Systemic AAV vectors for widespread and targeted gene delivery in rodents

We recently developed adeno-associated virus (AAV) capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. However, a detailed protocol for generating and systemically delivering novel AAV variants was not previously available. In this protocol, we describe how to produce and intravenously administer AAVs to adult mice to specifically label and/or genetically manipulate cells in the nervous system and organs, including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans 8 d, excluding the time required to assess gene expression, and can be readily adopted by researchers with basic molecular biology, cell culture, and animal work experience. We provide guidelines for experimental design and choice of the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing

Etude clinico-pathologique de l'audition chez un modèle canin de mucopolysaccharidose de type 1

La mucopolysaccharidose de type 1 (MPS 1) provoque chez l'enfant une surdité mixte dont la pathogénie est peu connue. Dans un modèle canin de MPS 1 nous avons 1/ étudié les lésions histopathologiques de l'oreille et des voies nerveuses auditives chez 10 chiens MPS 1, 2/ réalisé un suivi auditif par mesure des potentiels évoqués auditifs (PEA) pendant 4 mois chez 3 chiens MPS 1, et 3/ recherché une corrélation entre les lésions et les éventuelles modifications des PEA. L'oreille des chiens MPS 1 présente une surcharge cellulaire identique à celle rapportée chez l'enfant, validant ce modèle pour l'exploration de la fonction auditive. Les voies nerveuses auditives présentent une atteinte sélective avec une susceptibilité accrue apparente des neurones gabaergiques. L'analyse des PEA montre une surdité progressive principalement rétrocochléaire corrélée aux lésions du tronc cérébral, permettant d'envisager l'utilisation des PEA comme suivi clinique non invasif.NANTES-BU Médecine pharmacie (441092101) / SudocTOULOUSE-EN Vétérinaire (315552301) / SudocSudocFranceF

Měření a monitorování vybraných procesů dle ISO 9000:2000

Import 20/04/2006Prezenční výpůjčkaVŠB - Technická univerzita Ostrava. Ekonomická fakulta. Katedra (152) podnikohospodářsk

Colorectal Hamartomatous Polyposis and Ganglioneuromatosis in a Dog

International audienceA 5-month-old female Great Dane puppy was treated for hematochezia, tenesmus, and rectal prolapse by resection of a 10-cm-long segment of colon and rectum. Grossly, the colorectal segment had diffuse mucosal and submucosal thickening with multiple polypoid nodules. The histologic diagnosis was colorectal hamartomatous polyps with ganglioneuromatosis. Duplication of PTEN was detected by quantitative multiplex polymerase chain reaction testing. The presence of 2 hamartomatous colorectal lesions with PTEN mutation is similar to human Cowden syndrome

Toxicology Study of Intra-Cisterna Magna Adeno-Associated Virus 9 Expressing Human Alpha-L-Iduronidase in Rhesus Macaques

Mucopolysaccharidosis type I is a recessive genetic disease caused by deficiency of the lysosomal enzyme α-L-iduronidase, which leads to a neurodegenerative and systemic disease called Hurler syndrome in its most severe form. Several clinical trials are evaluating adeno-associated virus serotype 9 (AAV9) for the treatment of neurodegenerative diseases. Although these trials focus on systemic or lumbar administration, intrathecal administration via suboccipital puncture into the cisterna magna has demonstrated remarkable efficacy in large animals. We, therefore, conducted a good laboratory practice-compliant non-clinical study to investigate the safety of suboccipital AAV9 gene transfer of human α-L-iduronidase into nonhuman primates. We dosed 22 rhesus macaques, including three immunosuppressed animals, with vehicle or one of two doses of vector. We assessed in-life safety and immune responses. Animals were euthanized 14, 90, or 180 days post-vector administration and evaluated for histopathology and biodistribution. No procedure-related lesions or adverse events occurred. All vector-treated animals showed a dose-dependent mononuclear pleocytosis in the cerebrospinal fluid and minimal to moderate asymptomatic degeneration of dorsal root ganglia neurons and associated axons. These studies support the clinical development of suboccipital AAV delivery for Hurler syndrome and highlight a potential sensory neuron toxicity that warrants careful monitoring in first-in-human studies. Keywords: AAV9, intrathecal, MPS

Safe and Sustained Expression of Human Iduronidase After Intrathecal Administration of Adeno-Associated Virus Serotype 9 in Infant Rhesus Monkeys

Many neuropathic diseases cause early, irreversible neurologic deterioration, which warrants therapeutic intervention during the first months of life. In the case of mucopolysaccharidosis type I, a recessive lysosomal storage disorder that results from a deficiency of the lysosomal enzyme α-l-iduronidase (IDUA), one of the most promising treatment approaches is to restore enzyme expression through gene therapy. Specifically, administering pantropic adeno-associated virus (AAV) encoding IDUA into the cerebrospinal fluid (CSF) via suboccipital administration has demonstrated remarkable efficacy in large animals. Preclinical safety studies conducted in adult nonhuman primates supported a positive risk-benefit profile of the procedure while highlighting potential subclinical toxicity to primary sensory neurons located in the dorsal root ganglia (DRG). This study investigated the long-term performance of intrathecal cervical AAV serotype 9 gene transfer of human IDUA administered to 1-month-old rhesus monkeys (N = 4) with half of the animals tolerized to the human transgene at birth via systemic administration of an AAV serotype 8 vector expressing human IDUA from the liver. Sustained expression of the transgene for almost 4 years is reported in all animals. Transduced cells were primarily pyramidal neurons in the cortex and hippocampus, Purkinje cells in the cerebellum, lower motor neurons, and DRG neurons. Both tolerized and non-tolerized animals were robust and maintained transgene expression as measured by immunohistochemical analysis of brain tissue. However, the presence of antibodies in the non-tolerized animals led to a loss of measurable levels of secreted enzyme in the CSF. These results support the safety and efficiency of treating neonatal rhesus monkeys with AAV serotype 9 gene therapy delivered into the CSF