 5-((4-(6-Chlorothieno[2,3-d]pyrimidin-4-ylamino)piperidin-1-yl)methyl)-2-fluorobenzonitrile monofumarate (PRX-08066) is a selective 5-hydroxytryptamine receptor 2B (5-HT2BR) antagonist that causes selective vasodilation of pulmonary arteries. In the current study, the effects of PRX-08066 were assessed by using the monocrotaline (MCT)-induced PAH rat model. Male rats received 40 mg/kg MCT or phosphate-buffered saline and were treated orally twice a day with vehicle or 50 or 100 mg/kg PRX-08066 for 5 weeks. Pulmonary and cardiac functions were evaluated by hemodynamics, heart weight, magnetic resonance imaging (MRI), pulmonary artery (PA) morphology, and histology. Cardiac MRI demonstrated that PRX-08066 (100 mg/kg) significantly (P < 0.05) improved right ventricular ejection fraction. PRX-08066 significantly reduced peak PA pressure at 50 and 100 mg/kg (P < 0.05 and < 0.01, respectively) compared with MCT control animals. PRX-08066 therapy also significantly reduced right ventricle (RV)/body weight and RV/left ventricle + septum (P < 0.01 and < 0.001, respectively) compared with MCT-treated animals. Morphometric assessment of pulmonary arterioles revealed a significant reduction in medial wall thickening and lumen occlusion associated with both doses of PRX-08066 (P < 0.01). The 5-HT2BR antagonist PRX-08066 significantly attenuated the elevation in PA pressure and RV hypertrophy and maintained cardiac function. Pulmonary vascular remodeling was also diminished compared with MCT control rats. PRX-08066 prevents the severity of PAH in the MCT rat model.
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Generation of an anti-NAGase single chain antibody and its application in a biosensor-based assay for the detection of NAGase in milk.
J. Immunol. Methods
PUBLISHED: 04-13-2010
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Bovine mastitis, an inflammation of the mammary gland in cows, is a major challenge for the dairy industry worldwide as it lowers milk yield, reduces milk quality and increases overall production costs. Early diagnosis is of the utmost importance. N-acetyl-?-D-glucosaminidase (NAGase) is an enzyme released into milk during inflammation and acts as an early indicator of mastitis. This paper describes the selection of anti-NAGase single chain fragment variable antibodies (scFv) from naïve human antibody libraries and their incorporation into an automated optical biosensor-based immunoassay to detect NAGase in milk. The scFv with the highest affinity for NAGase was first characterized by inhibition ELISA, followed by further evaluation using a surface plasmon resonance platform. Purified NAGase was immobilized on the surface of a CM5 chip and spiked NAGase milk samples were analyzed. The limit of detection for the assay for the assay was determined as 1?g/ml.
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Intramyocardial injection of autologous bone marrow mononuclear cells for patients with chronic ischemic heart disease and left ventricular dysfunction (First Mononuclear Cells injected in the US [FOCUS]): Rationale and design.
Am. Heart J.
PUBLISHED: 03-16-2010
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The increasing worldwide prevalence of coronary artery disease (CAD) continues to challenge the medical community. Management options include medical and revascularization therapy. Despite advances in these methods, CAD is a leading cause of recurrent ischemia and heart failure, posing significant morbidity and mortality risks along with increasing health costs in a large patient population worldwide.
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Adeno-associated virus-mediated correction of a canine model of glycogen storage disease type Ia.
Hum. Gene Ther.
PUBLISHED: 02-19-2010
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Glycogen storage disease type Ia (GSDIa; von Gierke disease; MIM 232200) is caused by a deficiency in glucose-6-phosphatase-alpha. Patients with GSDIa are unable to maintain glucose homeostasis and suffer from severe hypoglycemia, hepatomegaly, hyperlipidemia, hyperuricemia, and lactic acidosis. The canine model of GSDIa is naturally occurring and recapitulates almost all aspects of the human form of disease. We investigated the potential of recombinant adeno-associated virus (rAAV) vector-based therapy to treat the canine model of GSDIa. After delivery of a therapeutic rAAV2/8 vector to a 1-day-old GSDIa dog, improvement was noted as early as 2 weeks posttreatment. Correction was transient, however, and by 2 months posttreatment the rAAV2/8-treated dog could no longer sustain normal blood glucose levels after 1 hr of fasting. The same animal was then dosed with a therapeutic rAAV2/1 vector delivered via the portal vein. Two months after rAAV2/1 dosing, both blood glucose and lactate levels were normal at 4 hr postfasting. With more prolonged fasting, the dog still maintained near-normal glucose concentrations, but lactate levels were elevated by 9 hr, indicating that partial correction was achieved. Dietary glucose supplementation was discontinued starting 1 month after rAAV2/1 delivery and the dog continues to thrive with minimal laboratory abnormalities at 23 months of age (18 months after rAAV2/1 treatment). These results demonstrate that delivery of rAAV vectors can mediate significant correction of the GSDIa phenotype and that gene transfer may be a promising alternative therapy for this disease and other genetic diseases of the liver.
