The infantile GM2 gangliosidoses are severe neurodegenerative disorders, caused by a defect in the β-hexosaminidase system. They are characterized by lysosomal accumulation of the substrate, GM2 ganglioside, which results in severe neuronal damage and death in the early years of life. Sandhoff mice deficient in both major hexosaminidase isozymes, Hex A and Hex B, mimic the disease severity in the human condition including the motor deterioration, histopathological findings, and premature death. To investigate the utility of systemic adeno-associated virus 9 (AAV9)-based gene delivery in treating GM2 gangliosidoses, we evaluated the therapeutic outcome of a single intravenous injection of recombinant AAV9 encoding the complementing Hexb gene in a Sandhoff mouse model. We showed prolonged survival, preserved motor function, and reduced GM2 ganglioside accumulation as well as inflammation when systemic AAV9 therapy was administered to 1-2 days old mice. However, the formation of liver or lung tumours accompanied the positive therapeutic effect.

La maladie de Sandhoff est une maladie génétique rare due à des mutations du gène HEXB. Elle se caractérise par un double déficit en hexosaminidase A (αβ) et B (ββ), responsable d’une accumulation de ganglioside GM2 essentiellement dans le système nerveux central (SNC). Cliniquement, la maladie débute dès les premiers mois de vie et le décès survient vers l’âge de 3 ans. A ce jour, aucun traitement n’est disponible pour cette maladie. Le modèle murin obtenu par invalidation du gène Hexb est un bon outil pour le développement d’approches thérapeutiques, car il présente un phénotype proche de la maladie humaine. Le but principal de mon projet de thèse était d’explorer une approche de transfert de gène dans le modèle murin de la maladie de Sandhoff en utilisant un vecteur scAAV9. Ce vecteur a la particularité de pouvoir traverser la barrière hématoencéphalique et de transduire le SNC après administration intraveineuse (IV). Un vecteur codant la chaîne β des hexosaminidases, appelé scAAV9-Hexb, a précédemment été administré par voie IV à des souris en période néonatale à une dose de 3,5 x 1013 vg/kg. Les souris traitées ont survécu comme les souris normales (>700 jours) sans développer d’atteinte neurologique, ni périphérique alors que les souris Sandhoff non traitées sont décédées vers l’âge de 4 mois. J’ai réalisé toutes les analyses à long terme des souris traitées en utilisant des tests de comportements, ainsi que des analyses tissulaires 24 mois après le traitement. Une analyse lipidique par HPTLC a montré que la surcharge en ganglioside GM2 est totalement absente au niveau du cerveau (4 mois après l'injection), alors que dans le cervelet cette accumulation est non significative, mais pas totalement absente. Aucun symptôme lié à cette surcharge n’a été mis en évidence chez les souris à 24 mois, mais nous nous sommes posé la question d’un possible effet délétère à long terme en cas d’extrapolation à la clinique. Nous avons donc décidé de tester une double administration IV + ICV (intracérébroventriculaire) en utilisant le même vecteur et la même dose globale de façon à mieux corriger le cervelet. Deux groupes de souris ont été injectés en période néonatale en utilisant des doses différentes dans les deux compartiments. Les analyses ont montré que dans le cerveau, à court terme, la restauration de l’activité enzymatique est partielle, mais significative. Par ailleurs, il existe une absence totale de surcharge en GM2, ainsi qu’une correction des biomarqueurs associés à la maladie. Dans le cervelet, l’efficacité du traitement a été montrée seulement pour le groupe traité avec la dose la plus importante en ICV, ce qui suggère qu’une dose minimale en ICV est nécessaire pour atteindre de manière globale le SNC. Ces résultats ont été confirmés par l’analyse à long terme. Concernant le foie, nos résultats ont montré qu’une dose IV minimale est nécessaire pour obtenir une baisse de l’accumulation lipidique. Ce travail a permis de définir les doses minimales nécessaires dans chaque compartiment (IV et ICV) et il montre que la double administration peut être avantageuse pour traiter toutes les régions du SNC et notamment les plus atteintes, comme le cervelet. Il va maintenant nous permettre de traiter de façon optimale