Long-term neurologic and cardiac correction by intrathecal gene therapy in Pompe disease.

Pompe disease is a lysosomal storage disorder caused by acid-α-glucosidase (GAA) deficiency, leading to glycogen storage. The disease manifests as a fatal cardiomyopathy in infantile form. Enzyme replacement therapy (ERT) has recently prolonged the lifespan of these patients, revealing a new natural history. The neurologic phenotype and the persistence of selective muscular weakness in some patients could be attributed to the central nervous system (CNS) storage uncorrected by ERT. GAA-KO 6neo/6neo mice were treated with a single intrathecal administration of adeno-associated recombinant vector (AAV) mediated gene transfer of human GAA at 1 month and their neurologic, neuromuscular, and cardiac function was assessed for 1 year. We demonstrate a significant functional neurologic correction in treated animals from 4 months onward, a neuromuscular improvement from 9 months onward, and a correction of the hypertrophic cardiomyopathy at 12 months. The regions most affected by the disease i.e. the brainstem, spinal cord, and the left cardiac ventricular wall all show enzymatic, biochemical and histological correction. Muscle glycogen storage is not affected by the treatment, thus suggesting that the restoration of muscle functionality is directly related to the CNS correction. This unprecedented global and long-term CNS and cardiac cure offer new perspectives for the management of patients.

Efficient CNS targeting in adult mice by intrathecal infusion of single-stranded AAV9-GFP for gene therapy of neurological disorders.

Adeno-associated virus (AAV) gene therapy constitutes a powerful tool for the treatment of neurodegenerative diseases. While AAVs are generally administered systemically to newborns in preclinical studies of neurological disorders, in adults the maturity of the blood-brain barrier (BBB) must be considered when selecting the route of administration. Delivery of AAVs into the cerebrospinal fluid (CSF) represents an attractive approach to target the central nervous system (CNS) and bypass the BBB. In this study, we investigated the efficacy of intra-CSF delivery of a single-stranded (ss) AAV9-CAG-GFP vector in adult mice via intracisternal (iCist) or intralumbar (it-Lumb) administration. It-Lumb ssAAV9 delivery resulted in greater diffusion throughout the entire spinal cord and green fluorescent protein (GFP) expression mainly in the cerebellum, cortex and olfactory bulb. By contrast, iCist delivery led to strong GFP expression throughout the entire brain. Comparison of the transduction efficiency of ssAAV9-CAG-GFP versus ssAAV9-SYN1-GFP following it-Lumb administration revealed widespread and specific GFP expression in neurons and motoneurons of the spinal cord and brain when the neuron-specific synapsin 1 (SYN1) promoter was used. Our findings demonstrate that it-Lumb ssAAV9 delivery is a safe and highly efficient means of targeting the CNS in adult mice.

Efficient central nervous system AAVrh10-mediated intrathecal gene transfer in adult and neonate rats.

Intracerebral administration of recombinant adeno-associated vector (AAV) has been performed in several clinical trials. However, delivery into the brain requires multiple injections and is not efficient to target the spinal cord, thus limiting its applications. To assess widespread and less invasive strategies, we tested intravenous (IV) or intrathecal (that is, in the cerebrospinal fluid (CSF)) delivery of a rAAVrh10-egfp vector in adult and neonate rats and studied the effect of the age at injection on neurotropism. IV delivery is more efficient in neonates and targets predominantly Purkinje cells of the cerebellum and sensory neurons of the spinal cord and dorsal root ganglia. A single intra-CSF administration of AAVrh10, single strand or oversized self-complementary, is efficient for the targeting of neurons in the cerebral hemispheres, cerebellum, brainstem and spinal cord. Green fluorescent protein (GFP) expression is more widespread in neonates when compared with adults. More than 50% of motor neurons express GFP in the three segments of the spinal cord in neonates and in the cervical and thoracic regions in adults. Neurons are almost exclusively transduced in neonates, whereas neurons, astrocytes and rare oligodendrocytes are targeted in adults. These results expand the possible routes of delivery of AAVrh10, a serotype that has shown efficacy and safety in clinical trials concerning neurodegenerative diseases.