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Gel-mediated delivery of AAV1 vectors corrects ventilatory function in Pompe mice with established disease.
Mol. Ther.
PUBLISHED: 01-26-2010
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Pompe disease is a muscular dystrophy that results in respiratory insufficiency. We characterized the outcomes of targeted delivery of recombinant adeno-associated virus serotype 1 (rAAV2/1) vector to diaphragms of Pompe mice with varying stages of disease progression. We observed significant improvement in diaphragm contractile strength in mice treated at 3 months of age that is sustained at least for 1 year and enhanced contractile strength in mice treated at 9 and 21 months of age, measured 3 months post-treatment. Ventilatory parameters including tidal volume/inspiratory time ratio, minute ventilation/expired CO2 ratio, and peak inspiratory airflow were significantly improved in mice treated at 3 months and tested at 6 months. Despite early improvement, mice treated at 3 months and tested at 1 year had diminished normoxic ventilation, potentially due to attenuation of correction over time or progressive degeneration of nontargeted accessory tissues. However, for all rAAV2/1-treated mice (treated at 3, 9, and 21 months, assayed 3 months later; treated at 3 months, assayed at 1 year), minute ventilation and peak inspiratory flows were significantly improved during respiratory challenge. These results demonstrate that gel-mediated delivery of rAAV2/1 vectors can significantly augment ventilatory function at initial and late phases of disease in a model of muscular dystrophy.
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IL-8 dictates glycosaminoglycan binding and stability of IL-18 in cystic fibrosis.
J. Immunol.
PUBLISHED: 12-21-2009
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Dysregulation of airway inflammation contributes to lung disease in cystic fibrosis (CF). Inflammation is mediated by inflammatory cytokines, including IL-8, which illustrates an increase in biological half-life and proinflammatory activity when bound to glycosaminoglycans (GAGs). The aim of this project was to compare IL-8 and IL-18 for their relative stability, activity, and interaction with GAGs, including chondroitin sulfate, hyaluronic acid, and heparan sulfate, present in high quantities in the lungs of patients with CF. Bronchoalveolar lavage fluid was collected from patients with CF (n = 28), non-CF controls (n = 14), and patients with chronic obstructive pulmonary disease (n = 12). Increased levels of IL-8 and reduced concentrations of IL-18 were detected in bronchial samples obtained from CF individuals. The low level of IL-18 was not a defect in IL-18 production, as the pro- and mature forms of the molecule were expressed and produced by CF epithelial cells and monocytes. There was, however, a marked competition between IL-8 and IL-18 for binding to GAGs. A pronounced loss of IL-18 binding capacity occurred in the presence of IL-8, which displaced IL-18 from these anionic-matrices, rendering the cytokine susceptible to proteolytic degradation by neutrophil elastase. As a biological consequence of IL-18 degradation, reduced levels of IL-2 were secreted by Jurkat T lymphocytes. In conclusion, a novel mechanism has been identified highlighting the potential of IL-8 to determine the fate of other inflammatory molecules, such as IL-18, within the inflammatory milieu of the CF lung.
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Expression of terminal alpha2-6-linked sialic acid on von Willebrand factor specifically enhances proteolysis by ADAMTS13.
Blood
PUBLISHED: 11-24-2009
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von Willebrand factor (VWF) multimeric composition is regulated in plasma by ADAMTS13. VWF deglycosylation enhances proteolysis by ADAMTS13. In this study, the role of terminal sialic acid residues on VWF glycans in mediating proteolysis by ADAMTS13 was investigated. Quantification and distribution of VWF sialylation was examined by sequential digestion and high-performance liquid chromatography analysis. Total sialic acid expression on VWF was 167nmol/mg, of which the majority (80.1%) was present on N-linked glycan chains. Enzymatic desialylation of VWF by alpha2-3,6,8,9 neuraminidase (Neu-VWF) markedly impaired ADAMTS13-mediated VWF proteolysis. Neu-VWF collagen binding activity was reduced to 50% (+/- 14%) by ADAMTS13, compared with 11% (+/- 7%) for untreated VWF. Despite this, Neu-VWF exhibited increased susceptibility to other proteases, including trypsin, chymotrypsin, and cathepsin B. VWF expressing different blood groups exhibit altered ADAMTS13 proteolysis rates (O > or = B > A > or = AB). However, ABO blood group regulation of ADAMTS13 proteolysis was ablated on VWF desialylation, as both Neu-O-VWF and Neu-AB-VWF were cleaved by ADAMTS13 at identical rates. These novel data show that sialic acid protects VWF against proteolysis by serine and cysteine proteases but specifically enhances susceptibility to ADAMTS13 proteolysis. Quantitative variation in VWF sialylation therefore represents a key determinant of VWF multimeric composition and, as such, may be of pathophysiologic significance.
