Thesis advisor: Thomas N. Seyfried / Sandhoff Disease (SD) is an autosomal recessive neurodegenerative disease caused by a mutation in the Hexb gene for the β-subunit of β-hexosaminidase A, resulting in the inability to catabolize ganglioside GM2 within the lysosomes. SD presents with an accumulation of GM2 and its asialo derivative GA2 primarily in the CNS. Myelin-enriched glycolipids, cerebrosides and sulfatides, are also decreased in SD corresponding with dysmyelination. At present, no treatment exists for SD. Previous studies have shown the therapeutic benefit of using adeno-associated virus (AAV) vector-mediated gene therapy in the treatment of SD in murine and feline models. In this study, CNS tissue was evaluated from SD cats (4-6 week old) treated with bilateral injections of AAVrh8 expressing feline β-hexosaminidase α and β into the thalamus and deep cerebellar nuclei (Thal/DCN) or into the thalamus combined with intracerebroventricular injections (Thal/ICV). Both groups of treated animals had previously shown improved quality of life and absence of whole-body tremors. The activity of β-hexosaminidase was significantly elevated whereas the content of GM2 and GA2 was significantly decreased in tissue samples taken from the cerebral cortex, cerebellum, thalamus, and cervical intumescence. Treatment also increased levels of myelin-enriched cerebrosides and sulfatides in the cortex and thalamus. This study demonstrates the therapeutic benefits of AAV treatment for feline SD and suggests a similar potential for human SD patients. / Thesis (MS) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Sandhoff disease (SD), a fatal lysosomal storage disease, results from a deficiency of the β-subunit of the β-hexosaminidase A and B enzymes. This deficiency leads to severe accumulation of GM2 gangliosides in lysosomes within the central nervous system (CNS) resulting in mass neuronal apoptosis. The mouse model of SD shows progressive neurodegeneration that closely resembles Sandhoff and Tay Sachs disease (TSD) in humans. SD and TSD consist of infantile, juvenile, and late-onset forms. These diseases can present with a multiplicity of symptoms including cognitive and speech impairments, ataxia, and lower motor neuron disease. Late-onset SD and TSD show motor neuron disease in over 40% of patients. In this study, we explore the role of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in the spinal cord during the development and progression of disease in Sandhoff mice. Using immunocytochemistry and western blotting, we analyzed the expression level and localization of several ER stress and cellular apoptosis markers within the cervical, thoracic, and lumbar regions of the spinal cord of Sandhoff mice. Our results revealed significant upregulation of several ER stress markers in motor neurons that appeared to coincide with significant lysosomal accumulations. In addition, we observed sequential and age-dependent expression changes in ATF6 and CHOP and their prominent nuclear localization within anterior horn motor neurons. Markers of apoptosis, caspases and PARP also appeared to be activated in the spinal cords of Sandhoff mice starting as early as 60 days. Interestingly, we noted more than 50% reduction in neuronal numbers in all regions of the spinal cord of Sandhoff mice between ages 80 and 120 days. Overall, this study provides strong evidence for the role of chronic ER stress and UPR activation in the spine pathophysiology of SD. / Thesis / Master of Science (MSc) / Lysosomal storage diseases are a rare group of inherited neurological disorders that are often fatal at a young age. Two diseases that fall within this category, Sandhoff and Tay Sachs disease, are similar in their cause and symptoms. Current research lacks a complete understanding of the mechanism behind these disorders making the development of new therapeutics challenging. This research highlights a group of cells in the spine that are vulnerable in these diseases. These cells show physical and functional changes in their structure as the diseases progress. We provide evidence of a new stress pathway which appears to be strongly implicated in the development and progression of these diseases. We also show an association between this pathway and the death of these vulnerable cells leading to the symptoms exhibited by patients. These findings expand our current knowledge of these disorders and open new avenues for therapeutic interventions.

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.