Chronic dietary exposure of zebrafish to PAH mixtures results in carcinogenic but not genotoxic effects.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants that can be present at high levels as mixtures in polluted aquatic environments. Many PAHs are potent mutagens and several are well-known carcinogens. Despite numerous studies on individual compounds, little is known about the toxicity of PAHs mixtures that are encountered in environmental situations. In the present work, zebrafish were continuously fed from 5 days post-fertilisation to 14 months post-fertilisation (mpf) with a diet spiked with fractions of either pyrolytic (PY), petrogenic light oil (LO), or petrogenic heavy oil (HO) origin at three concentrations. A decrease in survival was identified after 3 mpf in fish fed with the highest concentration of HO or LO, but not for PY. All PAH fractions caused preneoplastic and neoplastic disorders in long-term-exposed animals. Target tissues were almost exclusively of epithelial origin, with the bile duct epithelium being the most susceptible to chronic exposure to all PAH fractions, and with germ cells being the second most responsive cells. Significantly higher incidences of neoplasms were observed with increasing PAH concentration and exposure duration. The most severe carcinogenic effects were induced by dietary exposure to HO compared to exposure to LO or PY (45, 30 and 7 %, respectively, after 9 to 10 months of exposure to an intermediate concentration of PAHs). In contrast, earliest carcinogenic effects were detected as soon as 3 mpf after exposure to LO, including the lowest concentration, or to PY. PAH bioactivation and genotoxicity in blood was assessed by ethoxyresorufin-O-deethylase activity quantification and comet and micronuclei assays, respectively, but none of these were positive. Chronic dietary exposure of zebrafish to PAH mixtures results in carcinogenotoxic events that impair survival and physiology of exposed fish.

Intracisternal delivery of AAV9 results in oligodendrocyte and motor neuron transduction in the whole central nervous system of cats.

Systemic and intracerebrospinal fluid delivery of adeno-associated virus serotype 9 (AAV9) has been shown to achieve widespread gene delivery to the central nervous system (CNS). However, after systemic injection, the neurotropism of the vector has been reported to vary according to age at injection, with greater neuronal transduction in newborns and preferential glial cell tropism in adults. This difference has not yet been reported after cerebrospinal fluid (CSF) delivery. The present study analyzed both neuronal and glial cell transduction in the CNS of cats according to age of AAV9 CSF injection. In both newborns and young cats, administration of AAV9-GFP in the cisterna magna resulted in high levels of motor neurons (MNs) transduction from the cervical (84±5%) to the lumbar (99±1%) spinal cord, demonstrating that the remarkable tropism of AAV9 for MNs is not affected by age at CSF delivery. Surprisingly, numerous oligodendrocytes were also transduced in the brain and in the spinal cord white matter of young cats, but not of neonates, indicating that (i) age of CSF delivery influences the tropism of AAV9 for glial cells and (ii) AAV9 intracisternal delivery could be relevant for both the treatment of MN and demyelinating disorders.

Length variations in the NA stalk of an H7N1 influenza virus have opposite effects on viral excretion in chickens and ducks.

A deletion of ∼20 amino acids in the stalk of neuraminidase is frequently observed upon transmission of influenza A viruses from waterfowl to domestic poultry. A pair of recombinant H7N1 viruses bearing either a short- or long-stalk neuraminidase was genetically engineered. Inoculation of the long-stalk-neuraminidase virus resulted in a higher cloacal excretion in ducks and led conversely to lower-level oropharyngeal excretion in chickens, associated with a higher-level local immune response and better survival. Therefore, a short-stalk neuraminidase is a determinant of viral adaptation and virulence in chickens but is detrimental to virus replication and shedding in ducks.