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Limb-girdle muscular dystrophy type 2D gene therapy restores alpha-sarcoglycan and associated proteins.
Ann. Neurol.
PUBLISHED: 10-03-2009
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alpha-Sarcoglycan deficiency results in a severe form of muscular dystrophy (limb-girdle muscular dystrophy type 2D [LGMD2D]) without treatment. Gene replacement represents a strategy for correcting the underlying defect. Questions related to this approach were addressed in this clinical trial, particularly the need for immunotherapy and persistence of gene expression.
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Sustained transgene expression despite T lymphocyte responses in a clinical trial of rAAV1-AAT gene therapy.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 08-12-2009
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Alpha-1 antitrypsin (AAT) deficiency is well-suited as a target for human gene transfer. We performed a phase 1, open-label, dose-escalation clinical trial of a recombinant adeno-associated virus (rAAV) vector expressing normal (M) AAT packaged into serotype 1 AAV capsids delivered by i.m. injection. Nine AAT-deficient subjects were enrolled sequentially in cohorts of 3 each at doses of 6.9 x 10(12), 2.2 x 10(13), and 6.0 x 10(13) vector genome particles per patient. Four subjects receiving AAT protein augmentation discontinued therapy 28 or 56 days before vector administration. Vector administration was well tolerated, with only mild local reactions and 1 unrelated serious adverse event (bacterial epididymitis). There were no changes in hematology or clinical chemistry parameters. M-specific AAT was expressed above background in all subjects in cohorts 2 and 3 and was sustained at levels 0.1% of normal for at least 1 year in the highest dosage level cohort, despite development of neutralizing antibody and IFN-gamma enzyme-linked immunospot responses to AAV1 capsid at day 14 in all subjects. These findings suggest that immune responses to AAV capsid that develop after i.m. injection of a serotype 1 rAAV vector expressing AAT do not completely eliminate transduced cells in this context.
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Human RPE65 gene therapy for Leber congenital amaurosis: persistence of early visual improvements and safety at 1 year.
Hum. Gene Ther.
PUBLISHED: 07-09-2009
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Human gene therapy with rAAV2-vector was performed for the RPE65 form of childhood blindness called Leber congenital amaurosis. In three contemporaneous studies by independent groups, the procedure was deemed safe and there was evidence of visual gain in the short term. At 12 months after treatment, our young adult subjects remained healthy and without vector-related serious adverse events. Results of immunological assays to identify reaction to AAV serotype 2 capsid were unchanged from baseline measurements. Results of clinical eye examinations of study and control eyes, including visual acuities and central retinal structure by in vivo microscopy, were not different from those at the 3-month time point. The remarkable improvements in visual sensitivity we reported by 3 months were unchanged at 12 months. The retinal extent and magnitude of rod and cone components of the visual sensitivity between 3 and 12 months were also the same. The safety and efficacy of human retinal gene transfer with rAAV2-RPE65 vector extends to at least 1 year posttreatment.
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Analysis of regional congenital cardiac surgical outcomes in Florida using the Society of Thoracic Surgeons Congenital Heart Surgery Database.
Cardiol Young
PUBLISHED: 07-06-2009
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Florida is the fourth largest state in the United States of America. In 2004, 218,045 live babies were born in Florida, accounting for approximately 1744 new cases of congenital heart disease. We review the initial experience of The Society of Thoracic Surgeons Congenital Heart Surgery Database with a regional outcomes report, namely the Society of Thoracic Surgeons Florida Regional Report.
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Large-scale adeno-associated viral vector production using a herpesvirus-based system enables manufacturing for clinical studies.
Hum. Gene Ther.
PUBLISHED: 07-03-2009
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The ability of recombinant adeno-associated viral (rAAV) vectors to exhibit minimal immunogenicity and little to no toxicity or inflammation while eliciting robust, multiyear gene expression in vivo are only a few of the salient features that make them ideally suited for many gene therapy applications. A major hurdle for the use of rAAV in sizeable research and clinical applications is the lack of efficient and versatile large-scale production systems. Continued progression toward flexible, scalable production techniques is a prerequisite to support human clinical evaluation of these novel biotherapeutics. This review examines the current state of large-scale production methods that employ the herpes simplex virus type 1 (HSV) platform to produce rAAV vectors for gene delivery. Improvements have substantially advanced the HSV/AAV hybrid method for large-scale rAAV manufacture, facilitating the generation of highly potent, clinical-grade purity rAAV vector stocks. At least one human clinical trial employing rAAV generated via rHSV helper-assisted replication is poised to commence, highlighting the advances and relevance of this production method.