GM2 gangliosidoses are a family of lysosomal storage disorders that include both Tay-Sachs and Sandhoff diseases. These disorders result from deficiencies in the lysosomal enzyme β-N-acetylhexosaminidase (HexA). Impairment of HexA leads to accumulation of its substrate, GM2 ganglioside, in cells resulting in cellular dysfunction and death. There is currently no treatment for GM2 gangliosidoses. Patients primarily present with neurological dysfunction and degeneration. Here we developed a central nervous system gene therapy through direct injection that leads to long-term survival in the Sandhoff disease mouse model. We deliver an equal mixture of AAVrh8 vectors that encode for the two subunits (α and β) of HexA into the thalami and lateral ventricle. This strategy has also been shown to be safe and effective in treating the cat model of Sandhoff disease. We tested the feasibility and safety of this therapy in non-human primates, which unexpectedly lead to neurotoxicity in the thalami. We hypothesized that toxicity was due to high overexpression of HexA, which dose reduction of vector could not compensate for. In order to maintain AAV dose, and therefore widespread HexA distribution in the brain, six new vector designs were screened for toxicity in nude mice. The top three vectors that showed reduction of HexA expression with low toxicity were chosen and tested for safety in non-human primates. A final formulation was chosen from the primate screen that showed overexpression of HexA with minimal to no toxicity. Therapeutic efficacy studies were performed in Sandhoff disease mice to define the minimum effective dose.

<p><strong>Abstract</strong></p> <p>Sandhoff disease (SD) is a monogenic lysosomal storage disorder caused by a lack of a functional β-subunit of the beta-hexosaminidase A and B enzymes. The clinical phenotype of <em>Hexb</em>^-/-mouse model recapitulates the symptoms and signs of Tay-Sachs and Sandhoff diseases in human. To gain insight into the neuropathology of Sandhoff disease, we defined the role of TNFα in the development and progression of Sandhoff disease pathology in mice, by generating a <em>Hexb^-/-Tnf</em><em>a</em><em>^-/-</em> double knock-out mouse. Behavioural testing and immunostaining data revealed the neurodegenerative role of TNFα in disease pathology. Double knock-out mice showed ameliorated clinical course, with prolonged life span. TNFα-deficient Sandhoff mice also demonstrate decreased levels of astrogliosis, and reduced neuronal cell death. Deletion of <em>Tnfα</em> in Sandhoff mice inhibited JAK2/STAT3 pathway, implicating its role in glia cell activation. This result points to TNFa as a potential therapeutic target to attenuate neuro-pathogenesis.</p> <p>To investigate whether blood-derived or CNS-derived TNFα has the major impact on neurological function, we transplanted <em>Hexb^-/-Tnfα^+/+</em> with bone marrow from either <em>Hexb^-/-Tnfα^-/-</em>or <em>Hexb^-/-Tnf</em><em>a</em><em>^+/+</em> mice donors. Neurological tests shows a significant clinical improvement for Hexb<em>^-/-Tnfα^-/-</em> compared to <em>Hexb^-/-Tnf</em><em>a</em><em>^+/+</em> recipient, regardless the genotype of donor cells. These findings highlight the importance of resident-derived TNFα during the robust neurodegenerative consequences in Sandhoff disease. To understand of the role of microRNAs in Sandhoff pathology, we investigated the miRNA profile in Sandhoff brains. A pattern of dys-regulated microRNAs was evident in Sandhoff CNS. Microarray identified miR-210 and miR-96 dys-regulated pattern in the CNS of Sandhoff mice. Strikingly, neuronal pentraxin, a putative target gene for miR-210, was induced in Sandhoff brains.</p> <p>Taken together, this work establishes the proinflammatory role of TNFα in Sandhoff pathology, leading to massive neuro-apoptosis. Importantly, our studies propose that neuronal pentraxin as a novel target gene for microRNA-210 in Sandhoff brain samples, providing a potential modulator of neurodegeneration.</p> / Doctor of Philosophy (PhD)