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

Cardiac dysfunction and respiratory muscle weakness are primary features in patients with early onset Pompe disease. To reduce the progressive and rapid accumulation of glycogen resulting in cardiorespiratory dysfunction, adult Gaa-/- mice were administered a single systemic injection of rAAV2/9-DES-hGAA (AAV9) or bi-monthly injections of recombinant human GAA (ERT). Cardiac function and morphology was assessed one and three months after initiation of treatment while whole-body plethysmography and diaphragmatic contractile function was evaluated at three months post-treatment in all groups. Gaa-/- animals receiving either AAV9 or ERT demonstrated a significant improvement in cardiac function and diaphragmatic contractile function as compared to control animals. AAV9 treatment resulted in a significant reduction in cardiac dimension (end diastolic left ventricular mass/gram wet weight; EDMc) at three months post-injection. Neither AAV9 nor ERT therapy altered minute ventilation during quiet breathing (eupnea). However, breathing frequency and expiratory time were significantly improved in AAV9 animals. Glycogen deposition was significantly elevated in Gaa-/- and ERT but not AAV9 when compared to control animals. These results indicate systemic delivery of either strategy improves cardiac function but improvement in respiratory parameters and glycogen reduction are limited to AAV9 therapy at three-months post treatment in a murine model of Pompe disease.

Pompe disease is an autosomal recessive genetic disorder characterized by a deficiency of the enzyme responsible for degradation of lysosomal glycogen (acid α-glucosidase (GAA)). Cardiac dysfunction and respiratory muscle weakness are primary features of this disorder. To attenuate the progressive and rapid accumulation of glycogen resulting in cardiorespiratory dysfunction, adult Gaa (-/-) mice were administered a single systemic injection of rAAV2/9-DES-hGAA (AAV9-DES) or bimonthly injections of recombinant human GAA (enzyme replacement therapy (ERT)). Assessment of cardiac function and morphology was measured 1 and 3 months after initiation of treatment while whole-body plethysmography and diaphragmatic contractile function was evaluated at 3 months post-treatment in all groups. Gaa (-/-) animals receiving either AAV9-DES or ERT demonstrated a significant improvement in cardiac function and diaphragmatic contractile function as compared to control animals. AAV9-DES treatment resulted in a significant reduction in cardiac dimension (end diastolic left ventricular mass/gram wet weight; EDMc) at 3 months postinjection. Neither AAV nor ERT therapy altered minute ventilation during quiet breathing (eupnea). However, breathing frequency and expiratory time were significantly improved in AAV9-DES animals. These results indicate systemic delivery of either strategy improves cardiac function but AAV9-DES alone improves respiratory parameters at 3 months post-treatment in a murine model of Pompe disease.