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Neural deficits contribute to respiratory insufficiency in Pompe disease.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 05-27-2009
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Pompe disease is a severe form of muscular dystrophy due to glycogen accumulation in all tissues, especially striated muscle. Disease severity is directly related to the deficiency of acid alpha-glucosidase (GAA), which degrades glycogen in the lysosome. Respiratory dysfunction is a hallmark of the disease, muscle weakness has been viewed as the underlying cause, and the possibility of an associated neural contribution has not been evaluated previously. Therefore, we examined behavioral and neurophysiological aspects of breathing in 2 animal models of Pompe disease--the Gaa(-/-) mouse and a transgenic line (MTP) expressing GAA only in skeletal muscle, as well as a detailed analysis of the CNS in a Pompe disease patient. Glycogen content was elevated in the Gaa(-/-) mouse cervical spinal cord. Retrograde labeling of phrenic motoneurons showed significantly greater soma size in Gaa(-/-) mice vs. isogenic controls, and glycogen was observed in Gaa(-/-) phrenic motoneurons. Ventilation, assessed via plethysmography, was attenuated during quiet breathing and hypercapnic challenge in Gaa(-/-) mice (6 to >21 months of age) vs. controls. We confirmed that MTP mice had normal diaphragmatic contractile properties; however, MTP mice had ventilation similar to the Gaa(-/-) mice during quiet breathing. Neurophysiological recordings indicated that efferent phrenic nerve inspiratory burst amplitudes were substantially lower in Gaa(-/-) and MTP mice vs. controls. In human samples, we demonstrated similar pathology in the cervical spinal cord and greater accumulation of glycogen in spinal cord compared with brain. We conclude that neural output to the diaphragm is deficient in Gaa(-/-) mice, and therapies targeting muscle alone may be ineffective in Pompe disease.
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Tolerance induction to cytoplasmic beta-galactosidase by hepatic AAV gene transfer: implications for antigen presentation and immunotoxicity.
PLoS ONE
PUBLISHED: 05-20-2009
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Hepatic gene transfer, in particular using adeno-associated viral (AAV) vectors, has been shown to induce immune tolerance to several protein antigens. This approach has been exploited in animal models of inherited protein deficiency for systemic delivery of therapeutic proteins. Adequate levels of transgene expression in hepatocytes induce a suppressive T cell response, thereby promoting immune tolerance. This study addresses the question of whether AAV gene transfer can induce tolerance to a cytoplasmic protein.
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Antibody-based sensors: principles, problems and potential for detection of pathogens and associated toxins.
Sensors (Basel)
PUBLISHED: 04-07-2009
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Antibody-based sensors permit the rapid and sensitive analysis of a range of pathogens and associated toxins. A critical assessment of the implementation of such formats is provided, with reference to their principles, problems and potential for on-site analysis. Particular emphasis is placed on the detection of foodborne bacterial pathogens, such as Escherichia coli and Listeria monocytogenes, and additional examples relating to the monitoring of fungal pathogens, viruses, mycotoxins, marine toxins and parasites are also provided.
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Production, purification and preliminary X-ray crystallographic studies of adeno-associated virus serotype 9.
Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun.
PUBLISHED: 04-02-2009
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Adeno-associated virus (AAV) serotype 9, which is under development for gene-delivery applications, shows significantly enhanced capsid-associated transduction efficiency in muscle compared with other AAV serotypes. With the aim of characterizing the structural determinants of this property, the purification, crystallization and preliminary X-ray crystallographic analyses of the AAV9 viral capsid are reported. The crystals diffracted X-rays to 2.8 A resolution using synchrotron radiation and belonged to the trigonal space group P3(2), with unit-cell parameters a = b = 251.0, c = 640.0 A. There are three complete viral capsids in the crystal unit cell. The orientation and position of the asymmetric unit capsid have been determined by molecular-replacement methods and structure determination is in progress.
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Intracardiac foreign body in a dog.
J Vet Cardiol
PUBLISHED: 02-25-2009
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A dog that was referred to the University of Florida Veterinary Medical Center was discovered to have a bamboo skewer within the right atrium and right ventricle, traversing the tricuspid valve. The skewer was ingested approximately four months prior to referral and was partially removed via gastrotomy. The presenting complaint at the time of referral included coagulopathy, anemia and leukocytosis. A linear, hyperechoic structure was identified in the right heart during an echocardiogram. The foreign body was suspected to be a portion of the skewer that the patient had previously ingested. Cardiopulmonary bypass was performed and the foreign body was removed successfully. Complications following surgery included the development of tricuspid valve and ventricular wall thrombi, atrial flutter and amiodarone toxicity. Many indications have been described in the veterinary literature for cardiopulmonary bypass. However, to the best of the authors knowledge, successful removal of an intracardiac foreign body with cardiopulmonary bypass has not been reported in a veterinary patient. This represents a new indication for cardiopulmonary bypass in veterinary medicine.
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Rationale and design for TIME: A phase II, randomized, double-blind, placebo-controlled pilot trial evaluating the safety and effect of timing of administration of bone marrow mononuclear cells after acute myocardial infarction.
Am. Heart J.