les souris adultes. L’autre but de mon projet était d’explorer les défauts de signalisation et la physiopathologie cellulaire dans la maladie de Sandhoff en utilisant des études in vivo et in vitro. Les études in vitro ont été réalisées sur des fibroblastes de patients et des cellules embryonnaires murines (MEF) obtenues à partir des souris Hexb-/- et la surcharge lysosomale a été confirmée dans ces cellules. La voie mTOR (mammalian target of rapamycin) a été analysée et nous avons montré qu’elle était dérégulée. L’activité autophagique a aussi été étudiée et nous avons mis en évidence une augmentation du nombre d’autophagosomes chez les souris Hexb-/- suggérant un défaut de cette voie. (...) / Sandhoff disease (SD) is a genetic disorder due to mutations in the HEXB gene. It is characterized by a double Hex A (αβ) and B (ββ) deficiency, responsible for a GM2 accumulation, mainly in the central nervous system (CNS). Clinically, SD begins in the first months of life and culminates in death around 3 years of age. So far, no specific treatment is available for Sandhoff disease. The murine model obtained by invalidation of the Hexb gene is a useful tool for the development of therapeutic approaches, as it exhibits a phenotype quite close to the human disease. The main aim of my PhD project was to explore a gene transfer approach in Sandhoff mice using a specific scAAV9. This vector has the particularity to cross the blood-brain barrier after intravenous (IV) administration and to transduce brain. A vector encoding the hexosaminidases β chain, called scAAV9-Hexb, has been previously IV injected in neonatal Hexb-/- mice with a dose of 3.5 x 1013 vg/kg. I participated to the long-term analysis of the scAAV9-Hexb treated mice using behavioral tests and analysis of tissues at 24 months post-injection. Mice had a survival similar to normal mice (>700 days) without neurological sign and peripheral damage by comparison with naïve Sandhoff mice (death around 120 days). At 4 months post-treatment, lipid analysis using HPTLC showed that GM2 storage was absent in brain, but it was only decreased in cerebellum of treated mice. Even if no symptom was associated with this residual storage in mice at 2 years, we wondered if it could possibly be pathogenic at longer-term if extrapolated to patients. Therefore, we decide to test a combined way of administration i.e. intravenous (IV) + intracerebroventricular (ICV) using the same vector with the same final dose. Two groups of mice were injected using different doses in both compartments and treatment efficacy was evaluated at short- and long-term. In the cerebrum, at short-term, enzymatic activities were partially but significantly restored, GM2 accumulation was completely prevented and disease biomarkers corrected. In the cerebellum, a significant increase of enzymatic activity was only obtained for the group treated with the highest dose in the ICV compartment. Regarding GM2 analysis and long-term behavioral analysis, we confirmed that this dose is required to cure cerebellum. In liver, our results suggest that IV minimal dose is needed to obtain a decrease of lipid accumulation. Our results showed that minimal doses are required in ICV and IV to obtain a good efficacy in each compartments, and that combined administration permit a widespread correction in the CNS. These data will permit to treat adult mice with the optimal treatment. The other goal of my project was to explore signaling defects and cellular pathophysiology in Sandhoff disease using in vivo and in vitro studies. For in vitro studies, fibroblasts from Tay-Sachs and Sandhoff patients were analyzed and mouse embryonic fibroblasts (MEF) were obtained from the Hexb-/- murine model, lysosomal storage was confirmed. mTOR (mammalian target of rapamycin) pathway was studied showing signaling deregulation. Autophagy was analyzed in vitro and in vivo, as defect in this pathway has been reported in other lysosomal storage disorders. An increase of autophagosomes number was observed in Hexb-/- subjects suggesting a defect in autophagy. These results offer novel biomarkers of Sandhoff pathology which can be useful to test the efficacy of therapeutic approaches. They can also provide new therapeutic targets that could be tested in combination with gene transfer.