Human Gene Therapy Clinical DevelopmentVol. 25, No. 3 Clinical ProtocolsPhase I/II Trial of Diaphragm Delivery of Recombinant Adeno-Associated Virus Acid Alpha-Glucosidase (rAAV1-CMV-GAA) Gene Vector in Patients with Pompe DiseaseBarry J. Byrne, Barbara Smith, Cathryn Mah, Lee Ann Lawson, Saleem Islam, Manuela Corti, Daniel Martin, Nicole Dobija, Maria V. Irwin, Thomas Conlon, Brian Cleaver, Nathalie Clement, and Shelley W. CollinsBarry J. ByrneSearch for more papers by this author, Barbara SmithSearch for more papers by this author, Cathryn MahSearch for more papers by this author, Lee Ann LawsonSearch for more papers by this author, Saleem IslamSearch for more papers by this author, Manuela CortiSearch for more papers by this author, Daniel MartinSearch for more papers by this author, Nicole DobijaSearch for more papers by this author, Maria V. IrwinSearch for more papers by this author, Thomas ConlonSearch for more papers by this author, Brian CleaverSearch for more papers by this author, Nathalie ClementSearch for more papers by this author, and Shelley W. CollinsSearch for more papers by this authorPublished Online:19 Sep 2014https://doi.org/10.1089/humc.2014.2514AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited byHypertrophic Cardiomyopathy versus Storage Diseases with Myocardial Involvement26 August 2023 | International Journal of Molecular Sciences, Vol. 24, No. 17Monitoring and Management of Respiratory Function in Pompe Disease: Current Perspectives1 September 2023 | Therapeutics and Clinical Risk Management, Vol. Volume 19Immune Responses and Immunosuppressive Strategies for Adeno-Associated Virus-Based Gene Therapy for Treatment of Central Nervous System Disorders: Current Knowledge and Approaches Suyash Prasad, David P. Dimmock, Benjamin Greenberg, Jagdeep S. Walia, Chanchal Sadhu, Fatemeh Tavakkoli, and Gerald S. Lipshutz14 December 2022 | Human Gene Therapy, Vol. 33, No. 23-24What’s new and what’s next for gene therapy in Pompe disease?27 April 2022 | Expert Opinion on Biological Therapy, Vol. 22, No. 9Muscle‐directed gene therapy corrects Pompe disease and uncovers species‐specific GAA immunogenicity1 December 2021 | EMBO Molecular Medicine, Vol. 14, No. 1Hepatic expression of GAA results in enhanced enzyme bioavailability in mice and non-human primates4 November 2021 | Nature Communications, Vol. 12, No. 1Current Clinical Applications of In Vivo Gene Therapy with AAVsMolecular Therapy, Vol. 29, No. 2Preclinical Research in Glycogen Storage Diseases: A Comprehensive Review of Current Animal Models17 December 2020 | International Journal of Molecular Sciences, Vol. 21, No. 24Pompe Disease: New Developments in an Old Lysosomal Storage Disorder18 September 2020 | Biomolecules, Vol. 10, No. 9Molecular Approaches for the Treatment of Pompe Disease12 November 2019 | Molecular Neurobiology, Vol. 57, No. 2Advancements in AAV-mediated Gene Therapy for Pompe DiseaseJournal of Neuromuscular Diseases, Vol. 7, No. 1Next Generation of Adeno-Associated Virus Vectors for Gene Therapy for Human Liver DiseasesGastroenterology Clinics of North America, Vol. 48, No. 2Intravenous Injection of an AAV-PHP.B Vector Encoding Human Acid α-Glucosidase Rescues Both Muscle and CNS Defects in Murine Pompe DiseaseMolecular Therapy - Methods & Clinical Development, Vol. 12Capsid Modifications for Targeting and Improving the Efficacy of AAV VectorsMolecular Therapy - Methods & Clinical Development, Vol. 12Large-Scale Clinical Manufacturing of AAV Vectors for Systemic Muscle Gene Therapy31 March 2019Clinical Gene Therapy Trials for Pompe Disease31 March 2019Can an in vivo imaging system be used to determine localization and biodistribution of AAV5-mediated gene expression following subretinal and intravitreal delivery in mice?Experimental Eye Research, Vol. 176Innovative Therapieansätze bei hereditären neuromuskulären Erkrankungen31 August 2018 | Der Nervenarzt, Vol. 89, No. 10Pompe Disease: From Basic Science to Therapy16 August 2018 | Neurotherapeutics, Vol. 15, No. 4Gene Therapy With Regulatory T Cells: A Beneficial Alliance19 March 2018 | Frontiers in Immunology, Vol. 9Safety of Intradiaphragmatic Delivery of Adeno-Associated Virus-Mediated Alpha-Glucosidase (rAAV1-CMV-hGAA) Gene Therapy in Children Affected by Pompe Disease Manuela Corti, Cristina Liberati, Barbara K. Smith, Lee Ann Lawson, Ibrahim S. Tuna, Thomas J. Conlon, Kirsten E. Coleman, Saleem Islam, Roland W. Herzog, David D. Fuller, Shelley W. Collins, and Barry J. Byrne1 December 2017 | Human Gene Therapy Clinical Development, Vol. 28, No. 4Long-term neurologic and cardiac correction by intrathecal gene therapy in Pompe disease6 September 2017 | Acta Neuropathologica Communications, Vol. 5, No. 1Rescue of Pompe disease in mice by AAV-mediated liver delivery of secretable acid α-glucosidaseScience Translational Medicine, Vol. 9, No. 418Inspiratory muscle conditioning exercise and diaphragm gene therapy in Pompe disease: Clinical evidence of respiratory plasticityExperimental Neurology, Vol. 287In vivo tissue-tropism of adeno-associated viral vectorsCurrent Opinion in Virology, Vol. 21Copackaged AAV9 Vectors Promote Simultaneous Immune Tolerance and Phenotypic Correction of Pompe Disease Phillip A. Doerfler, Adrian G. Todd, Nathalie Clément, Darin J. Falk, Sushrusha Nayak, Roland W. Herzog, and Barry J. Byrne4 November 2015 | Human Gene Therapy, Vol. 27, No. 1Targeted approaches to induce immune tolerance for Pompe disease therapyMolecular Therapy - Methods & Clinical Development, Vol. 3Manufacturing of recombinant adeno-associated viral vectors for clinical trialsMolecular Therapy - Methods & Clinical Development, Vol. 3A scalable method for the production of high-titer and high-quality adeno-associated type 9 vectors using the HSV platformMolecular Therapy - Methods & Clinical Development, Vol. 3 Volume 25Issue 3Sep 2014 InformationCopyright 2014, Mary Ann Liebert, Inc.To cite this article:Barry J. Byrne, Barbara Smith, Cathryn Mah, Lee Ann Lawson, Saleem Islam, Manuela Corti, Daniel Martin, Nicole Dobija, Maria V. Irwin, Thomas Conlon, Brian Cleaver, Nathalie Clement, and Shelley W. Collins.Phase I/II Trial of Diaphragm Delivery of Recombinant Adeno-Associated Virus Acid Alpha-Glucosidase (rAAV1-CMV-GAA) Gene Vector in Patients with Pompe Disease.Human Gene Therapy Clinical Development.Sep 2014.134-163.http://doi.org/10.1089/humc.2014.2514Published in Volume: 25 Issue 3: September 19, 2014PDF download