PUBLISHED: 02-20-2009
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Several previous studies have demonstrated that administration of autologous bone marrow-derived mononuclear cells (BMMNCs) improves cardiac function in patients after acute myocardial infarction (AMI). However, optimum timing of administration has not been investigated in a clinical trial. The Cardiovascular Cell Therapy Research Network was developed and funded by the National Heart, Lung, and Blood Institute to address important questions such as timing of cell delivery and to accelerate research in the use of cell-based therapies. The TIME trial is a randomized, phase II, double-blind, placebo-controlled clinical trial. The 5 member clinical sites of the Cardiovascular Cell Therapy Research Network will enroll 120 eligible patients with moderate-to-large anterior AMIs who have undergone successful percutaneous coronary intervention of the left anterior descending coronary artery and have a left ventricular (LV) ejection fraction
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Recovery of urinary nanovesicles from ultracentrifugation supernatants.
Nephrol. Dial. Transplant.
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Urinary vesicles represent a newly established source of biological material, widely considered to faithfully represent pathological events in the kidneys and the urogenital epithelium. The majority of currently applied isolation protocols involve cumbersome centrifugation steps to enrich vesicles from urine. To date, the efficiency of these approaches has not been investigated with respect to performing quantitative and qualitative analyses of vesicle populations in the pellet and supernatant (SN) fractions.
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Tadalafil alleviates muscle ischemia in patients with Becker muscular dystrophy.
Sci Transl Med
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Becker muscular dystrophy (BMD) is a progressive X-linked muscle wasting disease for which there is no treatment. Like Duchenne muscular dystrophy (DMD), BMD is caused by mutations in the gene encoding dystrophin, a structural cytoskeletal protein that also targets other proteins to the muscle sarcolemma. Among these is neuronal nitric oxide synthase (nNOS?), which requires certain spectrin-like repeats in dystrophins rod domain and the adaptor protein ?-syntrophin to be targeted to the sarcolemma. When healthy skeletal muscle is subjected to exercise, sarcolemmal nNOS?-derived NO attenuates local ?-adrenergic vasoconstriction, thereby optimizing perfusion of muscle. We found previously that this protective mechanism is defective-causing functional muscle ischemia-in dystrophin-deficient muscles of the mdx mouse (a model of DMD) and of children with DMD, in whom nNOS? is mislocalized to the cytosol instead of the sarcolemma. We report that this protective mechanism also is defective in men with BMD in whom the most common dystrophin mutations disrupt sarcolemmal targeting of nNOS?. In these men, the vasoconstrictor response, measured as a decrease in muscle oxygenation, to reflex sympathetic activation is not appropriately attenuated during exercise of the dystrophic muscles. In a randomized placebo-controlled crossover trial, we show that functional muscle ischemia is alleviated and normal blood flow regulation is fully restored in the muscles of men with BMD by boosting NO-cGMP (guanosine 3,5-monophosphate) signaling with a single dose of the drug tadalafil, a phosphodiesterase 5A inhibitor. These results further support an essential role for sarcolemmal nNOS? in the normal modulation of sympathetic vasoconstriction in exercising human skeletal muscle and implicate the NO-cGMP pathway as a putative new target for treating BMD.
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Glycosylation-independent lysosomal targeting of acid ?-glucosidase enhances muscle glycogen clearance in pompe mice.
J. Biol. Chem.
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We have used a peptide-based targeting system to improve lysosomal delivery of acid ?-glucosidase (GAA), the enzyme deficient in patients with Pompe disease. Human GAA was fused to the glycosylation-independent lysosomal targeting (GILT) tag, which contains a portion of insulin-like growth factor II, to create an active, chimeric enzyme with high affinity for the cation-independent mannose 6-phosphate receptor. GILT-tagged GAA was taken up by L6 myoblasts about 25-fold more efficiently than was recombinant human GAA (rhGAA). Once delivered to the lysosome, the mature form of GILT-tagged GAA was indistinguishable from rhGAA and persisted with a half-life indistinguishable from rhGAA. GILT-tagged GAA was significantly more effective than rhGAA in clearing glycogen from numerous skeletal muscle tissues in the Pompe mouse model. The GILT-tagged GAA enzyme may provide an improved enzyme replacement therapy for Pompe disease patients.
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The modelling of blood coagulation using the quartz crystal microbalance.
J Biomech
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Blood is a clinically-important analytical matrix that is routinely selected for disease monitoring. Having a clear understanding of the mechanisms involved in blood coagulation is a key consideration in haemostasis, with modern clinical practices requiring rapid, miniaturised and informative diagnostic platforms to reliably study changes in viscoelasticity (VE). Oscillatory transducers such as the Quartz Crystal Microbalance (QCM) have considerable potential in this area, provided that they present simple, linear rheometric readings which can be adequately analysed and interpreted. Hence, integrating QCM data obtained in the laboratory with mathematical modelling of acoustic interactions between quartz crystal surfaces and coagulating blood is an important consideration for modelling thrombus formation. Here, we provide a comprehensive overview of experimental and theoretical applications currently being employed to monitor and model the VE properties of coagulating blood when applied to a QCM resonator, with key emphasis on data modelling and interpretation.