In my dissertation research we used use human wild type α-synuclein gene expression using an adeno-associated viral vector (AAV9) that induced a slowly progressive loss of dopamine (DA) neurons in the Substantia nigra (SN) as one of our animal model of Parkinson’s disease (PD). It is our hypothesis that neuroinflammation predisposes the brain to susceptibility to neurodegenerative diseases. Thus we examined the progression of a PD lesion and examined the manipulations of the immune system to understand further the inflammatory role when we administered exogenous soluble fractalkine. The specific etiology of neurodegeneration in PD is unknown, but the inflammatory mechanisms and free radicals have been postulated to play a central role. α-synuclein is believed to be the one of the main characteristic associated with PD. It has been found inside saclike structures, called lewy bodies. α-synuclein is believed to activate resident microglia worsening the degeneration of the nigrostriatal pathway due to its aggregation. Aggregation increases the production of reactive oxygen species (ROS) released from microglia. The constant release of these factors and prolonged activation of microglia could be the cause that leads to neurodegeneration in the SN. Spirulina, a blue - green algae, has been shown to have anti-oxidant and anti-inflammatory properties. For example, when rats received an intrastriatal injection of 6-OHDA and were fed a spirulina enriched diet for 4 weeks, there was a significant increase in regeneration of DA terminals into the Tyrosine Hydroxylase (TH) -negative zone of the striatum. This regeneration was accompanied by a decrease in microglia activation as determined by immunohistochemistry of major histo compatibility class II (MHC) (OX-6). This suggests that decreases in microglia activation modulate the beneficial effects of spirulina. Another important therapeutic tool we used was fractalkine as an anti-inflammatory treatment. It is known that fractalkine levels are reduced in the brain during aging. For this reason we administered exogenous fractalkine to 6-OHDA model of PD to test the hypothesis that it improved the microenvironment by reducing microglial activation.

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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of motor neurons, resulting in progressive muscle weakness, atrophy, paralysis and death within five years of diagnosis. About ten percent of cases are inherited, of which twenty percent are due to mutations in the superoxide dismutase 1 (SOD1) gene. Since the only FDA approved ALS drug prolongs survival by just a few months, new therapies for this disease are needed. Experiments in transgenic ALS mouse models have shown that decreasing levels of mutant SOD1 protein alters and in some cases entirely prevents disease progression. We explored this potential therapeutic approach by using a single stranded AAV9 vector encoding an artificial microRNA against human SOD1 injected bilaterally into the cerebral lateral ventricles of neonatal SOD1G93A mice. This therapy extended median survival from 135 to 206 days (a 50% increase) and delayed hind limb paralysis. Animals remained ambulatory until endpoint, as defined by a sharp drop in body weight. Treated animals had a reduction of mutant human SOD1 mRNA levels in upper and lower motor neurons. As compared to untreated SOD1G93A mice, the AAV9 treated mice also had significant improvements in multiple parameters including the number of motor neurons, diameter of ventral root axons, and degree of neuroinflammation in the spinal cord. These studies clearly show that an AAV9-delivered artificial microRNA is a translatable therapeutic approach for ALS.

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Mutations in the SOD1 gene are the best characterized genetic cause of amyotrophic lateral sclerosis (ALS) and account for ~20% of inherited cases and 1-3% of sporadic cases. The gene-editing tool Cas9 can silence mutant genes that cause disease, but effective delivery of CRISPR-Cas9 to the central nervous system (CNS) remains challenging. Here, I developed strategies using canonical Streptococcus pyogenes Cas9 to silence SOD1. In the first strategy, I demonstrate effectiveness of systemic delivery of guide RNA targeting SOD1 to the CNS in a transgenic mouse model expressing human mutant SOD1 and Cas9. Silencing was observed in both the brain and the spinal cord. In the second strategy, I demonstrate the effectiveness of delivering both guide RNA and Cas9 via two AAVs into the ventricles of the brain of SOD1G93A mice. Silencing was observed in the brain and in motor neurons within the spinal cord. For both strategies, treated mice had prolonged survival when compared to controls. Treated mice also had improvements in grip strength and rotarod function. For ICV treated mice, we detected a benefit of SOD1 silencing using net axonal transport assays, a novel method to detect motor neuron function in mice before onset of motor symptoms. These studies demonstrate that Cas9-mediated genome editing can mediate disease gene silencing in motor neurons and warrants further development for use as a therapeutic intervention for SOD1-linked ALS patients.