Pompe disease is a neuromuscular disorder due to mutations in the gene that encodes the acid alpha‐glucosidase (GAA) enzyme, which results in lysosomal glycogen accumulation in striated muscle and motor neurons. The severe infantile form leads to cardiac hypertrophy, respiratory failure and early mortality. An approved enzyme replacement therapy reduces the cardiomyopathy and increases survival, yet many patients eventually require mechanical ventilation (MV). Our objective is to investigate an alternative treatment, by correcting the defective gene in an open‐label, Phase I/II clinical study of AAV‐GAA therapy to the diaphragm. We hypothesized that retrograde transport of AAV‐GAA would promote diaphragm glycogen clearance to restore inspiratory motor function. To date, five children (ages 2‐15) with full‐time MV dependence have received intramuscular rAAV1‐CMV‐GAA delivery into the diaphragm and completed follow‐up ventilatory testing. Maximal pressure inspiratory generation did not improve one year after dosing, but maximum unassisted tidal volume increased significantly (median: 22% gain, range: 11‐77%). Moreover, all subjects generated improved spontaneous breathing without MV assistance (median: 586%, range: 192‐2901% improvement over baseline levels). We conclude these findings may be related to vector transduction properties, fiber atrophy from chronic MV, and properties of the postoperative muscle conditioning prescription.Grant Funding Source: K12 HD055929; CRB‐HLBI1‐S‐10‐00223

Pompe disease is a neuromuscular disease resulting from deficiency in acid a-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.

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.

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.

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.

Effective gene transfer with sustained gene expression is an important adjunct to the study of intestinal inflammation and future therapy in inflammatory bowel disease. Recombinant adeno-associated virus (AAV) vectors are ideal for gene transfer and long-term transgene expression. The purpose of our study was to identify optimal AAV pseudotypes for transduction of the epithelium in the small intestine and colon, which could be used for studies in experimental colitis. The tropism and transduction efficiencies of AAV pseudotypes 1-10 were examined in murine small intestine and colon 8 wk after administration by real-time PCR and immunohistochemistry. The clinical and histopathological effects of IL-10-mediated intestinal transduction delivered by AAVrh10 were examined in the murine IL-10⁻/⁻ enterocolitis model. Serum IL-10 levels and IL-10 expression were followed by ELISA and real-time PCR, respectively. AAV pseudotypes 4, 7, 8, 9, and 10 demonstrated optimal intestinal transduction. Transgene expression was sustained 8 wk after administration and was frequently observed in enteroendocrine cells. Long-term IL-10 gene expression and serum IL-10 levels were observed following AAV transduction in an IL-10-/- model of enterocolitis. Animals treated with AAVrh10-IL-10 had lower disease activity index scores, higher colon weight-to-length ratios, and lower microscopic inflammation scores. This study identifies novel AAV pseudotypes with small intestine and colon tropism and sustained transgene expression capable of modulating mucosal inflammation in a murine model of enterocolitis.

See also Miao CH. Tilt balance towards regulation: evolving new strategy for treatment of hemophilia inhibitors. This issue, pp 1521–3.DOI:10.1111/j.1538‐7836.2011.04351.x.

Barth's 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.

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-α. 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.