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The Barth Syndrome Registry: distinguishing disease characteristics and growth data from a longitudinal study.
Am. J. Med. Genet. A
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Barth syndrome (BTHS); MIM accession # 302060) is a rare X-linked recessive cardioskeletal mitochondrial myopathy with features of cardiomyopathy, neutropenia, and growth abnormalities. The objectives of this study were to further elucidate the natural history, clinical disease presentation, and course, and describe growth characteristics for males with BTHS. Patients with a confirmed genetic diagnosis of BTHS are referred to the BTHS Registry through the Barth Syndrome Foundation, self-referral, or physician referral. This study is based on data obtained from 73 subjects alive at the time of enrollment that provided self-reported and/or medical record abstracted data. The mean age at diagnosis of BTHS was 4.04 ± 5.45 years. While the vast majority of subjects reported a history of cardiac dysfunction, nearly 6% denied any history of cardiomyopathy. Although most subjects had only mildly abnormal cardiac function by echocardiography reports, 70% were recognized as having cardiomyopathy in the first year of life and 12% have required cardiac transplantation. Of the 73 enrolled subjects, there have been five deaths. Growth curves were generated demonstrating a shift down for weight, length, and height versus the normative population with late catch up in height for a significant percentage of cases. This data also confirms a significant number of patients with low birth weight, complications in the newborn period, failure to thrive, neutropenia, developmental delay of motor milestones, and mild learning difficulties. However, it is apparent that the disease manifestations are variable, both over time for an individual patient and across the BTHS population.
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MRI/MRS evaluation of a female carrier of Duchenne muscular dystrophy.
Neuromuscul. Disord.
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The purpose of this study was to evaluate skeletal muscle composition of lower extremity muscles in a manifesting female carrier of Duchenne muscular dystrophy (MFC(DMD)) using magnetic resonance imaging (MRI) and spectroscopy (MRS). MRI/MRS was performed on the lower extremities and heart of a MFC(DMD) (47 years, 51 kg) on four occasions within 21 months and in a control subject. Heterogeneity and asymmetry among muscles in the MFC(DMD) was observed in lipid fraction and mean transverse relaxation time (T(2)) of lower extremity muscles with some muscles presenting as unaffected (e.g., rectus femoris) and others showing substantial deterioration and lipid infiltration (e.g., vasti muscles). There was an association of abnormal MRI findings and strength and motor function. Over the 21 months a small decrease in CSA(max) and increase in lipid fraction and T(2) was observed in the MFC(DMD) in some muscles. In summary, this MFC(DMD) revealed significant imaging evidence of pathologic heterogeneity among muscles. Furthermore, this study shows the feasibility of combining various quantitative MRI and MRS approaches to monitor skeletal muscle involvement.
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A degradable, bioactive, gelatinized alginate hydrogel to improve stem cell/growth factor delivery and facilitate healing after myocardial infarction.
Med. Hypotheses
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Despite remarkable effectiveness of reperfusion and drug therapies to reduce morbidity and mortality following myocardial infarction (MI), many patients have debilitating symptoms and impaired left ventricular (LV) function highlighting the need for improved post-MI therapies. A promising concept currently under investigation is intramyocardial injection of high-water content, polymeric biomaterial gels (e.g., hydrogels) to modulate myocardial scar formation and LV adverse remodeling. We propose a degradable, bioactive hydrogel that forms a unique microstructure of continuous, parallel capillary-like channels (Capgel). We hypothesize that the innovative architecture and composition of Capgel can serve as a platform for endogenous cell recruitment and drug/cell delivery, therefore facilitating myocardial repair after MI.
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Biochemical and physical characterisation of urinary nanovesicles following CHAPS treatment.
PLoS ONE
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Urinary exosomes represent a precious source of potential biomarkers for disease biology. Currently, the methods for vesicle isolation are severely restricted by the tendency of vesicle entrapment, e.g. by the abundant Tamm-Horsfall protein (THP) polymers. Treatment by reducing agents such as dithiothreitol (DTT) releases entrapped vesicles, thus increasing the final yield. However, this harsh treatment can cause remodelling of all those proteins which feature extra-vesicular domains stabilized by internal disulfide bridges and have detrimental effects on their biological activity. In order to optimize exosomal yield, we explore two vesicle treatment protocols - dithiothreitol (DTT) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic (CHAPS) - applied to the differential centrifugation protocol for exosomal vesicle isolation. The results show that CHAPS treatment does not affect vesicle morphology or exosomal marker distribution, thus eliminating most of THP interference. Moreover, the recovery and preservation of catalytic activity of two trans-membrane proteases, dipeptidyl peptidase IV and nephrilysin, was examined and found to be clearly superior after CHAPS treatment compared to DTT. Finally, proteomic profiling by mass spectrometry (MS) revealed that 76.2% of proteins recovered by CHAPS are common to those seen for DTT treatment, which illustrates underlining similarities between the two approaches. In conclusion, we provide a major improvement to currently-utilized urinary vesicle isolation strategies to allow recovery of urinary vesicles without the deleterious interference of abundant urinary proteins, while preserving typical protein folding and, consequently, the precious biological activity of urinary proteins which serve as valuable biomarkers.
