Mai K. ElMallah

Pompe disease gene therapy

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.

The respiratory neuromuscular system in Pompe disease.

Pompe disease is due to mutations in the gene encoding the lysosomal enzyme acid α-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.

Intrapleural Administration of AAV9 Improves Neural and Cardiorespiratory Function in Pompe Disease

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

Sustained Correction of Motoneuron Histopathology Following Intramuscular Delivery of AAV in Pompe Mice

Pompe disease is an autosomal recessive disorder caused by mutations in the acid-α glucosidase (GAA) gene. Lingual dysfunction is prominent but does not respond to conventional enzyme replacement therapy (ERT). 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 threefold more GAA-positive XII motoneurons (P < 0.02 versus AAV1); the AAV9 group also gained more body weight over the course of the study (P < 0.05 versus 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.

Hypoglossal neuropathology and respiratory activity in Pompe mice

Pompe disease is a lysosomal storage disorder associated with systemic deficiency of acid α-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 μm) 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 < 0.01). Vagotomy caused a robust increase in XII inspiratory burst amplitude in B6/129 mice (239 ± 44% baseline; p < 0.01) but had little impact on burst amplitude in Gaa−/− mice (130 ± 23% baseline; 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.

Adeno-associated virus–delivered artificial microRNA extends survival and delays paralysis in an amyotrophic lateral sclerosis mouse model

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of motor neurons, resulting in progressive muscle weakness, paralysis, and death within 5 years of diagnosis. About 10% of cases are inherited, of which 20% are due to mutations in the superoxide dismutase 1 (SOD1) gene. Riluzole, the only US Food and Drug Administration–approved ALS drug, prolongs survival by only a few months. Experiments in transgenic ALS mouse models have shown decreasing levels of mutant SOD1 protein as a potential therapeutic approach. We sought to develop an efficient adeno‐associated virus (AAV)‐mediated RNAi gene therapy for ALS.

AAV delivered artificial microRNA extends survival and delays paralysis in an Amyotrophic Lateral Sclerosis mouse model

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of motor neurons, resulting in progressive muscle weakness, paralysis, and death within 5 years of diagnosis. About 10% of cases are inherited, of which 20% are due to mutations in the superoxide dismutase 1 (SOD1) gene. Riluzole, the only US Food and Drug Administration–approved ALS drug, prolongs survival by only a few months. Experiments in transgenic ALS mouse models have shown decreasing levels of mutant SOD1 protein as a potential therapeutic approach. We sought to develop an efficient adeno‐associated virus (AAV)‐mediated RNAi gene therapy for ALS.

Retrograde Gene Delivery to Hypoglossal Motoneurons Using Adeno-Associated Virus Serotype 9

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 β-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 β subunit, CT-β) 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-β.

Therapeutic rAAVrh10 Mediated SOD1 Silencing in Adult SOD1(G93A) Mice and Nonhuman Primates.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease; survival in ALS is typically 3–5 years. No treatment extends patient survival by more than three months. Approximately 20% of familial ALS and 1–3% of sporadic ALS patients carry a mutation in the gene encoding superoxide dismutase 1 (SOD1). In a transgenic ALS mouse model expressing the mutant SOD1G93A protein, silencing the SOD1 gene prolongs survival. One study reports a therapeutic effect of silencing the SOD1 gene in systemically treated adult ALS mice; this was achieved with a short hairpin RNA, a silencing molecule that has raised multiple safety concerns, and recombinant adeno-associated virus (rAAV) 9. We report here a silencing method based on an artificial microRNA termed miR-SOD1 systemically delivered using adeno-associated virus rAAVrh10, a serotype with a demonstrated safety profile in CNS clinical trials. Silencing of SOD1 in adult SOD1G93A transgenic mice with this construct profoundly delayed both disease onset and death in the SOD1G93A mice, and significantly preserved muscle strength and motor and respiratory functions. We also document that intrathecal delivery of the same rAAVrh10-miR-SOD1 in nonhuman primates significantly and safely silences SOD1 in lower motor neurons. This study supports the view that rAAVrh10-miR-SOD1 merits further development for the treatment of SOD1-linked ALS in humans.

Peripheral nerve and neuromuscular junction pathology in Pompe disease

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.