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Gene therapy approaches for lysosomal storage disease: next-generation treatment.
Hum. Gene Ther.
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Lysosomal storage diseases are a group of rare inborn errors of metabolism resulting from deficiency in normal lysosomal function. These diseases are characterized by progressive accumulation of storage material within the lysosomes of affected cells, ultimately leading to cellular dysfunction. Multiple tissues ranging from musculoskeletal and visceral to tissues of the central nervous system are typically involved in disease pathology. Since the advent of enzyme replacement therapy (ERT) to manage some LSDs, general clinical outcomes have significantly improved; however, treatment with infused protein is lifelong and continued disease progression is still evident in patients. Viral gene therapy may provide a viable alternative or adjunctive therapy to current management strategies for LSDs. In this review, we discuss the various viral vector systems that have been developed and some of the strategy designs for the treatment of LSDs.
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An acidic oligopeptide displayed on AAV2 improves axial muscle tropism after systemic delivery.
Genet Vaccines Ther
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The appropriate tropism of adeno-associated virus (AAV) vectors that are systemically injected is crucial for successful gene therapy when local injection is not practical. Acidic oligopeptides have been shown to enhance drug delivery to bones.
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Retrograde gene delivery to hypoglossal motoneurons using adeno-associated virus serotype 9.
Hum Gene Ther Methods
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Retrograde viral transport (i.e., muscle to motoneuron) enables targeted gene delivery to specific motor pools. Recombinant adeno-associated virus serotype 9 (AAV9) robustly infects motoneurons, but the retrograde transport capabilities of AAV9 have not been systematically evaluated. Accordingly, we evaluated the retrograde transduction efficiency of AAV9 after direct tongue injection in 129SVE mice as well as a mouse model that displays neuromuscular pathology (Gaa(-/-)). Hypoglossal (XII) motoneurons were histologically evaluated 8 weeks after tongue injection with AAV9 encoding green fluorescent protein (GFP) with expression driven by the chicken ?-actin promoter (1 × 10(11) vector genomes). On average, GFP expression was detected in 234 ± 43 XII motoneurons 8 weeks after AAV9-GFP tongue injection. In contrast, tongue injection with a highly efficient retrograde anatomical tracer (cholera toxin ? subunit, CT-?) resulted in infection of 818 ± 88 XII motoneurons per mouse. The retrograde transduction efficiency of AAV9 was similar between the 129SVE mice and those with neuromuscular disease (Gaa(-/-)). Routine hematoxylin and eosin staining and cluster of differentiation (CD) immunostaining for T cells (CD3) indicated no persistent inflammation within the tongue or XII nucleus after AAV9 injection. Additional experiments indicated no adverse effects of AAV9 on the pattern of breathing. We conclude that AAV9 can retrogradely infect a significant portion of a given motoneuron pool in normal and dystrophic mice, and that its transduction efficiency is approximately 30% of what can be achieved with CT-?.
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Transient B cell depletion or improved transgene expression by codon optimization promote tolerance to factor VIII in gene therapy.
PLoS ONE
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The major complication in the treatment of hemophilia A is the development of neutralizing antibodies (inhibitors) against factor VIII (FVIII). The current method for eradicating inhibitors, termed immune tolerance induction (ITI), is costly and protracted. Clinical protocols that prevent rather than treat inhibitors are not yet established. Liver-directed gene therapy hopes to achieve long-term correction of the disease while also inducing immune tolerance. We sought to investigate the use of adeno-associated viral (serotype 8) gene transfer to induce tolerance to human B domain deleted FVIII in hemophilia A mice. We administered an AAV8 vector with either human B domain deleted FVIII or a codon-optimized transgene, both under a liver-specific promoter to two strains of hemophilia A mice. Protein therapy or gene therapy was given either alone or in conjunction with anti-CD20 antibody-mediated B cell depletion. Gene therapy with a low-expressing vector resulted in sustained near-therapeutic expression. However, supplementary protein therapy revealed that gene transfer had sensitized mice to hFVIII in a high-responder strain but not in mice of a low-responding strain. This heightened response was ameliorated when gene therapy was delivered with anti-murine CD20 treatment. Transient B cell depletion prevented inhibitor formation in protein therapy, but failed to achieve a sustained hypo-responsiveness. Importantly, use of a codon-optimized hFVIII transgene resulted in sustained therapeutic expression and tolerance without a need for B cell depletion. Therefore, anti-CD20 may be beneficial in preventing vector-induced immune priming to FVIII, but higher levels of liver-restricted expression are preferred for tolerance.
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Gene therapy for aromatic L-amino acid decarboxylase deficiency.
Sci Transl Med
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Aromatic L-amino acid decarboxylase (AADC) is required for the synthesis of the neurotransmitters dopamine and serotonin. Children with defects in the AADC gene show compromised development, particularly in motor function. Drug therapy has only marginal effects on some of the symptoms and does not change early childhood mortality. Here, we performed adeno-associated viral vector-mediated gene transfer of the human AADC gene bilaterally into the putamen of four patients 4 to 6 years of age. All of the patients showed improvements in motor performance: One patient was able to stand 16 months after gene transfer, and the other three patients achieved supported sitting 6 to 15 months after gene transfer. Choreic dyskinesia was observed in all patients, but this resolved after several months. Positron emission tomography revealed increased uptake by the putamen of 6-[(18)F]fluorodopa, a tracer for AADC. Cerebrospinal fluid analysis showed increased dopamine and serotonin levels after gene transfer. Thus, gene therapy targeting primary AADC deficiency is well tolerated and leads to improved motor function.
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Substrate metabolism during basal and hyperinsulinemic conditions in adolescents and young-adults with Barth syndrome.
J. Inherit. Metab. Dis.
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Barth syndrome (BTHS) is a rare X-linked disorder that is characterized by mitochondrial abnormalities, infantile or childhood onset of cardioskeletal myopathy, and high mortality rates. It is currently unknown if BTHS related mitochondrial dysfunction results in substrate metabolism abnormalities and thereby contributes to cardioskeletal myopathy in patients with BTHS.
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Structural insight into the unique properties of adeno-associated virus serotype 9.
J. Virol.
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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, ?-barrel motif and the ?A 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.
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Mapping the T helper cell response to acid ?-glucosidase in Pompe mice.
Mol. Genet. Metab.
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Pompe disease is a neuromuscular disease caused by an inherited deficiency of the lysosomal enzyme acid ?-glucosidase (GAA). The resulting accumulation of glycogen causes muscle weakness with the severe form of the disease resulting in death by cardiorespiratory failure in the first year of life. The only available treatment, enzyme replacement therapy (ERT) with recombinant GAA (rhGAA), is severely hampered by antibody responses that reduce efficacy and cause immunotoxicities. Currently, Pompe mice represent the only pre-clinical model for development of new treatments and for immunological studies. While antibody formation following ERT in this model has been described, the underlying T cell response has not been studied. In order to define the T helper response to rhGAA in Pompe mice, immunodominant CD4(+) T cell epitopes were mapped in GAA(-/-) 129SVE mice using ELISpot. Additionally, cytokine responses and antibody formation against rhGAA during ERT were measured. Among the three CD4(+) T cell epitopes identified, only epitope IFLGPEPKSVVQ, predicted to be the strongest MHC II binder, consistently contributed to IL-4 production. Frequencies of IL-4 producing T cells were considerably higher than those of IL-17 or IFN-? producing cells, suggesting a predominantly Th2 cell mediated response. This is further supported by IgG1 being the prevalent antibody subclass against rhGAA during ERT and consistent with prior reports on IgE formation and anaphylaxis in this model. These results will facilitate mechanistic studies of the immune response to rhGAA in Pompe mice during development of new therapies and tolerance protocols.
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Treatment of congenital neurotransmitter deficiencies by intracerebral ventricular injection of an AAV9 vector.
Hum. Gene Ther.
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Dopamine and serotonin are produced by distinct groups of neurons in the brain, and gene therapies other than direct injection have not been attempted to correct congenital deficiencies in such neurotransmitters. In this study, we performed gene therapy to treat knock-in mice with dopamine and serotonin deficiencies caused by a mutation in the aromatic L-amino acid decarboxylase (AADC) gene (DdcKI mice). Intracerebral ventricular injection of neonatal mice with an adeno-associated virus (AAV) vector serotype 9 (AAV9) vector expressing the human AADC gene (AAV9-hAADC) resulted in widespread AADC expression in the brain. Without treatment, four-week-old DdcKI mice exhibited whole-brain homogenate dopamine and serotonin levels of 25% and 15% of normal, respectively. After gene therapy, the levels rose to 100% and 40% of normal, respectively. The gene therapy improved the growth rate and survival of DdcKI mice and normalized their hindlimb clasping and cardiovascular dysfunctions. The behavioral abnormalities of the DdcKI mice were partially corrected, and the treated DdcKI mice were slightly more active than normal mice. No immune reactions resulted from the treatment. Therefore, a congenital neurotransmitter deficiency can be treated safely through inducing widespread expression of the deficient gene in neonatal mice.
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